Molecular design, pharmacology and toxicology optimization of antimicrobial peptide from Hydrophis cyanocinctus, Hc-CATH / 生物工程学报

Based on the cathelicidin family antimicrobial peptide Hc-CATH derived from sea snake, the Hc-16 and Hc-15 of 16 and 15 amino acid residues, were designed. By using CCK8, minimal inhibitory concentration, ELISA and bio-layer interferometry assays, their cytotoxicity, antibacterial activity, anti-inflammatory activity, and LPS neutralization activity was examined. Compared with Hc-15, Hc-16 had lower cytotoxicity and better broad-spectrum antibacterial activity against pathogens including clinically resistant bacteria, with the minimum inhibitory concentration of only 4.69 μg/mL. Hc-16 inhibited the expression of inflammatory cytokines of TNF-α and IL-6 induced by LPS, so as to significantly reduce the inflammatory response induced by infection. In addition, structure-activity relationship studies have shown that the phenylalanine at the C- and N-terminals of Hc-16 played a crucial role in its antibacterial and anti-inflammatory activity. Altogether, the designed Hc-16 has an excellent prospect to be developed into a novel antibiotic.

Analysis of the efficacy of non-invasive prenatal testing in the system of prenatal screening and diagnosis / 中华医学遗传学杂志

OBJECTIVE@#To evaluate the efficacy of non-invasive prenatal testing (NIPT) in the prenatal screening and its role in the system of prenatal diagnosis.@*METHODS@#A total of 22 649 singleton pregnant women who were registered and finally delivered or had induced labor at Beijing Obstetrics and Gynecology Hospital of Capital Medical University were enrolled. The routes of prenatal screening were analyzed to evaluate the efficacy of prenatal screening. Meanwhile, 9268 pregnant women who underwent invasive prenatal diagnosis procedure were enrolled. The indications and results of prenatal diagnosis were analyzed to evaluate the effectiveness of prenatal screening.@*RESULTS@#60.24% of singleton pregnant women have opted for Down syndrome screening, and their age was mainly under 35. The proportion of women opted for NIPT was 34.74%, and were mainly between 35 and 39. The overall diagnostic rate of trisomy 21, 18 and 13 trisomy for those with high risk by NIPT was 0.89%, which yielded a positive predictive value of 75.71%. For those with moderate risk by serum screening, 0.30% was predicted with a high risk by NIPT. Among women undergoing prenatal diagnosis, 63.04% and 21.22% had the indication of advanced age or high risk by serum screening, and the positive predictive values were 5.1% and 5.13%, respectively. By contrast, 2.30% of women undergoing prenatal diagnosis had a high risk by NIPT, which yielded a positive predictive value of 54.46%.@*CONCLUSION@#With the change of the age composition of pregnant women and increase in the complexity of pregnancy in China, to build a prenatal screening system based on NIPT will be helpful to improve the efficiency of the current system of prenatal screening and diagnosis.

Progress of research on clinical use of non-invasive prenatal screening for special groups of pregnant women / 中华医学遗传学杂志

As a prenatal testing for chromosomal abnormalities, non-invasive prenatal testing (NIPT) has been integrated into prenatal healthcare service. NIPT has shown a high sensitivity and specificity for screening fetal trisomies 13, 18 and 21, and has attained excellent clinical results. With the propagation of the NIPT screening, international organizations have issued guidelines and comments for its clinical utility with regular updating. China has also developed guidelines for NIPT in 2016. NIPT guidelines in various countries have provided valuable guidance for its target diseases and suitable patient groups, but there has been few research data on its clinical application for special groups of patients. Based on the guidelines and comments of various professional bodies and published data on the clinical utility of NIPT, in addition with consideration of the conditions in China, clinical utility of NIPT for particular groups of pregnant women, including those with advanced maternal age, obesity, twin pregnancy and fetal ultrasonographic anomalies, are reviewed. The value of genetic counseling for NIPT is also emphasized, which is critical for the clinical application of NIPT.

Safe dose of 650 nm low-power semiconductor laser irradiation in chicken retina / 中华实验眼科杂志

Objective:To observe whether there was a chronic light damage after the irradiation of 650 nm semiconductor laser (power 2 mW) in chicken cone-rich-retina and discuss the safety of this laser for retina.Methods:Sixty 1-month-old chicken reared under natural light were divided into a normal control group, an irradiation 3-min group, an irradiation 6-min group and an irradiation 30-min group by using a random number table and 15 for each group.The chicken eyes were irradiated with 650 nm laser for different duration according to a grouping.Relative retina area was measured with optical coherence tomography (OCT) at 1 month (2-month-old chicken), 3 months (4-month-old chicken) and 6 months (7-month-old chicken) after laser irradiation.Chickens were sacrificed by overdose anesthesia and the histopathology of chiken retina was examined by hematoxylin-eosin staining.The apoptosis of the retinal cells was evaluated by TUNEL staining.Chicken retinal homogenate was prepared, and the content of malondialdehyde and activity of superoxide dismutase (SOD) in the retina were detected by TBA method and NBT method, respectively.Western blot was employed to detect the expression of L/M opsin and rhodopsin in the retina.The use and care of the animals complied with Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.Results:In 2-month-old chicken, the molar concentration of malondialdehyde in retina was significantly higher in the irradiation 30-min group compared with the normal control group ( P<0.05). In 4-month-old chicken, the molar concentration of malondialdehyde was statistically higher in the irradiation 6-min group and the irradiation 30-min group in comparison with the normal control group ( P=0.026, 0.003). In 7-month-old chicken, the concentrations of retinal malondialdehyde in the irradiation 3-min group, irradiation 6-min group and irradiation 30-min group were statistically higher than those of the normal control group( P=0.038, 0.032, <0.01). In 7-month-old chicken, the SOD activity and the relative expression of rhodopsin in the retina of the irradiation 30-min group were statistically reduced incomparison with the normal control group (SOD: [140.20±5.99][nmol/s·mg] vs.[160.57±3.13][nmol/s·mg]); Rhodopsin: 0.392±0.065 vs.0.566±0.072) (both at P<0.05). OCT showed that there was no significant difference in relative retinal area within 6 months among the four groups.Histopathological examination showed that the thickness of the retina in each irradiation group was close to the normal control group.TUNEL staining showed that the retinal cells were regularly arranged, and no TUNEL positive staining cells were found in all of the groups. Conclusions:Irradiation of a 650 nm semiconductor laser (2 mW) in chicken's eyes for 6 minutes is safe for retina within 6 months.The lasser irradiation 30 minutes for 6 months results in an increase of free radical content in the retina and a decrease in rhodopsin, suggesting the presence of photo damage.

Exploration of the process of prenatal screening and diagnosis for women with advanced maternal age based on results of 351 cases of fetal chromosomal abnormalities / 中华医学遗传学杂志

OBJECTIVE@#To explore the suitable process for prenatal screening and diagnosis for women with advanced maternal age.@*METHODS@#From January 2014 to November 2017, the indications and distributions of prenatal diagnosis for women with advanced maternal age only or accompanying with positive maternal serum test screening and non-invasive prenatal testing (NIPT), abnormal fetal ultrasound, one harboring chromosomal abnormalities or anomalous reproductive history were analyzed. The rate of fetal chromosomal abnormalities was compared between different groups.@*RESULTS@#The 351 pregnant women with fetal chromosomal abnormalities have included 196 cases with advanced maternal age, 26 with positive maternal serum test, 96 with high-risk by NIPT, 14 with abnormal fetal ultrasound, 15 with one partner harboring chromosomal abnormalities, and 4 with anomalous reproductive history. Assuming that all pregnant women had undergone maternal serum test screening or NIPT without amniocentesis, the detection rate of fetal chromosome abnormality would be 51.0% and 69.2%, respectively. However, should these women have received both tests, the detection rate would be as high as 84.6%. Should those with one partner harboring chromosomal abnormalities undergone maternal serum test screening or NIPT without amniocentesis, the detection rate of fetal chromosomal abnormality would only be 6.7%.@*CONCLUSION@#Should pregnant women with advanced maternal age undergo both maternal serum test and NIPT, the detection rate of fetal chromosomal abnormality will be higher than those receiving only maternal serum test screening or NIPT. Couples with one partner harboring chromosomal abnormalities should undergo prenatal diagnosis by amniocentesis.

Development of a quantitative serum assay of Golgi protein 73 in hepatocellular carcinoma using xMAP technology / 中华肿瘤杂志

Objective@#To establish a quantitative assay of serum Golgi protein 73 (GP73) using xMAP technology and evaluate its performance.@*Methods@#Monoclonal antibodies against GP73 were prepared and purified, and antibody pair screening was performed by double-antibody sandwich enzyme-linked immunosorbent assay. The screened antibodies were used to construct a Luminex liquid chip detection system, and the analysis performance of the detection system was evaluated. The serum levels of GP73 were detected in 90 clinical samples from healthy controls and patients with chronic hepatitis B infection (CHB) and hepatocellular carcinoma (HCC).@*Results@#Five anti-GP73 monoclonal antibodies were prepared and purified, and 5 antibody pairs were successfully screened. The Luminex liquid chip detection system of GP73 was successfully constructed using 8F10D1 and 10B9F11 antibody pairs. The analytical performance evaluation showed that the sensitivity of this system was 0.25 ng/ml and the dynamic range was 0.25-100 ng/ml. No cross reactivity was observed. The intra- and inter-assay variation for GP73 was <8% and <11%, respectively. The recovery was 83%-92%. The linear regression equation was y=1.141x+ 6.436 (r2=0.998 4, P<0.001). The GP73 concentrations in the serum samples of healthy control, CHB group, and HCC group were 42.8 (38.68, 55.90) ng/ml, 61.49 (43.59, 81) ng/ml, and 122.78 (49.36 liter, 264.55) ng/ml, respectively. The levels of GP73 in HCC group were significantly higher than those in CHB group and healthy controls (P<0.05). Moreover, the levels of GP73 in CHB group were significantly higher than those in healthy controls (P<0.05).@*Conclusions@#A liquid chip detection system of GP73 was successfully constructed. It provides a powerful tool for the clinical application of GP73 in the future.

Progress in the mechanisms of anticancer peptides / 生物工程学报

In recent years, cancer has become a major concern in relation to human morbidity and mortality. Anticancer peptides (ACPs) are the bioactive peptide with antitumor activity and found in many organisms, including mammals, amphibians, insects, plants and microorganisms. ACPs have been suggested as promising agents for antitumor therapy due to their numerous advantages over traditional chemical agents such as low molecular masses, relatively simple structures, greater tumor selectivity, fewer adverse reactions, ease of absorption, a variety of routes of administration and low risk for inducing multi-drug resistance. Combining with the related research in our group, we summarized the mechanisms of ACPs to provide some directions for research and development of peptide-based anticancer drugs.

Signaling pathway of early brain injury after subarachnoid hemorrhage / 国际脑血管病杂志

Early brain injury (EBI) refers to the direct damage and secondary pathophysiological changes of brain tissue within 72 h after aneurysmal subarachnoid hemorrhage. Studies have shown that a variety of signaling pathways are involved in the mechanism of EBI, such as ecoxy chloropropane Kelch sample related protein-1 (Keap1)-nuclear factor E2 related factor 2 (Nrf2)-antioxidant response element (ARE) pathway, Toll-like receptor 4 (TLR4)/nuclear factor-κB pathway, phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt) pathway. This article reviews the mechanisms of action of different signaling pathways involved in EBI.

Signaling pathway of early brain injury after subarachnoid hemorrhage / 国际脑血管病杂志

Early brain injury (EBI) refers to the direct damage and secondary pathophysiological changes of brain tissue within 72 h after aneurysmal subarachnoid hemorrhage.Studies have shown that a variety of signaling pathways are involved in the mechanism of EBI,such as ecoxy chloropropane Kelch sample related protein-1 (Keapl)-nuclear factor E2 related factor 2 (Nrf2)-antioxidant response element (ARE) pathway,Toll-like receptor 4 (TLR4)/nuclear factor-κB pathway,phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt) pathway.This article reviews the mechanisms of action of different signaling pathways involved in EBI.

The clinical discussion on diagnostic and treatment progress of metastatic spinal adrenal pheochromocytoma / 中华内分泌代谢杂志

Metastatic spinal adrenal pheochromocytoma is such a rare disease that its diagnosis is complicated and the treatment scheme has not reached a consensus at the international level. We should take the clinical manifestations, accessory examination, pathological diagnosis and gene tests into a full consideration to improve the accuracy of diagnosis and to choose reasonable treatment to improve the prognosis. The aim of this paper was to summarize the clinical characteristics, diagnostic basis, and treatment protocols of this disease, which may help to promote recognition of metastatic spinal adrenal pheochromocytoma.

Relationship between structure and function of cathelicidins and their molecular design: a review / 生物工程学报

Cathelicidins play critical roles in mammalian innate immune defense against invasive bacterial infection. In addition to their broad-spectrum bactericidal effect, cathelicidins are interesting peptide-based drug templates because they have multiple functions including anti-inflammatory, wound healing, and angiogenesis promotion. This article summarizes the aim and method of cathelicidin molecular designs. Residue mutation, fragment assembly, chemical modification, and construction of conjugates and dimers are usually used to increase the biological activities. Addition or deletion of certain residues, disruption of leucine zipper and phenylalanine zipper are used to reduce the hemolysis and cytotoxicity. By substituting L-amino acids with D-amino acids, circular constructions and immobilization, cathelicidins' in vitro and in vivo stability could be greatly enhanced, especially their proteinase resistance.

Role of M3 receptor in the effect of penehyclidine hydrochloride up-regulating β-arrestin-1 expression / 重庆医学

Objective To investigate the role of M3 receptor in the effect of penehyclidine hydrochloride(PHC) upregulating β-arrestin-1 expression in lipopolysaccharide(LPS)-induced human pulmonary microvascular endothelial cell(HPMVEC) injury.Methods.M3 shRNA transfected HPMVEC and normal HPMVEC cells were randomly divided into LPS group(A),LPS+pHC group(B),LPS+ M3 shRNA transfection group(C) and PHC+ LPS+ M3 shRNA transfection group(D).The cytoskeleton change was observed by laser scanning confocal.The LDH level in cellular supernate was detected.The VCAM 1 protein expression was examined by immunofluorescence chemistry.β-arrestin-1 protein expression was determined by Western blot and β-arrestin-1mRNA expression was measured by real-time PCR.Results Compared with the group A or C,F-actin cytoskeleton arrangement in the group B or D was neat,the LDH level and VCAM-1 protein expression were decreased,and β-arrestin-1 expression was increased;compared with group A or B,F-actin cytoskeleton arrangement in the group C or D was neat,the LDH level and VCAM-1 protein expression were decreased,while the β-arrestin-1 expression had no obvious change.Conclusion Silence M3 receptor is conducive to reduce LPS-induced HPMVEC injury.But the role of PHC up-regulating β-arrestin-1 expression has no necessary connection with M3 receptor.

Role of β-arrestin-1 in penehyclidine hydrochloride-induced inhibition of LPS-caused increase in pulmonary microvascular permeability in human pulmonary microvascular endothelial cells / 中华麻醉学杂志

Objective To evaluate the role of β-arrestin-1 in penehyclidine hydrochloride (PHC)-induced inhibition of lipopolysaccharide (LPS)-caused increase in pulmonary microvascular permeability in human pulmonary microvascular endothelial cells (PMVECs).Methods Human PMVECs were seeded in 6-well plates (2 ml/well) or in culture flasks (4 ml/flask) at the density of 1 × 105 cells/ml and divided into 5 groups (n=15 each) using a random number table:empty plasmid transfection group (group C),LPS plus empty plasmid transfection group (LPS group),PHC plus LPS plus empty plasmid transfection group (P+LPS group),LPS plus β-arrestin-1 short hairpin RNA (shRNA) transfection group (LPS+shRNA group) and PHC plus LPS plus β-arrestin-1 shRNA transfection group (P+LPS+shRNA group).In LPS and LPS+shRNA groups,the cells were transfected with empty plasmid 1.5 μg or with plasmid containing 15 nmol/L β-arrestin-1 shRNA,LPS with the final concentration of 0.1 μg/ml was added at 24 h of incubation,and the cells were then incubated for 1 h.In P+LPS and P+LPS+shRNA groups,the cells were transfected with empty plasmid 1.5 μg or with plasmid containing 15 nmol/L β-arrestin-1 shRNA,PHC with the final concentration of 2 μg/ml was added at 24 h of incubation,LPS with the final concentration of 0.1 μg/ml was added at 1 h of incubation,and the cells were then incubated for 1 h.The cell permeability was measured using Transwell chambers.The expression of heat shock protein (HSP27) was detected by immunofluorescence.The expression of β-arrestin-1,p38 mitogen-activated protein kinase (p38MAPK) and phosphorylated p38MAPK (p-p38MAPK) was detected by Western blot.The ratio of pp38MAPK/p38MAPK was calculated.Results Compared with group C,the cell permeability was significantly increased,the expression of HSP27 was up-regulated,p-p38MAPK/p38MAPK ratio was increased,and the expression of β-arrestin-1 was down-regulated in LPS,LPS + shRNA and P + LPS + shRNA groups (P＜0.05),and no significant change was found in the parameters mentioned above in group P+LPS (P＞ 0.05).Compared with group LPS,the cell permeability was significantly decreased,the expression of HSP27 was down-regulated,p-p38MAPK/p38MAPK ratio was decreased,and the expression of β-arrestin1 was up-regulated in group P +LPS,and p-p38MAPK/p38MAPK ratio was significantly increased (P＜0.05),and no significant change was found in the other parameters in group P+LPS+shRNA (P＞0.05).Compared with group P+LPS,the cell permeability was significantly increased,the expression of HSP27 was up-regulated,p-p38MAPK/p38MAPK ratio was increased,and the expression of β-arrestin-1 was down-regulated in group P+LPS+shRNA (P＜0.05).Conclusion The mechanism by which PHC inhibits LPS-induced increase in pulmonary microvascular permeability is totally related to β-arrestin-1 in human PMVECs.

Flexibility of the coronal lumbosacral region of congenital scoliosis: a three-dimensional finite element analysis / 中国组织工程研究

BACKGROUND: In the patients with congenital scoliosis, the spinal motor units exhibit developmental disorders and poor range of motion. It has been found that the compensation ability of coronal lumbosacral region (L4-S1) is associated with the occurrence of non-compensable trunk migration postoperatively.OBJECTIVE: To establish the three-dimensional finite element models of coronal lumbosacral region of normal and patients with congenital scoliosis and to compare the strain, displacement, stress and stiffness under different loading conditions among models.METHODS: One normal subject and two congenital scoliosis patients with different coronal lumbosacral region flexibility were selected, DICOM image datawere obtained by spiral CT scanning at the lumbosacral region, and then imported into MIMICS software, and a three-dimensional model was established according to the gray values of each tissue on CT,followed by simplified by GEOMAGIC, and finally imported into ABAQUS foftware to conduct a mechanic analysis under different loading conditions.RESULTS AND CONCLUSION: (1) Under different lateral forces, in the three models, the maximum stress mainly distributed on the frontal region of L4 cortical bone, and maximum displacement concentrated on L5. (2) There was no significant change in the stress distribution in the two scoliosis models, but the compensable model showed larger displacement change, and its stiffness was significantly less than that of the non-compensable model, indicating that the compensable model is easy to deform. (3) These findings suggest that three-dimensional finite element model is helpful to perform a biomechanical analysis for coronal lumbosacral region of congenital scoliosis, among which, a compensable model exhibits large displacement, suggesting a good flexibility.

Role of M3 receptor in penehyclidine hydrochloride-induced reduction of increased permeability of human pulmonary microvascular endothelial cells caused by endotoxin: the relationship with MAPK signaling pathway / 中华麻醉学杂志

Objective To evaluate the role of M3 receptor in penehyclidine hydrochloride(PHC)-induced reduction of increased permeability of human pulmonary microvascular endothelial cells (PMVECs) caused by endotoxin and the relationship with mitogen-activated protein kinase (MAPK) signaling pathway.Methods Human PMVECs were seeded in 6-well plates (2 ml/hole) or in culture flasks (4 ml/flask) at the density of 1 × 105 cells/ml and randomly divided into 6 groups (n=5 each):control group (group C),M3 receptor shRNA transfection group (group shRNA),lipopolysaccharide (LPS) group,penehyclidine plus LPS group (group P+LPS),LPS plus M3 receptor shRNA transfection group (group LPS+shRNA) and PHC plus LPS plus M3 shRNA transfection group (group P+LPS+shRNA).The cells were transfected with shRNA plasmid containing 2.5 nmol/L M3 receptors in shRNA,LPS+shRNA and P+LPS+shRNA groups.LPS at the final concentration of 0.1 μg/ml was added at 24 h of incubation and then cells were incubated for 1 h in LPS and LPS+shRNA groups.PHC at the final concentration of 2 μg/ml was added at 24 h of incubation,cells were incubated for 1 h,then LPS at the final concentration of 0.1 μg/ml was added,and cells were incubated for another l h in P+LPS and P+LPS+shRNA groups.The permeability of PMVECs was measured using Transwell assay.The expression of phosphorylated p38 MAPK (p-p38 MAPK)and phosphorylated extracellular signal-regulated protein kinase 1/2 (p-ERK1/2) was detected by Western blot,the expression of heat shock protein 27 (HSP27) using immunofluorescent staining,and the expression of M3receptor mRNA by real-time polymerase chain reaction.Results Compared with group C,M3 receptor mRNA expression was significantly down-regulated in group shRNA,and the permeability of cells was significantly increased,and the expression of p-p38 MAPK,p-ERK1/2,HSP27 and M3 receptor mRNA was up-regulated in group LPS (P＜0.05).The permeability of cells was significantly decreased,and the expression of p-p38 MAPK,p-ERK1/2,HSP27 and M3 receptor mRNA was down-regulated in P+ LPS,LPS+shRNA and P+LPS+shRNA groups as compared with group LPS,and in group P+LPS+shRNA as compared with group LPS+shRNA (P＜0.05).Conclusion The mechanism by which PHC reduces endotoxin-caused increased permeability of human PMVECs is related to inhibiting activation of MAPK signaling pathway after down-regulating M3 receptor.

Role of β-arrestin-1 in inhibition of endoxin-induced activation of MAPK signaling pathway in pulmonary microvascular endothelial cells by penehyclidine hydrochloride / 中华麻醉学杂志

Objective To investigate the role of β-arrestin-1 in inhibition of endoxin-induced activation of MAPK signaling pathway in pulmonary microvascular endothelial cells (PMVECs) by penehyclidine hydrochloride (PHC).Methods Human PMVECs were seeded in 6-well plates (2 ml/well) or in culture flasks (4 ml/flask) at the density of 1×105 cells/ml,and randomly divided into 5 groups (n=20 each) using a random number table:empty plasmid transfection group (group C),lipopolysaccharide (LPS) + empty plasmid transfection group (LPS group),PHC + LPS + empty plasmid transfection group (P + LPS group),LPS+β-arrestin-1 short hairpin RNA (shRNA) transfection group (LPS+shRNA group),and PHC + LPS+β-arrestin-1 shRNA transfection group (P+LPS+shRNA group).After the cells were transfected with empty plasmid 1.5 μg or with plasmid containing 15 nmol/L β-arrestin-1 shRNA,the cells were incubated for 24 h.At 24 h of incubation,LPS with the final concentration of 0.1 μg/ml was added,and the cells were then incubated for 1 h in LPS and LPS+ shRNA groups.In P+LPS and P+LPS+shRNA groups,PHC with the final concentration of 2 μg/ml was added,and the cells were incubated for 1 h,and then LPS with the final concentration of 0.1 μg/ml was added,and the cells were incubated for 1 h.The expression of filamentous actin (F-actin) was detected by flow cytometry.The expression of myosin light chain kinase (MLCK) and vascular endothelial-cadherin (VE-cadherin) was detected by immunofluorescence.The expression of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) and phosphorylated cJun N-terminal kinase (p-JNK) was determined by Western blot.The expression of β-arrestin-1 mRNA was determined by real-time polymerase chain reaction.Results Compared with group C,the expression of Factin,VE-cadherin and β-arrestin-1 mRNA was significantly down-regulated,and the expression of MLCK,p-ERK1/2 and p-JNK was up-regulated in group LPS,and the expression of p-ERK1/2 and p-JNK was significantly up-regulated (P＜0.05),and no significant change was found in the other parameters mentioned above in group P+LPS (P＞0.05).Compared with group LPS,the expression of F-actin,VE-cadherin and β-arrestin-1 mRNA was significantly up-regulated,and the expression of MLCK,p-ERK1/2 and p-JNK was down-regulated in group P+LPS,and the expression of F-actin,VE-cadherin and β-arrestin-1 mRNA was significantly down-regulated,and the expression of MLCK and p-JNK was up-regulated in group LPS+shRNA (P＜0.05).Compared with group P+LPS,the expression of F-actin,VE-cadherin and β-arrestin-1 mRNA was significantly down-regulated,and the expression of MLCK,p-ERK1/2 and p-JNK was up-regulated in group P+LPS+shRNA (P＜0.05).Conclusion The mechanism by which PHC inhibits endoxin-induced activation of MAPK signaling pathway in PMVECs is partially related to up-regulation of β-arrestin-1 expression.

Differential characteristics of AMA-M2 autoantibody in primary biliary cirrhosis and non-PBC patients / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To explore the differential characteristics of the AMA-M2 autoantibody in patients with primary biliary cirrhosis (PBC) and non-PBC patients.</p><p><b>METHODS</b>Patients with abnormal liver function at the Capital Medical University affiliated to Beijing You-an Hospital were enrolled in this study between January 2011 and December 2013. Serum levels of ANA, AMA and AMA-M2 were detected by indirect fluorescence assay and enzyme-linked immunosorbent assay. The patients' clinical data was obtained for retrospective analysis. Statistical analyses were performed using the SPSS 16.0 software. Enumeration data have been presented as numbers and percentages, and were analyzed using the chi-square test and one-way ANOVA test.</p><p><b>RESULTS</b>Of the 5315 patients with abnormal liver function, 15.3% (811/5315) were AMA-M2 positive patients; among those 811 patients, 78.4% (636) had PBC, 4.4% (36) had PBC overlapping with autoimmune hepatitis (AIH), 4.4% (36) had drug-induced liver injury, 6.5% (53) had hepatitis B, 3.3% (27) had hepatitis C, 0.6% (5) had hepatitis E, 0.9% (7) had alcoholic liver disease, 0.5% (4) had non-alcoholic fatty liver, 0.8% (6) had primary hepatic carcinoma, and 0.1% (1) had infectious mononucleosis. Serum AMA-M2 level was significantly higher in the PBC patients (vs. other groups, P less than 0.001) with the exception of the patients with PBC/AIH overlap syndrome. Among the 811 patients with AMA-M2 positivity, 88.5% (718) showed AMA positivity and 91.1% (739) showed ANA positivity. Serum alanine transferase (ALT) and aspartate transferase (AST) levels were significantly higher in the drag-induced liver injury patients (527.74+/-684.65 U/L, 490.60+/-716.89 U/L) and the hepatitis E patients (1015.94 ± 165.55 U/L, 665.4 ± 297.14 U/L) than in the PBC patients (96.02 ± 115.56 U/L, 94.82 ± 83.32 U/L) (ALT: F =8.041, P < 0.001, P < 0.001; AST: F =8.066, P < 0.001, P < 0.001). Serum alkaline phosphatase (ALP; 265.16 ± 179.08 U/L) and glutamyl transferase (GGT; 332.02 ± 279.29 U/L) were significantly higher in the PBC patients than in the hepatitis B patients (135.35 ± 123.17 U/L, 140.27 ± 229.24 U/L) and the hepatitis C patients (85.65 ± 27.77 U/L, 92.70 ± 125.72 U/L) (ALP: F=3.911, P =0.01, P=0.001; GGT: F=4.081, P <0.001, P < 0.001). The serum IgM level was significantly higher in the PBC patients (4.60 ± 2.67 g/L) than in the patients with drug-induced liver injury (1.76 ± 1.15 g/L), hepatitis B (2.02 ± 1.41 g/L), hepatitis C (1.48 ± 0.92 g/L), hepatitis E (1.40 ± 0.68 g/L), alcoholic liver disease (1.57 ± 1.07 g/L), non-alcoholic fatty liver (1.05 ± 0.72 g/L), and primary hepatic carcinoma (2.64 ± 2.26 g/L) (F=16.83, P < 0.001, P < 0.001, Probability value < 0.001, Probability value < 0.05, Probability value < 0.01, Probability value < 0.05 respectively).</p><p><b>CONCLUSION</b>Although detection of serum AMA-M2 is an important feature of PBC diagnostic testing,there is a high ratio of serum AMA-M2 detected in patients with drug-induced liver injury, hepatitis B, C and E, alcoholic liver disease, non-alcoholic fatty liver,and primary hepatic carcinoma. The AMA-M2 positive non-PBC patients still require close observation to watch for future development of PBC.</p>

Roles of β-arrestin-1 in inhibition of endotoxin-induced activation of NF-κB in human pulmonary microvascular endothelial cells by penehyclidine bydrochloride / 中华麻醉学杂志

Objective To evaluate the role of β-arrestin-1 in inhibition of endotoxin-induced activation of nuclear factor kappa B (NF-κB) in human pulmonary microvascular endothelial cells (HPM-VECs) by penehyclidine hydrochloride (PHC).Methods HPMVECs were seeded in 6-well plates (2 ml/hole) or in culture flasks (4 ml/flask) at the density of 1 × 105/ml,and were randomly divided into 5 groups (n =20 each) using a random number table:empty plasmid transfection group (group C),lipopolysaccharide (LPS) + empty plasmid transfection group (group LPS),PHC + LPS + empty plasmid transfection group (group P+LPS),LPS + β-arrestin-1 gene-shRNA transfection group (group LPS+shRNA) and PHC + LPS + β-arrestin-1 gene-shRNA transfection group (group P+LPS+shRNA).HPMVECs were transfected with empty plasmid 1.5 μg or with plasmid containing 15 nmol/L β-arrestin-1gene-shRNA.At 24 h of incubation,PHC with the final concentration of 2 μg/ml was added,the cells were incubated for 1 h,LPS with the final concentration of 0.1 μg/ml was then added,and the cells were continuously incubated for another 1 h.The supernatant was collected to measure the activity of lactic dehydrogenase (LDH).The cell suspension was collected for determination of vascular cell adhesion molecule-1 (VCAM-1) expression and NF-κB activities and NF-κB inhibitor I-κB and β-arrestin-1expression.Results Compared with group C,the activities of LDH in supernatant were increased,VCAM-1 expression was up-regulated,NF-κB activity was significantly increased,and I-κB and β-arrestin-1 expression was down-regulated in LPS and LPS+shRNA groups.Compared with group LPS,the activities of LDH in supernatant were decreased,VCAM-1 expression was down-regulated,NF-κB activity was significantly decreased,and I-κB and β-arrestin-l expression was up-regulated in group P+LPS,and no significant change was found in the parameters mentioned above in group P+LPS+shRNA.Compared with group P+LPS,the activities of LDH in supernatant were increased,VCAM-1 expression was up-regulated,NF-κB activity was significantly increased,and I-κB and β-arrestin-1 expression was down-regulated in group P+LPS+shRNA.Conclusion PHC inhibits endotoxin-induced activation of NF-κB in HPMVECs completely through up-regulating β-arrestin-1 expression.

Thickness and histopathology of levator palpebrae superioris muscle in patients with simplex congenital blepharoptosis / 中华实验眼科杂志

Background The simplex congenital blepharoptosis is the common blepharon motor dysfunction disease.Some researches have shown that congenital blepharoptosis is related to the hypoplasia of levator.Objective This study was to investigate the thickness and pathological features of levator palpebrae superioris aponeurosis in congenital blepharoptosis patients.Methods A prospective cohort study was carried out in Anyang Eye Hospital from March 2012 to April 2014.Eighty-five eyes of 56 patients with congenital blepharoptosis were divided into mild (15 eyes), moderate (25 eyes) and severe blepharoptosis (19 eyes) groups, and the fellow eyes of monocular blepharoptosis was used as fellow eye group (26 eyes).Twenty-six eyes of 13 normal subjects were recruited for the normal control group.The thickness of levator aponeurosis was measured by ultrasound biomicroscope (UBM) , and the shifting range of levator aponeurosis was detected by using measuring scale.Levator aponeurosis specimens were collected during the levator palpebrae superioris shortening surgery for the pathological examination.The study was approved by the medical ethics committee of Anyang Eye Hospital, and the patients or their guardian signed the informed consent.Results The thickness of levator aponeurosis was (0.331±0.018), (0.373±0.026), (0.539± 0.023) , (0.557 ± 0.024) and (0.547 ± 0.028) mm in the severe blepharoptosis group, moderate blepharoptosis group,mild blepharoptosis group, normal control group and fellow eye group, respectively, showing a significant difference among them (F =1.681, P =0.043).The thickness values of levator aponeurosis were considerably lower in the severe blepharoptosis group and moderate blepharoptosis group than those in the mild blepharoptosis group,fellow eye group and normal control group (all at P＜0.05) , and the thickness value of levator aponeurosis was significantly reduced in the severe blepharoptosis group compared with the moderate blepharoptosis group (P＜0.05).Pathological examination showed arranging disorder of muscle fibers,hyaline-like degeneration, connective tissue hyperplasia and interruption of endomysium.The number of eyes with severe hyaline-like degeneration and connective tissue hyperplasia was significantly increased in the severe blepharoptosis group than that in the moderate blepharoptosis group or the mild blepharoptosis group, as well as in the moderate blepharoptosis group than that in the mild blepharoptosis group(all at P＜0.01).The adipose cells in muscle in the mild blepharoptosis group, moderate blepharoptosis group and severe blepharoptosis group were (12.35±4.62), (17.58±7.46) and (26.19±10.81)/field,and adipose cells in the severe blepharoptosis group were significantly more than those in the mild and moderate blepharoptosis groups (t =5.60, P =0.00;t =2.71, P =0.01).A significant increase in the adipose cells also was seen in the moderate blepharoptosis group compared with the mild blepharoptosis group (t =2.44, P =0.02).Conclusions UBM can offer accurate thickness data of levator aponeurosis.The combination of thickness data and shifting range measurement of levator aponeurosis is helpful for the evaluation of muscle strength.The development of levator aponeurosis appears to be abnormal in congenital blepharoptosis patients.The histopathological change parallels to the severity of the disease.

Construction of novel thioredoxin fusion protein expression system and the production of recombinant Lf-CATH2 / 生物工程学报

The objective of this study was to construct an improved thioredoxin fusion protein expression system, and express the cathelicidin-derived peptide, Lf-CATH2. The improved fusion vector Lf-CATH2-pET32α(-TS) was successfully constructed by firstly deleting the thrombin site and S tag from the pET-32α vector, then inserting the Lf-CATH2 plus a thrombin site instead. Afterwards, Lf-CATH2 was expressed in Escherichia coli as fusion protein. After the cleavage by thrombin, Lf-CATH2 was released and subsequently separated using affinity chromatography. The antimicrobial activity of purified Lf-CATH2 was also examined. The improved expression vector significantly increased enzyme cleavage efficiency by 37%, and Lf-CATH2 could be expressed in high yield and maintain the biological activity. This novel thioredoxin fusion protein expression system enables a quick production of high-yield bioactive cationic peptides like cathelicidins.