Objective To establish a technical route for the efficient expression and purification of PprI protein from Deinococcus radiodurans R1 by using eukaryotic Pichia pastoris.Methods The encoding sequence of the Deinococcus radiodurans pprI gene was modified according to the preference of Pichia pastoris' codon.Modified pprI gene was fully synthesized with PCR and a 6 × His tag was added at its Nterminal.The PCR products were purified and then cloned into Pichia pastoris expression vector pHBM-905A.After utilizing Cop I and Not I double enzyme digestion and retrievering linear objective fragment,new pprI gene was transformed to the GS115 strain of Pichia pastoris.The obtained Pichia pastoris transformants were induced to express.Culture supernatants were detected by SDS-PAGE,Western blot,and mass spectrometry.A Ni-NTA column was uesd to purify the target protein and the BCA method was used to determine protein concentration.Results The coding sequence of new synthetic Deinococcus radiodurans pprI gene was correct.The purpose protein band of a molecular weight of 43 000 was detected in the culture supernatant of transformed Pichia pastoris strains by SDS-PAGE and Western blot.The mass spectrometry confirmed that it was the Deinococcus radiodurans PprI protein.When the concentration of imidazole was 250 mmol/L,the elution rate of PprI protein was the highest.The purified protein concentration was 0.35 mg/ml measured by BCA method.Conclusions This study has successfully constructed a new pprI gene and the recombinant strain of Pichia pastoris secreting PprI protein,and established a technical route for the efficient expression and purification of PprI protein.

The natriuretic peptide(NP) system is an endocrine system that maintains fluid and pressure homeostasis by modulating cardiac and renal function.NP levels are elevated in patients with heart failure(HF) and other cardiac diseases.They are early warning system to help to identify patients at high risk for cardiac events.Measurement of NPs may be used to aid diagnosis and prognosis.NPs also can exert important anti-proliferative,anti-fibrotic effects to prevent the remodification in the heart with myocardial infarction and advanced HF.Brain natriuretic peptide is an important biomarker in patients with HF and other cardiovascular diseases,such as pulmonary hypertension and atherosclerotic vascular disease.In addition,synthetic NPs such as nesiritide could be used to treat the patients with acutely congestive HF. These Recombinant drugs are also being investigated for myocardial and renal protection in the setting of cardiac surgery and for prevention of cardiac remodeling.

Objective To understand the epidemiologic characteristics and pathogenic virus of cases of viral diarrhea in sentinel hospitals in Liaoning Province.Methods From Jan 2009 to Dec 2011,639 stool samples from sentinel hospitals of Liaoning Province were collected.Rotavirus,human calicivirus,astrovirus and adenovirus were detected by polymerase chain reaction and reverse transcriptase-polymerase chain reaction.The data analysis used chi-squanetest and Fisher's exact test.Results Rotavirus,human calicivirus,astrovirus and adenovirus were detected in 15.96 ％,11.25 ％,1.25％ and 0.31％ of the 639 specimens,respectively.G3 was the most prevailing serotype and P[8] was the most common genotype among 101 group A rotavirus isolates.One strain of group C rotavirus was also detected,which was reported for the first time from Liaoning Province.Phylogenetic analysis showed that this group C rotavirus JX407109 in the present study had the closest genetic relationship with the outbreak strain AB648916 from Japan,with nucleotide sequence consistency of 99 ％.Among the 72 samples of human calicivirus,70 samples were norovirus with G Ⅱ/4 being the predominant genotype,and 2 samples were sapovirus.Astrovirus was detected in 8 samples,most of which were genotype 1.Adenovirus was detected in 2 samples,and both were genotype 41.High incidences of viral diarrhea were noted during the months from December to next year February,and children under 5 years of age had high incidence of rotavirus and astrovirus,while the incidence of calicivirus were similar among different age groups.Conclusions The predominant pathogens of viral diarrhea in Liaoning Province are group A rotavirus and calicivirus.Notably,the group C rotavirus in Liaoning Province shares high genetic consistency with the outbreak strain from Japan.

AlM: To explore the roles of neuronal axon-guidance molecules Slit3 and Robo4 receptor in corneal neovascularization ( CNV ) by study their expression in neovascularized cornea of rats.
METHODS: CNV models were established by implantation pellets containing basic fibroblast growth factor ( bFGF ) into corneal stroma. CNV models were measured by biomicroscopy photography. lmmunohistochemical staining and imaging analysis system were used to detect the expression of Slit3 and Robo4 in the models after 1, 4, 7, 10 and 14d.
RESULTS:The area of CNV and the expression of Slit3, Robo4 were increased in CNV models compared to that in normal cornea and reached highest level on 7d. And the expression level of Slit3 and Robo4 were significantly correlated with the size of CNV on every time point except 1d (r=0. 84-0. 91, all P<0. 05).
CONCLUSlON: The expression of Slit3 and Robo4 may be related to the CNV development. They are potential therapeutical target for CNV.

Objective Pemetrexed and β-elemene can inhibit the growth of tumor cells .This study was to investigate the effect of pemetrexed combined with β-elemene on the proliferation and apoptosis of cervical cancer HeLa cells. Methods Cervical cancer HeLa cells were treated with pemetrexed at the concentrations of 38, 76, 152, 228, and 304μg/mL, and at 24 and 48 hours of treatment subjected to MTT for detection of their proliferation .The experiment included four groups , with the cells treated with β-elemene ( 125μg/mL) , pemetrexed ( 76 μg/mL ) , β-elemene ( 125 μg/mL ) +pemetrexed (76μg/mL), and nothing (blank control) for 24 hours, followed by determination of their proliferation and apoptosis by MTT and flow cytometry, respectively. Results Pemetrexed at 38, 76, 152 and 228μg/mL inhibited the proliferation of the HeLa cells in a concentration-dependent manner, with the inhibition rates of (7.24 ±3.78), (7.94 ±4.37), (11.10 ±2.86) and (15.88 ± 3.38)%at 24 hours, and (16.69 ±0.95), (22.54 ±1.53), (24.48 ±0.92) and (25.54 ±3.61)%at 48 hours, both with statis-tically significant differences between any two groups (P<0.05).Significant differences were also found in the proliferation rate of the same concentration of pemetrexed at the two time points (P<0.05).The combination of pemetrexed and β-elemene showed an inhibi-tion rate of (49.95 ±5.76)%at 24 hours, remarkably higher than (24.36 ±5.59)%in theβ-elemene group and (10.69 ±1.37)%in the pemetrexed group (P<0.01). Conclusion Pemetrexed combined with β-elemene can significantly inhibit the proliferation and synergistically accelerate the apoptosis of HeLa cells .

A flavivirus, Culex flavivirus, was first isolated from Chinese mosquitoes with high sequences similarities to those of flaviviruses found in America and Japan. In this study, a total of 48 pools of field-collected mosquitoes were sampled from Dandong of Liaoning Province, China during July to September of 2011. Six isolated viruses showing cytopathic effect (CPE) in C6/C36 cells were tested by reverse transcription polymerase chain reaction(RT-PCR)using Flavivirus genus--specific primers and Culex flavivirus-specific primers and the positive PCR-product was sequenced and compared with the sequences of 10 isolates from GenBank. Phylogenetic tree of NS5 and enevelop genes of flavivirus were constructed. The GenBank accession numbers of NS5 gene were JQ409188, JQ409186, JQ409187, JQ409191, JQ409189 and JQ409190. The GenBank accession numbers of envelope gene were JQ065883, JQ065882, JQ065881, JQ065879,JQ065877 and JQ065878.
Animals ; Base Sequence ; Cell Line ; China ; Culex ; classification ; virology ; Flavivirus ; classification ; genetics ; isolation & purification ; Insect Vectors ; virology ; Molecular Sequence Data ; Phylogeny ; Viral Proteins ; genetics

Tumor brings great threat to human public health. In recent years, incidence rate and mortality of tumor were rapidly increased in the world. Anti-tumor therapies have undergone the development of cytotoxic therapy, targeted therapy, and immunotherapy. Among them, tumor immunotherapy is rapidly developed and becomes an important anti-tumor therapy in recent years, although it also brings some related side effects. Tumor microenvironment (TME) is composed of immune cells, vascular vessels, fibroblasts, the extracellular matrix, etc. TME significantly affects the efficacy of immunotherapy. Macrophages in the TME are named as tumor associated macrophages (TAMs). Recently, increasing studies have shown that TAMs play an important role in the regulation of tumor immunity, especially in tumor immune surveillance and immune escape. Currently, more and more anti-tumor immunotherapy strategies targeting TAMs are at the development stage. Based on the important role of TAMs in the TME and their potential as therapeutic targets in tumor immunotherapy, we first reviewed the subtypes and functions of TAMs, as well as the roles of TAMs in tumors. Furthermore, we summarized the research progress on anti-tumor strategies targeting TAMs and the current status of drug targeting TAMs. The current review will provide new ideas and novel insights for tumor immunotherapy.

OBJECTIVETo study the changes of immune function in patients with liver cancer after transcatheter arterial chemoembolizaton (TACE) combined with interstitial therapy.

METHODSForty patients with liver cancer were randomly divided into groups A and B to received TACE and TACE combined with percutaneous lipiodol and anti-cancer agent injection into the tumor. The T lymphocyte cell subsets in the peripheral blood before and one week after the operation were measured by flow cytometry, and the immunoglobulin contents determined by single radial immunodiffusion.

RESULTSCD3, CD4, and CD4/8 levels increased significantly after the operation in both groups A and B (P<0.05). The postoperative CD3 and CD4 levels, but not that of CD8, differed significantly between the two groups (P<0.05). The operations also resulted in an increase in the contents of the immunoglobulins and complements in the two groups, but the changes were not significant in group A (P>0.05); in group B, significant increases occurred in the immunoglobulin and complement levels (P<0.05) with the exception of C3.

CONCLUSIONThe combination of TACE and interstitial therapy with percutaneous intratumor injections of lipiodol and anti-cancer agents may better improve the cell-mediated immunity and humoral immune function of liver cancer patients.

Adult ; Aged ; Antineoplastic Combined Chemotherapy Protocols ; administration & dosage ; Carcinoma, Hepatocellular ; drug therapy ; immunology ; Chemoembolization, Therapeutic ; methods ; Ethiodized Oil ; administration & dosage ; Female ; Humans ; Immunoglobulins ; blood ; Injections, Intralesional ; Liver Neoplasms ; drug therapy ; immunology ; Male ; Middle Aged ; T-Lymphocyte Subsets ; immunology

OBJECTIVEDisturbances of the synthesis and breakdown of the extracellular matrix of arterial walls have emerged as key features of the atherosclerotic process. We observed the changes of circulating procollagen marker for type III collagen turnover rate, the N-terminal propeptide P III NP and vascular resistance in hypertensive patients treated with various antihypertensive regimens.

METHODA total of 130 light to moderate hypertensive patients were randomly assigned to receive enalapril (group B, n = 43), enalapril + spirolactone (20 mg/d, group A, n = 44) and anti-hypertensive drugs not directly affecting RAAS (calcium antagonist, beta-blocker, group C, n = 43) for 1 year. Target blood pressure is < 130/80 mm Hg.

RESULTSTarget blood pressure was reached in all treated patients and was similar among various groups. Under the same blood pressure controlling precondition, serum P III NP were similar at baseline among various groups and remained unchanged in group B [(3.4 +/- 0.3) microg/L vs. (3.7 +/- 0.3) microg/L, P > 0.05] and significantly decreased in group A [(2.3 +/- 0.2) microg/L vs. (3.8 +/- 0.2) microg/L, P < 0.05] while significantly increased in group C [(3.9 +/- 2.0) microg/L vs. (3.2 +/- 1.5) microg/L, P < 0.05]. Vascular resistance was similar among groups before therapy and all significantly decreased after 1 year antihypertensive therapy and the decrease was more significant in group A [(1064.3 +/- 158.6) dyn.s(-1).cm(-5)] than that in group B [(1200.8 +/- 298.7) dyn.s(-1).cm(-5)] and group C [(1205.1 +/- 206.4) dyn.s(-1).cm(-5)].

CONCLUSIONSpironolactone in conjunction with enalapril is a more favorable antihypertensive regimen in decreasing P III NP and improving vascular resistance than enalapril alone or antihypertensive drug regimens not directly affecting RAAS.

Adult ; Aged ; Angiotensin-Converting Enzyme Inhibitors ; therapeutic use ; Antihypertensive Agents ; therapeutic use ; Biomarkers ; Enalapril ; therapeutic use ; Humans ; Hypertension ; drug therapy ; metabolism ; physiopathology ; Middle Aged ; Procollagen ; blood ; Spironolactone ; therapeutic use ; Vascular Resistance

Background: Pseudorabies virus (PRV) infection leads to high mortality in swine. Despite extensive efforts, effective treatments against PRV infection are limited. Furthermore, the inflammatory response induced by PRV strain GXLB-2013 is unclear. Objectives: Our study aimed to investigate the inflammatory response induced by PRV strain GXLB-2013, establish an inflammation model to elucidate the pathogenesis of PRV infection further, and develop effective drugs against PRV infection. Methods: Kunming mice were infected intramuscularly with medium, LPS, and different doses of PRV-GXLB-2013. Viral spread and histopathological damage to brain, spleen, and lung were determined at 7 days post-infection (dpi). Immune organ indices, levels of reactive oxygen species (ROS), nitric oxide (NO), and inflammatory cytokines, as well as levels of activity of COX-2 and iNOS were determined at 4, 7, and 14 dpi. Results: At 105 –106 TCID50 PRV produced obviously neurological symptoms and 100% mortality in mice. Viral antigens were detectable in kidney, heart, lung, liver, spleen, and brain. In addition, inflammatory injuries were apparent in brain, spleen, and lung of PRVinfected mice. Moreover, PRV induced increases in immune organ indices, ROS and NO levels, activity of COX-2 and iNOS, and the content of key pro-inflammatory cytokines, including interleukin (IL)-1β, IL-6, tumor necrosis factor-α, interferon-γ and MCP-1. Among the tested doses, 10 2 TCID 50 of PRV produced a significant inflammatory mediator increase. Conclusions An inflammatory model induced by PRV infection was established in mice, and 102 TCID50 PRV was considered as the best concentration for the establishment of the model.