AIM:To explore the roles of Akt ( also called protein kinase B ) and active caspase-3 in the leptin-mediated chronic morphine antinociceptive tolerance in rats .METHODS: A model of chronic morphine antinociceptive tolerance was established in the SD rats .The protein levels of spinal Akt and cleaved caspase-3 were tested by Western blotting.The technique of immunohistochemical staining was used to detect the immunoreactivity positive cells of phospho -rylated ( p)-Akt and cleaved caspase-3 in the spinal cord .Double staining of immunohistochemistry was used to examine the cellular location of the p-Akt and cleaved caspase-3 positive cells.RESULTS: The chronic intrathecal injection of morphine (15 μg) for 7 d markedly upregulated the spinal protein levels of p-Akt and cleaved caspase-3 in the rats.Thirty min before injection of morphine , intrathecal injection of leptin antagonist (3μg) for 7 d significantly attenuated the upreg-ulation of the protein levels of p-Akt and cleaved caspase-3 induced by chronic morphine treatment .The p-Akt was exclu-sively observed in the spinal neurons .The cleaved caspase-3 was only localized with the spinal astrocytes .Intrathecal injec-ting the inhibitors of leptin , Akt and caspase-3 ameliorated the chronic antinociceptive tolerance .CONCLUSION: The spinal Akt pathway and active caspase-3 are involved in the leptin-mediated chronic morphine antinociceptive tolerance in rats.

BACKGROUND: Chitosan is one of the most significant non-viral vector materials with the advantages of outstanding biocompatibility. Quarternary chitosan derivatives can improve transfection efficiency and solubility of chitosan in a broader range of pH values. OBJECTIVE: To synthesize a new vector of quarternary chitosan and to study its complex conditions with plasmid and transfection efficiency compared with chitosan. DESIGN, TIME AND SETTING: A contrast observational study was performed in Second Affiliated Hospital of School of Medicine of Zhejiang University between August and October 2008. MATERIALS: Quarternary chitosan was synthesized in Polymer Materials Lab of Beijing Institute of Technology. Plasmid pEGFP-C1 was presented friendly by Mr. Zheng of School of Medicine of Zhejiang University. Hela cells were provided by Miss. Cheng of School of Medicine of Zhejiang University. METHODS: Quarternary chitosan was prepared according to mass concentration of 0.2 g/L, pH value 5.5 (or 6.9, 7.6) and sodium acetate concentration of 50 mmol/L, and rapidly mixed with pEGFP-C1. The mixture was swirled for 15-30 second and stood at room temperature for 30 minutes at least. MAIN OUTCOME MEASURES: The impacts of pH values and time on complex ability of quarternary chitosan and plasmid were studied by gel retardation test. Transfection efficiency of quarternary chitosan on Hela cells was observed by inversed fluorescence microscope and also compared with chitosan. RESULTS: Quarternaty chitosan could form complex with plasmid in acidic, neutral and basic conditions. It could be used in a broader range of pH values. In an acidic condition, the combination of quarternary chitosan with plasmid was superior to chitosan. A stable complex was formed via a combination of quarternary chitosan or chitosan with plasmid within 30 minutes, and the stability lasted for 12 hours. Transfection efficiency of quarternary chitosan on Hela cells demonstrated that transfection efficiency of quarternary chitosan was superior to chitosan. CONCLUSION: Quarternary chitosan has a broader range in use and higher transfection efficiency than chitosan; however, there is no significant difference in stability between quarternary chitosan and chitosan. Additionally, transfection efficiency of quarternary chitosan on Hela cells is superior to chitosan, which needs a further research.

Objective To investigate the association between chronic kidney dysfunction and the complexity of coronary artery disease in elderly patients.Methods A prospective study was conducted on 1380 consecutive patients underwent coronary angiography for the first time in our hospital and with angiographically diagnosed coronary artery disease from January 2011 to June 2012.The complexity of coronary artery disease were classified according to the American College of Cardiology/American Heart Association (ACC/AHA) grading system as types A,B1,B2,and C.Estimated glomerular filtration rate (eGFR) was calculated by the simplified Modification of Diet in Renal Disease(MDRD)equation.Patients were classified into 3 stages according to eGFR as follows:normal renalfunction(n=234,eGFR≥90 ml· min-1 · 1.73 m-2),mild renaldysfunction(n=881,60≤eGFR＜90 ml · min-1 · 1.73 m-2,and moderate or severe renaldysfunction(n=265,eGFR＜60ml · min-1 · 1.73 m-2).Ordinal logistic regression was used to analyze the association between chronic kidney dysfunction and the complexity of coronary artery disease.Results Patients with mild,moderate or severe renal dysfunction were older (F=56.82,P＜0.001),more predominantly female (x2 =66.29,P＜ 0.001) and more likely to have history of hypertension (x2 =17.57,P ＜ 0.001),diabetes (x2=20.97,P＜0.001) and hyperlipidemia (x2=10.48,P 0.005) than those with normal renal function.The percentage of lesions of types B2 or C in moderate or severe renal dysfunction group was higher than that in normal renal function group (x2=175.03,P＜0.001).The ordinal logistic regression showed that age,male,hypertension,diabetes,C-reactive protein and eGFR were independent risk factors for the ACC/AHA lesion classification.Conclusions Age,male,hypertension,diabetes,C-reactive protein and eGFR are independent risk factors for the complexity of coronary artery disease.

Objective To evaluate the effect of MRI in reflecting the pathological changes of VX2 carcinoma in rabbits after radiofrequency ablation (RFA) . Methods RFA was performed in the livers of 24 rabbits with planted VX2 carcinoma. The rabbits were divided into 4 groups. After RFA, the rabbits were killed after MR imaging on 0, 1, 2, and 4 weeks, respectively. The correlation between MRI and pathological findings was analyzed. Results In the acute phase, coagulative necrosis of the ablated tumors and inflammatory reaction with hyperemia around were detected at microscopic examination. The ablated tumor showed as hypointensity on T1WI and hyperintensity on T2WI, while rim of high signal intensity on T1WI and low signal intensity on T2WI was found. Gadolinium-enhanced MRI showed a thin high signal rim surrounding the central coagulative necrosis. In the subacute phase, extensive coagulative necrosis and marked infiltration by neutrophils, lymphocytes, macrophages and a peripheral fibrous generation rim were observed microscopically on the ablated tumor. The ablated tumor showed iso-or hyperintensity on T1WI and hypointensity on T2WI, while the periphery of ablated lesions was hypointensity on T1WI and hyperintensity on T2WI. There was prominent rim enhancement along the ablated margin. In the chronic phase, peripheral fibrous rim became obvious, more regular and thicker than at subacute phase as hypointensity on T1WI and T2WI, and unenhancement was observed. Residual or recurrence of tumor was found in 17 rabbits as hypointensity on T1WI and hyperintensity on T2WI, and irregular, thicker rim or nodular enhancing abnormalities. Conclusion MRI can effectively show the histopathological tissue changes of rabbit VX2 carcinoma after ablation and demonstrate the residual or recurrence of tumor.

Through introducing mutations into ribosomes by obtaining spontaneous drug resistance of microorganisms, ribosome engineering technology is an effective approach to develop mutant strains that overproduce secondary metabolites. In this study, ribosome engineering was used to improve the yield of butenyl-spinosyns produced by Saccharopolyspora pogona by screening streptomycin resistant mutants. The yields of butenyl-spinosyns were then analyzed and compared with the parent strain. Among the mutants, S13 displayed the greatest increase in the yield of butenyl-spinosyns, which was 1.79 fold higher than that in the parent strain. Further analysis of the metabolite profile of S13 by mass spectrometry lead to the discovery of Spinosyn α1, which was absent from the parent strain. DNA sequencing showed that there existed two point mutations in the conserved regions of rpsL gene which encodes ribosomal protein S12 in S13. The mutations occurred a C to A and a C to T transversion mutations occurred at nucleotide pair 314 and 320 respectively, which resulted in the mutations of Proline (105) to Gultamine and Alanine (107) to Valine. It also demonstrated that S13 exhibited genetic stability even after five passages.
Genetic Engineering ; Macrolides ; metabolism ; Point Mutation ; Ribosomal Proteins ; genetics ; Ribosomes ; metabolism ; Saccharopolyspora ; metabolism

Objective:To analyze the positive detection rate, main genotypes of β-thalassemia in western region of Guangxi Zhuang Autonomous Region (referred to as Guangxi).Methods:Retrospective analysis of 26 189 individuals who underwent gene testing for thalassemia at the Affiliated Hospital of Youjiang Medical University for Nationalities from January 2013 to December 2019. Using the crossing breakpoint PCR (Gap-PCR) and reverse dot blot (RDB) techniques to detect Chinese common type of 7 kinds of α-thalassemia and 17 kinds of β-thalassemia genotypes, high-throughput sequencing(Sanger) was performed for suspected rare β-thalassemia. Gap-PCR was used for suspected deletion β-thalassemia types.Results:β-thalassemia was diagnosed in 4 495 (17.16%) of 26 189 samples. A total of 6 177 alleles of 20 types of β-thalassemia were detected, mainly CD17 (2 712 cases, 43.90%) and CD41-42 (2 240 cases, 36.26%), including 7 rare alleles: Gγ +( Aγδβ) 0, SEA-HPFH, Hb New York, Hb G-Taipei, Hb Hezhou, Hb G-Coushatta and IVS-Ⅱ-81. There were 3 903 case (86.83%) heterozygous, 273 case (6.07%) double heterozygous, and 319 case (7.10%) homozygous among 4 495 β-thalassaemia subjects. A total of 48 genotypes were detected. The two most common genotypes were CD17/β N (1 890 cases, 42.05%) and CD41-42/β N (1 212 cases, 26.96%), accounted for 69.01% (3 102/4 495). Seven rare genotypes were detected: Gγ +( Aγδβ) 0/β N in 3 cases, Hb New York/β N in 3 cases, Hb G-Taipei/β N in 2 cases, SEA-HPFH/β N, Hb Hezhou/β N, Hb G-Coushatta/β N and IVS-Ⅱ-81/β N in 1 case each. A total of 1 041 cases (3.97%, 1 041/26 189) of 116 types of αβ-thalassemia were detected, mainly -- SEA/αα composite CD17/β N (144 cases, 13.83%), followed by -α 3.7/αα composite CD17/β N (112 cases, 10.76%). Conclusions:Western region of Guangxi is a high prevalence area of β-thalassemia, CD17/β N and CD41-42/β N are the main genotypes. The variation spectrum of β-thalassemia is complex and diverse, with rich genotype.

The current classification methods for traditional Chinese medicine （TCM） visceral syndrome differentiation suffer from excessive generalization， which hinders their clinical application. Based on the analysis of the pattern of "one syndrome corresponding to multiple formulas"， this paper focused on the principle of syndrome-formula correspondence， and proposed that formula-syndromes are the smallest units for refining visceral syndromes. By establishing the correspondence between formula-syndromes and visceral syndromes， this study aims to further clarify the refined categories of syndromes and their treatment patterns， providing a new perspective for the standardization and objectification of TCM syndromes.

The fcl gene encodes GDP-fucose synthase, which catalyzes two-step differential isomerase and reductase reactions in the synthesis of GDP-L-fucose from GDP-D-mannose. It also participates in the biosynthesis of amino sugar and ribose sugar, and is one of the key enzymes to regulate the metabolism of sugar and nucleotides in organisms. The presence of fcl gene in Saccharopolyspora pogona was found through sequencing result of genome. The mutant S. pogona-fcl and S. pogona-Δfcl were constructed by gene engineering technology. The results showed that the gene had an effects on growth and development, protein expression and transcriptional level, insecticidal activity, and biosynthesis of butenyl-spinosyn of Saccharopolyspora pogona. The results of HPLC analysis showed that the yield of butenyl-spinosyn in S. pogona-Δfcl was 130% compared with that in S. pogona, which reduced by 25% in S. pogona-fcl. The results of determination of insecticidal activity showed that S. pogona-Δfcl had a stronger insecticidal activity against Helicoverpa armigera than that of S. pogona, while the S. pogona-fcl had a lower insecticidal activity against Helicoverpa armigera compared with S. pogona. Scanning electron microscopy (SEM) was used to observe the morphology of the mycelia. It was found that the surface of the S. pogona-Δfcl was wrinkled, and the mycelium showed a short rod shape. There was no significant difference in mycelial morphology between S. pogona-fcl and S. pogona. Aboved all showed that deletion of fcl gene in S. pogona hindered the growth and development of mycelia, but was beneficial to increase the biosynthesis of butenyl-spinosyn and improve insecticidal activity. Whereas the fcl gene over-expression was not conducive to the biosynthesis of butenyl-spinosyn and reduced their insecticidal activity. SDS-PAGE results showed that the difference of protein expression among the three strains was most obvious at 96 hours, which was identified by real-time fluorescence quantitative polymerase chain reaction, the results showed that there were significant differences of related genes in transcriptional levels among the three strains. Based on the results of the study, a network metabolic control map was constructed to analyze the effect of fcl gene on growth and the regulation pathway of butenyl-spinosyn biosynthesis, which provided an experimental basis for revealing the regulation mechanism of butenyl-spinosyn biosynthesis and related follow-up studies.
Bacterial Proteins ; Genetic Engineering ; Insecticides ; Macrolides ; Saccharopolyspora