The article introduce a rapid method for preparation of fungal chromosome DNA In this method the quartz sand is used to break the fungal cell wall and the chromosome DNA is harvested rapidly in 1～2 h The method is applied successfully by the author to four kinds of fungi Neurospora crassa , Aspergillus oryzae , Morchella esculcnta , Saccharomyces cervisae All the chromosome DNA extracted has the fragment size lager than 20 kb and can be used directly for either digestion with restriction endoenzyme or PCR

Objective To investigate the incidence of occult pancreatobiliary reflux and to evaluate its relation to gallbladder epithelial dysplasia and cancer. Methods From July 2006 to Feb 2008,956 cases underwent selective biliary procedure or preoperative endoscopic retrograde cholangiopanereatography (ERCP), bile was collected and amylase was measured. All removed gallbladders were pathologically examined for dysplasia and cancer. Results Occult pancreatobihary reflux was found in 75 of 754 patients in this study, with an incidence of 9. 9%. The biliary amylase values in the patients with occult pancreatobiliary reflux and in controls were 7701±20 378 IU/L and 16±51 IU/L, respectively ( P <0. 01 ).Gallbladder dysplasia and cancer were found in 31.0% and 3.4% of the patients with occult pancreatobiliaryreflux, respectively, and both were higher than those in the patients without pancreatobiliary reflux ( P <0. 05). In the patients with occult pancreatobiliary reflux, the biliary amylase level with gallbladder dysplasia or cancer was 2388 ± 2745 IU/L and was higher than those without gallbladder dysplasia or cancer (P < 0. 01 ). Conclusions With an incidence of 9.9% in patients of normal pancreatobiliary junction,the occult pancreatobihary reflux may contribute to the pathogenesis of gallbladder epithelial dysplasia and cancer.

Glycerol from oil hydrolysis industry is being considered as one of the abundent raw materials for fermentation industry. In present study, the aerobic and anaerobic metabolism and growth properties on glycerol by Esherichia coli CICIM B0013-070, a D-lactate over-producing strain constructed previously, at different temperatures were investigated, followed by a novel fermentation process, named temperature-switched process, was established for D-lactate production from glycerol. Under the optimal condition, lactate yield was increased from 64.0% to 82.6%. Subsequently, the yield of D-lactate from glycerol was reached up to 88.9% while a thermo-inducible promoter was used to regulate D-lactate dehydrogenase transcription.
Aerobiosis ; Anaerobiosis ; Escherichia coli ; genetics ; metabolism ; Fermentation ; Glycerol ; metabolism ; L-Lactate Dehydrogenase ; metabolism ; Lactic Acid ; biosynthesis ; Promoter Regions, Genetic ; genetics ; Temperature

Shikimic acid (SA), as a hydroaromatic intermediate in the common pathway of aromatic amino acid biosynthesis, is the starting material for the synthesis of neuraminidase inhibitors and other useful compounds. The fermentative production of SA by metabolically engineered microorganisms is an excellent alternative to the extraction from fruits of the Illicium plant. In this study, Escherichia coli was metabolically engineered by rational design and genetic manipulation for fermentative production of SA. Firstly, blocking the aromatic amino acid pathway after the production of SA was carried out by deletion of aroL and aroK genes encoding SA kinase. Secondly, the ptsG gene encoding protein EIICBglc were removed in the aroL/aroK mutant strain to make the phosphotransferase system (PTS) system default. In the resulting strain, the phosphoenolpyruvate-dependent PTS pathway, a main pathway for glucose transport, were replaced by ATP-dependent GalP (galactose permease). Thus, more PEP flux was used to produce SA as a critical precursor of SA. Furthermore, ydiB gene (encoding quinic acid/SA dehydrogenase) was deleted to prevent SA precursors of 3-dehyroquinic acid into the byproduct of quinic acid. Thus, the engineered strain with four genes deletion was constructed and 576 mg/L SA was produced in the shake flask fermentation. Results show that SA produciton was increased 90 times compared to the parent strain E. coli CICIM B0013.
Escherichia coli ; enzymology ; genetics ; metabolism ; Gene Knockout Techniques ; Metabolic Engineering ; methods ; Recombinant Proteins ; genetics ; metabolism ; Shikimic Acid ; metabolism

Objective: To detect the expression of B cell transposition gene 3(BTG3) in pancreatic ductal adenocarcinoma(PDAC), and explore its relationship with postoperative recurrence and metastasis of tumor. Methods: Six self-paired frozen PDAC specimens and 3 normal pancreatic tissues from the Second Hospital of Jiaxing Affiliated to Jiaxing University were collected and the expression of BTG3 was detected by qPCR. Ten normal pancreatic tissues and 52 cases of PDAC tumor and paracarcinomatous tissues from the Second Hospital of Jiaxing Affiliated to Jiaxing University were collected from June 2009 to December 2016. The expression of BTG3 and relationship among BTG3 and clinicopathological characteristics of PDAC and patients′ prognosis were detected and analyzed using immunohistochemistry.χ² test, Kaplan-Meier method and Cox regression model were used to analyzed the data. Results: The results of qPCR showed that expression level of BTG3 in PDAC (0.63±0.17) was lower significantly than that in paracarcinomatous (0.96±0.04) and normal tissues (1.00)(t=4.673, 5.502; both P<0.05). Immunohistochemistrv showed that BTG3 mainly expressed in the cytoplasm.The high expression rate of BTG3 in PDAC tumor tissues was 25.0%(13/52), which was remarkably lower than that in paracarcinomatous tissues(65.4%) and normal liver tissues(7/10)(χ²=17.120 and 5.849, both P<0.05). The low expression of BTG3 in PDAC was correlated with primary tumor, and TNM stage(χ²=7.704, P=0.006; U=154.000, P=0.018, respectively). Survival analysis showed that disease free survival rate of patients with low expression of BTG3 was significantly less than that with high expression(χ²=192.493, P<0.01). The Cox multivariate analysis demonstrated that low expression of BTG3 was independent risk factors for disease free survival in patients with PDAC after a curative resection(RR=3.366, 95%CI: 1.040-10.889, P=0.043). Conclusion BTG3 may be involved in the occurence and development of tumor, and its low expression may be associated with poor prognosis in patients with PDAC.

When Escherichia coli CICIM B0013-030 (B0013, ack-pta, pps, pflB) was used for D-lactate production, succinate and acetate were the main byproducts (as much as 11.9 and 7.1% the amount of lactate respectively). In order to decrease the byproduct levels, we inactivated succinate and acetate synthesis in B0013-030. Two recombinant plasmids containing mutation cassettes of frdA::difGm and tdcDE::difGm respectively were constructed first. The mutation cassettes were used to delete the target genes on the chromosomal by Red recombination. Subsequently, the antibiotic resistance gene was excised from the chromosomal by Xer recombination. Thereby, mutants B0013-040B (B0013-030, frdA) and B0013-050B (B0013-040B, tdcDE) were produced. D-lactate producing abilities of the engineered strains were tested both in shake flasks and in bioreactors using two-phase fermentation (aerobic growth and anaerobic fermentation) with glucose as the sole carbon source. When fermentation was carried out in shake flasks, inactivation of frdA in B0013-030 to produce B0013-040B reduced succinate accumulation by 80.8%. When tested in a 7-liter bioreactor, B0013-040B accumulated 114.5 g/L D-lactate of over 99.9% optical purity. However, 1.0 g/L succinate and 5.4 g/L acetate still remained in the broth. Further inactivation of tdcD and tdcE genes in B0013-040B to produce B0013-050B decreased acetate and succinate accumulation to 0.4 g/L and 0.4 g/L respectively, and lactate titer was as much as 111.9 g/L (tested in the 7-liter bioreactor). In lightof the lower byproduct levels and high lactate production, strain B00 13-050B may prove useful for D-lactate production.
Acetates ; metabolism ; Escherichia coli ; genetics ; metabolism ; Fermentation ; Genetic Engineering ; Lactic Acid ; biosynthesis ; Metabolic Networks and Pathways ; genetics ; Mutation ; Plasmids ; genetics ; Succinic Acid ; metabolism

Objective To investigate the community structure and spatial distribution of wild myomorpha in the plague foci of Lianghe County,Yunnan Province.Methods From December 2015 to October 2016,in Mangdong,Hexi,Nangsong and other places in Lianghe County,forest landscape samples were randomly selected in different altitude gradients (1 000 ～,1 200 ～,1 400 ～,≥ 1 600 m).Peanut was used as a bait for trapping myomorpha,and the community structure of myomorpha was analyzed.Results A total of 663 myomorpha were collected and the density was 7.97％ (663/8 320),which were divided into 16 species,11 genus,5 families,and 3 orders.Rattus tanezumi (30.62％,203/663) was the dominant species.In the 1 000-＜ 1 200 m altitude gradient,the richness of myomorpha was the highest (13 kinds).In the 1 400-＜ 1 600 m altitude gradient,the index of diversity (2.094 2) was the highest.With the increase of altitude,the ecological dominance were increased,and the values were 0.672 2,0.774 2,0.842 8,and 0.882 4,respectively,whereas the evenness presented a decreasing trend,and the values were 0.269 2,0.197 1,0.142 8,and 0.162 0.Conclusions The Rattus tanezumi has presented a single dominant distribution at different elevation and the number and the species of the wild rodent with superior position have increased with the altitude increased in Lianghe focus.With the increase of altitude,the diversity and the evenness have increased,whereas the richness and the dominance have decreased.

Objective To investigate the geographic landscape features in Yulong plague natural focus.Methods Four altitudinal gradients (2 400-,2 600-,2 800-,≥3 000 m) were selected as study objects in Yulong plague natural focus from December 2015 to November 2016 to collect and analyze geographic landscape factors (topography and terrain,climate,water areas,vegetation and soil) based on the literatures about this plague focus.Results Yulong plague natural focus was an area with mid-mountain and subalpine topography in northwestern Yunnan Province,and its terrain had the character of lower in the southeast and higher in the northwest.This area belonged to plateau warm temperate monsoon climate of low latitude with an average month temperature 13.6 ℃ from 2005-2016,month air relative humidity averaged 59.4％,and rainfall averaged 884.5 mm a year,which concentrated mostly from June to September.About 98.1％ of the water area in Yulong County was controlled by Chin-sha River.The vegetation and soil were characterized by vertical distribution along with altitude gradients obviously.Conclusion The unique geographical landscape of the plague natural focus in Yulong County is one of important factors affecting the occurrence and epidemic of the plague.

Objective:To detect and analyze the contents of eight metal elements in water samples of plague foci in Yunnan Province.Methods:During the period from December 2015 to November 2016, the plague foci of Yulong, Jianchuan and Lianghe were selected as sampling sites, water samples were collected in areas with rodent activities in the 4 seasons of spring, summer, autumn and winter. The contents of eight metal elements calcium (Ca), iron (Fe), zinc (Zn), chromium (Cr), plumbum (Pb), manganese (Mn), cadmium (Cd) and cuprum (Cu) in water samples were measured by flame atomic absorption spectrometry (FAAS), and the data [median (interquartile distance)] were statistically analyzed.Results:Twenty-six, 58 and 54 water samples were collected from Yulong, Jianchuan and Lianghe plague foci, respectively. The contents of metal elements of Pb and Cd in water samples of the three plague foci [Yulong: 0.19 (0.78) and 0.08 (0.07) mg/L; Jianchuan: 0.23 (0.56) and 0.03 (0.06) mg/L; Lianghe: 0.13 (0.61) and 0.09 (0.08) mg/L] were higher than that of "Environmental Quality Standards for Surface Water" (Pb: 0.10 mg/L, Cd: 0.01 mg/L). There were significant differences in the contents of Ca and Cd elements among the three regions ( P < 0.05), but there was no significant difference in the contents of the other 6 metal elements among the three regions ( P > 0.05). The content of Ca element was the highest in Yulong plague foci, and the lowest in Lianghe plague foci ( P < 0.017). In the Yulong plague foci, there was no statistically significant difference in the content of Fe element in different seasons ( P > 0.05), and the differences in the contents of the other 7 metal elements were statistically significant ( P < 0.05). There was no significant difference in the content of Cr element in Jianchuan plague foci in different seasons ( P > 0.05), and the differences in the content of the other 7 metal elements were statistically significant ( P < 0.05). There was no significant difference in the content of Ca element in Lianghe plague foci in different seasons ( P > 0.05), and the differences in the content of the other 7 metal elements were statistically significant ( P < 0.05). Conclusion:The metal element contents of Pb and Cd are relatively abundant in water samples from 3 plague foci of Yunnan Province, and the seasonal variation trend of metal element content in water samples of Yulong and Jianchuan plague foci is similar.

Objective To compare and analyze the soil eight metal elements contents of rat holes of three plague foci in Yunnan Province.Methods The soils were collected from the rat holes in Yulong,Jianchuan,and Lianghe plague foci.Calcium (Ca),iron (Fe),zinc (Zn),chromium (Cr),lead (Pb),manganese (Mn),cadmium (Cd),and copper (Cu) were quantified with flame atomic absorption spectrometry (FAAS).Data input,calculation and collation were conducted through Excel 2007,and statistical software SPSS 20.0 was used to carried out statistical analysis through rank sum test.Results The medians of Ca content in the rat hole soil of Yulong County,Jianchuan County,and Lianghe County,respectively,were 85.40,308.56,and 0.00 mg/kg;the medians of Fe were 2 569.07,1 950.16,and 975.72 mg/kg;the medians of Zn were 5.64,4.02,and 2.04 mg/kg;the medians of Cr were 1.09,3.06,and 0.71 mg/kg;the medians of Pb were 3.61,29.56,and 22.80 mg/kg;the medians of Mn were 223.01,212.97,and 80.43 mg/kg;the medians of Cd were 0.00,2.02,and 2.94 mg/kg;and the medians of Cu were 1.92,1.08,and 0.51 mg/kg.The contents of eight metal elements were statistically different in the soil samples between Yulong,Jianchuan,and Lianghe plague foci (H =80.538,127.687,202.583,185.359,124.747,150.057,91.346,228.546,P ＜ 0.05).Conclusions The content of Fe in soil of the three plague foci in Yunnan Province is abundant.The contents of metal elements of Yulong County and Jianchuan County are similar.