Su L

References (27)

Title : Mitochondrial Esterase Activity Measured at the Single Organelle Level by Nano-flow Cytometry - Su_2024_Anal.Chem__
Author(s) : Su L , Gao K , Tian Y , Xiao X , Lu C , Xu J , Yan X
Ref : Analytical Chemistry , : , 2024
Abstract : Monitoring mitochondrial esterase activity is crucial not only for investigating mitochondrial metabolism but also for assessing the effectiveness of mitochondrial-targeting prodrugs. However, accurately detecting esterase activity within mitochondria poses challenges due to its ubiquitous presence in cells and the uncontrolled localization of fluorogenic probes. To overcome this hurdle and reveal variations among different mitochondria, we isolated mitochondria and preserved their activity and functionality in a buffered environment. Subsequently, we utilized a laboratory-built nano-flow cytometer in conjunction with an esterase-responsive calcein-AM fluorescent probe to measure the esterase activity of individual mitochondria. This approach enabled us to investigate the influence of temperature, pH, metal ions, and various compounds on the mitochondrial esterase activity without any interference from other cellular constituents. Interestingly, we observed a decline in the mitochondrial esterase activity following the administration of mitochondrial respiratory chain inhibitors. Furthermore, we found that mitochondrial esterase activity was notably higher in the presence of a high concentration of ATP compared to that of ADP and AMP. Additionally, we noticed a correlation between elevated levels of complex IV and increased mitochondrial esterase activity. These findings suggest a functional connection between the mitochondrial respiratory chain and mitochondrial esterase activity. Moreover, we detected an upsurge in mitochondrial esterase activity during the early stages of apoptosis, while cellular esterase activity decreased. This highlights the significance of analyzing enzyme activity within specific organelle subregions. In summary, the integration of a nano-flow cytometer and fluorescent dyes introduces a novel method for quantifying mitochondrial enzyme activity with the potential to uncover the alterations and unique functions of other mitochondrial enzymes.
ESTHER : Su_2024_Anal.Chem__
PubMedSearch : Su_2024_Anal.Chem__
PubMedID: 38173421

Title : ANGPTL3 accelerates atherosclerotic progression via direct regulation of M1 macrophage activation in plaque - Zhang_2024_J.Adv.Res__
Author(s) : Zhang Y , Yan C , Dong Y , Zhao J , Yang X , Deng Y , Su L , Yin J , Sun F , Feng Y
Ref : J Adv Res , : , 2024
Abstract : INTRODUCTION: The N-terminal domain of angiopoietin-like protein 3 (ANGPTL3) inhibits lipoprotein lipase activity. Its C-terminal fibrinogen-like (FBN) domain is a ligand of macrophage integrin alphavbeta3. OBJECTIVES: ANGPTL3 might home to plaque where it directly regulates macrophage function via integrin alphavbeta3 for atherosclerosis progression. METHODS: Ldlr(-/-) mice on a high-fat diet and ApoE(-/-) mice on a chow diet were received adeno-associated virus (AAV)-mediated Angptl3 gene transfer and followed up for 12 weeks. ApoE(-/-) mice were injected AAV containing FLAG-tagged Angptl3 cDNA for tracing. Atherosclerotic features were compared between Angptl3(-/-)ApoE(-/-) mice and ApoE(-/-) littermates. THP-1 cells were exposed to 0 or 50 microg/ml ANGPTL3 FBN domain for 24 h to evaluate Toll-like receptor (TLR)4 expression using western blot analysis and circulating cytokine and chemokine profiles by the MILLIPLEX MAP assay. Phospho-proteomic profile was established in ANGPTL3-treated macrophages. Integrin beta3 deficient THP-1 cells were obtained by sgRNAs targeting RGD sequence using Lentivirus-Cas9 system. RESULTS: Angptl3 overexpression increased atherosclerotic progression and CD68(+) macrophages in plaque (p < 0.05 for all). By immunostaining, FLAG(+) cells were identified in plaque of gene transferred ApoE(-/-) mice. Fluorescent immunostaining detected co-localisation of Angptl3 and CD68 in plaque macrophages. Phospho-proteomic analysis revealed that Angptl3 induced phosphorylation of proteins that were involved in the IL-17 signalling pathway in THP-1 cells. In vitro, ANGPTL3 treatment increased the production of interleukin (IL)-1beta and tumour necrosis factor-alpha in THP-1 cells (p < 0.05 for both). Exposure of ANGPTL3 to THP-1 cells induced Akt phosphorylation which was weakened in integrin beta3 deficient ones. ANGPTL3 elevated TLR4 expression via Akt phosphorylation. In response to lipopolysaccharide, nuclear factor-kappaB activity was 2.2-fold higher in THP-1 cells pre-treated with ANGPTL3 than in untreated cells (p < 0.05). CONCLUSIONS: Targeting ANGPTL3 could yield a dual benefit of lowering lipid levels in the blood and suppressing macrophage activation in plaque.
ESTHER : Zhang_2024_J.Adv.Res__
PubMedSearch : Zhang_2024_J.Adv.Res__
PubMedID: 38740260

Title : Engineering the stambomycin modular polyketide synthase yields 37-membered mini-stambomycins - Su_2022_Nat.Commun_13_515
Author(s) : Su L , Hotel L , Paris C , Chepkirui C , Brachmann AO , Piel J , Jacob C , Aigle B , Weissman KJ
Ref : Nat Commun , 13 :515 , 2022
Abstract : The modular organization of the type I polyketide synthases (PKSs) would seem propitious for rational engineering of desirable analogous. However, despite decades of efforts, such experiments remain largely inefficient. Here, we combine multiple, state-of-the-art approaches to reprogram the stambomycin PKS by deleting seven internal modules. One system produces the target 37-membered mini-stambomycin metabolites - a reduction in chain length of 14 carbons relative to the 51-membered parental compounds - but also substantial quantities of shunt metabolites. Our data also support an unprecedented off-loading mechanism of such stalled intermediates involving the C-terminal thioesterase domain of the PKS. The mini-stambomycin yields are reduced relative to wild type, likely reflecting the poor tolerance of the modules downstream of the modified interfaces to the non-native substrates. Overall, we identify factors contributing to the productivity of engineered whole assembly lines, but our findings also highlight the need for further research to increase production titers.
ESTHER : Su_2022_Nat.Commun_13_515
PubMedSearch : Su_2022_Nat.Commun_13_515
PubMedID: 35082289

Title : Isolation, sequencing of the HvnHID gene and its role in the purple-grain colour development in Tibetan hulless barley - Yao_2021_Czech.J.Genet.Plant.Breed__
Author(s) : Yao X , Su L , Yao Y , An L , Bai Y , Li X , Wu K
Ref : _Czech J Genet Plant Breed , : , 2021
Abstract : 2-hydroxyisoflavanone dehydratase (HID) plays an important role in isoflavone biosynthesis. In this study, HID was isolated from the seeds of the purple-grained Tibetan hulless barley variety Nerumuzha and the white-grained variety Kunlun 10. The HvnHID gene includes the 981 bp open reading frame and encodes a protein of 327 amino acids. It has a typical Abhydrolase_3 domain (78-306) and belongs to the carboxylesterase (CXE) family of the Abhydrolase_3 (alpha/beta hydrolase) superfamily. There are eight nucleotide differences in the HvnHID coding sequence and two amino acid differences (one in the Abhydrolase_3 domain) between Nerumuzha and Kunlun 10. The HvnHID of hulless barley has the closest relationship with the HID in Hordeum vulgare, and the most distant relationship in Panicum hallii. At the early-mid stage of the seed colour development, the HvnHID expression levels in the purple and black seeds were significantly higher than in the white and blue ones (P < 0.01). During the seed colour development of purple-grained hulless barley, the expression of the key genes (HvnF3'H, HvnDRF, HvnANT1, and HvnGT) in the anthocyanidin biosynthetic pathway increased significantly, while the HvnHID expression decreased significantly (P < 0.01). Thus, it is likely that HvnHID negatively regulates the anthocyanidin biosynthesis. This result provides an important basis for further study of the biological functions of HvnHID in the anthocyanidin biosynthetic pathway.
ESTHER : Yao_2021_Czech.J.Genet.Plant.Breed__
PubMedSearch : Yao_2021_Czech.J.Genet.Plant.Breed__
Gene_locus related to this paper: horvv-f2da29

Title : Structure-guided engineering of a Thermobifida fusca cutinase for enhanced hydrolysis on natural polyester substrate - Dong_2020_Bioresour.Bioprocess_7_37
Author(s) : Dong Q , Yuan S , Wu L , Su L , Zhao Q , Wu J , Huang W , Zhou J
Ref : Bioresour. Bioprocess , 7 :37 , 2020
Abstract : Cutinases could degrade insoluble polyester, including natural cutin and synthetic plastic. However, their turnover efficiency for polyester remains too low for industrial application. Herein, we report the 1.54-A resolution X-ray crystal structure of a cutinase from Thermobifida fusca and modeling structure in complex with a cutin mimic oligo-polyester C24H42O8. These efforts subsequently guided our design of cutinase variants with less bulky residues in the vicinity of the substrate binding site. The L90A and I213A variants exhibit increased hydrolysis activity (5- and 2.4-fold, respectively) toward cutin and also showed enhanced cotton scouring efficiency compared with the wild-type enzyme.
ESTHER : Dong_2020_Bioresour.Bioprocess_7_37
PubMedSearch : Dong_2020_Bioresour.Bioprocess_7_37
Gene_locus related to this paper: thefu-q6a0i4

Title : Design, synthesis and evaluation of diosgenin carbamate derivatives as multitarget anti-Alzheimer's disease agents - Yang_2020_Eur.J.Med.Chem_187_111913
Author(s) : Yang GX , Huang Y , Zheng LL , Zhang L , Su L , Wu YH , Li J , Zhou LC , Huang J , Tang Y , Wang R , Ma L
Ref : Eur Journal of Medicinal Chemistry , 187 :111913 , 2020
Abstract : In order to produce an effective and multi-targeted clinical drug that could prevent progressive neurodegeneration, a series of diosgenin carbamate derivatives were designed, synthesized and tested for their anti-inflammatory, antioxidant and anti-Abeta activities. The results demonstrated that compound M15 was the most promising derivative against inflammatory (NO inhibition 22.7 +/- 2.2%,10 muM) and cellular damage induced by H2O2 (SH-SY5Y cell protection = 75.3 +/- 3.4%, 10 muM) or Abeta (astrocytes protection = 70.2 +/- 6.5%, 10 muM). Molecular docking studies revealed the strong binding affinity of M15 to the active site of nNOS, Abeta42 and pro-inflammatory proteins. Western blot demonstrated that M15 decreased IL-1beta, IL-6 and TNF-alpha level, which may contribute to its anti-inflammatory effects. In addition, M15 maintained mitochondrial function as well as cell viability through reducing H2O2-induced ROS production. The results indicated that oral administration of M15 attenuated memory deficits and played a neuroprotective effect on subcutaneous (s.c.) D-gal aging mice. In summary, M15 could be considered as a potential multifunctional neuroprotective agent due to the effects of anti-inflammatory, antioxidant and anti-Abeta activities.
ESTHER : Yang_2020_Eur.J.Med.Chem_187_111913
PubMedSearch : Yang_2020_Eur.J.Med.Chem_187_111913
PubMedID: 31837501

Title : Enhanced activity towards polyacrylates and poly(vinyl acetate) by site-directed mutagenesis of Humicola insolens cutinase - Su_2020_Int.J.Biol.Macromol_162_1752
Author(s) : Su L , Hong R , Kong D , Wu J
Ref : Int J Biol Macromol , 162 :1752 , 2020
Abstract : Previous studies on the hydrolysis of polyacrylates by cutinase have found that cutinase from Humicola insolens can fulfill the requirement for a thermostable cutinase in the treatment of stickies from papermaking, but it has poor hydrolysis ability. To further improve its ability to hydrolyze the polymers in papermaking, we analyzed the structure of cutinase from H. insolens, and constructed three mutants L66A, I169A, and L66A/I169A to reduce the steric hindrance of the substrate binding region. The hydrolysis results for poly(methyl acrylate), poly(ethyl acrylate), and poly(vinyl acetate) showed the catalytic ability of the mutant L66A/I169A most significantly improved. Using polymer macroporous resin composites as substrate, the released products of L66A/I169A were 1.3-4.4 times higher than that of the wild-type enzyme. When polymer suspensions were no longer being deposited, that is, when the turbidity decrease was less than 1%, the amount of L66A/I169A added was reduced by 19%-51% compared with that of the wild-type enzyme. These results indicated that the removal of the gatekeeper structure above the substrate binding region of H. insolens cutinase enhances its ability to hydrolyze polymers, and provided a basis for the application of cutinase in the practical treatment of stickies.
ESTHER : Su_2020_Int.J.Biol.Macromol_162_1752
PubMedSearch : Su_2020_Int.J.Biol.Macromol_162_1752
PubMedID: 32771512
Gene_locus related to this paper: humin-cut

Title : In Situ Assessment of Donghu Lake China Using Rare Minnow (Gobiocypris rarus) - Xiong_2020_Arch.Environ.Contam.Toxicol_79_246
Author(s) : Xiong X , Qiu N , Su L , Hou M , Xu C , Xiong Y , Dong X , Song Z , Wang J
Ref : Archives of Environmental Contamination & Toxicology , 79 :246 , 2020
Abstract : In this work, rare minnow (Gobiocypris rarus) was applied as a sentinel organism and set in cages at control and test sampling sites in Donghu Lake for 4 weeks in March, June, September, and December 2016 to assess the biological toxicity of in situ water. Sampling for active biomonitoring and physicochemical variables was performed weekly. The control was obtained from the outdoor pool of the Institute of Hydrobiology, China. Superoxide dismutase, lipoperoxidation, metallothioneins, acetylcholinesterase activity, and Vtg mRNA expression were determined as biomarkers during the field exposure period. Survival and growth also were monitored to evaluate the overall physiological condition of the fish. The seasonal changes of organic pollutants and trace metals (As, Hg, Cr, Cu, Zn, Cd, Pb) in surface water were determined. The integrated biomarker response (IBR) index was applied to summarize biomarker responses and correlate stress levels with concentrations of organic pollutants and trace metals in the surface water. Results indicated that complex pollution by persistent organic pollutants and heavy metals was present in Donghu Lake and that the in situ exposed organisms were stressed. Moreover, the complex pollution of Donghu Lake in summer and autumn was more serious than that in spring and winter. Active biomonitoring combined with IBR analysis enabled good discrimination among different exposure seasons. The proposed protocol with caged rare minnow revealed marked biological effects caused by the investigated Lake and a useful approach that can easily be extended to monitor water pollution.
ESTHER : Xiong_2020_Arch.Environ.Contam.Toxicol_79_246
PubMedSearch : Xiong_2020_Arch.Environ.Contam.Toxicol_79_246
PubMedID: 32607658

Title : Carboxylesterase-Cleavable Biotinylated Nanoparticle for Tumor-Dual Targeted Imaging - Chen_2019_Theranostics_9_7359
Author(s) : Chen P , Kuang W , Zheng Z , Yang S , Liu Y , Su L , Zhao K , Liang G
Ref : Theranostics , 9 :7359 , 2019
Abstract : Near-infrared (NIR) nanoprobes with fluorescence "Turn-On" property are advantageous in cancer diagnosis but, to the best of our knowledge, "smart" nanoprobe that simultaneously targets both biotin receptor and carboxylesterase (CES) for HepG2 tumor-dual targeted imaging has not been reported. Methods: Using CBT-Cys click condensation reaction, we rationally designed a "smart" NIR fluorescence probe H2N-Cys(StBu)-Lys(Biotin)-Ser(Cy5.5)-CBT (NIR-CBT) and used it to facilely prepare the fluorescence-quenched nanoparticle NIR-CBT-NP. Results: In vitro results indicated that, after NIR-CBT-NP was incubated with CES for 6 h, its fluorescence was turned "On" by 69 folds. Cell experiments verified that NIR-CBT-NP was uptaken by HepG2 cells via biotin receptor-assisted endocytosis and its fluorescence was turned "On" by intracellular CES hydrolysis. Moreover, NIR-CBT-NP was successfully applied to image both biotin receptor- and CES-overexpressing HepG2 tumors. Conclusion: Fluorescence-quenched nanoparticle NIR-CBT-NP was facilely prepared to actively target biotin receptor-overexpressing HepG2 cancer cells and turn the fluorescence "On" by intracellular CES hydrolysis for tumor-dual targeted imaging. We anticipate that our fluorescence "Turn-On" nanoparticle could be applied for liver cancer diagnosis in clinic in the near future.
ESTHER : Chen_2019_Theranostics_9_7359
PubMedSearch : Chen_2019_Theranostics_9_7359
PubMedID: 31695773

Title : High-level expression of Humicola insolens cutinase in Pichia pastoris without carbon starvation and its use in cotton fabric bioscouring - Hong_2019_J.Biotechnol_304_10
Author(s) : Hong R , Sun Y , Su L , Gu L , Wang F , Wu J
Ref : J Biotechnol , 304 :10 , 2019
Abstract : Huimcola insolens cutinase (HiC) was heterologously expressed in Pichia pastoris. To avoid a carbon starvation step, fermentation was conducted using combinations of sorbitol with glycerol and methanol in the cell growth and induction phases, respectively. The cutinase productivity (27.71 U mL(-1) h(-1)) was 9.93 U mL(-1) h(-1) greater than that achieved using traditional two-phase methods, and a cutinase activity of 2660 U mL(-1), using p-nitrophenyl butyrate as substrate, was achieved after only 96 h in a 3-L bioreactor. Subsequently, the combination of HiC with Thermobifida fusca cutinase (TfC) in cotton fabric bioscouring was evaluated by monitoring the wettability and dyeability of the fabric. Treatment with 20 U mL(-1) of HiC at 80 degrees C for 5 min followed by 30 U mL(-1) of TfC at 50 degrees C for 1 h gave the best results. The total treatment time was shorter and performance was better than those seen with the alkali method.
ESTHER : Hong_2019_J.Biotechnol_304_10
PubMedSearch : Hong_2019_J.Biotechnol_304_10
PubMedID: 31400343

Title : Short-chain aliphatic ester synthesis using Thermobifida fusca cutinase - Su_2016_Food.Chem_206_131
Author(s) : Su L , Hong R , Guo X , Wu J , Xia Y
Ref : Food Chem , 206 :131 , 2016
Abstract : Short-chain aliphatic esters are commonly used as fruit flavorings in the food industry. In this study, Thermobifida fusca (T. fusca) cutinase was used for the synthesis of aliphatic esters, and the maximum yield of ethyl caproate reached 99.2% at a cutinase concentration of 50U/ml, 40 degrees C, and water content of 0.5%, representing the highest ester yield to date. The cutinase-catalyzed esterification displayed strong tolerance for water content (up to 8%) and acid concentration (up to 0.8M). At substrate concentrations 0.8M, the ester yield remained above 80%. Moreover, ester yields of more than 98% and 95% were achieved for acids of C3-C8 and alcohols of C1-C6, respectively, indicating extensive chain length selectivity of the cutinase. These results demonstrate the superior ability of T. fusca cutinase to catalyze the synthesis of short-chain esters. This study provides the basis for industrial production of short-chain esters using T. fusca cutinase.
ESTHER : Su_2016_Food.Chem_206_131
PubMedSearch : Su_2016_Food.Chem_206_131
PubMedID: 27041308

Title : Extracellular expression of natural cytosolic arginine deiminase from Pseudomonas putida and its application in the production of l-citrulline - Su_2015_Bioresour.Technol_196_176
Author(s) : Su L , Ma Y , Wu J
Ref : Bioresour Technol , 196 :176 , 2015
Abstract : The Pseudomonas putida arginine deiminase (ADI), a natural cytosolic enzyme, and Thermobifida fusca cutinase were co-expressed in Escherichia coli, and the optimized cutinase gene was used for increasing its expression level. 90.9% of the total ADI protein was released into culture medium probably through a nonspecific leaking mechanism caused by the co-expressed cutinase. The enzymatic properties of the extracellular ADI were found to be similar to those of ADI prepared by conventional cytosolic expression. Extracellular production of ADI was further scaled up in a 3-L fermentor. When the protein expression was induced by IPTG (25.0muM) and lactose (0.1gL-1h-1) at 30 degrees C, the extracellular ADI activity reached 101.2UmL-1, which represented the highest ADI production ever reported. In addition, the enzymatic synthesis of l-citrulline was performed using the extracellularly expressed ADI, and the conversion rate reached 100% with high substrate concentration at 650gL-1.
ESTHER : Su_2015_Bioresour.Technol_196_176
PubMedSearch : Su_2015_Bioresour.Technol_196_176
PubMedID: 26233330

Title : Extracellular expression of Thermobifida fusca cutinase with pelB signal peptide depends on more than type II secretion pathway in Escherichia coli - Su_2015_J.Biotechnol_204_47
Author(s) : Su L , Yu L , Xu C , Wu J
Ref : J Biotechnol , 204 :47 , 2015
Abstract : Our previous studies demonstrated that Thermobifida fusca cutinase is released into culture medium when expressed without a signal peptide in Escherichia coli, and this extracellular expression results from an enhanced membrane permeability caused by cutinase's phospholipid hydrolase activity. The present study investigated whether this phenomenon would also occur during the expression of cutinase fused to pelB signal peptide (pelB-cutinase). Secretion of fusion proteins of this type is generally believed to occur via type II secretion pathway. The results showed that when pelB-cutinase was expressed in a secB knockout strain, which has a defective type II secretion pathway, there was still a large amount of cutinase in the culture medium. Additional experiments confirmed that the periplasmic and cytoplasmic fractions of the expressing cells had hydrolytic activity toward phosphatidyl ethanolamine, and the recombinant cells showed correspondingly improved membrane permeability. All these phenomena were also observed in the parent E. coli strain. Moreover, the secretion efficiency of the inactive cutinase mutant was found to be significantly lower than that of pelB-cutinase in the parent E. coli. Based on these results, the phospholipid hydrolase activity of pelB-cutinase must play a larger role in its extracellular production than does type II secretion pathway.
ESTHER : Su_2015_J.Biotechnol_204_47
PubMedSearch : Su_2015_J.Biotechnol_204_47
PubMedID: 25863154

Title : Neuroimaging characteristics of dementia with Lewy bodies - Mak_2014_Alzheimers.Res.Ther_6_18
Author(s) : Mak E , Su L , Williams GB , O'Brien JT
Ref : Alzheimers Res Ther , 6 :18 , 2014
Abstract : This review summarises the findings and applications from neuroimaging studies in dementia with Lewy bodies (DLB), highlighting key differences between DLB and other subtypes of dementia. We also discuss the increasingly important role of imaging biomarkers in differential diagnosis and outline promising areas for future research in DLB. DLB shares common clinical, neuropsychological and pathological features with Parkinson's disease dementia and other dementia subtypes, such as Alzheimer's disease. Despite the development of consensus diagnostic criteria, the sensitivity for differential diagnosis of DLB in clinical practice remains low and many DLB patients will be misdiagnosed. The importance of developing accurate imaging markers in dementia is highlighted by the potential for treatments targeting specific molecular abnormalities as well as the responsiveness to cholinesterase inhibitors and marked neuroleptic sensitivity of DLB. We review various brain imaging techniques that have been applied to investigate DLB, including the characteristic nigrostriatal degeneration in DLB using positron emission tomography (PET) and single-photon emission computed tomography (SPECT) tracers. Dopamine transporter loss has proven to reliably differentiate DLB from other dementias and has been incorporated into the revised clinical diagnostic criteria for DLB. To date, this remains the 'gold standard' for diagnostic imaging of DLB. Regional cerebral blood flow, 18 F-fluorodeoxygluclose-PET and SPECT have also identified marked deficits in the occipital regions with relative sparing of the medial temporal lobe when compared to Alzheimer's disease. In addition, structural, diffusion, and functional magnetic resonance imaging techniques have shown alterations in structure, white matter integrity, and functional activity in DLB. We argue that the multimodal identification of DLB-specific biomarkers has the potential to improve ante-mortem diagnosis and contribute to our understanding of the pathological background of DLB and its progression.
ESTHER : Mak_2014_Alzheimers.Res.Ther_6_18
PubMedSearch : Mak_2014_Alzheimers.Res.Ther_6_18
PubMedID: 25031634

Title : Synthesis and characterization of 1H-phenanthro[9,10-d]imidazole derivatives as multifunctional agents for treatment of Alzheimer's disease - Liu_2014_Biochim.Biophys.Acta_1840_2886
Author(s) : Liu J , Qiu J , Wang M , Wang L , Su L , Gao J , Gu Q , Xu J , Huang SL , Gu LQ , Huang ZS , Li D
Ref : Biochimica & Biophysica Acta , 1840 :2886 , 2014
Abstract : BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative brain disorder that is characterized by dementia, cognitive impairment, and memory loss. Diverse factors are related to the development of AD, such as increased level of beta-amyloid (Abeta), acetylcholine, metal ion deregulation, hyperphosphorylated tau protein, and oxidative stress.
METHODS: The following methods were used: organic syntheses of 1H-phenanthro[9,10-d]imidazole derivatives, inhibition of self-mediated and metal-induced Abeta1-42 aggregation, inhibition studies for acetylcholinesterase and butyrylcholinesterase, anti-oxidation activity studies, CD, MTT assay, transmission electron microscopy, dot plot assay, gel electrophoresis, Western blot, and molecular docking studies.
RESULTS: We synthesized and characterized a new type of 1H-phenanthro[9,10-d]imidazole derivatives as multifunctional agents for AD treatment. Our results showed that most of these derivatives exhibited strong Abeta aggregation inhibitory activity. Compound 9g had 74% Abeta1-42 aggregation inhibitory effect at 10muM concentration with its IC50 value of 6.5muM for self-induced Abeta1-42 aggregation. This compound also showed good inhibition of metal-mediated (Cu2+ and Fe2+) and acetylcholinesterase-induced Abeta1-42 aggregation, as indicated by using thioflavin T assay, transmission electron microscopy, gel electrophoresis, and Western blot. Besides, compound 9g exhibited cholinesterase inhibitory activity, with its IC50 values of 0.86muM and 0.51muM for acetylcholinesterase and butyrylcholinesterase, respectively. In addition, compound 9g showed good anti-oxidation effect with oxygen radical absorbance capacity (ORAC) value of 2.29.
CONCLUSIONS: Compound 9g was found to be a potent multi-target-directed agent for Alzheimer's disease. GENERAL SIGNIFICANCE: Compound 9g could become a lead compound for further development as a multi-target-directed agent for AD treatment.
ESTHER : Liu_2014_Biochim.Biophys.Acta_1840_2886
PubMedSearch : Liu_2014_Biochim.Biophys.Acta_1840_2886
PubMedID: 24821011

Title : Cutinase: Characteristics, preparation, and application - Chen_2013_Biotechnol.Adv_31_1754
Author(s) : Chen S , Su L , Chen J , Wu J
Ref : Biotechnol Adv , 31 :1754 , 2013
Abstract : Cutinases (E.C. belong to the alpha/beta-hydrolase superfamily. They were initially discovered because they are secreted by fungi to hydrolyze the ester bonds of the plant polymer cutin. Since then, they have been shown to catalyze the hydrolysis of a variety of polymers, insoluble triacylglycerols, and low-molecular-weight soluble esters. Cutinases are also capable of catalyzing esterification and transesterification reactions. These relatively small, versatile, secreted catalysts have shown promise in a number of industrial applications. This review begins by describing the characteristics of cutinases, pointing out key differences among cutinases, esterases and lipases, and reviewing recent progress in engineering improved cutinases. It continues with a review of the methods used to produce cutinases, with the goal of obtaining sufficient quantities of material for use in industrial processes. Finally, the uses of cutinases in the textile industry are described. The studies presented here demonstrate that the cutinases are poised to become important industrial catalysts, replacing older technologies with more environmentally friendly processes.
ESTHER : Chen_2013_Biotechnol.Adv_31_1754
PubMedSearch : Chen_2013_Biotechnol.Adv_31_1754
PubMedID: 24055682

Title : Extracellular location of Thermobifida fusca cutinase expressed in Escherichia coli BL21(DE3) without mediation of a signal peptide - Su_2013_Appl.Environ.Microbiol_79_4192
Author(s) : Su L , Woodard RW , Chen J , Wu J
Ref : Applied Environmental Microbiology , 79 :4192 , 2013
Abstract : Cutinase is a multifunctional esterase with potential industrial applications. In the present study, a truncated version of the extracellular Thermobifida fusca cutinase without a signal peptide (referred to as cutinase(NS)) was heterologously expressed in Escherichia coli BL21(DE3). The results showed that the majority of the cutinase activity was located in the culture medium. In a 3-liter fermentor, the cutinase activity in the culture medium reached 1,063.5 U/ml (2,380.8 mg/liter), and the productivity was 40.9 U/ml/h. Biochemical characterization of the purified cutinase(NS) showed that it has enzymatic properties similar to those of the wild-type enzyme. In addition, E. coli cells producing inactive cutinase(NS)S130A were constructed, and it was found that the majority of the inactive enzyme was located in the cytoplasm. Furthermore, T. fusca cutinase was confirmed to have hydrolytic activity toward phospholipids, an important component of the cell membrane. Compared to the cells expressing the inactive cutinase(NS)S130A, the cells expressing cutinase(NS) showed increased membrane permeability and irregular morphology. Based on these results, a hypothesis of "cell leakage induced by the limited phospholipid hydrolysis of cutinase(NS)" was proposed to explain the underlying mechanism for the extracellular release of cutinase(NS).
ESTHER : Su_2013_Appl.Environ.Microbiol_79_4192
PubMedSearch : Su_2013_Appl.Environ.Microbiol_79_4192
PubMedID: 23603671

Title : A novel strategy for enhancing extracellular secretion of recombinant proteins in Escherichia coli - Su_2013_Appl.Microbiol.Biotechnol_97_6705
Author(s) : Su L , Xu C , Woodard RW , Chen J , Wu J
Ref : Applied Microbiology & Biotechnology , 97 :6705 , 2013
Abstract : Secretion of cytoplasmic expressed proteins into culture medium has significant commercial advantages in large-scale production of proteins. Our previous study demonstrated that the membrane permeability of Escherichia coli could be significantly improved when Thermobifida fusca cutinase, without a signal peptide, was expressed in cytoplasm. This study investigated the extracellular production of other recombinant proteins, including both secretory and cytosolic proteins, with co-expression of cutinase. When the secretory enzymes, xylanase and alpha-amylase, were co-expressed with cutinase, the culture period was shortened by half, and the productivity was 7.9 and 2.0-fold to that of their individual control without co-expression, respectively. When the normally cytosolic proteins, xylose isomerase and trehalose synthase, were co-expressed with cutinase, more than half of the target proteins were "secreted" into the culture medium. Moreover, by using beta-galactosidase to detect membrane leakage, the improved secretion of the above model proteins was confirmed not to be due to cell lysis. The study provides a novel strategy for enhancing extracellular secretion of recombinant proteins in E. coli.
ESTHER : Su_2013_Appl.Microbiol.Biotechnol_97_6705
PubMedSearch : Su_2013_Appl.Microbiol.Biotechnol_97_6705
PubMedID: 23722267

Title : Extracellular overexpression of recombinant Thermobifida fusca cutinase by alpha-hemolysin secretion system in E. coli BL21(DE3) - Su_2012_Microb.Cell.Fact_11_8
Author(s) : Su L , Chen S , Yi L , Woodard RW , Chen J , Wu J
Ref : Microb Cell Fact , 11 :8 , 2012
Abstract : BACKGROUND: Extracellular expression of proteins has an absolute advantage in a large-scale industrial production. In our previous study, Thermobifida fusca cutinase, an enzyme mainly utilized in textile industry, was expressed via type II secretory system in Escherichia coli BL21(DE3), and it was found that parts of the expressed protein was accumulated in the periplasmic space. Due to the fact that alpha-hemolysin secretion system can export target proteins directly from cytoplasm across both cell membrane of E. coli to the culture medium, thus in the present study we investigated the expression of cutinase using this alpha-hemolysin secretion system. RESULTS: T. fusca cutinase was fused with the specific signal peptide of alpha-hemolysin scretion system and expressed in E. coli BL21(DE3). In addition, HlyB and HlyD, strain-specific translocation components of alpha-hemolysin secretion system, were coexpressed to facilitate the enzyme expression. The cultivation of this engineered cell showed that cutinase activity in the culture medium reached 334 U/ml, which is 2.5 times that from type II secretion pathway under the same culture condition. The recombinant cutinase was further purified. Biochemical characterization of purified enzyme, which had an alpha-hemolysin secretion pathway signal peptide attached, had substrate specificity, pH and temperature profile, as well as application capability in bioscouring similar to that of wild-type cutinase. CONCLUSIONS: In the present study, T. fusca cutinase was successfully secreted to the culture media by alpha-hemolysin secretion system. This is the first report of cutinase being efficiently secreted by this pathway. Due to the limited cases of successful expression of industrial enzyme by E. coli alpha-hemolysin secretion system, our study further explored the utilization of this pathway in industrial enzymes.
ESTHER : Su_2012_Microb.Cell.Fact_11_8
PubMedSearch : Su_2012_Microb.Cell.Fact_11_8
PubMedID: 22239833

Title : Putative EPHX1 enzyme activity is related with risk of lung and upper aerodigestive tract cancers: a comprehensive meta-analysis - Li_2011_PLoS.One_6_e14749
Author(s) : Li X , Hu Z , Qu X , Zhu J , Li L , Ring BZ , Su L
Ref : PLoS ONE , 6 :e14749 , 2011
Abstract : BACKGROUND: EPHX1 is a key enzyme in metabolizing some exogenous carcinogens such as products of cigarette-smoking. Two functional polymorphisms in the EPHX1 gene, Tyr113His and His139Arg can alter the enzyme activity, suggesting their possible association with carcinogenesis risk, particularly of some tobacco-related cancers. METHODOLOGY/PRINCIPAL FINDINGS: A comprehensive systematic review and meta-analysis was performed of available studies on these two polymorphisms and cancer risk published up to November 2010, consisting of 84 studies (31144 cases and 42439 controls) for Tyr113His and 77 studies (28496 cases and 38506 controls) for His139Arg primarily focused on lung cancer, upper aerodigestive tract (UADT) cancers (including oral, pharynx, larynx and esophagus cancers), colorectal cancer or adenoma, bladder cancer and breast cancer. Results showed that Y113H low activity allele (H) was significantly associated with decreased risk of lung cancer (OR = 0.88, 95%CI = 0.80-0.96) and UADT cancers (OR = 0.86, 95%CI = 0.77-0.97) and H139R high activity allele (R) with increased risk of lung cancer (OR = 1.18, 95%CI = 1.04-1.33) but not of UADT cancers (OR = 1.05, 95%CI = 0.93-1.17). Pooled analysis of lung and UADT cancers revealed that low EPHX1 enzyme activity, predicted by the combination of Y113H and H139R showed decreased risk of these cancers (OR = 0.83, 95%CI = 0.75-0.93) whereas high EPHX1 activity increased risk of the cancers (OR = 1.20, 95%CI = 0.98-1.46). Furthermore, modest difference for the risk of lung and UADT cancers was found between cigarette smokers and nonsmokers both in single SNP analyses (low activity allele H: OR = 0.77/0.85 for smokers/nonsmokers; high activity allele R: OR = 1.20/1.09 for smokers/nonsmokers) and in combined double SNP analyses (putative low activity: OR = 0.73/0.88 for smokers/nonsmokers; putative high activity: OR = 1.02/0.93 for smokers/ nonsmokers). CONCLUSIONS/SIGNIFICANCE: Putative low EPHX1 enzyme activity may have a potential protective effect on tobacco-related carcinogenesis of lung and UADT cancers, whereas putative high EPHX1 activity may have a harmful effect. Moreover, cigarette-smoking status may influence the association of EPHX1 enzyme activity and the related cancer risk.
ESTHER : Li_2011_PLoS.One_6_e14749
PubMedSearch : Li_2011_PLoS.One_6_e14749
PubMedID: 21445251

Title : Biochemical characterization of the cutinases from Thermobifida fusca - Chen_2010_J.Mol.Catal.B.Enzym_63_121
Author(s) : Chen S , Su L , Billig S , Zimmermann W , Chen J , Wu J
Ref : J Mol Catal B Enzym , 63 :121 , 2010
Abstract : Thermobifida fusca produces two cutinases which share 93% identity in amino acid sequence. In the present study, we investigated the detailed biochemical properties of T. fusca cutinases for the first time. For a better comparison between bacterial and fungal cutinases, recombinant Fusarium solani pisi cutinase was subjected to the similar analysis. The results showed that both bacterial and fungal cutinases are monomeric proteins in solution. The bacterial cutinases exhibited a broad substrate specificity against plant cutin, synthetic polyesters, insoluble triglycerides, and soluble esters. In addition, the two isoenzymes of T. fusca and the F.solani pisi cutinase are similar in substrate kinetics, the lack of interfacial activation, and metal ion requirements. However, the T.fusca cutinases showed higher stability in the presence of surfactants and organic solvents. Considering the versatile hydrolytic activity, good tolerance to surfactants, superior stability in organic solvents, and thermostability demonstrated by T. fusca cutinases, they may have promising applications in related industries.
ESTHER : Chen_2010_J.Mol.Catal.B.Enzym_63_121
PubMedSearch : Chen_2010_J.Mol.Catal.B.Enzym_63_121
Gene_locus related to this paper: thefu-q6a0i4 , thefu-q6a0i3

Title : Imprinting analysis of the porcine MEST gene in 75 and 90 day placentas and prenatal tissues - Xu_2007_Acta.Biochim.Biophys.Sin.(Shanghai)_39_633
Author(s) : Xu C , Su L , Zhou Q , Li C , Zhao S
Ref : Acta Biochim Biophys Sin (Shanghai) , 39 :633 , 2007
Abstract : Imprinted genes play important roles in mammalian growth, development and behavior. Mouse mesoderm-specific transcript (MEST) has been identified as an imprinted gene and mapped to an imprinted region of mouse chromosome 6 (MMU6). It plays essential roles in embryonic and placental growth, and it is required for maternal behavior in adult female mouse. Here, we isolated the porcine MEST gene and detected a single nucleotide polymorphism in the 3 -untranslated region. The RsaI polymorphism was used to investigate the allele frequencies in different pig breeds and the imprinting status in prenatal porcine tissues. Allele frequencies were significantly different between the native Chinese and Landrace breeds, except that most of the native Yushan pigs (21/26) are heterozygous at this locus. The results indicate that MEST was imprinted in placentas on days 75 and 90 of gestation as well as in the 75 d fetal heart, muscle, kidney, lung and liver.
ESTHER : Xu_2007_Acta.Biochim.Biophys.Sin.(Shanghai)_39_633
PubMedSearch : Xu_2007_Acta.Biochim.Biophys.Sin.(Shanghai)_39_633
PubMedID: 17687499

Title : Microsomal epoxide hydrolase, endotoxin, and lung function decline in cotton textile workers - Hang_2005_Am.J.Respir.Crit.Care.Med_171_165
Author(s) : Hang J , Zhou W , Wang X , Zhang H , Sun B , Dai H , Su L , Christiani DC
Ref : American Journal of Respiratory & Critical Care Medicine , 171 :165 , 2005
Abstract : Occupational exposure to endotoxin in organic dust may induce lung function decline. Microsomal epoxide hydrolase (mEH) detoxifies reactive oxygen species generated by endotoxin exposure, and polymorphisms of the mEH gene are associated with altered enzyme activity. We investigated the associations between mEH polymorphisms, endotoxin exposure, and lung function decline in a 20-year prospective study of 265 workers exposed to endotoxin and 234 control subjects. mEH Tyr113His and His139Arg polymorphisms were genotyped by the 5' nuclease assay, and data were analyzed using multivariate linear regression models, adjusting for important covariates. Overall, the annual decline rate of FEV1 was 29.47 ml during the 20-year follow-up. Endotoxin exposure was associated with faster lung function decline among genotypes associated with slower enzyme activity: estimates (SE) of annual FEV1 decline rates for endotoxin exposure were -2.33 (2.07), -2.81 (1.66), and -6.73 (2.83) ml for Tyr/Tyr, Tyr/His, and His/His genotype groups, respectively, for the Tyr113His polymorphism; and -1.82 (2.58) and -4.27 (1.33) ml for Arg/Arg + His/Arg and His/His genotypes, respectively, for the His139Arg polymorphism. We conclude that mEH polymorphisms modify the association between occupational endotoxin exposure and longitudinal lung function decline.
ESTHER : Hang_2005_Am.J.Respir.Crit.Care.Med_171_165
PubMedSearch : Hang_2005_Am.J.Respir.Crit.Care.Med_171_165
PubMedID: 15531751

Title : Relationship between polymorphisms of genes encoding microsomal epoxide hydrolase and glutathione S-transferase P1 and chronic obstructive pulmonary disease - Xiao_2004_Chin.Med.J.(Engl)_117_661
Author(s) : Xiao D , Wang C , Du MJ , Pang BS , Zhang HY , Xiao B , Liu JZ , Weng XZ , Su L , Christiani DC
Ref : Chinese Medical Journal (Engl) , 117 :661 , 2004
Abstract : BACKGROUND: Cigarette smoking is the major risk factor for chronic obstructive pulmonary disease (COPD). However, only 10% - 20% of chronic heavy cigarette smokers develop symptomatic disease. COPD is most likely the result of complex interactions between environmental and genetic factors. Genetic susceptibility to COPD might depend on the variations in enzyme activities that detoxify cigarette smoke products, such as microsomal epoxide hydrolase (mEH) and glutathione S-transferase (GST). In this study, we investigated the relationship between polymorphisms in the genes encoding mEH and glutathione S-transferase P1 (GSTP1) and COPD in a Chinese population. METHODS: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to find mEH polymorphism in exon 3 (Tyr113-->His), exon 4 (His139-->Arg) and GSTP1 polymorphism in exon 5 (Ile105-->Val) in 100 COPD patients and 100 age- and sex-matched healthy controls. RESULTS: The proportion of mEH exon 3 heterozygotes was significantly higher in patients with COPD than that in the control subjects (42% vs 32%). The odds ratio (OR) adjusted by age, sex, body mass index (BMI) and cigarette years was 2.96 (95% CI 1.24 - 7.09). There was no marked difference in very slow activity genotype versus other genotypes between COPD patients and the controls. When COPD patients were non-smokers, the OR of very slow activity genotype versus other genotypes was more than 1.00; and when COPD patients were smokers (current smokers and ex-smokers), the OR was less than 1.00. There was no significant difference in GSTP1 polymorphism adjusted by age, sex, BMI and smoking between COPD patients and the controls.
CONCLUSIONS: mEH exon 3 heterozygotes might be associated with susceptibility to COPD in China. The interaction might exist between mEH genotype and smoke. The gene polymorphism for GSTP1 might not be associated with susceptibility to COPD in the Chinese population.
ESTHER : Xiao_2004_Chin.Med.J.(Engl)_117_661
PubMedSearch : Xiao_2004_Chin.Med.J.(Engl)_117_661
PubMedID: 15161530

Title : [Association between polymorphisms in the microsomal epoxide hydrolase (mEH) gene and chronic obstructive pulmonary disease] - Xiao_2003_Zhonghua.Yi.Xue.Za.Zhi_83_1782
Author(s) : Xiao D , Wang C , Du MJ , Pang BS , Zhang HY , Xiao B , Liu JZ , Weng XZ , Su L , Christiani DC
Ref : Zhonghua Yi Xue Za Zhi , 83 :1782 , 2003
Abstract : OBJECTIVE: To investigate the association between polymorphisms in the microsomal epoxide hydrolase (mEH) gene and susceptibility to chronic obstructive pulmonary disease (COPD) in a Chinese population. METHODS: Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) were performed to genotype mEH polymorphisms in exon3 (Tyr113-->His) and exon4 (His139-->Arg) in 100 COPD patients and 100 age and sex matched healthy controls. RESULTS: (1) The proportion of mEH heterozygotes in exon3 was significantly higher in the patients with COPD than that in the control subjects (42% vs 32%). The odds ratio (OR) adjusted by age, sex, body mass index (BMI) and cigarettes years was 2.96 (95% CI 1.24 - 7.09). (2) There was no marked difference in very slow activity genotype versus other genotypes between COPD patients and controls. (3) When COPD patients were nonsmokers, the OR of very slow activity genotype versus other genotypes was more than 1.00, and when COPD patients were smokers (Current smokers and ex-smokers), the OR was less than 1.00.
CONCLUSIONS: (1) mEH heterozygotes in exon3 might be associated with the susceptibility to COPD in China. (2) The interaction might be existed between mEH genotype and smoke.
ESTHER : Xiao_2003_Zhonghua.Yi.Xue.Za.Zhi_83_1782
PubMedSearch : Xiao_2003_Zhonghua.Yi.Xue.Za.Zhi_83_1782
PubMedID: 14642084

Title : Genetic polymorphisms in N-acetyltransferase-2 and microsomal epoxide hydrolase, cumulative cigarette smoking, and lung cancer - Zhou_2002_Cancer.Epidemiol.Biomarkers.Prev_11_15
Author(s) : Zhou W , Liu G , Thurston SW , Xu LL , Miller DP , Wain JC , Lynch TJ , Su L , Christiani DC
Ref : Cancer Epidemiol Biomarkers Prev , 11 :15 , 2002
Abstract : N-acetyltrasferase-2 (NAT2) and microsomal epoxide hydrolase (mEH) are polymorphic genes that metabolize different tobacco carcinogens. Smaller studies found inconsistent relationships between NAT2 or mEH polymorphisms and lung cancer risk. To determine whether there is gene-environment interaction between NAT2 polymorphisms, alone or in combination with mEH polymorphisms, and cumulative smoking exposure in the development of lung cancer, we conducted a case control study of 1115 Caucasian lung cancer patients and 1250 spouse and friend controls. The results were analyzed using generalized additive models and logistic regression, adjusting for relevant covariates. There was no overall relationship between NAT2 genotype and lung cancer risk; the adjusted odds ratio (OR) of the rapid versus slow acetylator genotypes was 0.96 [95% confidence interval (CI), 0.79-1.16]. However, gene-environment interaction analyses revealed that the adjusted ORs increased significantly as pack-years increased. For nonsmokers, the fitted OR was 0.66 (95% CI, 0.44-0.99), whereas for heavy smokers (80 pack-years), the OR increased to 1.22 (95% CI, 0.89-1.67). When comparing the extreme genotype combinations of the NAT2 rapid acetylator, higher mEH activity genotype to the NAT2 slow acetylator, and very low mEH activity genotype, the corresponding ORs at 0 and 80 pack-years were 0.30 (95% CI, 0.14-0.62) and 2.19 (95% CI, 1.26-3.81), respectively. Results were similar with ORs derived from stratified models. In conclusion, NAT2 rapid acetylator genotypes are protective against lung cancer in nonsmokers but are risk factors in heavy smokers. The joint effects of NAT2 and mEH polymorphisms are consistent with an independent, additive effect of these two genes, modified by smoking history.
ESTHER : Zhou_2002_Cancer.Epidemiol.Biomarkers.Prev_11_15
PubMedSearch : Zhou_2002_Cancer.Epidemiol.Biomarkers.Prev_11_15
PubMedID: 11815396

Title : The interaction between microsomal epoxide hydrolase polymorphisms and cumulative cigarette smoking in different histological subtypes of lung cancer - Zhou_2001_Cancer.Epidemiol.Biomarkers.Prev_10_461
Author(s) : Zhou W , Thurston SW , Liu G , Xu LL , Miller DP , Wain JC , Lynch TJ , Su L , Christiani DC
Ref : Cancer Epidemiol Biomarkers Prev , 10 :461 , 2001
Abstract : Microsomal epoxide hydrolase (mEH) is involved in the metabolism of environmental and tobacco carcinogens. Smaller studies found inconsistent results in the relationship between mEH polymorphisms and lung cancer risk. We investigated the two polymorphisms of mEH in 974 Caucasian lung cancer patients and 1142 controls using PCR-RFLP techniques. The results were analyzed using generalized additive models and logistic regression, adjusting for relevant covariates. There was no overall relationship between mEH genotypes and lung cancer risk. The adjusted odds ratio (OR) of the very low activity genotype versus that of other genotypes combined was 1.00 [95% confidence interval (CI), 0.74-1.34]. However, gene-environment interaction analyses revealed that the ORs decreased as cumulative smoking (defined as square root of pack-years) increased. When pack-years = 0, the OR was 1.89 (95% CI, 1.08-3.28). When pack-years = 28.5, the OR was 1.00 (95% CI, 0.76-1.32), and when pack-years = 80, the OR decreased to 0.65 (95% CI, 0.42-1.00). When cases were stratified according to histological subtypes, the interaction between mEH genotype and cumulative smoking was statistically significant (P < 0.01) for the 222 squamous cell carcinoma cases, whereas it was not significant (P = 0.18) for the 432 adenocarcinoma cases. In conclusion, cumulative cigarette smoking plays a pivotal role in the association between mEH polymorphisms and lung cancer risk, altering the direction of risk (in the case of the very low activity genotype) from a risk factor in nonsmokers to a relatively protective factor in heavy smokers.
ESTHER : Zhou_2001_Cancer.Epidemiol.Biomarkers.Prev_10_461
PubMedSearch : Zhou_2001_Cancer.Epidemiol.Biomarkers.Prev_10_461
PubMedID: 11352855