Meyer AS

References (13)

Title : Relationships of crystallinity and reaction rates for enzymatic degradation of poly (ethylene terephthalate), PET - Schubert_2024_ChemSusChem__e202301752
Author(s) : Schubert SW , Thomsen TB , Clausen KSR , Malmendal A , Hunt CJ , Borch K , Jensen K , Brask J , Meyer AS , Westh P
Ref : ChemSusChem , :e202301752 , 2024
Abstract : Biocatalytic degradation of plastic waste is anticipated to play an important role in future recycling systems. However, enzymatic degradation of crystalline poly (ethylene terephthalate) (PET) remains consistently poor. Herein, we employed functional assays to elucidate the molecular underpinnings of this limitation. This included utilizing complementary activity assays to monitor the degradation of PET disks with varying crystallinity (XC), as well as kinetic parameters for soluble PET fragments. The results indicate that a proficient PET-hydrolase, LCCICCG, operates through an endolytic mode of action, and that its activity is limited by conformational constraints in the PET polymer. Such constraints become more pronounced at high XC values, and this limits the density of productive sites on the PET surface. Endolytic chain-scissions are the dominant reaction type in the initial stage, and this means that little or no soluble organic product occurs here. However, endolytic cuts gradually and locally promote chain mobility and hence the density of attack sites on the surface. This leads to an upward concave progress curve; a behavior sometimes termed lag-phase kinetics.
ESTHER : Schubert_2024_ChemSusChem__e202301752
PubMedSearch : Schubert_2024_ChemSusChem__e202301752
PubMedID: 38252197
Gene_locus related to this paper: 9bact-g9by57

Title : Fungal feruloyl esterases can catalyze release of diferulic acids from complex arabinoxylan - Lin_2023_Int.J.Biol.Macromol__123365
Author(s) : Lin S , Hunt CJ , Holck J , Brask J , Krogh K , Meyer AS , Wilkens C , Agger JW
Ref : Int J Biol Macromol , :123365 , 2023
Abstract : Feruloyl esterases (FAEs, EC catalyze the hydrolytic cleavage of ester bonds between feruloyl and arabinosyl moieties in arabinoxylans. Recently, we discovered that two bacterial FAEs could catalyze release of diferulic acid moieties (diFAs) from highly substituted, cross-linked corn bran arabinoxylan. Here, we show that several fungal FAEs, notably AnFae1 (Aspergillus niger), AoFae1 (A. oryzae), and MgFae1 (Magnaporthe oryzae (also known as M. grisae)) also catalyze liberation of diFAs from complex arabinoxylan. By comparing the enzyme kinetics of diFA release to feruloyl esterase activity of the enzymes on methyl- and arabinosyl-ferulate substrates we demonstrate that the diFA release activity cannot be predicted from the activity of the enzymes on these synthetic substrates. A detailed structure-function analysis, based on AlphaFold2 modeled enzyme structures and docking with the relevant di-feruloyl ligands, reveal how distinct differences in the active site topology and surroundings may explain the diFA releasing action of the enzymes. Interestingly, the analysis also unveils that the carbohydrate binding module of the MgFae1 may play a key role in the diFA releasing ability of this enzyme. The findings contribute further understanding of the function of FAEs in the deconstruction of complex arabinoxylans and provide new opportunities for enzyme assisted upgrading of complex bran arabinoxylans.
ESTHER : Lin_2023_Int.J.Biol.Macromol__123365
PubMedSearch : Lin_2023_Int.J.Biol.Macromol__123365
PubMedID: 36690236
Gene_locus related to this paper: humin-HiFae1 , malci-McFae1 , 9zzzz-CE1.6RZN , 9zzzz-DAC80243 , 9pezi-a0a481sy08 , aspni-FAEA , aspor-q2uh24 , aspor-q2umx6 , aspor-q2unw5 , aspor-q2up89 , neucr-faeb , magoy-l7ic25

Title : Rate Response of Poly(Ethylene Terephthalate)-Hydrolases to Substrate Crystallinity: Basis for Understanding the Lag Phase - Thomsen_2023_ChemSusChem_16_e202300291
Author(s) : Thomsen TB , Schubert S , Hunt CJ , Borch K , Jensen K , Brask J , Westh P , Meyer AS
Ref : ChemSusChem , 16 :e202300291 , 2023
Abstract : The rate response of poly(ethylene terephthalate) (PET)-hydrolases to increased substrate crystallinity (X(C) ) of PET manifests as a rate-lowering effect that varies significantly for different enzymes. Herein, we report the influence of X(C) on the product release rate of six thermostable PET-hydrolases. All enzyme reactions displayed a distinctive lag phase until measurable product formation occurred. The duration of the lag phase increased with X(C) . The recently discovered PET-hydrolase PHL7 worked efficiently on "amorphous" PET disks (X(C) =10 %), but this enzyme was extremely sensitive to increased X(C) , whereas the enzymes LCC(ICCG) , LCC, and DuraPETase had higher tolerance to increases in X(C) and had activity on PET disks having X(C) of 24.4 %. Microscopy revealed that the X(C) -tolerant hydrolases generated smooth and more uniform substrate surface erosion than PHL7 during reaction. Structural and molecular dynamics analysis of the PET-hydrolyzing enzymes disclosed that surface electrostatics and enzyme flexibility may account for the observed differences.
ESTHER : Thomsen_2023_ChemSusChem_16_e202300291
PubMedSearch : Thomsen_2023_ChemSusChem_16_e202300291
PubMedID: 37073816

Title : New insights to diversity and enzyme-substrate interactions of fungal glucuronoyl esterases - Agger_2023_Appl.Microbiol.Biotechnol__
Author(s) : Agger JW , Madsen MS , Martinsen LK , Martins PA , Barrett K , Meyer AS
Ref : Applied Microbiology & Biotechnology , : , 2023
Abstract : Glucuronoyl esterases (GEs) (EC catalyze the cleavage of ester-linked lignin-carbohydrate complexes that has high impact on the plant cell wall integrity. The GEs are among the very few known types of hydrolytic enzymes that act at the interface of lignin, or which may potentially interact with lignin itself. In this review, we provide the latest update of the current knowledge on GEs with a special focus on the fungal variants. In addition, we have established the phylogenetic relationship between all GEs and this reveals that the fungal enzymes largely fall into one major branch, together with only a minor subset of bacterial enzymes. About 22% of the fungal proteins carry an additional domain, which is almost exclusively a CBM1 binding domain. We address how GEs may interact with the lignin-side of their substrate by molecular docking experiments based on the known structure of the Cerrena unicolor GE (CuGE). The docking studies indicate that there are no direct interactions between the enzyme and the lignin polymer, that the lignin-moiety is facing away from the protein surface and that an elongated carbon-chain between the ester-linkage and the first phenyl of lignin is preferable. Much basic research on these enzymes has been done over the past 15 years, but the next big step forward for these enzymes is connected to application and how these enzymes can facilitate the use of lignocellulose as a renewable resource. KEY POINTS: Fungal GEs are closely related and are sometimes linked to a binding module Molecular docking suggests good accommodation of lignin-like substructures GEs could be among the first expressed enzymes during fungal growth on biomass.
ESTHER : Agger_2023_Appl.Microbiol.Biotechnol__
PubMedSearch : Agger_2023_Appl.Microbiol.Biotechnol__
PubMedID: 37256329

Title : Standardized method for controlled modification of poly (ethylene terephthalate) (PET) crystallinity for assaying PET degrading enzymes - Thomsen_2022_MethodsX_9_101815
Author(s) : Thomsen TB , Hunt CJ , Meyer AS
Ref : MethodsX , 9 :101815 , 2022
Abstract : Poly(ethylene terephthalate) (PET) is a polyester plastic, which is widely used, notably as a material for single-use plastic bottles. Its accumulation in the environment now poses a global pollution threat. A number of enzymes are active on PET providing new options for industrial biorecycling of PET materials. The enzyme activity is strongly affected by the degree of PET crystallinity (X(C)), and the X(C) is therefore a relevant factor to consider in enzyme catalyzed PET recycling. Here, we present a new experimental methodology, based on systematic thermal annealing for controlled preparation of PET disks having different X(C), to allow systematic quantitative evaluation of the efficiency of PET degrading enzymes at different degrees of PET substrate crystallinity. We discuss the theory of PET crystallinity and compare PET crystallinity data measured by differential scanning calorimetry and attenuated Fourier transform infrared spectroscopy.This study introduces a simple method for controlling the crystallinity of PET samples via annealing in a heat block.The present methodology is not limited to the analytical methods included in the methods details.
ESTHER : Thomsen_2022_MethodsX_9_101815
PubMedSearch : Thomsen_2022_MethodsX_9_101815
PubMedID: 36039192

Title : Influence of substrate crystallinity and glass transition temperature on enzymatic degradation of polyethylene terephthalate (PET) - Thomsen_2022_N.Biotechnol__
Author(s) : Thomsen TB , Hunt CJ , Meyer AS
Ref : N Biotechnol , : , 2022
Abstract : This work examines the significance of the degree of polyethylene terephthalate (PET) crystallinity (X(C)) and glass transition temperature (T(g)) on enzymatic degradation of PET at elevated temperatures using two engineered, thermostable PET degrading enzymes: LCC(ICCG), a variant of the leaf-branch compost cutinase, and DuraPETase, evolved from the Ideonella sakaiensis PETase. X(C) was systematically varied by thermal annealing of PET disks ( 6mm, thickness 1mm). X(C) affected the enzymatic product release rate that essentially ceased at X(C) 22-27% for the LCC(ICCG) and at X(C) -17% for the DuraPETase. Scanning Electron Microscopy revealed that enzymatic treatment produced cavities on the PET surface when X(C) was >10% but resulted in a smooth surface on amorphous PET (X(C) -10%). The T(g) of amorphous PET disks decreased from 74 degreesC to 61 degreesC during 24h pre-soaking in water at 65 degreesC, while X(C) remained unchanged. Enzymatic reaction on pre-soaked disks at 65 degreesC, i.e. above the T(g), did not affect the enzymatic product release rate, but delayed the initiation of enzymatic attack despite the lower T(g) compared to enzymatic reaction on un-soaked samples. The data suggest that extended soaking of PET at 65 degreesC induces an increase in the rigid amorphous fraction (X(RAF)) that impedes the enzymatic attack. These findings improve the understanding of enzymatic PET degradation and have implications for development of efficient enzymatic PET upcycling processes.
ESTHER : Thomsen_2022_N.Biotechnol__
PubMedSearch : Thomsen_2022_N.Biotechnol__
PubMedID: 35247624

Title : Enzymatic Cleavage of Diferuloyl Cross-Links in Corn Bran Arabinoxylan by Two Bacterial Feruloyl Esterases - Lin_2022_J.Agric.Food.Chem_70_13349
Author(s) : Lin S , Brask J , Munk L , Holck J , KBRM , Meyer AS , Agger JW , Wilkens C
Ref : Journal of Agricultural and Food Chemistry , 70 :133349 , 2022
Abstract : Corn bran is an abundant coprocessing stream of corn-starch processing, rich in highly substituted, diferuloyl-cross-linked glucurono-arabinoxylan. The diferuloyl cross-links make the glucurono-arabinoxylan recalcitrant to enzymatic conversion and constitute a hindrance for designing selective enzymatic upgrading of corn glucurono-arabinoxylan. Here, we show that two bacterial feruloyl esterases, wtsFae1A and wtsFae1B, each having a carbohydrate-binding module of family 48, are capable of cleaving the ester bonds of the cross-linkages and releasing 5-5', 8-5', 8-5' benzofuran, and 8-O-4' diferulate from soluble and insoluble corn bran glucurono-arabinoxylan. All four diferulic acids were released at similar efficiency, indicating nondiscriminatory enzymatic selectivity for the esterified dimer linkages, the only exception being that wtsFae1B had a surprisingly high propensity for releasing the dimers, especially 8-5' benzofuran diferulate, indicating a potential, unique catalytic selectivity. The data provide evidence of direct enzymatic release of diferulic acids from corn bran by newly discovered feruloyl esterases, i.e., a new enzyme activity. The findings yield new insight and create new opportunities for enzymatic opening of diferuloyl cross-linkages to pave the way for upgrading of recalcitrant arabinoxylans.
ESTHER : Lin_2022_J.Agric.Food.Chem_70_13349
PubMedSearch : Lin_2022_J.Agric.Food.Chem_70_13349
PubMedID: 36205442
Gene_locus related to this paper: 9zzzz-CE1.6RZN , 9zzzz-DAC80243

Title : The structural basis of fungal glucuronoyl esterase activity on natural substrates - Ernst_2020_Nat.Commun_11_1026
Author(s) : Ernst HA , Mosbech C , Langkilde AE , Westh P , Meyer AS , Agger JW , Larsen S
Ref : Nat Commun , 11 :1026 , 2020
Abstract : Structural and functional studies were conducted of the glucuronoyl esterase (GE) from Cerrena unicolor (CuGE), an enzyme catalyzing cleavage of lignin-carbohydrate ester bonds. CuGE is an alpha/beta-hydrolase belonging to carbohydrate esterase family 15 (CE15). The enzyme is modular, comprised of a catalytic and a carbohydrate-binding domain. SAXS data show CuGE as an elongated rigid molecule where the two domains are connected by a rigid linker. Detailed structural information of the catalytic domain in its apo- and inactivated form and complexes with aldouronic acids reveal well-defined binding of the 4-O-methyl-a-D-glucuronoyl moiety, not influenced by the nature of the attached xylo-oligosaccharide. Structural and sequence comparisons within CE15 enzymes reveal two distinct structural subgroups. CuGE belongs to the group of fungal CE15-B enzymes with an open and flat substrate-binding site. The interactions between CuGE and its natural substrates are explained and rationalized by the structural results, microscale thermophoresis and isothermal calorimetry.
ESTHER : Ernst_2020_Nat.Commun_11_1026
PubMedSearch : Ernst_2020_Nat.Commun_11_1026
PubMedID: 32094331
Gene_locus related to this paper: cerui-gce

Title : Microbial enzymes catalyzing keratin degradation: Classification, structure, function - Qiu_2020_Biotechnol.Adv__107607
Author(s) : Qiu J , Wilkens C , Barrett K , Meyer AS
Ref : Biotechnol Adv , :107607 , 2020
Abstract : Keratin is an insoluble and protein-rich epidermal material found in e.g. feather, wool, hair. It is produced in substantial amounts as co-product from poultry processing plants and pig slaughterhouses. Keratin is packed by disulfide bonds and hydrogen bonds. Based on the secondary structure, keratin can be classified into alpha-keratin and beta-keratin. Keratinases (EC 3.4.-.- peptide hydrolases) have major potential to degrade keratin for sustainable recycling of the protein and amino acids. Currently, the known keratinolytic enzymes belong to at least 14 different protease families: S1, S8, S9, S10, S16, M3, M4, M14, M16, M28, M32, M36, M38, M55 (MEROPS database). The various keratinolytic enzymes act via endo-attack (proteases in families S1, S8, S16, M4, M16, M36), exo-attack (proteases in families S9, S10, M14, M28, M38, M55) or by action only on oligopeptides (proteases in families M3, M32), respectively. Other enzymes, particularly disulfide reductases, also play a key role in keratin degradation as they catalyze the breakage of disulfide bonds for better keratinase catalysis. This review aims to contribute an overview of keratin biomass as an enzyme substrate and a systematic analysis of currently sequenced keratinolytic enzymes and their classification and reaction mechanisms. We also summarize and discuss keratinase assays, available keratinase structures and finally examine the available data on uses of keratinases in practical biorefinery protein upcycling applications.
ESTHER : Qiu_2020_Biotechnol.Adv__107607
PubMedSearch : Qiu_2020_Biotechnol.Adv__107607
PubMedID: 32768519

Title : Novel xylanolytic triple domain enzyme targeted at feruloylated arabinoxylan degradation - Holck_2019_Enzyme.Microb.Technol_129_109353
Author(s) : Holck J , Djajadi DT , Brask J , Pilgaard B , Krogh K , Meyer AS , Lange L , Wilkens C
Ref : Enzyme Microb Technol , 129 :109353 , 2019
Abstract : A three catalytic domain multi-enzyme; a CE1 ferulic acid esterase, a GH62 alpha-l-arabinofuranosidase and a GH10 beta-d-1,4-xylanase was identified in a metagenome obtained from wastewater treatment sludge. The capability of the CE1-GH62-GH10 multi-enzyme to degrade arabinoxylan was investigated to examine the hypothesis that CE1-GH62-GH10 would degrade arabinoxylan more efficiently than the corresponding equimolar mix of the individual enzymes. CE1-GH62-GH10 efficiently catalyzed the production of xylopyranose, xylobiose, xylotriose, arabinofuranose and ferulic acid (FA) when incubated with insoluble wheat arabinoxylan (WAX-I) (kcat = 20.8 +/- 2.6 s(-1)). Surprisingly, in an equimolar mix of the individual enzymes a similar kcat towards WAX-I was observed (kcat = 17.3 +/- 3.8 s(-1)). Similarly, when assayed on complex plant biomass the activity was comparable between CE1-GH62-GH10 and an equimolar mix of the individual enzymes. This suggests that from a hydrolytic point of view a CE1-GH62-GH10 multi-enzyme is not an advantage. Determination of the melting temperatures for CE1-GH62-GH10 (71.0 +/- 0.05 degrees C) and CE1 (69.9 +/- 0.02), GH62 (65.7 +/- 0.06) and GH10 (71 +/- 0.05 degrees C) indicates that CE1 and GH62 are less stable as single domain enzymes. This conclusion was corroborated by the findings that CE1 lost 50% activity within 2 h, while GH62 retained 50% activity after 24 h, whereas CE1-GH62-GH10 and GH10 retained 50% activity for 72 h. GH62-GH10, when appended to each other, displayed a higher specificity constant (kcat/Km = 0.3 s(-1) mg(-1) ml) than the individual GH10 (kcat/Km = 0.12 s(-1) +/- 0.02 mg(-1) ml) indicating a synergistic action between the two. Surprisingly, CE1-GH62, displayed a 2-fold lower kcat towards WAX-I than GH62, which might be due to the presence of a putative carbohydrate binding module appended to CE1 at the N-terminal. Both CE1 and CE1-GH62 released insignificant amounts of FA from WAX-I, but FA was released from WAX-I when both CE1 and GH10 were present, which might be due to GH10 releasing soluble oligosaccharides that CE1 can utilize as substrate. CE1 also displayed activity towards solubilized 5-O-trans-feruloyl-alpha-l-Araf (kcat = 36.35 s(-1)). This suggests that CE1 preferably acts on soluble oligosaccharides.
ESTHER : Holck_2019_Enzyme.Microb.Technol_129_109353
PubMedSearch : Holck_2019_Enzyme.Microb.Technol_129_109353
PubMedID: 31307573
Gene_locus related to this paper: 9zzzz-DAC80243

Title : A carbohydrate-binding family 48 module enables feruloyl esterase action on polymeric arabinoxylan - Holck_2019_J.Biol.Chem_294_17339
Author(s) : Holck J , Fredslund F , Moller MS , Brask J , Krogh K , Lange L , Welner DH , Svensson B , Meyer AS , Wilkens C
Ref : Journal of Biological Chemistry , 294 :17339 , 2019
Abstract : Feruloyl esterases (EC, belonging to carbohydrate esterase family 1 (CE1), hydrolyze ester bonds between ferulic acid (FA) and arabinose moieties in arabinoxylans. Recently, some CE1 enzymes identified in metagenomics studies have been predicted to contain a family 48 carbohydrate-binding module (CBM48), a CBM family associated with starch binding. Two of these CE1s, wastewater treatment sludge (wts) Fae1A and wtsFae1B isolated from wastewater treatment surplus sludge, have a cognate CBM48 domain and are feruloyl esterases, and wtsFae1A binds arabinoxylan. Here, we show that wtsFae1B also binds to arabinoxylan and that neither binds starch. Surface plasmon resonance analysis revealed that wtsFae1B's Kd for xylohexaose is 14.8 mum and that it does not bind to starch mimics, beta-cyclodextrin, or maltohexaose. Interestingly, in the absence of CBM48 domains, the CE1 regions from wtsFae1A and wtsFae1B did not bind arabinoxylan and were also unable to catalyze FA release from arabinoxylan. Pretreatment with a beta-d-1,4-xylanase did enable CE1 domain-mediated FA release from arabinoxylan in the absence of CBM48, indicating that CBM48 is essential for the CE1 activity on the polysaccharide. Crystal structures of wtsFae1A (at 1.63 A resolution) and wtsFae1B (1.98 A) revealed that both are folded proteins comprising structurally-conserved hydrogen bonds that lock the CBM48 position relative to that of the CE1 domain. wtsFae1A docking indicated that both enzymes accommodate the arabinoxylan backbone in a cleft at the CE1-CBM48 domain interface. Binding at this cleft appears to enable CE1 activities on polymeric arabinoxylan, illustrating an unexpected and crucial role of CBM48 domains for accommodating arabinoxylan.
ESTHER : Holck_2019_J.Biol.Chem_294_17339
PubMedSearch : Holck_2019_J.Biol.Chem_294_17339
PubMedID: 31558605
Gene_locus related to this paper: 9zzzz-CE1.6RZN , 9zzzz-DAC80243

Title : The natural catalytic function of CuGE glucuronoyl esterase in hydrolysis of genuine lignin-carbohydrate complexes from birch - Mosbech_2018_Biotechnol.Biofuels_11_71
Author(s) : Mosbech C , Holck J , Meyer AS , Agger JW
Ref : Biotechnol Biofuels , 11 :71 , 2018
Abstract : BACKGROUND: Glucuronoyl esterases belong to carbohydrate esterase family 15 and catalyze de-esterification. Their natural function is presumed to be cleavage of ester linkages in lignin-carbohydrate complexes particularly those linking lignin and glucuronoyl residues in xylans in hardwood. RESULTS: Here, we show for the first time a detailed product profile of aldouronic acids released from birchwood lignin by a glucuronoyl esterase from the white-rot fungus Cerrena unicolor (CuGE). CuGE releases substrate for GH10 endo-xylanase which results in significantly increased product release compared to the action of endo-xylanase alone. CuGE also releases neutral xylo-oligosaccharides that can be ascribed to the enzymes feruloyl esterase side activity as demonstrated by release of ferulic acid from insoluble wheat arabinoxylan. CONCLUSION: The data verify the enzyme's unique ability to catalyze removal of all glucuronoxylan associated with lignin and we propose that this is a direct result of enzymatic cleavage of the ester bonds connecting glucuronoxylan to lignin via 4-O-methyl glucuronoyl-ester linkages. This function appears important for the fungal organism's ability to effectively utilize all available carbohydrates in lignocellulosic substrates. In bioprocess perspectives, this enzyme is a clear candidate for polishing lignin for residual carbohydrates to achieve pure, native lignin fractions after minimal pretreatment.
ESTHER : Mosbech_2018_Biotechnol.Biofuels_11_71
PubMedSearch : Mosbech_2018_Biotechnol.Biofuels_11_71
PubMedID: 29560026
Gene_locus related to this paper: cerui-gce

Title : A New Functional Classification of Glucuronoyl Esterases by Peptide Pattern Recognition - Agger_2017_Front.Microbiol_8_309
Author(s) : Agger JW , Busk PK , Pilgaard B , Meyer AS , Lange L
Ref : Front Microbiol , 8 :309 , 2017
Abstract : Glucuronoyl esterases are a novel type of enzymes believed to catalyze the hydrolysis of ester linkages between lignin and glucuronoxylan in lignocellulosic biomass, linkages known as lignin carbohydrate complexes. These complexes contribute to the recalcitrance of lignocellulose. Glucuronoyl esterases are a part of the microbial machinery for lignocellulose degradation and coupling their role to the occurrence of lignin carbohydrate complexes in biomass is a desired research goal. Glucuronoyl esterases have been assigned to CAZymes family 15 of carbohydrate esterases, but only few examples of characterized enzymes exist and the exact activity is still uncertain. Here peptide pattern recognition is used as a bioinformatic tool to identify and group new CE15 proteins that are likely to have glucuronoyl esterase activity. 1024 CE15-like sequences were drawn from GenBank and grouped into 24 groups. Phylogenetic analysis of these groups made it possible to pinpoint groups of putative fungal and bacterial glucuronoyl esterases and their sequence variation. Moreover, a number of groups included previously undescribed CE15-like sequences that are distinct from the glucuronoyl esterases and may possibly have different esterase activity. Hence, the CE15 family is likely to comprise other enzyme functions than glucuronoyl esterase alone. Gene annotation in a variety of fungal and bacterial microorganisms showed that coprophilic fungi are rich and diverse sources of CE15 proteins. Combined with the lifestyle and habitat of coprophilic fungi, they are predicted to be excellent candidates for finding new glucuronoyl esterase genes.
ESTHER : Agger_2017_Front.Microbiol_8_309
PubMedSearch : Agger_2017_Front.Microbiol_8_309
PubMedID: 28293230