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Article ; Online: Evidence that Xrn1 is in complex with Gcn1, and is required for full levels of eIF2α phosphorylation.

Shanmugam, Renuka / Anderson, Reuben / Schiemann, Anja H / Sattlegger, Evelyn

2024 Volume 481, Issue 7, Page(s) 481–498

Abstract: The protein kinase Gcn2 and its effector protein Gcn1 are part of the general amino acid control signalling (GAAC) pathway best known in yeast for its function in maintaining amino acid homeostasis. Under amino acid limitation, Gcn2 becomes activated, ... ...

Abstract	The protein kinase Gcn2 and its effector protein Gcn1 are part of the general amino acid control signalling (GAAC) pathway best known in yeast for its function in maintaining amino acid homeostasis. Under amino acid limitation, Gcn2 becomes activated, subsequently increasing the levels of phosphorylated eIF2α (eIF2α-P). This leads to the increased translation of transcriptional regulators, such as Gcn4 in yeast and ATF4 in mammals, and subsequent re-programming of the cell's gene transcription profile, thereby allowing cells to cope with starvation. Xrn1 is involved in RNA decay, quality control and processing. We found that Xrn1 co-precipitates Gcn1 and Gcn2, suggesting that these three proteins are in the same complex. Growth under starvation conditions was dependent on Xrn1 but not on Xrn1-ribosome association, and this correlated with reduced eIF2α-P levels. Constitutively active Gcn2 leads to a growth defect due to eIF2α-hyperphosphorylation, and we found that this phenotype was independent of Xrn1, suggesting that xrn1 deletion does not enhance eIF2α de-phosphorylation. Our study provides evidence that Xrn1 is required for efficient Gcn2 activation, directly or indirectly. Thus, we have uncovered a potential new link between RNA metabolism and the GAAC.
MeSH term(s)	Amino Acids/metabolism ; Eukaryotic Initiation Factor-2/genetics ; Eukaryotic Initiation Factor-2/metabolism ; Mammals/metabolism ; Peptide Elongation Factors/genetics ; Peptide Elongation Factors/metabolism ; Phosphorylation ; Protein Serine-Threonine Kinases/genetics ; Protein Serine-Threonine Kinases/metabolism ; Saccharomyces cerevisiae/genetics ; Saccharomyces cerevisiae/metabolism ; Saccharomyces cerevisiae Proteins/genetics ; Saccharomyces cerevisiae Proteins/metabolism ; Exoribonucleases/genetics ; Exoribonucleases/metabolism
Chemical Substances	Amino Acids ; Eukaryotic Initiation Factor-2 ; Peptide Elongation Factors ; Protein Serine-Threonine Kinases (EC 2.7.11.1) ; Saccharomyces cerevisiae Proteins ; XRN1 protein, S cerevisiae (EC 3.1.11.-) ; GCN1 protein, S cerevisiae ; Exoribonucleases (EC 3.1.-)
Language	English
Publishing date	2024-02-19
Publishing country	England
Document type	Journal Article
ZDB-ID	2969-5
ISSN	1470-8728 ; 0006-2936 ; 0306-3275 ; 0264-6021
ISSN (online)	1470-8728
ISSN	0006-2936 ; 0306-3275 ; 0264-6021
DOI	10.1042/BCJ20220531
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Article ; Online: Domain II of the translation elongation factor eEF1A is required for Gcn2 kinase inhibition.

Ramesh, Rashmi / Sattlegger, Evelyn

FEBS letters

2020 Volume 594, Issue 14, Page(s) 2266–2281

Abstract: The signalling pathway governing general control nonderepressible (Gcn)2 kinase allows cells to cope with amino acid shortage. Under starvation, Gcn2 phosphorylates the translation initiation factor eukaryotic translation initiation factor (eIF)2α, ... ...

Abstract	The signalling pathway governing general control nonderepressible (Gcn)2 kinase allows cells to cope with amino acid shortage. Under starvation, Gcn2 phosphorylates the translation initiation factor eukaryotic translation initiation factor (eIF)2α, triggering downstream events that ultimately allow cells to cope with starvation. Under nutrient-replete conditions, the translation elongation factor eEF1A binds Gcn2 to contribute to keeping Gcn2 inactive. Here, we aimed to map the regions in eEF1A involved in binding and/or regulating Gcn2. We find that eEF1A amino acids 1-221 and 222-315, containing most of domains I and II, respectively, bind Gcn2 in vitro. Overexpression of eEF1A lacking or containing domain III impairs eIF2α phosphorylation. While the latter reduces growth under starvation similarly to eEF1A lacking domain I, the former enhances growth in a Gcn2-dependent manner. Our studies suggest that domain II is required for Gcn2 inhibition and that eEF1A lacking domain III mainly affects the Gcn2 response pathway downstream of Gcn2.
MeSH term(s)	Amino Acids/metabolism ; Chemical Precipitation ; Drug Resistance, Fungal/genetics ; Eukaryotic Initiation Factor-2/chemistry ; Eukaryotic Initiation Factor-2/metabolism ; Peptide Elongation Factor 1/chemistry ; Peptide Elongation Factor 1/genetics ; Peptide Elongation Factor 1/metabolism ; Peptide Fragments/chemistry ; Peptide Fragments/genetics ; Peptide Fragments/metabolism ; Phosphorylation ; Protein-Serine-Threonine Kinases/antagonists & inhibitors ; Protein-Serine-Threonine Kinases/chemistry ; Protein-Serine-Threonine Kinases/metabolism ; Saccharomyces cerevisiae/drug effects ; Saccharomyces cerevisiae/enzymology ; Saccharomyces cerevisiae/genetics ; Saccharomyces cerevisiae/metabolism ; Saccharomyces cerevisiae Proteins/antagonists & inhibitors ; Saccharomyces cerevisiae Proteins/chemistry ; Saccharomyces cerevisiae Proteins/genetics ; Saccharomyces cerevisiae Proteins/metabolism ; Sulfonylurea Compounds/pharmacology ; Triazoles/pharmacology
Chemical Substances	Amino Acids ; Eukaryotic Initiation Factor-2 ; Peptide Elongation Factor 1 ; Peptide Fragments ; Saccharomyces cerevisiae Proteins ; Sulfonylurea Compounds ; TEF2 protein, S cerevisiae ; Triazoles ; GCN2 protein, S cerevisiae (EC 2.7.11.1) ; Protein-Serine-Threonine Kinases (EC 2.7.11.1) ; sulfometuron methyl (JLY5D60J1A)
Language	English
Publishing date	2020-05-22
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	212746-5
ISSN	1873-3468 ; 0014-5793
ISSN (online)	1873-3468
ISSN	0014-5793
DOI	10.1002/1873-3468.13803
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Article: Domain II of the translation elongation factor eEF1A is required for Gcn2 kinase inhibition

Ramesh, Rashmi / Sattlegger, Evelyn

FEBS letters. 2020 July, v. 594, no. 14

2020

Abstract	The signalling pathway governing general control nonderepressible (Gcn)2 kinase allows cells to cope with amino acid shortage. Under starvation, Gcn2 phosphorylates the translation initiation factor eukaryotic translation initiation factor (eIF)2α, triggering downstream events that ultimately allow cells to cope with starvation. Under nutrient‐replete conditions, the translation elongation factor eEF1A binds Gcn2 to contribute to keeping Gcn2 inactive. Here, we aimed to map the regions in eEF1A involved in binding and/or regulating Gcn2. We find that eEF1A amino acids 1–221 and 222–315, containing most of domains I and II, respectively, bind Gcn2 in vitro. Overexpression of eEF1A lacking or containing domain III impairs eIF2α phosphorylation. While the latter reduces growth under starvation similarly to eEF1A lacking domain I, the former enhances growth in a Gcn2‐dependent manner. Our studies suggest that domain II is required for Gcn2 inhibition and that eEF1A lacking domain III mainly affects the Gcn2 response pathway downstream of Gcn2.
Keywords	amino acids ; peptide elongation factors ; phosphorylation ; starvation
Language	English
Dates of publication	2020-07
Size	p. 2266-2281.
Publishing place	John Wiley & Sons, Ltd
Document type	Article
Note	JOURNAL ARTICLE
ZDB-ID	212746-5
ISSN	1873-3468 ; 0014-5793
ISSN (online)	1873-3468
ISSN	0014-5793
DOI	10.1002/1873-3468.13803
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Article: Enterotoxigenicity and genetic relatedness of Staphylococcus aureus in a commercial poultry plant and poultry farm

Qian, Cheng / Castañeda-Gulla, Kristine / Sattlegger, Evelyn / Mutukumira, Anthony N.

International journal of food microbiology. 2022 Feb. 16, v. 363

2022

Abstract: Raw (fresh) and frozen poultry products are frequently associated with Staphylococcus aureus contamination. New Zealand is among the developed countries with high incidences of staphylococcal food poisoning. The study investigated the S. aureus isolates ... ...

Abstract	Raw (fresh) and frozen poultry products are frequently associated with Staphylococcus aureus contamination. New Zealand is among the developed countries with high incidences of staphylococcal food poisoning. The study investigated the S. aureus isolates obtained from various stages of poultry production, to determine the primary source of contamination. Viable cell counts of S. aureus were enumerated using Petrifilm™ Staph Express Count Plates, and the isolates were confirmed by Gram-stain and coagulase-positive test. Sixty S. aureus isolates were further confirmed by PCR. The PCR analysis used primers that specifically amplifies a fragment of the femA gene, unique to S. aureus. The confirmed S. aureus strains were further examined for enterotoxigenicity by PCR. Multilocus Sequence Typing (MLST) was then used to identify sequence types (STs) of the sixty isolates of S. aureus. The relatedness of the sequence types was investigated by eBURST. In this study, it was observed that all samples from the processing plant and live chickens at the farm were contaminated by S. aureus. Fifty-nine (59) of the 60 isolates were enterotoxigenic carrying enterotoxin genes: seg, sei, seh, sek, sel, sem, sen, or seo. The sixty isolates were categorised into six different sequence types: ST5, ST2594, ST101, ST83, ST398, ST1; where ST5, ST83 and ST2594 belonged to the Clonal Complex (CC) 5 with ST5 being the clonal ancestor. The sources of S. aureus contamination in the final poultry products were linked to fresh mechanically separated meat, fresh skin, fresh skin-on-breast fillet, rubber fingers on mechanical pluckers, and live chickens at the farm. The skin of live chickens at the farm was most likely the origin of S. aureus contamination on equipment and final products. Not all identified S. aureus strains at the farm were observed in the final products. Therefore, further investigation on other potential contamination sources such as gloves and knives used at the processing plant, and feeders and drinkers at the farm level is recommended.
Keywords	Staphylococcus aureus ; ancestry ; cell viability ; enterotoxins ; farms ; fillets ; food microbiology ; genes ; meat ; multilocus sequence typing ; polymerase chain reaction ; poultry production ; rubber ; New Zealand
Language	English
Dates of publication	2022-0216
Publishing place	Elsevier B.V.
Document type	Article
ZDB-ID	87122-9
ISSN	1879-3460 ; 0168-1605
ISSN (online)	1879-3460
ISSN	0168-1605
DOI	10.1016/j.ijfoodmicro.2021.109454
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Article ; Online: Enterotoxigenicity and genetic relatedness of Staphylococcus aureus in a commercial poultry plant and poultry farm.

Qian, Cheng / Castañeda-Gulla, Kristine / Sattlegger, Evelyn / Mutukumira, Anthony N

International journal of food microbiology

2021 Volume 363, Page(s) 109454

Abstract	Raw (fresh) and frozen poultry products are frequently associated with Staphylococcus aureus contamination. New Zealand is among the developed countries with high incidences of staphylococcal food poisoning. The study investigated the S. aureus isolates obtained from various stages of poultry production, to determine the primary source of contamination. Viable cell counts of S. aureus were enumerated using Petrifilm™ Staph Express Count Plates, and the isolates were confirmed by Gram-stain and coagulase-positive test. Sixty S. aureus isolates were further confirmed by PCR. The PCR analysis used primers that specifically amplifies a fragment of the femA gene, unique to S. aureus. The confirmed S. aureus strains were further examined for enterotoxigenicity by PCR. Multilocus Sequence Typing (MLST) was then used to identify sequence types (STs) of the sixty isolates of S. aureus. The relatedness of the sequence types was investigated by eBURST. In this study, it was observed that all samples from the processing plant and live chickens at the farm were contaminated by S. aureus. Fifty-nine (59) of the 60 isolates were enterotoxigenic carrying enterotoxin genes: seg, sei, seh, sek, sel, sem, sen, or seo. The sixty isolates were categorised into six different sequence types: ST5, ST2594, ST101, ST83, ST398, ST1; where ST5, ST83 and ST2594 belonged to the Clonal Complex (CC) 5 with ST5 being the clonal ancestor. The sources of S. aureus contamination in the final poultry products were linked to fresh mechanically separated meat, fresh skin, fresh skin-on-breast fillet, rubber fingers on mechanical pluckers, and live chickens at the farm. The skin of live chickens at the farm was most likely the origin of S. aureus contamination on equipment and final products. Not all identified S. aureus strains at the farm were observed in the final products. Therefore, further investigation on other potential contamination sources such as gloves and knives used at the processing plant, and feeders and drinkers at the farm level is recommended.
MeSH term(s)	Animals ; Chickens ; Enterotoxins ; Farms ; Multilocus Sequence Typing ; Poultry ; Staphylococcal Infections ; Staphylococcus aureus/genetics
Chemical Substances	Enterotoxins
Language	English
Publishing date	2021-10-23
Publishing country	Netherlands
Document type	Journal Article
ZDB-ID	87122-9
ISSN	1879-3460 ; 0168-1605
ISSN (online)	1879-3460
ISSN	0168-1605
DOI	10.1016/j.ijfoodmicro.2021.109454
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Article ; Online: A genetic approach to identify amino acids in Gcn1 required for Gcn2 activation.

Gottfried, Susanne / Koloamatangi, Siaosi M B M J / Daube, Clement / Schiemann, Anja H / Sattlegger, Evelyn

PloS one

2022 Volume 17, Issue 11, Page(s) e0277648

Abstract: The protein kinase Gcn2 is present in virtually all eukaryotic cells. It is best known for its role in helping cells cope with amino acid starvation. Under starvation, Gcn2 phosphorylates the α subunit of the eukaryotic translation initiation factor 2 ( ... ...

Abstract	The protein kinase Gcn2 is present in virtually all eukaryotic cells. It is best known for its role in helping cells cope with amino acid starvation. Under starvation, Gcn2 phosphorylates the α subunit of the eukaryotic translation initiation factor 2 (eIF2α), to stimulate a signal transduction pathway that allows cells to cope and overcome starvation. Gcn2 has been implicated in many additional biological functions. It appears that for all functions, Gcn2 must directly bind to its effector protein Gcn1, mediated via a region in Gcn1 called the RWD binding domain (RWDBD). Arg-2259 in this region is important for Gcn2 binding. Overexpression of a Gcn1 fragment only encompassing the RWDBD binds Gcn2, thereby disrupting endogenous Gcn1-Gcn2 interaction which dampens Gcn2 activation. Consequently, cells are unable to increase eIF2α phosphorylation under starvation conditions, visible by impaired growth. This dominant negative phenotype is reverted by the R2259A substitution, again allowing Gcn1-Gcn2 interaction and enhanced eIF2α phosphorylation. We have found that the amino acid substitutions, R2289A, R2297A, and K2301A, also reverted the dominant negative phenotype as well as allowed enhanced eIF2α phosphorylation, as found previously for the R2259A substitution. This suggests that the respective amino acids are relevant for the overexpressed RWDBD to disrupt Gcn1-Gcn2 interaction and impair Gcn2 activation, supporting the idea that in Gcn1 these amino acids mediate Gcn2-binding. Our findings suggest that two helices in Gcn1 constitute a Gcn2 binding site. We serendipitously found amino acid substitutions that enhanced the dominant negative phenotype that correlated with a further reduction in eIF2α-P levels, suggesting that the respective RWDBD variants are more potent in disrupting Gcn1-Gcn2 interaction.
MeSH term(s)	Amino Acid Substitution ; Amino Acids ; Eukaryotic Initiation Factor-2/genetics ; Peptide Elongation Factors/genetics ; Peptide Elongation Factors/metabolism ; Protein Serine-Threonine Kinases/genetics ; Protein Serine-Threonine Kinases/metabolism ; RNA-Binding Proteins/metabolism ; Saccharomyces cerevisiae ; Saccharomyces cerevisiae Proteins/genetics ; Saccharomyces cerevisiae Proteins/metabolism ; Trans-Activators/metabolism
Chemical Substances	Amino Acids ; Eukaryotic Initiation Factor-2 ; GCN1 protein, S cerevisiae ; GCN2 protein, S cerevisiae (EC 2.7.11.1) ; Peptide Elongation Factors ; Protein Serine-Threonine Kinases (EC 2.7.11.1) ; RNA-Binding Proteins ; Saccharomyces cerevisiae Proteins ; Trans-Activators
Language	English
Publishing date	2022-11-28
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2267670-3
ISSN	1932-6203 ; 1932-6203
ISSN (online)	1932-6203
ISSN	1932-6203
DOI	10.1371/journal.pone.0277648
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Article ; Online: Rapid yeast-based screen for Functionally Relevant Amino Acids (RS-FRAA) in a protein.

Ghuge, Aditi A / Anderson, Reuben A / Gottfried, Susanne / Daube, Clément / Koloamatangi, Siaosi M B M J / Schiemann, Anja H / Sattlegger, Evelyn

STAR protocols

2023 Volume 4, Issue 1, Page(s) 101545

Abstract: Here, we describe a fast and cost-effective procedure to generate a large array of mutant proteins and immediately screen for those with altered protein function. This protocol is a modification from three existing approaches: fusion PCR, Saccharomyces ... ...

Abstract	Here, we describe a fast and cost-effective procedure to generate a large array of mutant proteins and immediately screen for those with altered protein function. This protocol is a modification from three existing approaches: fusion PCR, Saccharomyces cerevisiae in-yeast recombination, and semi-quantitative growth assays. We also describe a mating step to reduce the occurrence of false positive findings due to ectopic mutations. The only requirement is that the protein elicits a phenotype in Saccharomyces cerevisiae.
MeSH term(s)	Saccharomyces cerevisiae/genetics ; Saccharomyces cerevisiae/metabolism ; Amino Acids/genetics ; Amino Acids/metabolism ; Saccharomyces cerevisiae Proteins/genetics ; Saccharomyces cerevisiae Proteins/metabolism ; Phenotype
Chemical Substances	Amino Acids ; Saccharomyces cerevisiae Proteins
Language	English
Publishing date	2023-01-25
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	2666-1667
ISSN (online)	2666-1667
DOI	10.1016/j.xpro.2022.101545
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Article ; Online: Persistent contamination of

Castañeda-Gulla, Kristine / Sattlegger, Evelyn / Mutukumira, Anthony N

Canadian journal of microbiology

2019 Volume 66, Issue 3, Page(s) 171–185

Abstract: Intensive poultry production due to public demand raises the risk of contamination, creating potential foodborne hazards to consumers. The prevalence and microbial load of the ... ...

Abstract	Intensive poultry production due to public demand raises the risk of contamination, creating potential foodborne hazards to consumers. The prevalence and microbial load of the pathogens
MeSH term(s)	Animal Husbandry/statistics & numerical data ; Animals ; Campylobacter/classification ; Campylobacter/genetics ; Campylobacter/isolation & purification ; Chickens ; Escherichia coli/classification ; Escherichia coli/genetics ; Escherichia coli/isolation & purification ; Farms/statistics & numerical data ; Food Contamination/analysis ; New Zealand/epidemiology ; Poultry Diseases/epidemiology ; Poultry Diseases/microbiology ; Salmonella/classification ; Salmonella/genetics ; Salmonella/isolation & purification ; Staphylococcus aureus/classification ; Staphylococcus aureus/genetics ; Staphylococcus aureus/isolation & purification
Language	English
Publishing date	2019-11-13
Publishing country	Canada
Document type	Journal Article
ZDB-ID	280534-0
ISSN	1480-3275 ; 0008-4166
ISSN (online)	1480-3275
ISSN	0008-4166
DOI	10.1139/cjm-2019-0280
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Article: A Rapid Extraction Method for mammalian cell cultures, suitable for quantitative immunoblotting analysis of proteins, including phosphorylated GCN2 and eIF2α

Silva, Richard C / Castilho, Beatriz A / Sattlegger, Evelyn

MethodsX. 2018, v. 5

2018

Abstract: Many studies require the detection and relative quantitation of proteins from cell culture samples using immunoblotting. Limiting factors are the cost of protease inhibitors, the time required to break cells and generate samples, as well as the high risk ...

Abstract	Many studies require the detection and relative quantitation of proteins from cell culture samples using immunoblotting. Limiting factors are the cost of protease inhibitors, the time required to break cells and generate samples, as well as the high risk of protein loss during cell breakage procedures. In addition, a common problem is the viscosity of lysed samples due to the released genomic DNA. As a consequence, the DNA needs to be broken down prior to denaturing polyacrylamide protein gel electrophoresis (SDS-PAGE), e.g. by passing the sample through a syringe gauge needle, sonication, or DNase treatment. In a quest to find a more cost-effective, fast, and yet robust procedure, we found that cell lysis, protein denaturation, and DNA fragmentation can be done in only two steps: harvesting followed by a simple non-laborious 2nd step. Similarly to many pre-existing cell breakage procedures, in our Rapid Protein Extraction (RPE) method, proteins liberated from cells are immediately exposed to a denaturing environment. However, advantages of our method are:No breaking buffer is needed, instead proteins are liberated directly into the denaturing protein loading buffer used for SDS-PAGE. Consequently, our RPE method does not require any expensive inhibitors. The RPE method does not involve post-lysis centrifugation steps; instead all cell material is dissolved during the 2nd step, the mixing-heat-treatment step which is new to this method. This prevents potential protein loss that may occur during centrifugation. In addition, this 2nd step simultaneously shears the genomic DNA, making an additional step for DNA fragmentation unnecessary. The generated samples are suitable for high-quality quantitative immunoblotting. With our RPE method we successfully quantified the phosphorylated forms of protein kinase GCN2 and its substrate eIF2α. In fact, the western signals were stronger and with less background, as compared to samples generated with a pre-existing method.
Keywords	cell culture ; centrifugation ; cost effectiveness ; deoxyribonucleases ; DNA ; DNA fragmentation ; harvesting ; immunoblotting ; mammals ; polyacrylamide ; polyacrylamide gel electrophoresis ; protein denaturation ; protein depletion ; protein kinases ; proteinase inhibitors ; proteins ; risk ; sonication ; viscosity
Language	English
Size	p. 75-82.
Publishing place	Elsevier B.V.
Document type	Article
ISSN	2215-0161
DOI	10.1016/j.mex.2017.10.008
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Article: Persistent contamination of Salmonella, Campylobacter, Escherichia coli, and Staphylococcus aureus at a broiler farm in New Zealand

Castañeda-Gulla, Kristine / Sattlegger, Evelyn / Mutukumira, Anthony N

Canadian journal of microbiology. 2020, v. 66, no. 3

2020

Abstract: Intensive poultry production due to public demand raises the risk of contamination, creating potential foodborne hazards to consumers. The prevalence and microbial load of the pathogens Campylobacter, Salmonella, Staphylococcus aureus, and Escherichia ... ...

Abstract	Intensive poultry production due to public demand raises the risk of contamination, creating potential foodborne hazards to consumers. The prevalence and microbial load of the pathogens Campylobacter, Salmonella, Staphylococcus aureus, and Escherichia coli was determined by standard methods at the farm level. After disinfection, swab samples collected from wall crevices, drinkers, and vents were heavily contaminated, as accumulated organic matter and dust likely protected the pathogens from the disinfectants used. The annex floor also showed high microbial concentrations, suggesting the introduction of pathogens from external environments, highlighting the importance of erecting hygiene barriers at the entrance of the main shed. Therefore, pathogen control measures and proper application of disinfectants are recommended as intervention strategies. Additionally, quantitative polymerase chain reaction (qPCR) was evaluated as a quantification tool. qPCR showed limitations with samples containing low microbial counts because of the low detection limit of the method. Thus, bacterial pre-enrichment of test samples may be necessary to improve the detection of pathogens by qPCR.
Keywords	Campylobacter ; Escherichia coli ; Salmonella ; Staphylococcus aureus ; control methods ; detection limit ; disinfectants ; disinfection ; dust ; farms ; hygiene ; microbial detection ; microbial load ; organic matter ; pathogens ; poultry production ; quantitative polymerase chain reaction ; risk ; New Zealand
Language	English
Size	p. 171-185.
Publishing place	NRC Research Press
Document type	Article
ZDB-ID	280534-0
ISSN	1480-3275 ; 0008-4166
ISSN (online)	1480-3275
ISSN	0008-4166
DOI	10.1139/cjm-2019-0280
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