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https://openalex.org/W3160813570
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https://journal.ikipsiliwangi.ac.id/index.php/project/article/download/4288/pdf
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English
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AN ANALYSIS OF ILLOCUTIONARY AND PERLOCUTIONARY SPEECH ACTS OF AN INSTAGRAM ONLINE SHOPPING ACCOUNT
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Project (Professional Journal of English Education)
| 2,021
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cc-by-sa
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PROJECT
(Professional Journal of English Education)
Volume 4, No. 3, May 2021 p–ISSN 2614-6320
e–ISSN 2614-6258 Abstract Language is important in human’s life because we cannot communicate with one another without it. Therefore, it is appealing to study speech acts since we can understand how utterances are reflected in
actions. It is also interesting to analyze the speech acts used in Instagram captions. This study aims to
analyze speech acts of an online shopping account on Instagram that is an international brand N.Y.X. Illocutionary speech acts aims to state something to influence the listener to do activities according to
what the speaker means. Based on the data obtained, the illocutionary act of the online shopping account
@nyxcosmetics is in the form of directive and expressive acts. The directives illocution acts are ordering
and demanding. On the other hand, the perlocutionary aims to portray the act of offering someone. Keywords: Speech Acts, Illocutionary Acts, Perlocutionary Acts AN ANALYSIS OF ILLOCUTIONARY AND
PERLOCUTIONARY SPEECH ACTS OF AN INSTAGRAM
ONLINE SHOPPING ACCOUNT Cahya Wati Dewi 1
1 IKIP Siliwangi
1 cahyawatidewi@student.ikipsiliwangi.ac.id Pragmatics on Social Media g
There are many social media users who use Instagram accounts as a tool of building
communications. The interesting fact of Instagram is that it is not only as a communication tool
but also it can be used as a promotion media. Instagram has features that can help users to
communicate with one another, including captions, comments and personal messages. Actually,
Instagram users take advantage of these features to perform branding on their products or
services. Based on the phenomenon above, the researchers, thus, were interested to do speech
act analysis on the Instagram users' captions since captions describe performative language. When speaking, individuals not only mention one sentence that has a grammatical structure and
words, but also carry out activities when they speak. Speech acts are thus also to be distinguished from performatives. ‘Performative’ is another
technical term; it refers to the first instance of a sentence. A performative sentence is in the first
person, present tense, indicative mood, active voice, that describes its speaker as performing a
speech act. We may also define a performative utterance as an utterance of a performative
sentence that is also a speech act (Green, 2017). Language is important in our life because
without language we cannot communicate with other people. According to Huang (2007) one of the understanding or principles of logical positivism is
descriptive fallacy, an opinion that assumes that the function of language from a philosophical
point of view is to create statements that are true or false (Huang, 2007). On such a view,
sentences that are used by the Instagram users for branding are simply meaningless because
they are not used to verify or falsify propositions. Data of speech acts were found on the
Instagram captions according to the theory of speech act by Austin (1955). Austin has produced two important studies. Firstly, that some common sentences are not used
to make statements, and therefore, it is not mentioned whether the sentence is true or false. Secondly, there are decralative sentences which are also important to note, which reject the
conditional truth analysis in the same way. Illocutionary and Perlocutionary Acts In this study, illocutionary and perlocutionary acts are interesting to analyze. Utterance can be
used to do something besides its functions to say or inform something. Thus, if that happens,
then the speech act on the speech is illocutionary. Illocutionary acts are also referred to as "the
act of doing something." Based on the description, the researchers found an illocutionary act
utterance on the online shopping account of Instagram. Furthermore, the researchers also
discovered data phenomena in the form of acts of perlocution arising from the speech. Therefore, it is interesting to study speech act because we can know how the utterances are
reflected in actions. It is also interesting to analyze the speech act used in social media. Many
people use social media to build communication or market share. This study analyzes how to
use speech acts on captions of an instagram online shopping account. INTRODUCTION On daily communications, language carries out an important role. When people practice
language, they can express what they have in mind. However, when people communicate with
one another, sometimes one does not understand what the other speaker means, or the speaker's
meaning sounds absurd. Therefore, this research aims to analyze what the speaker means. This
research demonstrates how language is adopted by the user in pragmatics. Pragmatics is a study
about the meaning of a speaker's speech. Pragmatics is also about the relationship between
linguistics forms and the users of those forms (Zulfa, 2018). Thus, pragmatics is not only
studying linguistics forms but equally considering the speaker's meaning and context. Pragmatics is a part of linguistics that study the meaning of a context in a speech. According to
Yule “pragmatics is a study about the speaker's meaning, contextual meaning, and what more
to get communicated than what the speaker has stated; and the study of the relation of relative
distance” (Tutuarima, Nuraeningsih, & Rusiana, 2018). The focus of Pragmatics is on the speaker's meaning based on the context and the situation. In
this research, the researchers focus on speech acts, especially in illocutionary acts. J. L. Austin
is the first person who introduces the theory of speech acts (Tutuarima et al., 2018). The Speech
act exists in our daily activity, and it has been practised to makeonversations among people. Speech act theory was foreshadowed by Austin views on how to do things with words (Huang,
2007). Afterward, Austin’s ideas were refined, systemized, and advanced especially by Searle. Searle’s view of speech act theory described it as stating one sentence that is part of the
framework of a socially agreed institution and convention. Based on Searle’s view, this research
conducted an analysis of illocutionary and perlocutionary speech acts of an online shopping
account on Instagram. In fact, Instagram account has a broad appealing as a social media. An Analysis of Illocutionary and Perlocutionary Speech Acts of an Instagram Online Shopping
Account |353 Volume 4, No. 3, May 2021 pp 353-359 Pragmatics on Social Media METHOD This research used a qualitative approach. A qualitative approach aims to build a knowledge
statement based on a constructive or perspective participatory (Budiasih, 2016). This study
intends to do an analysis of speech acts of an online shopping account on an international brand
N.Y.X cosmetics Instagram. Primary data were collected in the following steps: 1) collecting
the contents of N.Y.X cosmetics Instagram on March, 2) making the data korpus. A validity of nalysis of Illocutionary and Perlocutionary Speech Acts of an Instagram Online Shopping 354 | An Analysis of Illocutionary and Perlocutionary Speech Acts of an Instagram Online Shopping
Account Volume 4, No. 3, May 2021 pp 353-359
the data (content analysis) is a guarantee of stability summary and interpretations of meanings
as a result of research. Volume 4, No. 3, May 2021 pp 353-359 Volume 4, No. 3, May 2021 pp 353-359 the data (content analysis) is a guarantee of stability summary and interpretations of meanings
as a result of research. Results Austin defined the speech act as actions that we perform when we produce utterances like;
giving suggestion, promising, inviting, requesting, forbidding, and so on (Sumarsono, 2017). Speech act is utterances which can be categorized into three types i.e. locutionary act,
illocutionary act, and perlocutionary act (Fala & Kumala, 2018). In this section, the researchers
only analyzed illocutionary act and perlocutinary act. The reason was that the utterances which
were found in instagram online shopping have meanings on illocutionary act and perlocutionary
act. Based on the data above, the researchers concluded that speech act is a statement that
contains action as a functional unity in communications considering situation aspect. According
to Austin (1955) speech act can be categorized into three types (Fala & Kumala 2018): a) Illocutionary Act
The illocutionary act is the process of making statements, offerings, and promises
through utterances according to conventional aspects relating either explicitly or
performatively. This Act is also interpreted as taking an action to convey a purpose. For
example: “It close to seven o’clock”. This sentence is uttered by a husband to his wife in
the morning. He not only says the words but also reminds his wife that he has to go to his
office in that moment. The Illocutionary Act is one of the Speech Acts which helps people
do something while saying words. b) Perlocutinary Act )
y
The perlocutionary act is the impact of the words conveyed by the speaker to the
listener. These types of acts are words that can affect others. For example: “There is a
snake next to you!”, if we say this utterance to someone, there will be some effects
caused by that utterance. If the hearer heard that utterance, he/she might run. The perlocutionary act is the impact of the words conveyed by the speaker to the
listener. These types of acts are words that can affect others. For example: “There is a
snake next to you!”, if we say this utterance to someone, there will be some effects
caused by that utterance. If the hearer heard that utterance, he/she might run. Based on the data, the researchers identified utterances into five kinds of function
according to Searle’s classification. The majority of the data are identified as the 1) assertive,
2) directive, 3) declarative, 4) commissive, and 5) expressive. Discussion The following is the descriptions of data put on online shopping account on international brand
N.Y.X Instagram: The following is the descriptions of data put on online shopping account on international brand
N.Y.X Instagram: An Analysis of Illocutionary and Perlocutionary Speech Acts of an Instagram Online Shopping
Account|355
a. Illocutionary Act
1) Directive
Directive is the type of speech act that aims to get listener to do something
(Sembiring & Ambalegin, 2019). The functions of directive acts is commanding,
requesting, inviting, forbidding, and suggesting. The types of directive as explained
above can be seen from the data below:
Data 1
Post ( March 2020)
caption:
nyxcosmetics: what are your tips and tricks to make your shadow POP?! @kimcreates_ uses our #JumboEyeShadow pencil in ‘Milk’#nyxcosmeics
#nyxprofessionalmakeup
comment: An Analysis of Illocutionary and Perlocutionary Speech Acts of an Instagram Online Shopping
Account|355
a. Illocutionary Act
1) Directive
Directive is the type of speech act that aims to get listener to do something
(Sembiring & Ambalegin, 2019). The functions of directive acts is commanding,
requesting, inviting, forbidding, and suggesting. The types of directive as explained
above can be seen from the data below:
Data 1
Post ( March 2020)
caption:
nyxcosmetics: what are your tips and tricks to make your shadow POP?! @kimcreates_ uses our #JumboEyeShadow pencil in ‘Milk’#nyxcosmeics
#nyxprofessionalmakeup
comment: a. Illocutionary Act
1) Directive
Directive is the type of speech act that aims to get listener to do something
(Sembiring & Ambalegin, 2019). The functions of directive acts is commanding,
requesting, inviting, forbidding, and suggesting. The types of directive as explained
above can be seen from the data below:
Data 1
Post ( March 2020)
caption:
nyxcosmetics: what are your tips and tricks to make your shadow POP?! @kimcreates_ uses our #JumboEyeShadow pencil in ‘Milk’#nyxcosmeics
#nyxprofessionalmakeup
comment: a. Illocutionary Act
1) Directive
Directive is the type of speech act that aims to get listener to do something
(Sembiring & Ambalegin, 2019). The functions of directive acts is commanding,
requesting, inviting, forbidding, and suggesting. The types of directive as explained
above can be seen from the data below: caption:
nyxcosmetics: what are your tips and tricks to make your shadow POP?! @kimcreates_ uses our #JumboEyeShadow pencil in ‘Milk’#nyxcosmeics
#nyxprofessionalmakeup @kimcreates_ uses our #JumboEyeShadow pencil in ‘Milk’#nyxcosmeics
#nyxprofessionalmakeup comment: comment: @makeuped_._: so pretty omg @anggelss: beautiful makeup love this purple shades The utterances were taken from nyxcosmetics Instagram. These utterances show that
the admin of nyxcosmetics did a directive illocutionary act that is ordering. It shows
utterances on captions that has meaning that one of the followers of @nyxcosmetics uses
their product. Furthemore, the utterance that has meanings that other followers to order
@nyxcosmetics as their product. 2) Expressive comment: An Analysis of Illocutionary and Perlocutionary Speech Acts of an Instagram Online Shopping
Account|355 An Analysis of Illocutionary and Perlocutionary Speech Acts of an Instagram Online Shopping
Account|355 Volume 4, No. 3, May 2021 pp 353-359 Volume 4, No. 3, May 2021 pp 353-359 @corpse.garden: I used either Analogos or Complimentary color schemes. I love
the liquid eyeshadow, it blends really well. @rabianunesxox: Stunning eye makeup
@thivibeauty: Soooo pretty @corpse.garden: I used either Analogos or Complimentary color schemes. I love
the liquid eyeshadow, it blends really well. @rabianunesxox: Stunning eye makeup
@thivibeauty: Soooo pretty The utterances were taken from nyxcosmetics Instagram. These utterances show that
the admin of nyxcosmetics did a directive illocutinary act that are advising, demanding
and ordering. The utterances on the captions that describe meaning of demanding were
on a sentence like what are your tips and tricks to make your shadow POP?!. The
utterances also portray a meaning of advising on a sentence like @kimcreates_ uses
our #JumboEyeShadow pencil in ‘Milk’#nyxcosmeics. So, the captions show the
sentences to invite followers of @nyxcosmetics like #JumboEyeShadow pencil in
‘Milk’#nyxcosmeics. Furthermore, directive illocutionary act that has ordering meaning
is shown on a sentence like uses our #JumboEyeShadow pencil in ‘Milk’#nyxcosmeics. Data 2
Post ( March 2020)
caption:
nyxcosmetics: @anggelzz applies our Jumbo Eye Pencil in ‘Milk’ as a base for this
poppin’ pastel #EOTD #nyxcosmetics #nxprofessionalmakep comment:
@r.m.s.k.h: I love this shade
@anggelss: the best
@ kimgoodyear37: love your lip color, what is it? The utterances were taken from nyxcosmetics Instagram. These utterances show that
the admin of nyxcosmetics did a directive illocutionary act that is ordering. It shows
utterances on captions that has meaning that one of the followers @nyxcosmetics uses
their product. Furthemore, the utterance has meaning that other followers were ordering
@nyxcosmetics as their product. Data 3
Post ( March 2020)
caption:
nyxcosmetics: @makeupbyevva applies our best selling #epicInkLiner as a base for
this #flawless lilac liner. nyxcosmetics: @makeupbyevva applies our best selling #epicInkLiner as a base for
this #flawless lilac liner. 2) Expressive 2) Expressive 356 | An Analysis of Illocutionary and Perlocutionary Speech Acts of an Instagram Online Shopping
Account An Analysis of Illocutionary and Perlocutionary Speech Acts of an Instagram Online Shopping
unt Account Volume 4, No. 3, May 2021 pp 353-359 Volume 4, No. 3, May 2021 pp 353-359 Searle proposed the types of expressive are apologizing, thanking, congratulating,
condoling, deploring and welcoming (Sembiring and Ambeligin, 2019). Expressive
illocutionary act found in the caption of an online shopping account on @nyxcosmetics. Searle proposed the types of expressive are apologizing, thanking, congratulating,
condoling, deploring and welcoming (Sembiring and Ambeligin, 2019). Expressive
illocutionary act found in the caption of an online shopping account on @nyxcosmetics. Data 4
Post ( March 2020)
caption:
nyxcosmetics: Nothing is more satisfying than applying the perfect lippie
@mellowyellowpay wears our new #shoutloud Satin Lipsstick in Desert Rose. Which lippie takes your day from 0-100 real wuick? Data 4
Post ( March 2020)
caption:
nyxcosmetics: Nothing is more satisfying than applying the perfect lippie
@mellowyellowpay wears our new #shoutloud Satin Lipsstick in Desert Rose. Which lippie takes your day from 0-100 real wuick? p
nyxcosmetics: Nothing is more satisfying than applying the perfect lippie
@mellowyellowpay wears our new #shoutloud Satin Lipsstick in Desert Rose. Which lippie takes your day from 0-100 real wuick? comment:
@brookesmakeup444: O pretty in pink
@michelle_m_k_f: beautiful girl and make up comment:
@brookesmakeup444: O pretty in pink
@michelle_m_k_f: beautiful girl and make up comment: The speech act contains expressive illocution. The caption like Nothing is more
satisfying than applying the perfect lippie shows expressive utterance. The speaker
explained how pretty and perfect lippie looks. The utterances were taken from
nyxcosmetics Instagram. Those utterances show that the admin of nyxcosmetics did an
expressive illocutionary act that is advising their followers. Data 5
Post ( March 2020)
caption:
nyxcosmetics: Liner on point achieve fire line every single time with our bes-selling
#EpicInkLiner
comment:
@blakk.mua: best luber eeever
@luv4kai: best liner ever!!! onl one I use comment:
@blakk.mua: best luber eeever
@luv4kai: best liner ever!!! onl one I use The utterances were taken from nyxcosmetics Instagram. These utterances show that
the admin of nyxcosmetics did an expressive illocutionary act that is to command their
followers to use their product. It can be can be seen from the follower’s comment like
@blakk.mua: best luber eeever @luv4kai: best liner ever!!! onl one I use. That comment
shows expressive utterance. An Analysis of Illocutionary and Perlocutionary Speech Acts of an Instagram Online Shopping
Account|357 comment: comment:
@juspaid_: Aloha wahines’s
@traganellabialderosyxmac: Hello nyx in Argentina please, I love the brand. @wendigobeauty: #shoutlod to @kenadrogan one of the best friends I’ve ever had
and to @shannoxo m favorite youtuber and the woman I go to before buing any
higher end products. The data (7) is also a form of a perlocutionary act, in which the speech of the speaker
has an influence on the listener to shout their opinions. For example, Help us #shoutloud
b tagging a beaut who inspire you! and the hearer answer @juspaid_: Aloha wahines’s,
@traganellabialderosyxmac: Hello nyx in Argentina please, I love the brand.,
@wendigobeauty: #shoutlod to @kenadrogan one of the best friends I’ve ever had and
to @shannoxo m favorite youtuber and the woman I go to before buing any higher end
products. CONCLUSION The illocutionary speech act aims to state something in order to influence the listener to do
something according to what the speaker means. It is found that the illocutionary act on online
shopping of @nyxcosmetics Instagram account is the form of acts of directive and expressive. The directive illocution data are ordering and demanding. On the contrary, the perlocutionary
speech act aims to affect someone. ACKNOWLEDGMENTS Alhamdulillah, thank you Allah, my family, my lecturer and those who have supported this
research. 2) Expressive b. Perlocutionary Act
The perlocutionary act is the effect caused by some utterances that are uttered by the
speaker to the listener. It can be said that this act is the act of affecting someone (Fala
et al., 2019). A perlocutionary act is an act that is carried out by saying one sentence in
a specific context. This Act represents the changes that occur each in that context. This
Act is an additional output from the act of communication, explaining something,
encouraging someone to be angry, making others entertained, etc. This Act shows words
that are not supplemented by special impacts or purposes. (Fala et al., 2019). Data 6
Post ( March 2020)
caption: An Analysis of Illocutionary and Perlocutionary Speech Acts of an Instagram Online Shopping
Account|357 Volume 4, No. 3, May 2021 pp 353-359 Volume 4, No. 3, May 2021 pp 353-359 She’s backkkk mark your calenders, beuties. The final Restock of our limited edition. @sabrinanetflix collection will be wednesday 3/18. Available exclusively on
nyxcosmetics.com She’s backkkk mark your calenders, beuties. The final Restock of our limited edition. @sabrinanetflix collection will be wednesday 3/18. Available exclusively on
nyxcosmetics.com comment:
@tatusmakeup: Ok I nedd this
@nolwee_legoff: I want it but I live in France comment:
@tatusmakeup: Ok I nedd this
@nolwee_legoff: I want it but I live in France comment:
@tatusmakeup: Ok I nedd this
@nolwee_legoff: I want it but I live in France comment:
@tatusmakeup: Ok I nedd this
@nolwee_legoff: I want it but I live in France comment: The data (6), it appears that the speech on the nyxcosmetics instagram caption is a a
perlocutinary act. This is identified by the intention of the speaker to give effect or
influence. So, in the comment section of other speakers, it gives effect to the other
speakers. The effect is that the followers of nyxcosmetics give positive respons. This
act is the act of affecting someone. Data 7
Post ( March 2020)
caption:
Help us #shoutloud b tagging a beaut who inspire you! Data 7
Post ( March 2020)
caption:
Help us #shoutloud b tagging a beaut who inspire you! Data 7
Post ( March 2020)
caption:
Help us #shoutloud b tagging a beaut who inspire you! comment:
@juspaid_: Aloha wahines’s
@traganellabialderosyxmac: Hello nyx in Argentina please, I love the brand. @wendigobeauty: #shoutlod to @kenadrogan one of the best friends I’ve ever had
and to @shannoxo m favorite youtuber and the woman I go to before buing any
higher end products. REFERENCES Austin, J. L. (1955). How To Do Things With Words. London: Oxford University Press. 358 | An Analysis of Illocutionary and Perlocutionary Speech Acts of an Instagram Online Shopping
Account Volume 4, No. 3, May 2021 pp 353-359 Fala, A., Intan, K., Z, T., W. A, S., U.A, D., & N, W. (2019). An Analysis of Speech Acts in
the Croods Movie, 7(1), 277–284. https://doi.org/https://doi.org/10.22216/jk.v3i1.3161 Fala, A., & Kumala, I. (2018). An Analysis of Speech Acts in the Croods Movie. Kumala
/Academic J Edu. Sci, 1(1), 1–7. https://doi.org/10.22341/ajes.00101p001 Green, M. (2017). Speech Acts (Stanford Encyclopedia of Philosophy). (E. N. Zalta, Ed.)
(Winter 201). Retrieved from https://plato.stanford.edu/entries/speech-acts/#Int Huang, Y. (2007). Pragmatics. New York: Oxford Universit Press. Sembiring, W. A., & Ambalegin, A. (2019). Illocutionary Acts on Aladdin Movie 2019. JURNAL BASIS, 6(2), 277–284. https://doi.org/10.33884/basisupb.v6i2.1419 Sumarsono. (2017). Sosiolinguistik Yogyakarta. Yogyakarta: SABDA (Lembaga Studi Agama,
Budaya dan Perdamaian). Tri Budiasih, L. (2016). Illocution on Speech Acts of Foreign Students in Indonesian Learning. PAROLE:
Journal
of
Linguistics
and
Education,
6(2),
41–48. https://doi.org/10.14710/parole.v6i2.41-48 Tutuarima, Z., Nuraeningsih, N., & Rusiana, R. (2018). An Analysis of Speech Act Used in
London Has Fallen Movie. Vision: Journal for Language and Foreign Language
Learning. https://doi.org/10.21580/vjv7i23022 Zulfa, A. F. (2018). A Graduating Paper, The Types of Illocutionary Acts on the Hackerman’s
Utterances in Cyberbully Movie. State Islamic University Sunan Kalijaga. An Analysis of Illocutionary and Perlocutionary Speech Acts of an Instagram Online Shopping
Account|359 An Analysis of Illocutionary and Perlocutionary Speech Acts of an Instagram Online Shopping
Account|359
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https://openalex.org/W4234563227
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https://www.qeios.com/read/M7659W/pdf
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English
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Petroleum Hydrocarbon Compound
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Definitions
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cc-by
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Qeios · Definition, February 2, 2020 Open Peer Review on Qeios Petroleum Hydrocarbon Compound National Cancer Institute Qeios ID: M7659W · https://doi.org/10.32388/M7659W Source National Cancer Institute. Petroleum Hydrocarbon Compound. NCI Thesaurus. Code
C122495. Any chemical compound that is derived from crude oil. Petroleum hydrocarbon
compounds can enter the environment through accidents, industrial releases, or as
byproducts of commercial or private uses. Qeios ID: M7659W · https://doi.org/10.32388/M7659W 1/1
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https://openalex.org/W2888524538
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https://hal.inria.fr/hal-02177858/file/472851_1_En_35_Chapter.pdf
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English
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Predictive Maintenance Platform Based on Integrated Strategies for Increased Operating Life of Factories
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IFIP advances in information and communication technology
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cc-by
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To cite this version: Gökan May, Nikos Kyriakoulis, Konstantinos Apostolou, Sangje Cho, Konstantinos Grevenitis, et
al.. Predictive Maintenance Platform Based on Integrated Strategies for Increased Operating Life of
Factories. IFIP International Conference on Advances in Production Management Systems (APMS),
Aug 2018, Seoul, South Korea. pp.279-287, 10.1007/978-3-319-99707-0_35. hal-02177858 Predictive Maintenance Platform Based on Integrated
Strategies for Increased Operating Life of Factories
Gökan May, Nikos Kyriakoulis, Konstantinos Apostolou, Sangje Cho,
Konstantinos Grevenitis, Stefanos Kokkorikos, Jovana Milenkovic, Dimitris
Kiritsis Distributed under a Creative Commons Attribution 4.0 International License gokan.may@epfl.ch Abstract. Process output and profitability of the operations are mainly deter-
mined by how the equipment is being used. The production planning, operations
and machine maintenance influence the overall equipment effectiveness (OEE)
of the machinery, resulting in more ‘good parts’ at the end of the day. The target
of the predictive maintenance approaches in this respect is to increase efficiency
and effectiveness by optimizing the way machines are being used and to decrease
the costs of unplanned interventions for the customer. To this end, development
of ad-hoc strategies and their seamless integration into predictive mainte-
nance systems is envisaged to bring substantial advantages in terms of produc-
tivity and competitiveness enhancement for manufacturing systems, representing
a leap towards the real implementation of the Industry 4.0 vision. Inspired by this
challenge, the study provides an approach to develop a novel predictive mainte-
nance platform capable of preventing unexpected-breakdowns based on inte-
grated strategies for extending the operating life span of production systems. The
approach and result in this article are based on the development and implemen-
tation in a large collaborative EU-funded H2020 research project entitled Z-
Bre4k, i.e. Strategies and predictive maintenance models wrapped around physi-
cal systems for zero-unexpected-breakdowns and increased operating life of fac-
tories. Keywords: Industry 4.0, predictive maintenance, big data, asset management,
smart factories, sustainable manufacturing, industrial production HAL Id: hal-02177858
https://inria.hal.science/hal-02177858v1
Submitted on 9 Jul 2019 L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d’enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés. HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Predictive maintenance platform based on integrated
strategies for increased operating life of factories Gökan May1, Nikos Kyriakoulis2, Konstantinos Apostolou3, Sangje Cho1, Konstanti-
nos Grevenitis3, Stefanos Kokkorikos2, Jovana Milenkovic3, Dimitris Kiritsis1 Gökan May1, Nikos Kyriakoulis2, Konstantinos Apostolou3, Sangje Cho1, Konstanti-
nos Grevenitis3, Stefanos Kokkorikos2, Jovana Milenkovic3, Dimitris Kiritsis1 1EPFL, ICT for Sustainable Manufacturing, EPFL SCI-STI-DK, Station 9, CH-1015 Lausanne,
Switzerland; 2Core Innovation and Technology O.E, Athens, Greece; 3ATLANTIS Engineering
SA, Thessaloniki, Greece 1
Introduction & State-of-the-art 1 The requirement of competitiveness is a constant objective of manufacturers [1]. For a
successful shift toward Industry 4.0, companies must constantly innovate and imple-
ment new methods and approaches enabling them to reduce operating costs and in-
crease the availability and reliability of their production equipment [2]. For this reason,
any downtime due to technical issues with the equipment needs to be avoided and, if
occurring, decreased as much as possible [3]. On that vein, maintenance in general and 2 2 predictive maintenance strategies in particular, are now facing significant challenges to
deal with the evolution of the equipment, instrumentation and manufacturing processes
they should support. Preventive maintenance strategies designed for traditional highly
repetitive and stable mass production processes based on predefined components and
machine behaviour models are no longer valid and more predictive-prescriptive mainte-
nance strategies are needed. The success of those adaptive and responsive maintenance
strategies highly depends on real-time and operation-synchronous information from the
production system, the production process and the individual product, which should
enrich and extend more traditional techniques and models. To meet the requirements mentioned above and aligned with the Industry 4.0 key
objectives toward eco-factories of the future [4, 5], this study provides a holistic frame-
work and a comprehensive set of integrated strategies encompassing the whole manu-
facturing line for addressing the issue of asset management in smart factories of indus-
try 4.0 in order to extend the life of production systems. Doing so, the research aims at
providing an answer as to what could be the proper strategies and associated technolo-
gies to effectively minimize downtimes of manufacturing systems. A large collabora-
tive EU-funded H2020 research project entitled Z-Bre4k [6] has been the main driver
of the described approach and is designed for its validation. The project consortium is
formed by 17 organisations across Europe including industrial pilot plants, academic
institutions and technology providing companies. To this end, novel strategies are designed in this research to be deployed at the field
in order to prevent/predict/diagnose/remediate failures, estimate remaining useful life
(RUL) of assets, manage alarms and mitigation actions, and synchronise with shop-
floor operations and plant management systems while ensuring the safety of workers. 1
Introduction & State-of-the-art The ultimate aim is to introduce and apply a holistic approach via integrated strategies
to increase maintainability, accurately predict the condition and the RUL of networked
machines, and adapt the performance to increase the operating life span of production
systems. 2
Strategies for increased operating life of production systems The innovative synergies between online data gathering systems, real-time simulation
models, data-based models and the knowledge management system form the main strat-
egies which contribute to achieve zero breakdowns in manufacturing. In this context,
the proposed solution comprises the introduction of eight (8) scalable strategies at com-
ponent, machine and system level, all of which can be applied in the existing manufac-
turing plants with minimum interventions, targeting (1) the prediction occurrence of
failure (Z-PREDICT), (2) the early detection of current or emerging failure (Z-
DIAGNOSE), (3) the prevention of failure occurrence, building up, or even propaga-
tion in the production system (Z-PREVENT), (4) the estimation of the RUL of assets
(Z-ESTIMATE), (5) the management of the aforementioned strategies through event
modelling, KPI monitoring and real-time decision support (Z-MANAGE), (6) the re-
placement, reconfiguration, re-use, retirement, and recycling of components/assets (Z-
REMEDIATE), (7) synchronizing remedy actions, production planning and logistics 3 (Z-SYNCHRONISE), (8) preserving the safety, health, and comfort of the workers (Z-
SAFETY). Each of the developed strategies are triggered based on predicting, detecting
and assessing the impact of system level events that cause low performances, generate
failures, and increase the costs. Figure 1 highlights the synergies and interactions be-
tween the eight Z-Strategies for building a novel predictive maintenance platform and
the role of each strategy is further explained below. (Z-SYNCHRONISE), (8) preserving the safety, health, and comfort of the workers (Z-
SAFETY). Each of the developed strategies are triggered based on predicting, detecting
and assessing the impact of system level events that cause low performances, generate
failures, and increase the costs. Figure 1 highlights the synergies and interactions be-
tween the eight Z-Strategies for building a novel predictive maintenance platform and
the role of each strategy is further explained below. Fig. 1. Synergies and interactions between the eight Z-Strategies Fig. 1. Synergies and interactions between the eight Z-Strategies Z-PREDICT: The events detected from the physical layer of the system are engineered
into high value data that stipulates new and more accurate process models. Such an
unbiased systems behaviour monitoring and analysis provides the basis for enriching
the existing knowledge of the system (experience) learning new patterns, raising atten-
tion towards behaviour that cause operational and functional discrepancies (e.g. alarms
for predicted failures) and the general trends in the shop-floor. The more the data pool
is being increased the more precise (repeatability) and accurate the predictions will be. 2
Strategies for increased operating life of production systems The estimations for the future states involve the whole production line – network of
machines and components. The system can thus predict with high confidence the ex-
pected performance of components and their maintenance needs, predicting current or
emerging failures, allowing better production planning and decision making on their
RUL. Hence, the ability to optimise the manufacturing processes according to the RUL,
production needs, and the maintenance operations is the key innovation to fulfil the
industrial requirements. Z-PREVENT: The prevention of failure occurrence strategy is based on the predic-
tion strategy (i.e. degraded performance of assets or failure) realised across the shop-
floor for condition monitoring of machinery and respective produced quality. The Z-
PREDICT is predecessor of Z- PREVENT. The initial estimation of the future states is
based on the simulation and modelling of the parameters. For each predicted failure or
low performance (e.g. due to fatigue, wear), the responsible factors are identified and
flagged through the FMEA system. The system analyses these factors based on an ini-
tial estimation, which after the simulation these are updated recursively. The result of
this process is to avoid the building up or even propagation of a failure that leads to
breakdown based on each recorded event both from previous and current states. The
strategy thus prevents multiple alarm activations on similar failures. 4 Z-DIAGNOSE: This strategy is invoked when a current or an emerging failure is
detected considering the condition at all three levels – machine, product, shop-floor. In
such a scenario, an alarm is being triggered to flag the events that resulted in a failure
or system performance degradation. By mapping the true reasons, the system is then
able to avoid generating the failure or its emergence by weighting the system model. The strategy also involves more actions and processes to deal both with the generation
of the diagnosed failure, and its severity increase to the next iterations as well as its
impact to the production line. Depending on the criticality of the generated failure, the
system can either adapt its parameters to prolong the RUL until the next maintenance,
or plan to the production for maintenance. The final decision on the actions is based on
the Z-MANAGE strategy. Z-ESTIMATE: This strategy combines the information from the Z-DIAGNOSE
and Z-PREDICT estimating the RUL of the assets. 2
Strategies for increased operating life of production systems The estimated values are also com-
bined with the information from the maintenance operations (physical examination
from operators) as well as from the specifications provided from the manufacturer. The
latter is used as the starting point for the estimation process, which after each iteration
the deviation of the real-model from the physical model is reduced having an accurate
virtual-model wrapped around the actual state of each machine and its components. The
trends for the fatigue and wear rates provide a confident RUL estimation. Z-MANAGE: This strategy is executing the overall supervision and optimisation of
the system. The failures are processed with the Decision Support System (DSS) tools
and are interfaced with Manufacturing Execution Systems (MES). False positives and
false negatives are clustered within the Z-PREDICT and Z-PREVENT Strategies. To
achieve so, the previous acquired knowledge and incidents are also processed to fine
tune the system’s performance. Additionally, the production is optimised by better
scheduling (Z-SYNCHRONISE), taking into account the impact of each failure. The
optimised scheduling and adaptability of the manufacturing improves the overall flex-
ibility, placing a premium on the production systems, extending their operating life,
while preserve increased machinery availability. Z-REMEDIATE: This strategy involves the decision making in the event of a fail-
ure, which classifies and categorises the input in terms of criticality, type, etc. Based
on the component/assets types (repairable-non repairable) and their RUL the strategy
decides for the following: (1) replace, (2) reconfigure and/or re-use, (3) retire, and (4)
recycle. This strategy triggers the Z-SYNCHRONISE and Z-SAFETY strategies from
which the maintenance actions can be planned and organized. Z-SYNCHRONISE: The predecessor Z-REMEDIATE strategy identifies the type
of action required for diagnosed failures which are then fused with the Z-MANAGE
output. This strategy synchronises all the remedy actions with internal and external
supply-chain tiers, as well as with production planning and logistics. It is therefore re-
sponsible to shift the production from one machine to another due to failure or deterio-
rated condition/performance, acting as the “end-effector” thus leading to optimised
scheduling and reduced costs by carrying out maintenance activities on time. Z-SAFETY: This strategy is invoked to increased Health & Safety during Z-Bre4k
shop-floor operations. Since most of the accidents occur during maintenance actions,
the Z-SAFETY prevents any activation to the machine that is under investigation or 5 5 repair. 2
Strategies for increased operating life of production systems The “Safety-Mode” lifts any unauthorised control from the personnel for the
whole duration of the maintenance. Apart from reducing the accidents Z-SAFETY also
takes into account the comfort of the human personnel on the shop-floor, e.g. extreme
heat or noise may be tolerable for the machines but not for humans. Therefore, the
health & safety procedures are also taken into account towards the operation feedback
of the whole production line. 3
Predictive maintenance platform based on Z-Strategies Manufacturing enterprises are pushed to take local actions: thinking globally however
staying economically compatible within the local context. In order to achieve high pre-
cision manufacturing of complex products, there has to be a fundamental rethink on
how to improve the operation of machines and improved controls. The improvement
should not only concern the individual machines as isolated islands but encompass the
totality of production process as a system of interrelated elements that seek to maximise
efficiency, productivity, customer satisfaction; whilst at the same time eliminating
waste and excess inventory. For that purpose, aligned with the Z-Strategies, a set of
technologies and overall system architecture have been identified as a part of the pro-
posed approach, following the method and procedures developed and proposed by May
et al. (2017) [7]. The first high-level description to lead to the definition of the predictive maintenance
platform consisted in identifying and classifying all components that can be called as
the tools’ landscape and logical architecture, i.e. conceptual view. Figure 2 presents this
landscape by proposing a compact representation of the involved tools. Fig. 2. Tools’ landscape and logical architecture of the predictive maintenance platform Fig. 2. Tools’ landscape and logical architecture of the predictive maintenance platform 6 Based on the proposed approach and defined conceptual view of the system, in Z-
Bre4k a novel predictive maintenance platform will be developed and demonstrated in
three pilot plans proving its universal applicability for the achievement of zero break-
downs in manufacturing. Therefore, the predictive maintenance platform will: Introduce a novel design for predictive maintenance based on three levels: machine
(network of components), product, and shop-floor (network of machines). It will
reconfigure the system to increase its performance (shorter cycles), increase its qual-
ity, and its availability by the employment of eight strategies to maximise these fac-
tors. Make accurate predictions for the future states of the components/machines/systems
by the employment of intelligent and adaptive simulators forecasting the generation
of failures, the fatigue and wear levels, estimating the RUL triggering respective
remedy actions. The condition monitoring will provide data about the actual status
which will update the simulation results, increasing its accuracy. The DSS will syn-
chronise the plans for maintenance, production and logistics. Estimate the RUL through its simulation capabilities, calling for maintenance and
suggesting the optimal times to place orders for spare parts, reducing the related
costs. 3
Predictive maintenance platform based on Z-Strategies The increased predictability of the system and the failure prevention actions
will reduce the number of failures, maximise the performance, decrease the repair/re-
cover times reducing further the costs. Optimise the performance of the machines, based on the current and predicted fa-
tigue/wear levels allowing actions to maintain and increase the operating life of these
assets, as well as to reduce the unexpected failures and breakdowns. Following the development of the conceptual view, the required components have
been highlighted in a preliminary architectural view, identifying services and depend-
encies within the Z-Bre4k platform. Later, new components were added in order to
cover all the required functionalities of the resulting predictive maintenance platform. As a result, definitions, identifications and classification of the system principle and its
process is presented in Figure 3. Besides the defined overall architecture, Table 1 pre-
sents initial links on how these strategies are integrated within the overall architecture
and how they are associated with each component of Z-Bre4k platform. Z-Bre4k components, their functionality and their interactions are thus described in
the overall architecture (Figure 3). Initial data, generated by shop-floor assets (i.e. sen-
sors, cameras, industrial/IoT devices, etc.) is collected by Condition Monitoring, Cog-
nitive Embedded Condition Monitoring and Machine Simulators components. All data
is sent to the Industrial Data Spaces (IDS) reaching the overall Z-Bre4k platform after
which they are homogenized by the Semantic Framework and used by the components
in a unified manner. Within the platform, users/employees are using HMI in order to
add data and/or parameterize components. Specifically, FMECA (Failure mode, ef-
fects, and criticality analysis) component needs these inputs to calculate risks, Risk
Priority Numbers (RPNs), criticality matrices and alerts in order to send data to DSS,
that further generates and delivers strategies, recommendations, notifications, reports 7 and updated schedules. Finally, M3 Gage, M3 Software and VRfx components are re-
lated to XYZ cloud points, 3D representations and visualization data of physical ob-
jects. and updated schedules. Finally, M3 Gage, M3 Software and VRfx components are re-
lated to XYZ cloud points, 3D representations and visualization data of physical ob-
jects. jects. Fig. 3. Overall architecture of the predictive maintenance platform j
Fig. 3. Overall architecture of the predictive maintenance platform Fig. 3. Overall architecture of the predictive maintenance platform Table 1. Z-Strategy and component association
No. Z-Strategy
Components
1
Z-PREDICT
Condition monitoring,
Machine simulators, VRfx, Predictive Maintenance. 3
Predictive maintenance platform based on Z-Strategies 2
Z-PREVENT
Machine simulators, Predictive Maintenance, FMECA. 3
Z-DIAGNOSE
Predictive Maintenance, FMECA. 4
Z-ESTIMATE
Machine simulators, VRfx, Predictive Maintenance. 5
Z-MANAGE
Predictive Maintenance, FMECA, DSS. 6
Z-REMEDIATE
FMECA, DSS. 7
Z-SYNCHRONIZE
AUTOWARE Communication Middleware. 8
Z-SAFETY
DSS. Table 1. Z-Strategy and component association Table 1. Z-Strategy and component association The resulting Z-Bre4k system will be demonstrated in three key sectors with the
strongest SME presence (i.e. automotive, food and beverage, consumer electronics) for
a wide range of components and machines with different operational requirements and
behaviours, illustrating the potential and full value of Z-Bre4k as a holistic framework
to address predictive maintenance strategies for operation in high diversity of machin-
ery (e.g. robotic systems, inline quality control equipment, injection moulding, stamp-
ing press, high performance smart tooling/dies and fixtures), including highly challeng-
ing and sometimes critical manufacturing processes (e.g. automated packaging indus-
try, multi-stage zero-defect adaptive manufacturing of structural light-weight compo-
nent for automotive industry, short-batch mass customised production process for con-
sumer electronics and health sector). 8 4
Discussion and concluding remarks The main goal of the predictive maintenance approach and implementation is to provide
machine builders and designers (OEMs), industrial component suppliers and engineer-
ing software developers with novel solutions which will: (1) improve the performance
of the manufacturing processes; (2) increase the machine maintainability; (3) provide
predictions on damages/failures; (4) understand and interpret the source of the failures
enhancing eventually the design process; (5) increase the availability of the machine
builders while making them cost effective; and thus (6) increase OEE. In this context, the study will: (a) act as a comprehensive and practical guide for
optimizing production machineries and processes by implementing predictive mainte-
nance principles; (b) transform machine tool and process related data into useful infor-
mation that could support machinery prognosis and optimization strategies by enabling
model-based control of machine tools based on actual machine life-cycle parameters. Accordingly, the expected impacts are highlighted as follows: (1) improved predic-
tive maintenance and system adaptability for manufacturing systems and processes; (2)
new maintainability concepts based on predictive maintenance with improved machine
reliability (MTBF) and reduced maintenance costs; and (3) incorporating intelligent
systems and data analysis methods for achieving smart factories of Industry 4.0. Future work will focus on implementing and validating the proposed approach on
several use cases in different industries, demonstrating its ability to support major ac-
tors of the manufacturing sector to take advantage of the digital transformation. Acknowledgements. This work has been carried out in the framework of Z-Bre4k Pro-
ject, which has received funding from the European Union’s Horizon 2020 research
and innovation programme under grant agreement Nº 768869. 1. Swanson, L.: Linking maintenance strategies to performance. International Journal of Pro-
duction Economics 70(3), 237–244 (2001) References 1. Swanson, L.: Linking maintenance strategies to performance. International Journal of Pro-
duction Economics 70(3), 237–244 (2001) 1. Swanson, L.: Linking maintenance strategies to performance. International Journal of Pro-
duction Economics 70(3), 237–244 (2001) 2. Wang, S., Wan, J., Li, D., & Zhang, C.: Implementing Smart Factory of Industrie 4.0: An
Outlook. International Journal of Distributed Sensor Networks, 12(1), 3159805 (2016). 2. Wang, S., Wan, J., Li, D., & Zhang, C.: Implementing Smart Factory of Industrie 4.0: An
Outlook. International Journal of Distributed Sensor Networks, 12(1), 3159805 (2016). 3. Lee, J., Kao, H.-A., & Yang, S.: Service Innovation and Smart Analytics for Industry 4.0
and Big Data Environment. Procedia CIRP 16, 3–8 (2014). 4. Lee, J., Bagheri, B., & Kao, H.-A.: A Cyber-Physical Systems architecture for Industry 4.0-
based manufacturing systems. Manufacturing Letters 3, 18–23 (2015). 5. May, G., Stahl, B., Taisch, M.: Energy management in manufacturing: Toward eco-factories
of the future – A focus group study. Applied Energy 164, 628–638 (2016). 6. Z-Bre4k Project Homepage, https://www.z-bre4k.eu, last accessed 2018/03/21 7. May, G., Ioannidis, D., Metaxa, I. N., Tzovaras, D., & Kiritsis, D.: An Approach to Devel-
opment of System Architecture in Large Collaborative Projects. IFIP Advances in Infor-
mation and Communication Technology, 67–75 (2017)
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Cardiovascular magnetic resonance imaging to assess myocardial fibrosis in valvular heart disease
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The international journal of cardiovascular imaging
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cc-by
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Cardiovascular magnetic resonance imaging to assess myocardial
fibrosis in valvular heart disease The present review article summa-
rizes the current status of CMR techniques to assess myo-
cardial fibrosis and appraises the current evidence on the Keywords Cardiovascular magnetic resonance · Valvular
heart disease · Mitral regurgitation · Aortic stenosis ·
Aortic regurgitation · Late gadolinium enhancement · T1
mapping · Tagging · Feature tracking Keywords Cardiovascular magnetic resonance · Valvular
heart disease · Mitral regurgitation · Aortic stenosis ·
Aortic regurgitation · Late gadolinium enhancement · T1
mapping · Tagging · Feature tracking Keywords Cardiovascular magnetic resonance · Valvular
heart disease · Mitral regurgitation · Aortic stenosis ·
Aortic regurgitation · Late gadolinium enhancement · T1
mapping · Tagging · Feature tracking Abbreviations
AR
Aortic regurgitation
AS
Aortic stenosis
CMR
Cardiovascular magnetic resonance
ECV
Extracellular volume
iECV
Indexed extracellular volume
LGE
Late gadolinium enhancement
MR
Mitral regurgitation
LV
Left ventricle/left ventricular
VHD
Valvular heart disease Abbreviations
AR
Aortic regurgitation
AS
Aortic stenosis
CMR
Cardiovascular magnetic resonance
ECV
Extracellular volume
iECV
Indexed extracellular volume
LGE
Late gadolinium enhancement
MR
Mitral regurgitation
LV
Left ventricle/left ventricular
VHD
Valvular heart disease Int J Cardiovasc Imaging (2018) 34:97–112
DOI 10.1007/s10554-017-1195-y ORIGINAL PAPER ORIGINAL PAPER Cardiovascular magnetic resonance imaging to assess myocardial
fibrosis in valvular heart disease Tomaz Podlesnikar1 · Victoria Delgado1 · Jeroen J. Bax1 Received: 8 March 2017 / Accepted: 13 June 2017 / Published online: 22 June 2017
© The Author(s) 2017. This article is an open access publication use of these techniques for risk stratification of patients
with severe aortic stenosis or regurgitation and mitral
regurgitation. Abstract The left ventricular (LV) remodeling process
associated with significant valvular heart disease (VHD)
is characterized by an increase of myocardial interstitial
space with deposition of collagen and loss of myofibers. These changes occur before LV systolic function dete-
riorates or the patient develops symptoms. Cardiovascular
magnetic resonance (CMR) permits assessment of reac-
tive fibrosis, with the use of T1 mapping techniques, and
replacement fibrosis, with the use of late gadolinium con-
trast enhancement. In addition, functional consequences
of these structural changes can be evaluated with myocar-
dial tagging and feature tracking CMR, which assess the
active deformation (strain) of the LV myocardium. Several
studies have demonstrated that CMR techniques may be
more sensitive than the conventional measures (LV ejec-
tion fraction or LV dimensions) to detect these structural
and functional changes in patients with severe left-sided
VHD and have shown that myocardial fibrosis may not be
reversible after valve surgery. More important, the pres-
ence of myocardial fibrosis has been associated with lesser
improvement in clinical symptoms and recovery of LV sys-
tolic function. Whether assessment of myocardial fibrosis
may better select the patients with severe left-sided VHD
who may benefit from surgery in terms of LV function and
clinical symptoms improvement needs to be demonstrated
in prospective studies. 1
Department of Cardiology, Heart and Lung Center, Leiden
University Medical Center, Albinusdreef 2 2333 ZA, Leiden,
The Netherlands * Jeroen J. Bax
j.j.bax@lumc.nl Introduction Valvular heart disease (VHD) is an important public-health
problem with an increasing prevalence along with ageing
of the population [1]. Moderate and severe VHD on echo-
cardiography affects 2.5% of the population of the United
States and increases up to 11.7% in the group of patients
aged 75 and older [2]. The decision to operate in patients
with severe VHD is frequently complex and relies on an
individual risk–benefit analysis. In general, improvement
in prognosis compared with natural history of the disease
should outweigh the risk of intervention and its potential
late consequences, particularly prosthesis-related com-
plications. Current guidelines recommend to intervene in 1
Department of Cardiology, Heart and Lung Center, Leiden
University Medical Center, Albinusdreef 2 2333 ZA, Leiden,
The Netherlands (0121 3456789)
3 3456789)
3 Int J Cardiovasc Imaging (2018) 34:97–112 98 patients with symptomatic severe VHD and in asympto-
matic patients with reduced left ventricular (LV) ejection
fraction, LV dilatation, pulmonary hypertension, right ven-
tricular dilatation and dysfunction and presence of atrial
fibrillation [1, 3]. However, most of these adverse conse-
quences of severe VHD are observed in advanced stages
of the disease and are partially irreversible after interven-
tion, leading to suboptimal long-term clinical outcomes [4]. Therefore, additional markers that identify early structural
and functional consequences of severe VHD before irre-
versible damage of the myocardium occurs would help to
redefine the optimal timing for intervention. Table 1 Cardiovascular magnetic resonance techniques to assess myocardial fibrosis valvular heart disease
CMR cardiovascular magnetic resonance, ECV extracellular volume, SSFP steady state free precession, VHD valvular heart disease
CMR technique
Availability Fibrosis specificity Advantages
Limitations
Experi-
ence in
VHD
T1 mapping (native T1 and
ECV quantification)
++
+++
Assessment of diffuse fibrosis, early disease changes
(preclinical stages). Introduction Quantification of the degree of
fibrosis
Multiple methodologies, no standardized reference
values, overlap between normal and diseased myocar-
dium
++
Late gadolinium enhancement +++
+++
Reference standard for assessment of replacement
fibrosis
Focal fibrosis assessment only
+++
Molecular imaging
±
++++
Improved visualization of fibrosis, investigation of
underlying processes (necrosis, apoptosis, inflamma-
tion, scar maturation…)
Experimental technique, animal studies only
–
CMR tagging
++
+
Current gold standard for myocardial deformation
assessment, high reproducibility of the results
Expertise, additional scan sequences, time consuming
post-processing, tag fading through cardiac cycle
(only with some techniques), limited in assessment of
thin myocardium
++
Feature tracking CMR
+++
+
Post-processing of SSFP cines (no additional scan
sequences), relatively fast post-processing, high
feasibility
Susceptible to through-plane motion artifacts, limited
inter-vendor agreement
+ i
Chronic pressure and volume overload caused by severe
left-sided VHD results in LV remodeling. Changes in the
extracellular matrix with deposition of collagen I and loss
of myofibers at a later stage result in myocardial fibrosis,
the hallmark of LV remodeling [5, 6]. Cardiovascular mag-
netic resonance (CMR) imaging techniques permit direct
and indirect assessment of myocardial fibrosis. T1 mapping
and late gadolinium enhancement (LGE) permit myocardial
tissue characterization and provide measures of direct myo-
cardial fibrosis whereas CMR tagging and feature track-
ing CMR allow for assessment of myocardial deforma-
tion (strain), a functional parameter that indirectly reflects
myocardial fibrosis. In addition, advances in molecular
CMR imaging provide high-specificity tools for detection
of myocardial fibrosis. This article provides an overview
of current CMR techniques to assess myocardial fibrosis in
patients with left-sided VHD. CMR techniques for direct assessment
of myocardial fibrosis LV remodeling in response to chronic pressure and volume
overload caused by VHD is characterized by progressive
increase of the interstitial space with increased collagen
volume fraction (reactive fibrosis) and eventually apopto-
sis of myocardial cells which are replaced by firm fibrous
tissue (replacement fibrosis or scar). T1 mapping and LGE
CMR techniques are currently the most frequently used
techniques to directly assess myocardial fibrosis (Table 1). CMR T1 mapping 1, panels A and B). The T1 time can be
obtained for any myocardial segment and T1 maps can be
generated by determining the T1 time at each pixel loca-
tion (Fig. 1, panel C). Three T1 mapping-derived metrics
have been proposed as markers of increased myocardial
fibrosis: the native T1 time, the post-contrast T1 time
and the myocardial extracellular volume (ECV). With
the increase of interstitial fibrosis, the native T1 values
(without the use of gadolinium contrast) become longer
whereas the post-contrast T1 values become shorter. By
combining them, myocardial ECV fraction can be com-
puted, which quantifies the extracellular matrix space. In
the absence of amyloid deposition or edema, collagen I
is the main component of the extracellular matrix space
and therefore the myocardial ECV fraction is considered
a robust marker of myocardial fibrosis [8–10]. The added
value of these metrics over LGE is the ability to quantify
the degree of fibrosis and, particularly, to detect diffuse
interstitial fibrosis, often associated with early stages of
the disease. generated (Fig. 1, panels A and B). The T1 time can be
obtained for any myocardial segment and T1 maps can be
generated by determining the T1 time at each pixel loca-
tion (Fig. 1, panel C). Three T1 mapping-derived metrics
have been proposed as markers of increased myocardial
fibrosis: the native T1 time, the post-contrast T1 time
and the myocardial extracellular volume (ECV). With
the increase of interstitial fibrosis, the native T1 values
(without the use of gadolinium contrast) become longer
whereas the post-contrast T1 values become shorter. By
combining them, myocardial ECV fraction can be com-
puted, which quantifies the extracellular matrix space. In
the absence of amyloid deposition or edema, collagen I
is the main component of the extracellular matrix space
and therefore the myocardial ECV fraction is considered
a robust marker of myocardial fibrosis [8–10]. The added
value of these metrics over LGE is the ability to quantify
the degree of fibrosis and, particularly, to detect diffuse
interstitial fibrosis, often associated with early stages of
the disease. CMR T1 mapping The longitudinal magnetization relaxation time of the
myocardium, so-called T1 time, is highly sensitive to
processes that increase the interstitial space and can be
quantified with various techniques [7]. One of the most
commonly used in clinical practice is the modified
Look-Locker pulse sequence where multiple single-shot
images are acquired intermittently in diastole during
9–17 cardiac cycles and the inversion recovery curves are 1 3 3 Int J Cardiovasc Imaging (2018) 34:97–112 99 Fig. 1 Modified Look-Locker (MOLLI) technique for myocardial
T1 mapping. After radiofrequency inversion pulse, myocardial tis-
sue longitudinal magnetization in a stable magnetic field returns to
the equilibrium and a series of images are acquired in diastole over
several heart beats (A). The images are sorted in order of increasing
T1 times and the T1 recovery curve is obtained by plotting respec-
tive signal intensities against T1 time (B). The T1 map is obtained by
applying this technique for all pixels in the image (C). Reproduced
with permission from Taylor et al. [7] T1 times and the T1 recovery curve is obtained by plotting respec-
tive signal intensities against T1 time (B). The T1 map is obtained by
applying this technique for all pixels in the image (C). Reproduced
with permission from Taylor et al. [7] Fig. 1 Modified Look-Locker (MOLLI) technique for myocardial
T1 mapping. After radiofrequency inversion pulse, myocardial tis-
sue longitudinal magnetization in a stable magnetic field returns to
the equilibrium and a series of images are acquired in diastole over
several heart beats (A). The images are sorted in order of increasing T1 times and the T1 recovery curve is obtained by plotting respec-
tive signal intensities against T1 time (B). The T1 map is obtained by
applying this technique for all pixels in the image (C). Reproduced
with permission from Taylor et al. [7] However, it should be noted that the cut-off values of
the T1 mapping-derived metrics to define fibrosis cannot
be currently established since the values show considerable
overlap in normal and diseased myocardium [11]. Moreo-
ver, neither of the techniques is entirely specific to myo-
cardial fibrosis; abnormal myocardial ECV fraction can
be observed in infiltrative diseases (i.e., amyloidosis) and
edema, while native T1 values may also be altered in iron
deposition and diffuse fat infiltration [12]. Furthermore,
standardization of CMR T1 mapping techniques is neces-
sary to obtain reproducible measurements across different
vendors and institutions. generated (Fig. Late gadolinium contrast enhanced CMR LGE CMR is considered the reference standard to quantify
myocardial replacement fibrosis and scar. The increased
extracellular space and decreased capillary density of the
fibrous tissue result in increased volume of distribution
and prolonged wash-out of gadolinium in comparison to 1 3 Int J Cardiovasc Imaging (2018) 34:97–112 100 remodeling in a mice model of acute myocardial infarction
[22]. Several other molecular probes have been synthesized
to study individual processes involved in fibrosis formation,
like necrosis, apoptosis, inflammation and scar maturation
[23]. Further efficacy and safety studies are needed before
clinical implementation. However, the current evidence is
promising for future improvements in fibrosis detection and
monitoring of molecular processes associated with myocar-
dial remodeling. the normal myocardium [13]. 10–20 min after intravenous
administration of gadolinium, inversion recovery images
are acquired in mid to late diastole. The inversion time is
chosen to null the normal myocardium and provide the
best tissue contrast between fibrous tissue, which appears
bright, and normal myocardium, which appears black. Distinct patterns of LGE have been described in various
cardiac diseases and associated with adverse prognosis
[14–19] (Fig. 2). CMR techniques for indirect assessment
of myocardial fibrosis Molecular magnetic resonance imaging with the use of col-
lagen-specific contrast agents is a new experimental method
for the assessment of myocardial fibrosis. These novel con-
trast agents have shown to improve visualization of scar and
perfusion defects in animal models of myocardial infarction
[20, 21]. Furthermore, an elastin/tropoelastin-targeting con-
trast agent has provided interesting insights into the patho-
physiology of remote myocardium extracellular matrix The functional consequences of myocardial fibrosis such
as increased LV stiffness, impaired LV diastolic and sys-
tolic function, can be evaluated with CMR tagging and
feature tracking CMR (Table 1). These techniques evalu-
ate the active deformation (strain) of the myocardium in
three orthogonal directions: radial, circumferential and Fig. 2 Patterns of late gadolinium enhancement (LGE). A shows no
LGE, no focal replacement fibrosis. B–E demonstrate different pat-
terns of non-infarct myocardial fibrosis: B diffuse patchy LGE of the
anterior and lateral wall (arrows); C focal nodular LGE of the infe-
rior wall (arrow); D focal LGE of the anterior and inferior right ven-
tricular insertion points (arrows) and E linear midwall septal LGE
with additional foci at the right ventricular insertion points (arrows). In F, typical infarct-type subendocardial LGE distribution is shown
(arrows) Fig. 2 Patterns of late gadolinium enhancement (LGE). A shows no
LGE, no focal replacement fibrosis. B–E demonstrate different pat-
terns of non-infarct myocardial fibrosis: B diffuse patchy LGE of the
anterior and lateral wall (arrows); C focal nodular LGE of the infe-
tricular insertion points
with additional foci at th
In F, typical infarct-type
(arrows) tricular insertion points (arrows) and E linear midwall septal LGE
with additional foci at the right ventricular insertion points (arrows). In F, typical infarct-type subendocardial LGE distribution is shown
(arrows) Fig. 2 Patterns of late gadolinium enhancement (LGE). A shows no
LGE, no focal replacement fibrosis. B–E demonstrate different pat-
terns of non-infarct myocardial fibrosis: B diffuse patchy LGE of the
anterior and lateral wall (arrows); C focal nodular LGE of the infe-
rior wall (arrow); D focal LGE of the anterior and inferior right ven- 1 3 3 3 101 Int J Cardiovasc Imaging (2018) 34:97–112 longitudinal. In patients with VHD, the measurement of LV
ejection fraction, which merely reflects the change in LV
volumes between systole and diastole, may be misleading. Feature tracking CMR Feature tracking CMR is based on post-processing of
standard steady state free precession cine images, similar
to echocardiographic speckle tracking. Feature tracking
CMR algorithms focus on the endo- and epicardial borders
and detect the in- and outward motion of the cavity-tissue
interface [27, 28]. Global and segmental LV longitudinal,
circumferential and radial strain, strain-rates, and LV rota-
tional mechanics can be derived from standard long- and
short-axis views (Fig. 3). Global rather than segmental CMR techniques for indirect assessment
of myocardial fibrosis For example, in patients with mitral regurgitation, LV ejec-
tion fraction may be preserved for long time since the LV
is emptying in a low-pressure chamber (left atrium) while
myocardial longitudinal strain may be impaired [24]. In
patients with severe aortic stenosis, the LV hypertrophy,
developed in response to the pressure overload, reduces the
wall stress and maintains the LV ejection fraction. How-
ever, myocardial longitudinal strain may be impaired [25]. CMR tagging and feature tracking CMR track distinctive
features of the myocardium throughout the cardiac cycle
and calculate mechanical indices, such as strain, strain-rate,
twist and torsion. employed. Recent developments in pulse sequences and
image processing have resulted in a plethora of new tag-
ging techniques [26]. The main advantage of CMR tagging
over feature tracking CMR is that the imposed tags are
more clearly defined and easier tracked than the natural fea-
tures and are not subjected to through plane displacements,
thereby providing more reproducible measurements [27]. The main shortcomings of this technique are the need for
additional, elaborate scan sequences with limited accuracy
when applied to thin myocardium (such as the remodeled,
thinned-wall LV, the right ventricle and the atria) and the
time-consuming post-processing. CMR tagging This method is based on alteration of the myocardial tis-
sue magnetization to create trackable markers within the
myocardium which are visualized as dark lines in the form
of a grid pattern. This allows immediate visual assess-
ment of myocardial deformation, but for a more objective
approach and quantification additional post-processing is Fig. 3 Feature tracking cardiovascular magnetic resonance (CMR) in
a patient with severe aortic stenosis. A Long-axis (top) and a mid-
cavity short-axis (bottom) end-diastolic steady state free precession
images. Left ventricular endo- and epicardium are contoured (red
and green lines) and the anterior right ventricular insertion point is
marked in short-axis (blue dot). B Fully automated feature tracking
analysis is performed by tracking distinctive features along the out-
lined myocardium borders. C The derived time-strain curves show
a wide variation in segmental longitudinal strain (top) and normal
global peak circumferential strain (bottom). The purple colored curve
corresponds to the anteroseptal segment. D The 16-segment bullseye
plots for longitudinal (top) and circumferential (bottom) left ventric-
ular strain, showing impaired myocardial deformation of the basal
interventricular septum. (Feature tracking analysis was performed
with cvi42 v5.3, Circle Cardiovascular Imaging, Calgary, Canada) a wide variation in segmental longitudinal strain (top) and normal
global peak circumferential strain (bottom). The purple colored curve
corresponds to the anteroseptal segment. D The 16-segment bullseye
plots for longitudinal (top) and circumferential (bottom) left ventric-
ular strain, showing impaired myocardial deformation of the basal
interventricular septum. (Feature tracking analysis was performed
with cvi42 v5.3, Circle Cardiovascular Imaging, Calgary, Canada) a wide variation in segmental longitudinal strain (top) and normal
global peak circumferential strain (bottom). The purple colored curve
corresponds to the anteroseptal segment. D The 16-segment bullseye
plots for longitudinal (top) and circumferential (bottom) left ventric-
ular strain, showing impaired myocardial deformation of the basal
interventricular septum. (Feature tracking analysis was performed
with cvi42 v5.3, Circle Cardiovascular Imaging, Calgary, Canada) Fig. 3 Feature tracking cardiovascular magnetic resonance (CMR) in
a patient with severe aortic stenosis. A Long-axis (top) and a mid-
cavity short-axis (bottom) end-diastolic steady state free precession
images. Left ventricular endo- and epicardium are contoured (red
and green lines) and the anterior right ventricular insertion point is
marked in short-axis (blue dot). B Fully automated feature tracking
analysis is performed by tracking distinctive features along the out-
lined myocardium borders. C The derived time-strain curves show Fig. Aortic stenosis The pressure overload caused by AS increases LV wall
stress and as a consequence the myocardium responds
with myocyte hypertrophy to maintain LV systolic func-
tion. This myocardial hypertrophy is characterized by an
increased muscle fiber diameter with parallel addition of
new myofibrils [37]. Furthermore, there is an increase of
interstitial fibrosis and myocyte apoptosis, partially as a
consequence of oxygen supply–demand mismatch and
myocardial ischemia [37–39]. At a late stage in the natural
history of severe AS, the LV myocardium is characterized
by large areas of myocyte loss and replacement fibrosis
causing LV systolic dysfunction and associated with poor
prognosis [38]. The early changes in the interstitial space with increased
deposition of collagen I can be assessed with CMR T1
mapping (Table 2) [8, 34, 40–46]. Several studies have
validated LV native T1 values and myocardial ECV frac-
tion against histology in patients with AS undergoing aor-
tic valve replacement [8, 34, 40, 41]. In 109 patients with
moderate and severe AS, Bull and colleagues showed
that LV native T1 values were significantly higher among
patients with symptomatic severe AS compared with mod-
erate and asymptomatic severe AS (1014 ± 38 vs. 955 ± 30
and 972 ± 33 ms, respectively; p < 0.05) (Fig. 4) [40]. A
significant correlation was observed between native T1
values and collagen volume fraction assessed on myocar-
dial biopsies (R = 0.65, p = 0.002). Similarly, Flett and
coworkers validated the measurement of myocardial ECV
fraction in 18 patients with severe AS [8]. ECV strongly
correlated with the histological collagen volume fraction
(R2 = 0.86; p < 0.001). Although still not implemented in
routine clinical practice, the measurement of myocardial
ECV in patients with AS has important clinical implica-
tions [34, 43–46]. Increased ECV has been associated with In addition, LGE is an important prognostic marker
in patients with AS [19, 32, 33]. In 143 patients
with moderate and severe AS who were followed for
2.0 ± 1.4 years, the presence of LGE was associated with
an increase in all-cause and cardiac mortality (every 1%
increase in LGE mass was associated with 5% increased
risk of all-cause mortality; p = 0.005) [19]. CMR tagging 3 Feature tracking cardiovascular magnetic resonance (CMR) in
a patient with severe aortic stenosis. A Long-axis (top) and a mid-
cavity short-axis (bottom) end-diastolic steady state free precession
images. Left ventricular endo- and epicardium are contoured (red
and green lines) and the anterior right ventricular insertion point is
marked in short-axis (blue dot). B Fully automated feature tracking
analysis is performed by tracking distinctive features along the out-
lined myocardium borders. C The derived time-strain curves show 1 3 Int J Cardiovasc Imaging (2018) 34:97–112 102 symptoms, worse LV systolic and diastolic function, higher
levels of cardiac troponin T and ECG strain [34, 43–46]. Recently, Chin et al. reported the prognostic implications
of myocardial ECV fraction corrected for LV end-dias-
tolic myocardial volume normalized to the body surface
area (iECV) in 166 patients with mild to severe AS [34]. Patients with increased myocardial iECV (≥22.5 ml/m2)
but without LGE (replacement fibrosis) showed signifi-
cantly higher all-cause mortality and AS-related mortality
rates (36 per 1000 patients-year for both) as compared to
the patients with normal myocardium (iECV < 22.5 ml/m2,
8 and 0 deaths/1000 patient-years) (Fig. 5).i strain values appear the most reproducible [29–31]. Addi-
tional methodology standardization is an important prereq-
uisite for wider dissemination of this technique in clinical
practice. CMR left ventricular myocardial fibrosis
assessment in VHD: clinical evidence Accumulating evidence on the deleterious impact of LV
myocardial fibrosis on clinical outcomes after surgical
treatment of left-sided VHD has raised interest on tissue
characterization and LV strain with CMR techniques [19,
32–36]. This evidence is summarized for aortic stenosis
(AS) and regurgitation (AR) and for mitral regurgitation
(MR) in the following sections. p
y
) (
g
)
LGE, myocardial replacement fibrosis, is detected in
19–62% of patients with severe AS [19, 32, 47, 48]. Two
forms of LGE can be observed: the ischemic and the non-
ischemic pattern. The ischemic pattern is characterized by
subendocardial LGE along specific coronary artery terri-
tories whereas in the non-ischemic pattern the distribution
of LGE can be diffuse, (multi)focal or linear, confined or
patchy, and is predominantly located in the midwall myo-
cardial layer and does not correspond to a specific coronary
artery territory (Fig. 2) [19, 32, 47, 48]. The presence and
the extent of LGE have been associated with increased LV
mass, worse LV ejection fraction, the presence of symp-
toms, markers of myocardial injury such NT-pro-brain
natriuretic peptide and high-sensitivity cardiac troponins
and ECG strain (Table 2) [19, 32, 45, 46, 48, 49]. How-
ever, LGE was not significantly associated with transaortic
gradients or the aortic valve area, common indices of AS
severity [19, 32, 48], suggesting that there is different indi-
vidual susceptibility to develop LV hypertrophy and myo-
cardial fibrosis, likely influenced by multiple factors such
as advanced age, male sex, obesity and certain genetic vari-
ants [50]. Aortic stenosis [40]
109
AS
Native T1 mapping
Native T1 values increased along
with hemodynamic severity of AS
and correlated with the degree of
biopsy-quantified fibrosis (R = 0.65;
p = 0.002; N = 23)
Lee et al. [41]
80
AS
Native T1 mapping
Native T1 values at 3T CMR were
significantly longer in asympto-
matic patients with moderate to
severe AS compared to normal
controls
Flett et al. [8]
18
AS
ECV
ECV correlated strongly with col-
lagen volume fraction on histology
(R2 = 0.86; p < 0.001)
Dusenbery et al. [44]
35
AS
ECV
ECV was significantly higher in
patients with congenital AS than in
normal subjects
Flett et al. [43]
66
AS
ECV
Patients with severe AS had higher
ECV than normal controls
Chin et al. [34]
166
AS
iECV, LGE
Increased iECV was associated with
increased all-cause mortality com-
pared to patients with normal iECV
(36 vs. 8 deaths/1000 patient-years,
respectively)
Chin et al. [45]
122
AS
ECV, LGE
ECV and percent of midwall replace-
ment fibrosis (LGE) were associ-
ated with increased high-sensitivity
cardiac troponin I levels
Shah et al. [46]
102
AS
ECV, LGE
LGE and ECV were associated with
ECG strain in patients with mild to
severe AS
Debl et al. [47]
22
AS
LGE
LGE was associated with severe LV
hypertrophy
Rudolph et al. [48]
21
AS
LGE
LGE was associated with increased
LV mass index and LV end-dias-
tolic volume index. LGE was not
associated with the severity of AS
Dweck et al. [19]
143
AS
LGE
Midwall fibrosis on LGE CMR was
associated with higher mortality
than infarct-type LGE (HR 8.59;
95% CI 1.97–37.38; p = 0.004
and HR 6.46; 95% CI 1.39-30.00;
p = 0.017, respectively)
Barone-Rochette et al. [32]
154
AS
LGE
LGE was an independent predictor of
all-cause and cardiovascular mor-
tality in patients with severe AS
undergoing surgical valve replace-
ment (HR for all-cause mortality:
2.8; 95% CI 1.3–6.9; p = 0.025)
Weidemann et al. [49]
58
AS
LGE
The extent of LGE in patients with
symptomatic severe AS undergo-
ing aortic valve surgery correlated
with biopsy-quantified myocardial
fibrosis and remained unchanged at
9 months after surgery Increased iECV was associated with
increased all-cause mortality com-
pared to patients with normal iECV
(36 vs. Aortic stenosis When divid-
ing the population according to the pattern of LGE,
patients with midwall fibrosis (N = 54) had higher mor-
tality than patients with infarct-type LGE (N = 40) (HR
8.59; 95% CI 1.97–37.38; p = 0.004 and HR 6.46; 95% CI
1.39–30.00; p = 0.017, respectively). Furthermore, in 154
patients with severe AS undergoing surgical aortic valve
replacement, the presence of LGE was an independent
predictor of all-cause and cardiovascular mortality (HR
for all-cause mortality: 2.8; 95% CI 1.3–6.9; p = 0.025)
[32]. Importantly, after aortic valve replacement, LGE
does not completely regress and has been associated with
incomplete LV functional recovery, worse New York 1 3 3 103 Int J Cardiovasc Imaging (2018) 34:97–112 Table 2 CMR studies to detect myocardial fibrosis in valvular heart disease
Study
No. of patients Valve disease CMR technique
Bull et al. [40]
109
AS
Native T1 mapping
Lee et al. [41]
80
AS
Native T1 mapping
Flett et al. [8]
18
AS
ECV
Dusenbery et al. [44]
35
AS
ECV
Flett et al. [43]
66
AS
ECV
Chin et al. [34]
166
AS
iECV, LGE
Chin et al. [45]
122
AS
ECV, LGE
Shah et al. [46]
102
AS
ECV, LGE
Debl et al. [47]
22
AS
LGE
Rudolph et al. [48]
21
AS
LGE
Dweck et al. [19]
143
AS
LGE
Barone-Rochette et al. [32]
154
AS
LGE
Weidemann et al. [49]
58
AS
LGE Table 2 CMR studies to detect myocardial fibrosis in valvular heart disease
Study
No. of patients Valve disease CMR technique
Bull et al. [40]
109
AS
Native T1 mappin
Lee et al. [41]
80
AS
Native T1 mappin
Flett et al. [8]
18
AS
ECV
Dusenbery et al. [44]
35
AS
ECV
Flett et al. [43]
66
AS
ECV
Chin et al. [34]
166
AS
iECV, LGE
Chin et al. [45]
122
AS
ECV, LGE
Shah et al. [46]
102
AS
ECV, LGE
Debl et al. [47]
22
AS
LGE
Rudolph et al. [48]
21
AS
LGE
Dweck et al. [19]
143
AS
LGE
Barone-Rochette et al. [32]
154
AS
LGE
Weidemann et al. [49]
58
AS
LGE Table 2 CMR studies to detect myocardial fibrosis in valvular heart disease Table 2 CMR studies to detect myocardial fibrosis in valvular heart disease
Study
No. of patients Valve disease CMR technique
Main findings
Bull et al. Aortic stenosis [55]
30, 18
AS
CMR tagging, feature tracking
CMR
Reasonable agreement between both
techniques, but feature tracking
CMR yielded higher strain values
than CMR tagging
Mahmod et al. [57]
39
AS
CMR tagging
Patients with AS had impaired LV
strain compared to controls
Al Musa et al. [56]
42
AS
CMR tagging, feature tracking
CMR
Longitudinal strain rate was impaired
in symptomatic vs. asymptomatic
patients with severe AS and pre-
served LVEF (−83.4 ± 24.8%/s and
− 106.3 ± 43.3%/s, respectively;
P = 0.048)
Musa et al. [36]
98
AS
CMR tagging
Impaired mid-LV circumferential
strain was associated with all-cause
mortality after aortic valve replace-
ment (HR 1.03; 95% CI 1.01–1.05;
p = 0.009)
Meyer et al. [58]
44
AS
Feature tracking CMR
Peak systolic LV strain of the api-
cal segments was significantly
impaired in transapical versus
transfemoral transcatheter aortic
valve replacement
Sparrow et al. [62]
8
AR
T1 mapping
Post-contrast T1 values in abnor-
mally contracting segments were
prolonged compared to controls
(532 vs. 501 ms, respectively;
p = 0.002)
de Meester de Ravenstein [63]
9
AR
ECV
ECV measured on 3T CMR was
strongly correlated with the extent
of interstitial fibrosis on histol-
ogy in patients with severe AR
(r = 0.79, p = 0.011)
Pomerantz et al. [64]
14
AR
Myocardial tagging
Global longitudinal and circumfer-
ential strain were decreased 2 years
after aortic valve replacement,
despite an improvement in LVEF
and LV size
Ungacta et al. [65]
8
AR
Myocardial tagging
Posterior wall circumferential strain
was decreased 6 months after Singh et al. [51]
174
AS
LGE
Schneeweis et al. [54], Singh et al. [55]
30, 18
AS
CMR tagging, feature tracking
CMR
Mahmod et al. [57]
39
AS
CMR tagging
Al Musa et al. [56]
42
AS
CMR tagging, feature tracking
CMR
Musa et al. [36]
98
AS
CMR tagging
Meyer et al. [58]
44
AS
Feature tracking CMR
Sparrow et al. [62]
8
AR
T1 mapping
de Meester de Ravenstein [63]
9
AR
ECV
Pomerantz et al. [64]
14
AR
Myocardial tagging
Ungacta et al. [65]
8
AR
Myocardial tagging
Edwards et al. [68]
35
MR
ECV, native T1 mapping, LGE Singh et al. [51] Patients with AS had impaired LV
strain compared to controls Longitudinal strain rate was impaired
in symptomatic vs. Aortic stenosis 8 deaths/1000 patient-years,
respectively) ECV and percent of midwall replace-
ment fibrosis (LGE) were associ-
ated with increased high-sensitivity
cardiac troponin I levels LGE and ECV were associated with
ECG strain in patients with mild to
severe AS LGE was associated with severe LV
hypertrophy LGE was associated with increased
LV mass index and LV end-dias-
tolic volume index. LGE was not
associated with the severity of AS Midwall fibrosis on LGE CMR was
associated with higher mortality
than infarct-type LGE (HR 8.59;
95% CI 1.97–37.38; p = 0.004
and HR 6.46; 95% CI 1.39-30.00;
p = 0.017, respectively) LGE was an independent predictor of
all-cause and cardiovascular mor-
tality in patients with severe AS
undergoing surgical valve replace-
ment (HR for all-cause mortality:
2.8; 95% CI 1.3–6.9; p = 0.025) The extent of LGE in patients with
symptomatic severe AS undergo-
ing aortic valve surgery correlated
with biopsy-quantified myocardial
fibrosis and remained unchanged at
9 months after surgery 1 3 Int J Cardiovasc Imaging (2018) 34:97–112 104 104
Table 2 (continued)
Study
No. of patients Valve disease CMR technique
Azevedo et al. [33]
54
AS + AR
LGE
Singh et al. [51]
174
AS
LGE
Schneeweis et al. [54], Singh et al. [55]
30, 18
AS
CMR tagging, feature tracking
CMR
Mahmod et al. [57]
39
AS
CMR tagging
Al Musa et al. [56]
42
AS
CMR tagging, feature tracking
CMR
Musa et al. [36]
98
AS
CMR tagging
Meyer et al. [58]
44
AS
Feature tracking CMR
Sparrow et al. [62]
8
AR
T1 mapping
de Meester de Ravenstein [63]
9
AR
ECV
Pomerantz et al. [64]
14
AR
Myocardial tagging
Ungacta et al. [65]
8
AR
Myocardial tagging
Edwards et al. [68]
35
MR
ECV, native T1 mapping, LGE Table 2 (continued)
Study
No. of patients Valve disease CMR technique
Main findings
Azevedo et al. [33]
54
AS + AR
LGE
LGE correlated with the extent of
fibrosis on histology (r = 0.69,
p < 0.001) and demonstrated sig-
nificant inverse correlation with the
LVEF improvement after surgery
(r=-0.47, p = 0.02)
LGE was associated with worse long-
term survival (Chi square = 5.85;
p = 0.02)
Singh et al. [51]
174
AS
LGE
Patients with asymptomatic moderate
and severe AS who presented with
valve related complications during
follow-up showed comparable
extent of LGE than patients who
remained asymptomatic
Schneeweis et al. [54], Singh et al. Aortic stenosis AS
aortic stenosis, LGE late gadolinium enhancement, ns non-signifi-
cant. Adapted with permission from Bull et al. [40] Fig. 4 Native T1 mapping in aortic stenosis. A Color maps of T1
values of mid-ventricular short-axis slices (top row) and correspond-
ing LGE images (bottom row) of normal controls and patients with
moderate and severe AS. The left column shows a normal volunteer
(T1 = 944 ms), the middle column a patient with moderate AS and
moderate left ventricular hypertrophy (T1 = 951 ms) and the right
column shows a patient with severe AS with severe left ventricular hypertrophy (T1 = 1020 ms). B Whisker-plots of myocardial T1 val-
ues of normal controls and of patients with moderate AS, asympto-
matic severe AS and symptomatic severe AS. The between-group
comparisons with the corresponding p-values are also presented. AS
aortic stenosis, LGE late gadolinium enhancement, ns non-signifi-
cant. Adapted with permission from Bull et al. [40] Fig. 4 Native T1 mapping in aortic stenosis. A Color maps of T1
values of mid-ventricular short-axis slices (top row) and correspond-
ing LGE images (bottom row) of normal controls and patients with
moderate and severe AS. The left column shows a normal volunteer
(T1 = 944 ms), the middle column a patient with moderate AS and
moderate left ventricular hypertrophy (T1 = 951 ms) and the right
column shows a patient with severe AS with severe left ventricular
hypertrophy (T1 = 1020 ms). B Whisker-plots of myocardial T1 val-
ues of normal controls and of patients with moderate AS, asympto-
matic severe AS and symptomatic severe AS. The between-group
comparisons with the corresponding p-values are also presented. AS
aortic stenosis, LGE late gadolinium enhancement, ns non-signifi-
cant. Adapted with permission from Bull et al. [40] Fig. 4 Native T1 mapping in aortic stenosis. A Color maps of T1
values of mid-ventricular short-axis slices (top row) and correspond-
ing LGE images (bottom row) of normal controls and patients with
moderate and severe AS. The left column shows a normal volunteer
(T1 = 944 ms), the middle column a patient with moderate AS and
moderate left ventricular hypertrophy (T1 = 951 ms) and the right
column shows a patient with severe AS with severe left ventricular hypertrophy (T1 = 1020 ms). B Whisker-plots of myocardial T1 val-
ues of normal controls and of patients with moderate AS, asympto-
matic severe AS and symptomatic severe AS. Aortic stenosis asymptomatic
patients with severe AS and pre-
served LVEF (−83.4 ± 24.8%/s and
− 106.3 ± 43.3%/s, respectively;
P = 0.048) Impaired mid-LV circumferential
strain was associated with all-cause
mortality after aortic valve replace-
ment (HR 1.03; 95% CI 1.01–1.05;
p = 0.009) Peak systolic LV strain of the api-
cal segments was significantly
impaired in transapical versus
transfemoral transcatheter aortic
valve replacement Post-contrast T1 values in abnor-
mally contracting segments were
prolonged compared to controls
(532 vs. 501 ms, respectively;
p = 0.002) ECV measured on 3T CMR was
strongly correlated with the extent
of interstitial fibrosis on histol-
ogy in patients with severe AR
(r = 0.79, p = 0.011) 1 105 Int J Cardiovasc Imaging (2018) 34:97–112 Table 2 (continued)
Study
No. of patients Valve disease CMR technique
Main findings
Han et al. [69]
25
MR
LGE
LGE of the papillary muscles was
present in 63% of patients with MV
prolapse
Chaikriangkrai et al. [35]
48
MR
LGE
The presence of LV LGE in chronic
severe MR was associated with
worse clinical outcomes (HR 4.8;
95% CI 1.1–20.7; p = 0.037)
Maniar et al. [70]
15
MR
CMR tagging
Patients with chronic moderate and
severe MR and preserved LVEF
had impaired septal LV strain val-
ues compared to normal controls
Mankad et al. [71]
7
MR
CMR tagging
Patients with severe MR and
preserved LVEF had reduced
circumferential strain compared
to controls (12 ± 6 vs. 21 ± 6%,
respectively; p ≤ 0.001)
Ahmed et al. [72], Schiros et al. [73], Ahmed et al. [74]
27
35
22
MR
CMR tagging
Global longitudinal and circum-
ferential strain parameters were
decreased after MV repair
AS aortic stenosis, AR aortic regurgitation, CMR cardiovascular magnetic resonance, ECV extracellular volume, HR hazard ratio, ICU intensive
care unit, iECV indexed extracellular volume, LGE late gadolinium enhancement, LV left ventricle, LVEF left ventricular ejection fraction, MR
mitral regurgitation AS aortic stenosis, AR aortic regurgitation, CMR cardiovascular magnetic resonance, ECV extracellular volume, HR hazard ratio, ICU intensive
care unit, iECV indexed extracellular volume, LGE late gadolinium enhancement, LV left ventricle, LVEF left ventricular ejection fraction, MR
mitral regurgitation hypertrophy (T1 = 1020 ms). B Whisker-plots of myocardial T1 val-
ues of normal controls and of patients with moderate AS, asympto-
matic severe AS and symptomatic severe AS. The between-group
comparisons with the corresponding p-values are also presented. Aortic stenosis There was a stepwise increase in: B severity of
valve narrowing; C degree of left ventricular (LV) hypertrophy; D
myocardial injury, assessed by high-sensitivity troponin I concentra-
tion (hsTni); E LV diastolic dysfunction; and F all-cause-mortality
with increased diffuse myocardial fibrosis and replacement fibrosis. Adapted with permission from Chin et al. [34] Fig. 5 Prognostic implications of interstitial and replacement fibro-
sis in aortic stenosis. A Patients with mild to severe aortic stenosis
were categorized into three groups based upon cardiovascular mag-
netic resonance assessments of myocardial fibrosis: normal myo-
cardium [indexed extracellular volume (iECV) < 22.5 ml/m2, no
late gadolinium enhancement (LGE)], diffuse myocardial fibrosis
(iECV ≥ 22.5 ml/m2, no LGE) and replacement fibrosis (presence of midwall LGE). There was a stepwise increase in: B severity of
valve narrowing; C degree of left ventricular (LV) hypertrophy; D
myocardial injury, assessed by high-sensitivity troponin I concentra-
tion (hsTni); E LV diastolic dysfunction; and F all-cause-mortality
with increased diffuse myocardial fibrosis and replacement fibrosis. Adapted with permission from Chin et al. [34] aortic valve replacement [57]. Similarly, LV circumferen-
tial strain by CMR tagging was significantly associated
with all-cause mortality in 98 severe AS patients under-
going surgical and transcatheter aortic valve replacement
(HR per each 1% deterioration of circumferential strain:
1.03; 95% CI 1.01–1.05; p = 0.009) [36]. Furthermore, the
effect of procedural access (transfemoral vs. transapical)
on LV mechanics was studied with CMR feature track-
ing in 44 patients undergoing transcatheter aortic valve
replacement [58]. The transapical approach was associ-
ated with impaired peak systolic longitudinal strain of the
apical segments as compared to the transfemoral approach
(−8.9 ± 5.3 vs. −16.9 ± 4.3%, respectively; p < 0.001),
while there were no differences in LV ejection fraction and
peak systolic longitudinal strain of the basal and midven-
tricular segments between both approaches (Fig. 7). Interstitial and replacement myocardial fibrosis lead
to impaired LV myocardial deformation which can be
detected with strain imaging. Myocardial tagging and fea-
ture tracking CMR demonstrated that global as well as
regional LV strains were significantly correlated with LGE
extent in patients with hypertrophic cardiomyopathy, who
exhibit a similar pattern of midwall fibrosis to patients with
AS [52, 53]: global and regional LV strain values impair as
LGE increases. Aortic stenosis The between-group
comparisons with the corresponding p-values are also presented. AS
aortic stenosis, LGE late gadolinium enhancement, ns non-signifi-
cant. Adapted with permission from Bull et al. [40] Heart Association functional class and worse survival
(Fig. 6) [32, 33, 49]. However, detection of LV myocar-
dial fibrosis in patients with asymptomatic moderate and
severe AS seems insufficient to identify the patients who
will present valve related complications. In the prog-
nostic importance of microvascular dysfunction in aor-
tic stenosis (PRIMID AS) study, including 174 patients with asymptomatic moderate to severe AS, the group of
patients who presented with cardiovascular death, major
adverse cardiovascular events and development of typi-
cal AS symptoms, necessitating referral for aortic valve
replacement, showed comparable extent of LGE than
patients who remained asymptomatic or free of valve
related complications during follow-up [51]. Heart Association functional class and worse survival
(Fig. 6) [32, 33, 49]. However, detection of LV myocar-
dial fibrosis in patients with asymptomatic moderate and
severe AS seems insufficient to identify the patients who
will present valve related complications. In the prog-
nostic importance of microvascular dysfunction in aor-
tic stenosis (PRIMID AS) study, including 174 patients 1 3 Int J Cardiovasc Imaging (2018) 34:97–112 106 Fig. 5 Prognostic implications of interstitial and replacement fibro-
sis in aortic stenosis. A Patients with mild to severe aortic stenosis
were categorized into three groups based upon cardiovascular mag-
netic resonance assessments of myocardial fibrosis: normal myo-
cardium [indexed extracellular volume (iECV) < 22.5 ml/m2, no
late gadolinium enhancement (LGE)], diffuse myocardial fibrosis
(iECV ≥ 22.5 ml/m2, no LGE) and replacement fibrosis (presence
of midwall LGE). There was a stepwise increase in: B severity of
valve narrowing; C degree of left ventricular (LV) hypertrophy; D
myocardial injury, assessed by high-sensitivity troponin I concentra-
tion (hsTni); E LV diastolic dysfunction; and F all-cause-mortality
with increased diffuse myocardial fibrosis and replacement fibrosis. Adapted with permission from Chin et al. [34] Fig. 5 Prognostic implications of interstitial and replacement fibro-
sis in aortic stenosis. A Patients with mild to severe aortic stenosis
were categorized into three groups based upon cardiovascular mag-
netic resonance assessments of myocardial fibrosis: normal myo-
cardium [indexed extracellular volume (iECV) < 22.5 ml/m2, no
late gadolinium enhancement (LGE)], diffuse myocardial fibrosis
(iECV ≥ 22.5 ml/m2, no LGE) and replacement fibrosis (presence of midwall LGE). Aortic stenosis Head-to-head comparisons between tagged
and feature tracking CMR in moderate to severe AS have
shown reasonable agreement for LV strain measurement,
albeit feature tracking provided systematically higher val-
ues than CMR tagging [54, 55]. The correlation between
CMR LV circumferential and longitudinal strain and strain
rate and symptomatic status of patients with severe AS and
preserved LV ejection fraction was demonstrated by Al
Musa et al. [56]. LV longitudinal strain rate was the most
sensitive parameter to discriminate between asymptomatic
versus symptomatic patients (−106.3 ± 43.3%/s in patients
with “no/mild” symptoms vs. −83.4 ± 24.8%/s in moder-
ate and severely symptomatic patients; P = 0.048). The
association between LV myocardial strain and outcomes
after surgical or transcatheter treatment was demonstrated
in two studies [36, 57]. Mahmod and coworkers showed
that global LV circumferential, but not longitudinal strain
measured on CMR significantly improved at 8 months after Aortic regurgitation compared myocardial T1 values meas-
ured with a modified Look-Locker technique before and
after gadolinium contrast in eight patients with severe AR
and 15 normal controls [62]. Patients with AR had signif-
icantly prolonged post-contrast T1 values in abnormally
contracting segments compared to the controls (532 vs. 501 ms, respectively; p = 0.002), suggesting increased
interstitial fibrosis. Furthermore, in nine patients with
severe AR who underwent surgical aortic valve replace-
ment, ECV measured on 3T CMR was strongly corre-
lated with the extent of interstitial fibrosis on histology
(r = 0.79, p = 0.011) [63]. Replacement fibrosis has been
also described in 26 patients with severe AR by Azevedo
et al. [33]. The authors reported a 69% prevalence of
LGE, mostly following a multifocal pattern. The corre-
lation between myocardial replacement fibrosis assessed
with LGE and histopathology was good (r = 0.70, p < 0.001). Moreover, in a combined cohort of 26 patients
with severe AR and 28 patients with severe AS, the
amount of myocardial fibrosis was inversely correlated
with LV functional improvement (r = −0.47; p = 0.02) and
was associated with worse long-term survival after aortic
valve replacement surgery (Chi square = 5.85; p = 0.02)
(Fig. 6) [33]. Furthermore, in 14 patients with chronic
severe AR, myocardial CMR tagging showed an impair-
ment in global longitudinal and circumferential strain at
2 years after aortic valve replacement (p < 0.03 for both),
despite an improvement in LV ejection fraction and a
decrease in LV size (Fig. 8) [64]. Similarly, Ungacta et al. showed a decrease in posterior wall circumferential strain
in patients with AR 6 months after valve replacement
[65]. These findings suggest that the presence of LV myo-
cardial fibrosis in patients with AR is a marker of adverse
remodeling that may lead to further deterioration in LV
strain and poor prognosis after aortic valve surgery. also evaluated myocardial fibrosis with CMR [33, 62,
63]. Sparrow et al. compared myocardial T1 values meas-
ured with a modified Look-Locker technique before and
after gadolinium contrast in eight patients with severe AR
and 15 normal controls [62]. Patients with AR had signif-
icantly prolonged post-contrast T1 values in abnormally
contracting segments compared to the controls (532 vs. 501 ms, respectively; p = 0.002), suggesting increased
interstitial fibrosis. Aortic regurgitation In aortic regurgitation (AR), pressure and volume over-
load induce growth of cardiomyocytes with addition of
new sarcomeres in series and interstitial fibrosis, charac-
terized by increased fibronectin and non-collagen com-
ponents [59]. Several clinical studies have histologically
proven pronounced myocardial fibrosis in severe AR at
the time of valve surgery [37, 60, 61]. A few studies have 1 3 3 Int J Cardiovasc Imaging (2018) 34:97–112 107 Fig. 6 Prognostic implications of late gadolinium enhancement
(LGE) cardiovascular magnetic resonance (CMR) in patients with
severe aortic stenosis and aortic regurgitation after aortic valve
replacement surgery. Linear regression graphs illustrate the inverse
relationship between the degree of left ventricular ejection fraction
improvement and the amount of myocardial fibrosis by histopathol-
ogy (A) and by LGE CMR (B). The Kaplan–Meier graphs demon-
strate significantly worse survival after aortic valve replacement in
patients with larger myocardial fibrosis assessed by histopathology
(C) or LGE (D). Reproduced with permission from Azevedo et al. [33] ce-MRI contrast-enhanced magnetic resonance imaging, EF
ejection fraction, MF myocardial fibrosis ogy (A) and by LGE CMR (B). The Kaplan–Meier graphs demon-
strate significantly worse survival after aortic valve replacement in
patients with larger myocardial fibrosis assessed by histopathology
(C) or LGE (D). Reproduced with permission from Azevedo et al. [33] ce-MRI contrast-enhanced magnetic resonance imaging, EF
ejection fraction, MF myocardial fibrosis ogy (A) and by LGE CMR (B). The Kaplan–Meier graphs demon-
strate significantly worse survival after aortic valve replacement in
patients with larger myocardial fibrosis assessed by histopathology
(C) or LGE (D). Reproduced with permission from Azevedo et al. [33] ce-MRI contrast-enhanced magnetic resonance imaging, EF
ejection fraction, MF myocardial fibrosis Fig. 6 Prognostic implications of late gadolinium enhancement
(LGE) cardiovascular magnetic resonance (CMR) in patients with
severe aortic stenosis and aortic regurgitation after aortic valve
replacement surgery. Linear regression graphs illustrate the inverse
relationship between the degree of left ventricular ejection fraction
improvement and the amount of myocardial fibrosis by histopathol- ogy (A) and by LGE CMR (B). The Kaplan–Meier graphs demon-
strate significantly worse survival after aortic valve replacement in
patients with larger myocardial fibrosis assessed by histopathology
(C) or LGE (D). Reproduced with permission from Azevedo et al. [33] ce-MRI contrast-enhanced magnetic resonance imaging, EF
ejection fraction, MF myocardial fibrosis also evaluated myocardial fibrosis with CMR [33, 62,
63]. Sparrow et al. Aortic regurgitation Furthermore, in nine patients with
severe AR who underwent surgical aortic valve replace-
ment, ECV measured on 3T CMR was strongly corre-
lated with the extent of interstitial fibrosis on histology
(r = 0.79, p = 0.011) [63]. Replacement fibrosis has been
also described in 26 patients with severe AR by Azevedo
et al. [33]. The authors reported a 69% prevalence of
LGE, mostly following a multifocal pattern. The corre-
lation between myocardial replacement fibrosis assessed
with LGE and histopathology was good (r = 0.70, 1 3 3 108 Int J Cardiovasc Imaging (2018) 34:97–112 Fig. 7 The impact of tran-
scatheter aortic valve implan-
tation on the left ventricular
(LV) mechanics, assessed
with feature tracking cardio-
vascular magnetic resonance
(CMR). A Systolic CMR cine
frames derived from four- (top
row), three- (middle row), and
two-chamber (bottom row) LV
views of a patient before and
after transfemoral (TF) access
(left two columns) as well as
from a patient before and after
transapical (TA) access (right
two columns). The green arrows
represent velocity vectors illus-
trating systolic inward motion. The TA transcatheter aortic
valve implantation (TAVI)
patient shows reduced systolic
deformation of the apical LV
segments 3 months after the
procedure. B Average peak
systolic radial strain values of
49 analyzed segments obtained
from all TF-TAVI patients (blue
line) and all TA-TAVI patients
(red line). The apical segments
are displayed in the middle,
while the basal segments are
displayed on the left and on the
right side of the graph. There is
a reduction in peak radial strain
of the apical segments after TA-
TAVI. Adapted with permission
from Meyer et al. [58] while the basal segments are
displayed on the left and on the
right side of the graph. There is
a reduction in peak radial strain
of the apical segments after TA-
TAVI. Adapted with permission
from Meyer et al. [58] Mitral regurgitation A tagging pattern in the form
of parallel lines was used for the long-axis cines and a grid pattern for
the short-axis cines. Dedicated software was employed for the myo-
cardial deformation analysis. C At an average of 28 ± 11 months after
aortic valve replacement global and regional LV longitudinal and cir-
cumferential strain decreased (p < 0.05 for both global strain values)
despite an improvement in LV ejection fraction and a decrease in LV
size, which might imply an ongoing myocardial fibrosis after valve
surgery. Adapted with permission from Pomerantz et al. [64]. AI aor-
tic insufficiency, Ant anterior, Lat lateral, Post posterior, preop preop-
erative, postop postoperative, Sept septal
Fig. 9 Cardiovascular magnetic resonance (CMR) myocardial fibro-
sis assessment in primary degenerative mitral regurgitation (MR). A
Late gadolinium enhanced CMR images (top) and native T1 maps
(bottom) in patients with MR. The arrows indicate the presence of
midwall replacement fibrosis in the inferolateral wall. The native T1
values were increased in corresponding areas (Hash 1045 ms and
Asterisk 1102 ms). B Left ventricular fibrosis demonstrated on his-
tology: replacement fibrosis can be well-delineated (upper plot) or
patchy (lower plot). C Individual patient data presented in the scatter
plot demonstrate a wide overlap of the extracellular volume (ECV)
values in patients with MR and controls. However, the mean and the
standard error of the mean (error bars) were significantly larger in
patients with MR as compared to the controls. Adapted with permis-
sion from Edwards et al. [68] Fig. 8 CMR tagging in patients with chronic severe aortic regurgita-
tion. Left ventricular (LV) long-axis (top row) and short-axis (bottom
row) cardiovascular magnetic resonance (CMR) tagging images at
end-diastole (A) and at end-systole (B). A tagging pattern in the form
of parallel lines was used for the long-axis cines and a grid pattern for
the short-axis cines. Dedicated software was employed for the myo-
cardial deformation analysis. C At an average of 28 ± 11 months after
aortic valve replacement global and regional LV longitudinal and cir-
cumferential strain decreased (p < 0.05 for both global strain values)
despite an improvement in LV ejection fraction and a decrease in LV
size, which might imply an ongoing myocardial fibrosis after valve
surgery. Adapted with permission from Pomerantz et al. [64]. Mitral regurgitation AI aor-
tic insufficiency, Ant anterior, Lat lateral, Post posterior, preop preop-
erative, postop postoperative, Sept septal aortic valve replacement global and regional LV longitudinal and cir-
cumferential strain decreased (p < 0.05 for both global strain values)
despite an improvement in LV ejection fraction and a decrease in LV
size, which might imply an ongoing myocardial fibrosis after valve
surgery. Adapted with permission from Pomerantz et al. [64]. AI aor-
tic insufficiency, Ant anterior, Lat lateral, Post posterior, preop preop-
erative, postop postoperative, Sept septal Fig. 8 CMR tagging in patients with chronic severe aortic regurgita-
tion. Left ventricular (LV) long-axis (top row) and short-axis (bottom
row) cardiovascular magnetic resonance (CMR) tagging images at
end-diastole (A) and at end-systole (B). A tagging pattern in the form
of parallel lines was used for the long-axis cines and a grid pattern for
the short-axis cines. Dedicated software was employed for the myo-
cardial deformation analysis. C At an average of 28 ± 11 months after Fig. 9 Cardiovascular magnetic resonance (CMR) myocardial fibro-
sis assessment in primary degenerative mitral regurgitation (MR). A
Late gadolinium enhanced CMR images (top) and native T1 maps
(bottom) in patients with MR. The arrows indicate the presence of
midwall replacement fibrosis in the inferolateral wall. The native T1
values were increased in corresponding areas (Hash 1045 ms and
Asterisk 1102 ms). B Left ventricular fibrosis demonstrated on his-
tology: replacement fibrosis can be well-delineated (upper plot) or
patchy (lower plot). C Individual patient data presented in the scatter
plot demonstrate a wide overlap of the extracellular volume (ECV)
values in patients with MR and controls. However, the mean and the
standard error of the mean (error bars) were significantly larger in
patients with MR as compared to the controls. Adapted with permis-
sion from Edwards et al. [68] Fig. 9 Cardiovascular magnetic resonance (CMR) myocardial fibro-
sis assessment in primary degenerative mitral regurgitation (MR). A
Late gadolinium enhanced CMR images (top) and native T1 maps
(bottom) in patients with MR. The arrows indicate the presence of
midwall replacement fibrosis in the inferolateral wall. The native T1
values were increased in corresponding areas (Hash 1045 ms and
Asterisk 1102 ms). B Left ventricular fibrosis demonstrated on his- tology: replacement fibrosis can be well-delineated (upper plot) or
patchy (lower plot). Mitral regurgitation exhibited a non-infarct LGE pattern on CMR. Patients who
had non-infarct type LGE presented with higher ECV val-
ues compared to MR patients without LGE (0.35 ± 0.02 vs. 0.27 ± 0.03, p < 0.01). The ECV values correlated with LV
end-systolic volume, measures of systolic and diastolic LV
dysfunction as well as with peak oxygen consumption on
treadmill testing. The distribution of LGE in patients with
MR varies significantly. Han et al. demonstrated the pres-
ence of LGE of the papillary muscles in 63% of patients
with MV prolapse [69] whereas Chaikriangkrai et al. observed LV replacement fibrosis in 40% of patients with
chronic severe MR [35]. The presence of LV LGE was
associated with worse clinical outcomes in terms of inten-
sive care unit readmission, incidence of permanent pace-
maker implantation and rehospitalization (HR 4.775; 95%
CI 1.100–20.729; p = 0.037) [35]. Mitral regurgitation (MR) is a heterogeneous disease,
broadly classified as organic (primary) or functional (sec-
ondary) based on the underlying mechanism. Organic MR
is due to intrinsic valvular disease whereas functional MR
is caused by regional and/or global LV remodeling without
structural abnormalities of the mitral valve [66]. Degenera-
tive mitral valve disease (myxomatous disease and fibroe-
lastic deficiency) is the most frequent etiology of primary
MR in developed countries. The indication for mitral valve
repair/replacement is determined by the presence of symp-
toms or LV function deterioration and LV remodeling [1,
3]. However, LV remodeling and myocardial fibrosis may
occur before the development of symptoms. Chronic LV
volume overload associated with MR leads to myocardial
hypertrophy and increased interstitial fibrosis [67]. In 35
asymptomatic patients with moderate to severe primary
MR, Edwards et al. demonstrated higher ECV on CMR as
compared to controls (0.32 ± 0.07 vs. 0.25 ± 0.02, p < 0.01)
(Fig. 9) [68]. Furthermore, 31% of patients with MR These structural changes of the LV myocardium may
be associated with subtle functional abnormalities. In 15
patients with chronic moderate and severe MR and pre-
served LV ejection fraction who underwent CMR with
tissue tagging, Maniar et al. demonstrated preserved 1 3 109 Int J Cardiovasc Imaging (2018) 34:97–112 g g
Fig. 8 CMR tagging in patients with chronic severe aortic regurgita-
tion. Left ventricular (LV) long-axis (top row) and short-axis (bottom
row) cardiovascular magnetic resonance (CMR) tagging images at
end-diastole (A) and at end-systole (B). Future perspectives Tissue characterization and strain imaging with CMR have
provided new insights into the pathophysiology of VHD. Current guidelines recommend valve surgery in severe
symptomatic VHD or when LV function decreases [1, 3]. However, early detection of LV structural and functional
changes may help to identify patients who may benefit
from early surgery. It is conceivable that early relief of the
pressure or volume overload would result in less damage
to the LV and better outcome at follow-up. However, there
are currently no prospective data to evaluate whether early
surgical valve treatment results in better prognosis in VHD. It may be challenging as well to define the cut-off values of
ECV, T1 times, LGE and LV myocardial strains for thera-
peutic intervention. Standardization in data acquisition and
analysis are important issues to be resolved. J Am Coll Cardiol 63:e57–e185. doi:10.1016/j.jacc.2014.02.536 J Am Coll Cardiol 63:e57–e185. doi:10.1016/j.jacc.2014.02.536 j j
4. Enriquez-Sarano M, Sundt TM, 3rd (2010) Early surgery is rec-
ommended for mitral regurgitation. Circulation 121:804–811. doi:10.1161/CIRCULATIONAHA.109.868083 (discussion 812) 4. Enriquez-Sarano M, Sundt TM, 3rd (2010) Early surgery is rec-
ommended for mitral regurgitation. Circulation 121:804–811. doi:10.1161/CIRCULATIONAHA.109.868083 (discussion 812) 5. Villari B, Campbell SE, Hess OM, Mall G, Vassalli G, Weber
KT, Krayenbuehl HP (1993) Influence of collagen network on
left ventricular systolic and diastolic function in aortic valve dis-
ease. J Am Coll Cardiol 22:1477–1484 6. Mewton N, Liu CY, Croisille P, Bluemke D, Lima JA (2011)
Assessment of myocardial fibrosis with cardiovascular mag-
netic resonance. J Am Coll Cardiol 57:891–903. doi:10.1016/j. jacc.2010.11.013 7. Taylor AJ, Salerno M, Dharmakumar R, Jerosch-Herold M
(2016) T1 mapping: basic techniques and clinical applica-
tions. JACC Cardiovasc Imaging 9:67–81. doi:10.1016/j. jcmg.2015.11.005 8. Flett AS, Hayward MP, Ashworth MT et al (2010) Equi-
librium contrast cardiovascular magnetic resonance for the
measurement of diffuse myocardial fibrosis: preliminary vali-
dation in humans. Circulation 122:138–144. doi:10.1161/
circulationaha.109.930636 The early valve replacement guided by biomarkers of
left ventricular decompensation in asymptomatic patients
with advanced aortic stenosis (EVOLVED) is the first
multicenter randomized controlled clinical trial that
will investigate whether the early valve intervention in
patients with asymptomatic severe AS and midwall fibro-
sis on CMR improves patients’ clinical outcomes com-
pared to the standard care (NCT03094143). The results
of this study may have an impact on future guidelines and
recommendations on treatment of VHD. 9. References 1. Joint Task Force on the Management of Valvular Heart Disease
of the European Society of Cardiology, European Association for
Cardio-Thoracic Surgery, Vahanian A et al. (2012) Guidelines
on the management of valvular heart disease (version 2012). Eur
Heart J 33:2451–2496. doi:10.1093/eurheartj/ehs109 2. Nkomo VT, Gardin JM, Skelton TN, Gottdiener JS, Scott CG,
Enriquez-Sarano M (2006) Burden of valvular heart diseases: a
population-based study. Lancet 368:1005–1011. doi:10.1016/
S0140-6736(06)69208-8 3. Nishimura RA, Otto CM, Bonow RO et al (2014) 2014 AHA/
ACC guideline for the management of patients with valvular
heart disease: a report of the American College of Cardiology/
American Heart Association Task Force on Practice Guidelines. ll
di l 6
5
5 d i
6 j j
5 6 Future perspectives Miller CA, Naish JH, Bishop P et al (2013) Comprehensive vali-
dation of cardiovascular magnetic resonance techniques for the
assessment of myocardial extracellular volume. Circ Cardiovasc
Imaging 6:373–383. doi:10.1161/circimaging.112.000192 10. Fontana M, White SK, Banypersad SM et al (2012) Comparison
of T1 mapping techniques for ECV quantification. Histological
validation and reproducibility of ShMOLLI versus multibreath-
hold T1 quantification equilibrium contrast CMR. J Cardiovasc
Magn Reson 14:88. doi:10.1186/1532-429x-14-88 g
11. Bulluck H, Maestrini V, Rosmini S et al (2015) Myocardial T1
mapping. Circ J 79:487–494. doi:10.1253/circj.CJ-15-0054 Mitral regurgitation Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted
use, distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
made. Mitral regurgitation C Individual patient data presented in the scatter
plot demonstrate a wide overlap of the extracellular volume (ECV)
values in patients with MR and controls. However, the mean and the
standard error of the mean (error bars) were significantly larger in
patients with MR as compared to the controls. Adapted with permis-
sion from Edwards et al. [68] 1 3 1 3 110 Int J Cardiovasc Imaging (2018) 34:97–112 global longitudinal and circumferential strain but abnor-
mal regional strain values: the septal LV segments exhib-
ited impaired strain whereas the lateral segments showed
compensatory hyper-contractility [70]. Similarly, Mankad
et al. showed with CMR tagging abnormal regional
strain patterns in patients with severe MR and preserved
LV ejection fraction: while radial strain was increased
(19 ± 9 vs. 16 ± 6%, p = 0.003), circumferential strain
was reduced (12 ± 6 vs. 21 ± 6%, p ≤ 0.001) as compared
to healthy controls [71]. Several authors have demon-
strated a decrease in global longitudinal and circumfer-
ential strain parameters on CMR tagging in patients with
severe degenerative MR after mitral valve repair, which
might imply an ongoing myocardial fibrosis after surgery
[72–74]. global longitudinal and circumferential strain but abnor-
mal regional strain values: the septal LV segments exhib-
ited impaired strain whereas the lateral segments showed
compensatory hyper-contractility [70]. Similarly, Mankad
et al. showed with CMR tagging abnormal regional
strain patterns in patients with severe MR and preserved
LV ejection fraction: while radial strain was increased
(19 ± 9 vs. 16 ± 6%, p = 0.003), circumferential strain
was reduced (12 ± 6 vs. 21 ± 6%, p ≤ 0.001) as compared
to healthy controls [71]. Several authors have demon-
strated a decrease in global longitudinal and circumfer-
ential strain parameters on CMR tagging in patients with
severe degenerative MR after mitral valve repair, which
might imply an ongoing myocardial fibrosis after surgery
[72–74]. Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted
use, distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
made. Compliance with ethical standards 12. Moon JC, Messroghli DR, Kellman P et al (2013) Myocar-
dial T1 mapping and extracellular volume quantification: a
Society for Cardiovascular Magnetic Resonance (SCMR) and
CMR Working Group of the European Society of Cardiol-
ogy consensus statement. J Cardiovasc Magn Reson 15:92. doi:10.1186/1532-429x-15-92 Conflict of interest The Department of Cardiology of the Leiden
University Medical Center receives research grants from Biotronik,
Boston Scientific, Medtronic, Edwards Lifesciences. Tomaz Podlesni-
kar has been awarded with a training grant of the European Society
of Cardiology. Victoria Delgado received speaker fees from Abbott
Vascular. Jeroen J Bax has no disclosures. 13. Croisille P, Revel D, Saeed M (2006) Contrast agents and cardiac
MR imaging of myocardial ischemia: from bench to bedside. Eur
Radiol 16:1951–1963. doi:10.1007/s00330-006-0244-z 1 3 111 Int J Cardiovasc Imaging (2018) 34:97–112 an intermodality comparison. Echocardiography 30:203–210. doi:10.1111/echo.12016 14. Kim RJ, Wu E, Rafael A et al (2000) The use of contrast-
enhanced magnetic resonance imaging to identify revers-
ible myocardial dysfunction. N Engl J Med 343:1445–1453. doi:10.1056/nejm200011163432003 30. Morton G, Schuster A, Jogiya R, Kutty S, Beerbaum P, Nagel
E (2012) Inter-study reproducibility of cardiovascular magnetic
resonance myocardial feature tracking. J Cardiovasc Magn Reson
14:43. doi:10.1186/1532-429x-14-43 j
15. Wu E, Ortiz JT, Tejedor P et al (2008) Infarct size by contrast
enhanced cardiac magnetic resonance is a stronger predictor of
outcomes than left ventricular ejection fraction or end-systolic
volume index: prospective cohort study. Heart 94:730–736. doi:10.1136/hrt.2007.122622 31. Augustine D, Lewandowski AJ, Lazdam M et al (2013) Global
and regional left ventricular myocardial deformation measures
by magnetic resonance feature tracking in healthy volunteers:
comparison with tagging and relevance of gender. J Cardiovasc
Magn Reson 15:8. doi:10.1186/1532-429x-15-8 16. Kwong RY, Chan AK, Brown KA, Chan CW, Reynolds HG,
Tsang S, Davis RB (2006) Impact of unrecognized myocar-
dial scar detected by cardiac magnetic resonance imaging on
event-free survival in patients presenting with signs or symp-
toms of coronary artery disease. Circulation 113:2733–2743. doi:10.1161/CIRCULATIONAHA.105.570648 32. Barone-Rochette G, Pierard S, De Meester de Ravenstein C et al
(2014) Prognostic significance of LGE by CMR in aortic steno-
sis patients undergoing valve replacement. J Am Coll Cardiol
64:144–154. doi:10.1016/j.jacc.2014.02.612 17. Assomull RG, Prasad SK, Lyne J et al (2006) Cardiovascular
magnetic resonance, fibrosis, and prognosis in dilated cardio-
myopathy. J Am Coll Cardiol 48:1977–1985. doi:10.1016/j. jacc.2006.07.049 33. Compliance with ethical standards Azevedo CF, Nigri M, Higuchi ML et al (2010) Prognostic sig-
nificance of myocardial fibrosis quantification by histopathology
and magnetic resonance imaging in patients with severe aortic
valve disease. J Am Coll Cardiol 56:278–287. doi:10.1016/j. jacc.2009.12.074 18. O’Hanlon R, Grasso A, Roughton M et al (2010) Prognos-
tic significance of myocardial fibrosis in hypertrophic car-
diomyopathy. J Am Coll Cardiol 56:867–874. doi:10.1016/j. jacc.2010.05.010i 34. Chin CW, Everett RJ, Kwiecinski J et al (2016) Myocardial
fibrosis and cardiac decompensation in aortic stenosis. JACC
Cardiovasc Imaging. doi:10.1016/j.jcmg.2016.10.007 19. Dweck MR, Joshi S, Murigu T et al (2011) Midwall fibrosis
is an independent predictor of mortality in patients with aor-
tic stenosis. J Am Coll Cardiol 58:1271–1279. doi:10.1016/j. jacc.2011.03.064 35. Chaikriangkrai K, Lopez-Mattei JC, Lawrie G et al (2014) Prog-
nostic value of delayed enhancement cardiac magnetic resonance
imaging in mitral valve repair. Ann Thorac Surg 98:1557–1563. doi:10.1016/j.athoracsur.2014.06.049 20. Helm PA, Caravan P, French BA et al (2008) Postinfarction
myocardial scarring in mice: molecular MR imaging with use
of a collagen-targeting contrast agent. Radiology 247:788–796. doi:10.1148/radiol.2473070975 36. Musa TA, Uddin A, Swoboda PP et al (2017) Cardiovascu-
lar magnetic resonance evaluation of symptomatic severe aor-
tic stenosis: association of circumferential myocardial strain
and mortality. J Cardiovasc Magn Reson 19:13. doi:10.1186/
s12968-017-0329-7 21. Spuentrup E, Ruhl KM, Botnar RM et al (2009) Molecular mag-
netic resonance imaging of myocardial perfusion with EP-3600,
a collagen-specific contrast agent: initial feasibility study in
a swine model. Circulation 119:1768–1775. doi:10.1161/
circulationaha.108.826388 37. Krayenbuehl HP, Hess OM, Monrad ES, Schneider J, Mall G,
Turina M (1989) Left ventricular myocardial structure in aortic
valve disease before, intermediate, and late after aortic valve
replacement. Circulation 79:744–755 22. Protti A, Lavin B, Dong X et al (2015) Assessment of myocar-
dial remodeling using an elastin/tropoelastin specific agent with
high field magnetic resonance imaging (MRI). J Am Heart Assoc
4:e001851. doi:10.1161/jaha.115.001851 38. Hein S, Arnon E, Kostin S et al (2003) Progression from com-
pensated hypertrophy to failure in the pressure-overloaded
human heart: structural deterioration and compensatory mecha-
nisms. Circulation 107:984–991 23. Jivraj N, Phinikaridou A, Shah AM, Botnar RM (2014) Molecu-
lar imaging of myocardial infarction. Basic Res Cardiol 109:397. doi:10.1007/s00395-013-0397-2 39. Rakusan K, Flanagan MF, Geva T, Southern J, Van Praagh R
(1992) Morphometry of human coronary capillaries during nor-
mal growth and the effect of age in left ventricular pressure-over-
load hypertrophy. Circulation 86:38–46 24. Compliance with ethical standards Mannacio V, Guadagno E, Mannacio L et al (2015) Comparison
of left ventricular myocardial structure and function in patients
with aortic stenosis and those with pure aortic regurgitation. Car-
diology 132:111–118. doi:10.1159/000431283 j
46. Shah AS, Chin CW, Vassiliou V et al (2014) Left ventricu-
lar hypertrophy with strain and aortic stenosis. Circulation
130:1607–1616. doi:10.1161/circulationaha.114.011085 62. Sparrow P, Messroghli DR, Reid S, Ridgway JP, Bainbridge G,
Sivananthan MU (2006) Myocardial T1 mapping for detection
of left ventricular myocardial fibrosis in chronic aortic regur-
gitation: pilot study. AJR Am J Roentgenol 187:W630–W635. doi:10.2214/ajr.05.1264 47. Debl K, Djavidani B, Buchner S et al (2006) Delayed hyperen-
hancement in magnetic resonance imaging of left ventricular
hypertrophy caused by aortic stenosis and hypertrophic cardio-
myopathy: visualisation of focal fibrosis. Heart 92:1447–1451. doi:10.1136/hrt.2005.079392 63. de Meester de Ravenstein C, Bouzin C, Lazam S et al (2015)
Histological Validation of measurement of diffuse interstitial
myocardial fibrosis by myocardial extravascular volume frac-
tion from modified look-locker imaging (MOLLI) T1 map-
ping at 3 T. J Cardiovasc Magn Reson 17:48. doi:10.1186/
s12968-015-0150-0 48. Rudolph A, Abdel-Aty H, Bohl S, Boye P, Zagrosek A, Dietz R,
Schulz-Menger J (2009) Noninvasive detection of fibrosis apply-
ing contrast-enhanced cardiac magnetic resonance in different
forms of left ventricular hypertrophy relation to remodeling. J
Am Coll Cardiol 53:284–291. doi:10.1016/j.jacc.2008.08.064 64. Pomerantz BJ, Wollmuth JR, Krock MD et al (2005) Myocar-
dial systolic strain is decreased after aortic valve replacement
in patients with aortic insufficiency. Ann Thorac Surg 80:2186–
2192. doi:10.1016/j.athoracsur.2005.05.095 j j
49. Weidemann F, Herrmann S, Stork S et al (2009) Impact
of myocardial fibrosis in patients with symptomatic severe
aortic
stenosis. Circulation
120:577–584. doi:10.1161/
circulationaha.108.847772i 65. Ungacta FF, Davila-Roman VG, Moulton MJ et al (1998) MRI-
radiofrequency tissue tagging in patients with aortic insuffi-
ciency before and after operation. Ann Thorac Surg 65:943–950 50. Dweck MR, Boon NA, Newby DE (2012) Calcific aortic steno-
sis: a disease of the valve and the myocardium. J Am Coll Car-
diol 60:1854–1863. doi:10.1016/j.jacc.2012.02.093 66. Lancellotti P, Moura L, Pierard LA et al (2010) European asso-
ciation of echocardiography recommendations for the assess-
ment of valvular regurgitation. Part 2: mitral and tricuspid regur-
gitation (native valve disease). Eur J Echocardiogr 11:307–332. doi:10.1093/ejechocard/jeq031 j j
51. Compliance with ethical standards Singh A, Greenwood JP, Berry C et al (2017) Comparison
of exercise testing and CMR measured myocardial perfusion
reserve for predicting outcome in asymptomatic aortic stenosis:
the prognostic importance of microvascular dysfunction in aortic
stenosis (PRIMID AS) Study. Eur Heart J. doi:10.1093/eurheartj/
ehx001 67. Fuster V, Danielson MA, Robb RA, Broadbent JC, Brown AL
Jr, Elveback LR (1977) Quantitation of left ventricular myocar-
dial fiber hypertrophy and interstitial tissue in human hearts with
chronically increased volume and pressure overload. Circulation
55:504–508 52. Kim YJ, Choi BW, Hur J et al (2008) Delayed enhancement in
hypertrophic cardiomyopathy: comparison with myocardial tag-
ging MRI. J Magn Reson Imaging 27:1054–1060. doi:10.1002/
jmri.21366 68. Edwards NC, Moody WE, Yuan M, Weale P, Neal D, Townend
JN, Steeds RP (2014) Quantification of left ventricular intersti-
tial fibrosis in asymptomatic chronic primary degenerative mitral
regurgitation. Circ Cardiovasc Imaging 7:946–953. doi:10.1161/
CIRCIMAGING.114.002397 53. Bogarapu S, Puchalski MD, Everitt MD, Williams RV, Weng
HY, Menon SC (2016) Novel cardiac magnetic resonance feature
tracking (CMR-FT) analysis for detection of myocardial fibro-
sis in pediatric hypertrophic cardiomyopathy. Pediatr Cardiol
37:663–673. doi:10.1007/s00246-015-1329-8 69. Han Y, Peters DC, Salton CJ et al (2008) Cardiovascular mag-
netic resonance characterization of mitral valve prolapse. JACC
Cardiovasc Imaging 1:294–303. doi:10.1016/j.jcmg.2008.01.013 54. Schneeweis C, Lapinskas T, Schnackenburg B et al (2014) Com-
parison of myocardial tagging and feature tracking in patients
with severe aortic stenosis. J Heart Valve Dis 23:432–440 70. Maniar HS, Brady BD, Lee U, Cupps BP, Kar J, Wallace KM,
Pasque MK (2014) Early left ventricular regional contractile
impairment in chronic mitral regurgitation occurs in a consist-
ent, heterogeneous pattern. J Thorac Cardiovasc Surg 148:1694–
1699. doi:10.1016/j.jtcvs.2014.05.088 55. Singh A, Steadman CD, Khan JN et al (2015) Intertechnique
agreement and interstudy reproducibility of strain and diastolic
strain rate at 1.5 and 3 tesla: a comparison of feature-tracking
and tagging in patients with aortic stenosis. J Magn Reson Imag-
ing 41:1129–1137. doi:10.1002/jmri.24625 71. Mankad R, McCreery CJ, Rogers WJ Jr, Weichmann RJ, Savage
EB, Reichek N, Kramer CM (2001) Regional myocardial strain
before and after mitral valve repair for severe mitral regurgita-
tion. J Cardiovasc Magn Reson 3:257–266 56. Al Musa T, Uddin A, Swoboda PP et al (2017) Myocardial strain
and symptom severity in severe aortic stenosis: insights from
cardiovascular magnetic resonance. Quant Imaging Med Surg
7:38–47. doi:10.21037/qims.2017.02.05 72. Compliance with ethical standards Marciniak A, Claus P, Sutherland GR et al (2007) Changes in
systolic left ventricular function in isolated mitral regurgita-
tion. A strain rate imaging study. Eur Heart J 28:2627–2636. doi:10.1093/eurheartj/ehm072 40. Bull S, White SK, Piechnik SK et al (2013) Human non-contrast
T1 values and correlation with histology in diffuse fibrosis. Heart
99:932–937. doi:10.1136/heartjnl-2012-303052 25. Delgado V, Tops LF, van Bommel RJ et al (2009) Strain analysis
in patients with severe aortic stenosis and preserved left ventric-
ular ejection fraction undergoing surgical valve replacement. Eur
Heart J 30:3037–3047. doi:10.1093/eurheartj/ehp351 41. Lee SP, Lee W, Lee JM, Park EA, Kim HK, Kim YJ, Sohn DW
(2015) Assessment of diffuse myocardial fibrosis by using MR
imaging in asymptomatic patients with aortic stenosis. Radiol-
ogy 274:359–369. doi:10.1148/radiol.14141120 42. Chin CW, Semple S, Malley T et al (2014) Optimization and
comparison of myocardial T1 techniques at 3T in patients with
aortic stenosis. Eur Heart J Cardiovasc Imaging 15:556–565. doi:10.1093/ehjci/jet245f 26. Ibrahim el SH (2011) Myocardial tagging by cardiovascular
magnetic resonance: evolution of techniques–pulse sequences,
analysis algorithms, and applications. J Cardiovasc Magn Reson
13:36. doi:10.1186/1532-429x-13-36 27. Pedrizzetti G, Claus P, Kilner PJ, Nagel E (2016) Principles of
cardiovascular magnetic resonance feature tracking and echocar-
diographic speckle tracking for informed clinical use. J Cardio-
vasc Magn Reson 18:51. doi:10.1186/s12968-016-0269-7 43. Flett AS, Sado DM, Quarta G et al (2012) Diffuse myocardial
fibrosis in severe aortic stenosis: an equilibrium contrast car-
diovascular magnetic resonance study. Eur Heart J Cardiovasc
Imaging 13:819–826. doi:10.1093/ehjci/jes102 44. Dusenbery SM, Jerosch-Herold M, Rickers C, Colan SD, Geva
T, Newburger JW, Powell AJ (2014) Myocardial extracellular
remodeling is associated with ventricular diastolic dysfunction in
children and young adults with congenital aortic stenosis. J Am
Coll Cardiol 63:1778–1785. doi:10.1016/j.jacc.2013.11.066 28. Schuster A, Hor KN, Kowallick JT, Beerbaum P, Kutty S (2016)
Cardiovascular magnetic resonance myocardial feature track-
ing: concepts and clinical applications. Circ Cardiovasc Imaging
9:e004077. doi:10.1161/circimaging.115.004077 29. Padiyath A, Gribben P, Abraham JR et al (2013) Echocardiog-
raphy and cardiac magnetic resonance-based feature tracking in
the assessment of myocardial mechanics in tetralogy of Fallot: 45. Chin CW, Shah AS, McAllister DA et al (2014) High-sensi-
tivity troponin I concentrations are a marker of an advanced 1 3 1 112 Int J Cardiovasc Imaging (2018) 34:97–112 hypertrophic response and adverse outcomes in patients with
aortic stenosis. Eur Heart J 35:2312–2321. doi:10.1093/
eurheartj/ehu189 61. Compliance with ethical standards Ahmed MI, Gladden JD, Litovsky SH et al (2010) Increased
oxidative stress and cardiomyocyte myofibrillar degeneration in
patients with chronic isolated mitral regurgitation and ejection
fraction > 60%. J Am Coll Cardiol 55:671–679. doi:10.1016/j. jacc.2009.08.074 57. Mahmod M, Bull S, Suttie JJ et al (2013) Myocardial steato-
sis and left ventricular contractile dysfunction in patients with
severe aortic stenosis. Circ Cardiovasc Imaging 6:808–816. doi:10.1161/circimaging.113.000559i 58. Meyer CG, Frick M, Lotfi S et al (2014) Regional left ventricu-
lar function after transapical vs. transfemoral transcatheter aortic
valve implantation analysed by cardiac magnetic resonance fea-
ture tracking. Eur Heart J Cardiovasc Imaging 15:1168–1176. doi:10.1093/ehjci/jeu103i 73. Schiros CG, Dell’Italia LJ, Gladden JD et al (2012) Magnetic
resonance imaging with 3-dimensional analysis of left ventric-
ular remodeling in isolated mitral regurgitation: implications
beyond dimensions. Circulation 125:2334–2342. doi:10.1161/
circulationaha.111.073239 j
j
59. Borer JS, Truter S, Herrold EM et al (2002) Myocardial fibrosis
in chronic aortic regurgitation: molecular and cellular responses
to volume overload. Circulation 105:1837–1842 74. Ahmed MI, Guichard JL, Rajasekaran NS et al (2016) Dis-
ruption of desmin-mitochondrial architecture in patients with
regurgitant mitral valves and preserved ventricular function. J
Thorac Cardiovasc Surg 152(1059–1070):e1052. doi:10.1016/j. jtcvs.2016.06.017 60. Piper C, Schultheiss HP, Akdemir D, Rudolf J, Horstkotte D,
Pauschinger M (2003) Remodeling of the cardiac extracellular
matrix differs between volume- and pressure-overloaded ventri-
cles and is specific for each heart valve lesion. J Heart Valve Dis
12:592–600 1 3
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https://openalex.org/W4293541695
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https://dimensi.petra.ac.id/index.php/ars/article/view/18627/18389
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Indonesian
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KAJIAN POLA PERMUKIMAN KHAS KAMPUNG LENGKONG ULAMA, SERPONG, BANTEN
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DOAJ (DOAJ: Directory of Open Access Journals)
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DIMENSI (Journal of Architecture and Built Environment), Vol. 39, No. 1, July 2012, 31-36
ISSN 0126-219X DIMENSI (Journal of Architecture and Built Environment), Vol. 39, No. 1, July 2012, 31-36
ISSN 0126-219X KAJIAN POLA PERMUKIMAN KHAS KAMPUNG LENGKONG ULAMA,
SERPONG, BANTEN KHAMDEVI, Muhammar
Email: arch_fiqh@yahoo.com ABSTRACT Who knows about Kampong Lengkong Ulama, Serpong? Perhaps it has been known by "the sarong people", because
it echoed only in the religious media. In 1983, there was a history book about this village, included essays and translations
from ancient texts, written by local leaders; Ustadz Mukri Mian, without published. In 2009, there was a book written by
Indonesian archaeologist; Uka Tjandrasasmita. Until now, he is the only person who researches about this village from
archaeology perspective. He did not mention about the typical pattern of this village, as Ustadz Mukri Mian described in his
book, that the position of the buildings follows the Qibla. Is the settlement pattern of Kampong Lengkong Ulama follows the
Qibla? This paper is a new research to study the pattern of this village. This study uses qualitative method from architecture
perspective. The research conclusion is that the pattern of Kampong Lengkong Ulama really follows the Qibla direction and
situated by the river that also follows the Qibla. In addition, the placement of the tomb of the founder of this village on the hill
has the same concept with the general concept in Javanese community and kingdoms about the world and the hereafter. Keywords: Historic village, conservation, rural urban. ABSTRAK Siapa yang tahu tentang Kampung Lengkong Ulama, Serpong? Mungkin dikenal oleh “kaum bersarung” saja, karena
bergaung hanya di media-media agama. Tahun 1983, muncullah buku sejarah kampung ini karangan sekaligus termasuk
terjemahan dari naskah kuno kampung ini oleh tokoh setempat; Ustadz Mukri Mian, namun tidak dipenerbitkan. Pada tahun
2009, hadir satu buku pencerah karangan arkeolog Indonesia; alm. Uka Tjandrasasmita. Hingga kini, baru beliaulah orang
pertama yang meneliti kampung ini dari sudut pandang arkeologi, dengan kajian terhadap naskah dan peninggalan berupa
makam. Namun beliau tidak menyebutkan tentang pola kampung ini, sebagaimana Ustadz Mukri Mian gambarkan dalam
bukunya, bahwa posisi bangunan-bangunannya mengikuti posisi arah kiblat. Apakah pola permukiman Kampung Lengkong
Ulama mengikuti posisi arah kiblat? Penelitian ini adalah penelitian baru yang bertujuan untuk mengkaji dan mengetahui
pola kampung ini dan bagaimana proses terbentuknya. Penelitian ini menggunakan metode kualitatif dari sudut pandang
arsitektur. Dari hasil penelitian dapat disimpulkan, bahwa pola Kampung Lengkong Ulama mengikuti posisi arah kiblat dan
secara unik terletak pada bibir sungai yang juga mengikuti posisi arah kiblat. Selain itu, penempatan makam pendiri
kampung ini di atas bukit memiliki benang merah dengan kebiasaan yang terjadi pada masyarakat dan kerajaan-kerajaan
jawa pada umumnya mengenai dunia dan akhirat. Kata kunci: Kampung bersejarah, konservasi, desa kota. Keywords: Historic village, conservation, rural urban. Kata kunci: Kampung bersejarah, konservasi, desa kota. PENDAHULUAN Naskah ini tidak pernah dikem-
balikan, namun arkeolog; Uka Tjandrasasmita,
memastikan bahwa naskah itu sudah pasti asli, dan
patut ditelusuri ke mana sekarang naskah itu berada. (Tjandrasasmita, 2009). Padahal berdasarkan analisis sejarah, arkeologis,
tradisi setempat, bahkan dari segi geografis serta
lingkungannya, yang dilakukan oleh Bapak Arkeolog
Islam Indonesia, Uka Tjandrasasmita, Kampung
Lengkong Sumedang atau Lengkong Ulama,
merupakan kampung bersejarah atau situs bersejarah
(historical site) yang perlu diperhatikan kelestariannya
(Tjandrasasmita, 2009). Sejak berdirinya kota Tangerang Selatan, Kabu-
paten Tangerang tidak memiliki makam pahlawan,
karena Makam Seribu Pahlawan beralih ke Kota
Tangerang Selatan. Sehingga Pemerintah Kabupaten
Tangerang merenovasi komplek makam Kampung
Lengkong Ulama sebagai Makam Pahlawan
Kabupaten Tangerang, yang diresmikan pada tanggal
17 Agustus 2012 (Bharatanews, 2012). Untungnya naskah tersebut sempat diterjemah-
kan ke dalam Bahasa Indonesia dalam sebuah buku
yang diproduksi untuk kalangan terbatas oleh Ustadz
Mukri Mian pada tahun 1983. Uka Tjandrasasmita
memastikan bahwa data-data dalam buku tersebut
dapat diabsahkan, dengan berdasarkan kepada
gambaran fisik naskah aslinya (Tjandrasasmita,
2009). Kampung Lengkong Ulama telah dilalui oleh
banyak peristiwa sejarah, terutama mewarnai sejarah
Tangerang. Dari semenjak zaman kerajaan Mataram,
lalu perjuangan Sultan Ageng Tirtayasa terhadap
VOC Belanda (Tjandrasasmita, 2009), hingga juga
pada masa kemerdekaan; terutama menjadi salah satu
basis Masyumi (Mian, 1983) dan juga menjadi area
pertempuran Mayor Daan Mogot, yang dikenal
sebagai “Pertempuran Lengkong” (Seno, 2011). Itu
dikarenakan kampung ini terletak secara strategis di
Sungai Cisadane untuk syiar Islam dan perdagangan. Dalam buku tersebut tercantum tentang pola
kampung yang mengikuti arah kiblat. Hal ini
diperkuat lagi dengan wawancara terhadap informan
kunci, yakni Ustadz Mukri Mian, dan terhadap
informan lainnya, yakni para Kyai Kampung Leng-
kong Ulama. Karena belum ada yang meneliti tentang
hal ini, termasuk Bapak Arkeologi Islam Indonesia,
Uka Tjandrasasmita, sehingga lokasi dan kasus kajian
ini menjadi penting dan menarik untuk diadakan
penelitian terhadap pola kampung ini secara ilmiah. Dari dulu hingga sekarang kampung ini telah
mencetak banyak ulama. Tak heran dalam satu
kampung terdapat banyak Kyai yang memiliki
pengikut yang tersebar di wilayah Tangerang pada
saat ini (Mian, 2001). Selain itu para ulama dan
santrinya memiliki keterampilan dalam kaligrafi yang
diakui hingga dunia internasional (Septayuda, 2011). Apakah pola Kampung Lengkong Ulama meng-
ikuti arah kiblat? Makalah ini mencoba membahas
pola Kampung Lengkong Ulama dari perspektif
arsitektur. Dengan kajian ini, diharapkan bertambah
lagi penelitian baru mengenai kampung ini dan
membuka
peluang
untuk
penelitian-penelitian
selanjutnya. Sayangnya hingga kini, peneliti yang mengkaji
kampung ini barulah seorang, yakni Uka Tjandra-
sasmita. PENDAHULUAN Arya Wangsakara adalah seorang ulama dan dapat
dipastikan adalah Pangeran Arya Wiraraja II yang
berasal dari Sumedang, yang pindah dari Sumedang
ke Banten untuk menghindari dari tekanan Kerajaan
Mataram dan dari Pemberontakan Dipati Ukur
(Tjandrasasmita, 2009). Selain itu kata Lengkong ini
juga menunjukkan bahwa lokasi kampung ini berada
pada sebuah lingkung air; sungai (Mian, 1983). Arya Wangsakara adalah seorang ulama dan dapat
dipastikan adalah Pangeran Arya Wiraraja II yang
berasal dari Sumedang, yang pindah dari Sumedang
ke Banten untuk menghindari dari tekanan Kerajaan
Mataram dan dari Pemberontakan Dipati Ukur
(Tjandrasasmita, 2009). Selain itu kata Lengkong ini
juga menunjukkan bahwa lokasi kampung ini berada
pada sebuah lingkung air; sungai (Mian, 1983). Selain dikenal sebagai Kampung Lengkong
Kyai, seperti yang tertera pada signage yang saya
temui di sana, kampung ini juga dikenal dengan
banyak nama, seperti; Lengkong Sumedang atau
Lengkong Ulama (Septayuda, 2011) dan bahkan
Lengkong Alit atau Lengkong Kecil (Mukri, 1983). Bahkan pada peta topografi hanya disebut dengan
Lengkong saja (Tjandrasasmita, 2009). Lain halnya
yang disebutkan di majalah Media Dakwah, yakni
disebut sebagai Lengkong Kulon (Dakwah, 1992). Kata Lengkong ini diambil dari asal pendiri kampung
ini, Raden Arya Wangsa Di Kara, di Sumedang
(Mian, 1983). Raden Wangsa Di Kara, atau Raden Kampung Lengkong Ulama terletak di jantung
kota Serpong, berdampingan dengan pengembangan
kota Bumi Serpong Damai (BSD). Eksistensinya
semakin dikhawatirkan, karena makin lama kampung-
kampung disekitarnya mulai pudar tergantikan dengan
perumahan dan fungsi baru. Apalagi kekhawatiran itu
makin bertambah, sejak salah satu jembatan peng- 31 Khamdevi hubung wilayah barat dan timur Sungai Cisadane
mulai terealisasikan, Juli 2010 (Seno, 2011), yang
sudah satu dekade tertunda akibat ketidak setujuan
warga setempat. Tentu saja hal ini akan memberikan
dampak yang besar bagi eksistensi kampung ini, yang
sejak lama tertutup dan tersembunyi secara alamiah,
akan terekspos dengan perubahan yang cenderung
akan ekstrim. 17) dan letaknya berada di atas bukit yang meng-
ingatkan pada model makam para Sunan dan Sultan
di Jawa, makam Kyai Mustaqim yang bertipe antefik
(abad 18-19), bekas rumah istri kedua Raden Arya
Wangsakara dan Masjid Raden Arya Wangsakara
yang sekarang sudah dirombak habis menjadi lebih
modern. Sebenarnya ada bukti tertulis dari kampung ini,
naskah asli yang pernah disimpan oleh almarhum
Kyai Muchtar dan Bapak Yasin, yang ditulis dalam
huruf pegon pada kertas daluwang dengan tinta cina. Naskah asli tersebut pernah dipinjamkan kepada
dosen dan beberapa mahasiswa yang melakukan
Kuliah Kerja Nyata. PENDAHULUAN Beliau menggali kesejarahan kampung ini
dengan pendekatan arkeologi, yakni dari objek
naskah dan peninggalan makam. Menurut beliau
berdasar babad-babad jawa terutama Babad Cirebon,
pendiri kampung ini, Raden Arya Wangsakara dapat
dipastikan adalah Pangeran Arya Wiraraja II yang
berasal dari Sumedang, yang memiliki benang merah
dengan Kesultanan Cirebon. METODE PENELITIAN Penelitian ini menggunakan metode kualitatif
dengan pendekatan analisis deskriptif. Data primer
dikumpulkan secara empirik melalui pengamatan
lapangan, pengambilan foto dan pembuatan sketsa,
serta wawancara yang mendalam untuk menggali
informasi dari responden sebagai informan kunci. Informan kunci dipilih berdasarkan otoritas penuh
yang diberikan warga sebagai juru bicara mengenai Data arkeologis kampung ini adalah makam
Raden Arya Wangsakara yang bertipe arkaik (Abad 32 Kajian Pola Permukiman Khas Kampung Lengkong Ulama, Serpong, Banten Kajian Pola Permukiman Khas Kampung Lengkong Ulama, Serpong, Banten Ketika wafatnya Raden Arya Wangsakara,
beliau dimakamkan di bukit yang bersebelahan
dengan kampung. Penempatan makam ini mirip
dengan makam-makan sultan maupun sunan di
masyarakat jawa pada umumnya (Tjandrasasmita,
2009). seluk beluk kampungnya. Sedangkan informan yang
lain digunakan sebagai bahan pembanding dan
pemeriksaan. Data sekunder diperoleh dari referensi pustaka,
peta-peta dan dokumen-dokumen pendukung dan
pembanding yang relevan untuk memperkuat argu-
mentasi. Karena baru satu peneliti yang mengkaji
kampung ini, maka data-data studi kasus yang mirip
sangat diperlukan sebagai data pembanding. Penempatan makam islam di jawa merupakan
akulturasi dengan agama-agama sebelumnya, seperti:
animisme, hindu dan budha. Penempatan makam
biasanya diletakkan; pertama, di tempat yang lebih
tinggi (gunung atau bukit). Biasanya untuk para raja. Contoh: Komplek Makam Sunan Gunung Jati dan
Makam raja-raja Mataram di Bukit Imogiri. Kedua,
dekat dengan masjid. Biasanya para wali dan ulama. Contoh: Masjid Demak, Kadilangu dan Sendang
Duwur (Mustopo dkk., 2003). Data-data yang telah dikumpulkan dari segala
sumber tersebut diperiksa, dipilah, diolah lalu di-
analisis. Analisis dilakukan pertama-tama terhadap
kondisi eksisting, lalu diuji dengan teori pembentuk
kota Kevin Lynch untuk mengidentifikasi pembentuk
citra kawasan dan selanjutnya dikaji lagi dengan teori
figure ground untuk mengidentifikasi pola tata ruang
dan hubungan antar massa atau ruang (Zahnd, 1999). Bagi masyarakat jawa, gunung atau bukit
menyimbolkan status yang tinggi sekaligus merupa-
kan upaya mendekatkan diri dengan Yang Maha
Kuasa. Orang Jawa percaya gunung adalah tempat
sakral dan biasanya didiami oleh mahluk halus, roh-
roh leluhur atau dewa. Gunung atau bukit me-
lambangkan hubungan antara dunia manusia atau
bumi dan akhirat atau kahyangan (Walsh, 2000). Keberadaan masjid pada proses permulaan ter-
bentuknya kampung yang dibangun oleh pendiri
kampung hingga makam pendiri di bukit menunjuk-
kan sebuah hubungan kesatuan timeline cerita sejarah
dan menggambarkan hubungan dunia dan akhirat
tersebut. Proses Terbentuknya Kampung Setelah berpindah-pindah beberapa kali, akibat
ancaman dari VOC, akhirnya Raden Arya Wangsa-
kara mendapatkan lokasi yang tepat. Lokasi kampung
ini strategis tersembunyi dan terlindungi oleh alam
(hutan bambu) dan dilingkungi Sungai Cisadane dan
kali kecil. Dan uniknya, penentuan lokasi yang
dilakukan beliau berdasarkan pemilihan bagian alur
Sungai Cisadane yang secara kebetulan menghadap
kiblat. Hal ini mempengaruhi posisi bangunan rumah-
rumah santri yang berupa gubug panggung yang
mengikuti posisi kiblat, serta bagian memanjang
rumah tersebut menghadap sungai dan bukit. Bangunan yang pertama kali dibangun adalah masjid
yang menjorok ke dekat sungai, lalu gubug-gubug
para santri (Mian, 1983). Eksisting Kampung Dilihat dari lingkungan alamiah eksistingnya, posisi
alur Sungai Cisadane yang menjadi lokasi Kampung
Lengkong Ulama memang terlihat secara kebetulan
sesuai dengan arah kiblat, yakni 25 derajat dari barat
ke utara (BADILAG, 2007). Dari data vegetasi dan
topografi, kampung ini benar-benar tersembunyi dan
tertutup dari lingkungan lainnya, sehingga dapat
menjadi tempat untuk berlindung dari tekanan VOC
dan juga melaksanakan pengajaran Islam dengan
lebih nyaman dan aman. Hal ini menunjukkan
pemilihan lokasi oleh Raden Arya Wangsakara
memang sudah diperhitungkan dengan matang. Hal ini serupa dengan proses terbentuknya
pondok pesantren tradisional pada umumnya. Suatu
pondok pesantren berawal dari adanya seorang kyai
di suatu tempat, kemudian datang santri yang ingin
belajar agama kepadanya. Setelah semakin hari
semakin banyak santri yang datang, timbullah inisiatif
untuk mendirikan pondok atau asrama di samping
rumah kyai. Para santri selanjutnya memopulerkan
keberadaan pondok pesantren tersebut, contohnya
seperti pada pondok-pondok yang timbul pada zaman
Walisongo (Hielmy, 2000). Selain fungsi hunian, terdapat fasilitas-fasilitas
umum berupa; masjid, musholla, majelis ta’lim,
makam, pesantren dan madrasah. Objek-objek sejarah
yang ada di kampung ini, antara lain: Masjid Al
Muttaqin, Makam Raden Arya Wangsakara, Makam
Kyai Mustaqim, Makam Kyai Azhari, Musholla
Azhari dan Rumah-rumah kuno yang bertipe mirip
rumah Betawi atau Banten. Akhirnya, lambat laun beberapa santri ada yang
bermukim dan berkeluarga di sana. Gubug-gubug
santri yang bertipe panggung dengan atap julang
ngapak berubah menjadi rumah-rumah hingga mem-
bentuk sebuah permukiman kampung (Mian, 1983). 33 Khamdevi Selain fungsi hunian, terdapat fasilitas-fasilitas umum berupa; masjid,
Masjid
dan
Makam
Musholla
Makam
S. Cisadane
K. Cipicung
Madrasah
Pesantren
Baru
M. Ta’lim
Kiblat
Sumber: Google Map
Gambar 1. Peta Satelit Kampung Lengkong Ulama, Serpong, Banten Musholla Pesantren
Baru K. Cipicung Sumber: Google Map Sumber: Google Map unian terdapat fasilitas-fasilitas umum be
Gambar 1. Peta Satelit Kampung Lengkong Ulama, Serpong, Banten Sumber: Jarot dan Purwono, Geografi UI
Gambar 2. Peta Kampung Lengkong Ulama, Serpong, Banten Sumber: Jarot dan Purwono, Geografi UI Gambar 2. Peta Kampung Lengkong Ulama, Serpong, Banten 34 Kajian Pola Permukiman Khas Kampung Lengkong Ulama, Serpong, Banten Sumber: Jarot dan Purwono, Geografi UI DAFTAR PUSTAKA selatan; sungai dan bukit (makam). Hal ini bisa
disebabkan, bahwa pada mula berdirinya, kampung
ini merupakan pesantren yang terdiri dari masjid dan
pondok (gubug) santri. Dengan berdirinya masjid,
posisi pondok dan bangunan pendukung lainnya pun
praktis mengikuti posisi masjid. Maka dengan makin
berkembangnya pesantren dan santri-santrinya me-
netap, maka yang tadinya hanyalah sebuah pesantren,
justru berkembang menjadi sebuah kampung baru. Badilag (2007). Daftar Arah Kiblat Kota-Kota di
Indonesia. Direktorat Jenderal Badan Peradilan
Agama, Mahkamah Agung Republik Indonesia. http://www.badilag.net/data/hisabrukyat/Daftar
Koord. Geo&Arah Kiblat.pdf Bintarto, R. (1983). Interaksi Desa-Kota Dan Perma-
salahannya. Yogyakarta: Ghalia I Indonesia. Dari analisis ini juga terlihat, bahwa semakin
keluar, ada beberapa posisi bangunan mulai tidak
mengikuti pola sebelumnya, seakan terjadi inkonsis-
tensi. Menurut penuturan Ustadz Mukri Mian (2001),
bahwa pola ini tidaklah mengikat dan tergantung
bentuk kavling yang dimiliki. Walau begitu, ini justru
memudahkan dalam menentukan mana yang merupa-
kan kampung bagian yang lama dan mana yang baru. Hielmy, Irfan (2000). Wacana Islam. Ciamis: Pusat
Informasi Pesantren. Mian, M. (1983). Sejarah Kampung Lengkong. Lengkong: tanpa penerbit. Mustopo, Habib (2003). Sejarah dan Budaya: Dari
Masa Kuno Sampai Kontemporer. Malang: UM
Press. Nuryanto & I. Machpudin (2007). Kajian Pola
Kampung dan Rumah Tinggal: Warga Kasepuh-
an Kesatuan Adat Banten Kidul di Sukabumi
Selatan–Jawa Barat. Artikel Hasil Penelitian
Arsitektur Tradisional Sunda. Bandung: Univer-
sitas Pendidikan Indonesia. Elemen Pembentuk Kampung Pintu masuk ke lokasi kampung ini bisa melalui
jalur sungai dengan ditandai dengan alat transportasi
getek yang berada pada sisi timur kampung dan juga
bisa melalui jalur darat yang berada pada sisi barat
kampung ini, di mana pintu masuk ini ditandai
dengan turunan yang curam, hutan yang lebat dan
jembatan Kali Cipicung. Kampung, dalam hal ini desa, adalah merupakan
suatu hasil perpaduan antara kegiatan kelompok
manusia dengan lingkungannya. Pola ruang desa,
terutama di pulau Jawa, dapat dibedakan menjadi
empat macam (Bintarto, 1983). Pertama, memusat;
bentuk perdesaan ini terpencar menyendiri. Kedua,
linier; bentuk perdesaan ini memanjang mengikuti
jalur jalan raya, alur sungai atau garis pantai. Ketiga,
terpencar; bentuk perdesaan yang terpencar cenderung
menyendiri. Keempat, mengelilingi fasilitas; bentuk
perdesaan mengelilingi fasilitas-fasilitas umum. Sedangkan batas fisik kawasan kampung ini
secara dominan dibatasi oleh elemen alamiah yang
melingkungi kampung, seperti: sungai, hutan dan
bukit. Namun selain batas fisik, kampung ini me-
miliki batas non fisik, berupa makam yang disakral-
kan. Batas non fisik sulit dibuktikan, karena berkaitan
dengan keyakinan warga, tetapi dapat dirasakan,
seperti: takut, angker, seram, hormat dan lain-lain
(Nuryanto, 2007). Dari analisis figure ground, dapat diketahui
bahwa pola kampung ini memiliki pola linear dan
terpusat ke Masjid. Namuin pola liniear ini justru
tidak umum seperti pola desa yang ada di Indonesia
umumnya. Seharusnya dengan kampung ini berada di
sisi sungai, maka ia harus mengikuti pola alur
sungainya. Namun ini tidak, justru ia menentang pola
lingkungan alamiahnya. Landmark kawasan kampung ini adalah Masjid
dan Makam. Jaringan jalannya juga terbentuk secara
organik dengan pola grid-iron (papan catur), di mana
pola jalan terjadi disebabkan bentukan dari per-
mukiman. Pola jalan dan permukiman ini mirip
dengan pola permukiman Kampung Kauman Sura-
karta (Setyaningsih, 2007). Dari sinipun diketahui pula, bahwa pola-pola
bangunan secara umum, selain Masjid, sepertinya
memang mengikuti posisi kiblat, yakni 25 derajat dari
arah barat ke utara (BADILAG, 2007), dengan sisi
yang memanjang menghadap ke arah utara dan Sumber: Jarot dan Purwono, Geografi UI
Gambar 3. Peta Kampung Lengkong Ulama, Serpong, Banten Sumber: Jarot dan Purwono, Geografi UI Gambar 3. Peta Kampung Lengkong Ulama, Serpong, Banten 35 Khamdevi KESIMPULAN Dari riset ini, maka disimpulkan bahwa pola
kampung Lengkong Ulama adalah linier dan meng-
ikuti posisi arah kiblat, dimana bagian memanjang
bangunan, terutama hunian, menghadap utara dan
selatan; sungai dan bukit. Kampung ini dilingkungi
oleh objek alamiah, berupa; sungai, kali, hutan bambu
dan bukit, sehingga kampung ini tertutup dari wilayah
sekitarnya. Akses masuk ke kampung ada di sisi barat
(darat) dan pada sisi timur (air/ getek). N.n. (1992). Lengkong Kulon: Kampung Ulama yang
Nyaris Punah. Jakarta: Media Dakwah. N.n. (2012). Makam Pendiri Tangerang Gantikan
Taman Makam Pahlawan Seribu. Bharatanews, 2012. (http://bharatanews.com/berita-1717-
makam-pendiri-tangerang-gantikan-taman-makan-
pahlawan-seribu.html) Dari hasil analisis ini juga diketahui bahwa
kampung lama berada pada lapis pertama yang dekat
dengan bibir Sungai Cisadane. Pola dan posisi
bangunan kampung ini berpola linear dan terpusat,
dimana hampir 100% mengikuti posisi arah kiblat. Bagian yang memanjang dari bangunannya meng-
hadap bukit dan sungai. Bukit – makam dan sungai –
masjid merepresentasikan hubungan timeline sejarah
dan juga hubungan analogi dunia–akhirat dalam
kaitannya dengan Ketuhanan yang berlaku umum
pada masyarakat jawa. Seno (2011). Kelak, Makam Pahlawan Kab. Tange-
rang Ada di Lengkong Kulon. Jakarta: Kompa-
siana.com. (http://sejarah.kompasiana.com/2011/
07/24/kelak-makam-pahlawan-kab-tangerang-
ada-di-lengkong-kulon/). Seno (2011). Lengkong Kulon, Desa yang Nyaris
Hilang. Jakarta: Kompasiana.com. (http://sejarah. kompasiana.com/2011/07/24/lengkong-kulon-
desa-yang-nyaris-hilang/). Dengan potensi yang begitu beragam ini, baik
secara fisik maupun budaya, kampung ini sangat
cocok sekali menjadi kawasan wisata religi dan
budaya, seperti yang juga diusulkan oleh Uka
Tjandrasasmita, yang menyatakan bahwa kampung ni
adalah kampung bersejarah. Sehingga Pemerintah
Kabupaten Tangerang sebaiknya tidak hanya me-
netapkannya makam pendiri kampung sebagai
Makam Pahlawan Kabupaten Tangerang saja, tapi
Kapung Lengkong Ulama juga sebagai kampung
bersejarah yang memiliki objek wisata. Potensi wisata
yang mungkin bisa diangkat adalah wisata ziarah,
wisata santri (belajar ilmu agama), wisata kaligrafi,
wisata alam dan wisata sejarah. Namun perlu dilaku-
kan upaya penataan untuk merevitalisasi kampung
ini. Ini menjadi bahan riset berikut peneliti, selain
melengkapi riset saat ini dengan data segar. Septayuda, T. (2011). Jejak Arkeologis Penyebaran
Agama Islam di Tangerang. Ponorogo: Majalah
Gontor. Setyaningsih, W. (2007). Potensi Spasial Fisik Kampung
Kauman Surakarta sebagai Kawasan Budaya
dan Religi. Gema Teknik–Nomor 2/Tahun X
Juli 2007. Tjandrasasmita, U. (2009). Arkeologi Islam Nusan-
tara. Jakarta: Kepustakaan Populer Gramedia. Walsh, D. (2000). Kepercayaan Masyarakat Jawa
Terhadap Gunung. Thesis. http://www.acicis. murdoch.edu.au/hi/field_topics/dylan.html Zahnd, M. (1999). Perancangan Kota Secara Ter-
padu. Yogyakarta: Kanisius. 36
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Distribution of Selenium in Soils and Human Health
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Chapter
Distribution of Selenium in Soils
and Human Health
Muhammad Imran, Zhikun Chen, Ayaz Mehmood, Shah Rukh,
Wang Weixie, Waleed Asghar and Farhan Iftikhar
Abstract
Selenium (Se) is essential as well as a toxic element for humans and animals if it
exceeds a certain limit. Soil selenium plays an important role through the food chain.
Total selenium in world soils ranges from 0.125 to 0.3 mg kg 1 and varies with the
soils’ nature. High and low selenium in soils also poses serious environmental and
health risks. However, in addition to selenium's overall quantity in soil, selenium
reactivity, and bioavailability also depend on its chemical structure. The amount of
available selenium in the soil varies depending on its oxidation state since selenium
species include selenide (Se2 ), elemental selenium (Se0), selenite (Seo23 Þ, selenate
(Seo24 Þ. The pH, soil texture, amount of organic matter, and the presence of competing ions are the four most significant soil characteristics that affect Se availability.
Similarly, selenium uptake and accumulation are influenced by the crop type whether
it is an accumulator or not. The selenium environmental and health risk assessment is
necessary to evaluate in soils with high selenium contents and crops with higher
selenium uptake. Whereas in areas where selenium deficiency is observed or vulnerable to selenium, deficiency needs to be supplemented through Se inputs. The selenium deficiency and toxicity areas should be monitored carefully from a health
perspective.
Keywords: total soil selenium, selenium species, selenium transformation, selenium
bioavailability, selenium risk assessment
1. Introduction
A vital element for both humans and animals, selenium (Se) is a metalloid that lies
in the middle of the metal and non-metal. Selenium plays a crucial role in the biological processes of human and animals body. Its high concentrations make it poisonous,
and a lack of it can have catastrophic consequences on human and animal health [1].
Despite the fact that selenium has a wide range of important advantages, selenium
insufficiency is becoming a widespread issue around the world. A health danger exists
when selenium intake is excessive. Moreover, type II diabetes risk may be increased
by a diet high in selenium [2]. A high selenium intake may enhance the expression of
the transcription coactivator peroxisome proliferator-activated receptor-coactivator
1
Selenium and Human Health
(PGC-1), which is important in cellular energy metabolism and may result in hyperglycemia [3]. Excessive selenium consumption results in loss of hair and nails, damage
to the neurological system, paralysis, and even death [4]. The daily selenium consumption dosage so has significance. Selenium 40 μg d 1 the recommended daily
allowance (RDA) suggested by the WHO [5]. An overdose occurs when the consumption for men is greater than 60 μg d 1 and for women is greater than 53 μg d 1
[6]. Responses varied when referring to various forms of selenium, and this is for the
total amount of selenium.
Selenium availability from soil affects the food chain selenium level. Three major
selenium mineral i.e. tiemannite (HgSe), clausthalite (PbSe), and naumannite ((Ag,
Pb) Se) contains selenium and is present in soils [7]. The soil’s total selenium depends
on the type of parent materials and the soil-forming processes which redistribute
selenium [8]. Overall, total selenium in world soils ranges from 0.125 to 0.3 mg kg 1
and varies with the soils’ nature [9]. The essential level of selenium for animals ranges
from 0.04 to 0.1 mg kg 1, while a concentration exceeding 3.5 to 5 mg kg 1 in their
food may cause harmful impacts [4, 10]. Human activities including fossil fuel and
coal burning, metal smelting, inorganic, and organic fertilizer application, lime,
manure, and solid sewage waste disposal cause Se accumulation in soils [11]. The
selenium accumulation in soils poses serious threats to the agroecosystem via
bioaccumulation [11, 12]. Selenium toxicity in soil and food chains depends on its
forms and distribution rather than its total contents [13]. Total selenium concentration in soils derived from various sources ranges from 0.27 to 7.05 mg kg 1 [8].
Yet in addition to the amount of selenium in the soil as a whole, selenium reactivity
and bioavailability also depend on the chemical form of the element. Several forms of
selenium, including selenide, elemental selenium, selenite, selenate, and organic selenium, are found in soil, depending on its oxidation state [14]. The replenishment of
selenium in soil solution is also aided by selenium that is contained in or bonded to
various fractions in soils. Typically, there are five different selenium fractions: ionexchangeable or calcium-bound selenium, oxides-bound selenium (iron and aluminum oxides), organic and humic-bound selenium, sulfide-bound selenium, and residual selenium. Thus, it’s critical to keep an eye on the type and amount of selenium
exposure through different foods grown under different soils.
2. Selenium species in soils
There are a variety of selenium species that can be found in soil solution.
Selenate ðSe VIÞ ¼ Seo24 , HSeo4 , H2 Seo4
Selenite ðSeIVÞ ¼ Seo23 , HSeo3 , H2 Seo03
SelenateðIIÞ ¼ Se2 , ðHSe Þ, H2 Se0
Depending on the characteristics of the environment or the soil, several species of
selenium can be found in the form of selenide (Se2 ), elemental selenium (Se0),
selenite (Seo23 Þ, selenate (Seo24 Þ. Selenate (Seo24 Þ: Under conditions in which it is
thoroughly oxidized, selenate maintains its stability. Selenate is not absorbed by soil
elements with the same level of strength as selenite [15, 16], and the transformation of
selenate into less mobile forms of selenite or elemental Se) is a long process [17].
2
Distribution of Selenium in Soils and Human Health
DOI: http://dx.doi.org/10.5772/intechopen.110636
Selenate is the form of selenium that may be taken up by plants in the greatest
quantity [18, 19]. Selenite (Seo23 Þ: Selenite is a can be found in settings that are only
slightly oxidized. Selenous acid is a weak acid that can only be protonated in conditions where the pH values range from acidic to neutral. Microorganisms in acidic
settings [20] or moderately reducing agents in neutral or alkaline environments [21]
can convert selenite to elemental selenium. Selenite possesses a significant propensity
for sorption, in particular by oxides of iron and aluminum [17, 18]. Whereas the
adsorption of selenite (Seo23 Þ depends on pH, and the concentration of competing
anions such as phosphate (PO34 Þ [22]. Selenide: Selenide (Se2 ) typically exists in
reducing environments as metal selenides and hydrogen selenide (H2Se) a poisonous
gas with a bad smell. In water, it readily oxidizes to elemental Se [23]. Se-sulfides and
metal selenides often have very low solubility [24]. Besides that, microbial activities
also result in the production of dissolved organic selenide molecules or volatile methylated derivatives of selenium such as dimethyl diselenide [25, 26]. Elemental Selenium (Se0): Elemental selenium (Se0) exists in reduced conditions in the form of
crystalline or amorphous. Red crystalline Se is alpha- and beta-monoclinic Se.
Whereas the amorphous form is Red and glassy or black [27]. Elemental Se oxidize or
reduce slowly and extremely insoluble in water. Specific microorganisms can oxidize
elemental Se to selenite (Seo23 Þ, and selenate (Seo24 Þ [23].
3. Selenium solubility and transformation
Selenate and selenite are the major forms of Se in cultivated soils. The mole
fractions of Se species were used to calculate the total soluble Se supported by eight
selenate and selenite minerals, which might be present in soils. The effect of redox on
total soluble Se at which these minerals can form in neutral soils. None of these
minerals are expected to form in normally cultivated soils. Only manganese selenite
(MnSeO3) is sufficiently stable that it might precipitate in strongly acidic environments. Ferric selenite was included because several investigators reported that it
might be formed in acid soils. Decreasing pH has a negative effect on the solubility of
both minerals which also suggests that Fe2(SeO3)3 is unstable with respect to MnSeO3.
At pH 4, Fe2(SeO3)3 and MnSeo3 can maintain 10 1.5 and 10 6.7 M of Se in the
solution. The previous studies’ reported for soluble Se in acid soils appeared to be close
to the solubility of MnSeO3. Drastic changes in pH have strong effects on precipitation/dissolution and adsorption/desorption processes in soils, and disturbed soil systems may need much longer time than pure systems to re-attain equilibrium. The
concept that Se in soils is governed by an adsorption type of mechanism rather than by
precipitation/dissolution reactions is accepted by most soil scientists. The sorption of
Se in acid soils was related to sesquioxides.
The majority of the Se in agricultural soils exists as selenate or selenite. The sum of
soluble Se can be calculated through the Se supplied through selenate and selenite
minerals that could be calculated in soils by using the mole fractions of Se species.
Changes in the amount of total soluble Se that are necessary for mineralization in
neutral soils as a result of redox conditions. In typical agricultural soils, none of these
minerals would be expected to occur. However, only manganese selenite (MnSeO3) is
stable enough to possibly precipitate in highly acidic conditions. Many researchers
suggested that ferric selenite could be generated in acid soils, so it was included. Both
minerals become less soluble as pH decreases, which is more evidence that Fe2(SeO3)3
3
Selenium and Human Health
is more unstable than MnSeO3 in acidic conditions. The compounds Fe2(SeO3)3 and
MnSeO3 supply 101.5 and 106.7 M of Se, respectively, in soil solution at 4 pH. It
appeared that the solubility of MnSeO3 in acid soils was close to that reported in prior
investigations. It may take significantly longer for disturbed soil systems to re-attain
than pure systems, as large shifts in pH have profound effects on precipitation/dissolution and adsorption/desorption processes in soils. Most soil scientists agree that Se in
soils is controlled by an adsorption mechanism rather than precipitation/dissolution
events and sesquioxides are considered to play a significant role in the sorption of Se
in acid soils.
Soil selenium can be found in a variety of oxidation states, including 2, 4, and 6.
The chemical speciation and environmental stability of selenium compounds are
largely controlled by redox potential and pH. The selenate species predominates
throughout a wide pH range at high redox. At the middle of the redox scale, biselenite
or selenite dominates depending on the pH. We anticipate the presence of elemental
Se and selenide species only at low redox. The amount of Se in the liquid phase of acid
soils may be regulated by adsorption and desorption processes. The chemical forms of
an element in soil are regulated by the redox potential (Eh) and pH. It has been shown
Figure 1 [28] that when elemental Se is given to soils, some of it is rapidly oxidized to
selenite, and that the rate of transfer from selenite to selenate and selenate to elemental Se is considerably slower. There was no correlation between soil pH and the rate of
oxidation of elemental Se, but this oxidation rate did vary. In alkaline soils, selenite
can be easily oxidized to selenate, while in acid soils, this process can be somewhat
challenging [16]. Of all the Se oxides, selenium dioxide has the highest degree of
Figure 1.
Selenium forms at different pE-pH levels (adopted from Seby et al. 2001).
4
Distribution of Selenium in Soils and Human Health
DOI: http://dx.doi.org/10.5772/intechopen.110636
stability. Selenium dioxide is readily reduced to elemental Se by mild reducing agents
[29]. Easily dissolving in water, selenium dioxide reacts with hydrogen peroxide to
produce a weak acid called selenious acid. With the help of mild reducing agents like
sulfur dioxide, selenite can be quickly converted to elemental Se in acidic circumstances [25]. In dry, alkaline conditions, elemental Se persists in the form of sandstones. it has been claimed that some soils contain elemental Se because they contain
bacteria and fungi that can break down selenite and selenate into elemental Se. In
addition, bacteria are capable of converting elemental Se into selenite or selenate
under the right conditions.
4. Selenium bioavailability
The subject of selenium (Se) uptake by plants always remained an important
aspect to study, because of its direct relation to human and animal health through the
food chain. The high Se concentrations in food cause adverse health effects for
humans [10]. While Se plays a crucial part in a variety of biological processes, which
makes it a necessary trace element for both humans and other animals. Se insufficiency has been associated with multiple ailments, both in humans and in livestock.
Due to the fact that Se is both an important nutrient and an element that can be
poisonous, a substantial amount of study has been done to both enhance and decrease
the amount of se that is present in plants.
The selenium uptake and accumulation by plants is a major concern because of its
deficiency or toxicity through crops. To meet human and animal nutritional requirements it is very important to carefully consider the soil and crop factors to understand
how uptake and accumulation in crop plants are influenced. In Se deficient regions
how to increase Se concentrations in plant tissue and ultimately in the food chain.
Whereas the Se-rich areas or seleniferous soils how can reduce the selenium uptake or
accumulation in plants. Plant species also vary in Se accumulation. Unlike normal
agricultural crops, which accumulate very little selenium, selenium accumulator
plants can accumulate exceptionally high quantities of Se when cultivated in seleniferous soils [18, 23]. Yet the essential role of se for plants is not known. Plants uptake
selenate (Se6+) many folds greater than selenite (Se4+). whereas the elemental Se (Se0)
is difficult or impossible for plants to obtain. Plants’ ability to absorb selenium is also
impacted by the chemical and physical properties of the soil, including pH level, soil
texture, amount of organic matter, and the presence of ions like PO4- and SO4-. The
concentration of selenium (Se) in soils and plants that are poor in Se can be increased
by applying selenium (Se) to the soil, the seed, and the plant leaves.
4.1 Influencing factors on selenium bioavailability
The presence of competitive ions, electrical conductivity (Eh), pH, soil texture,
and organic matter content are the five most critical soil variables that influence the
availability of selenium. The Eh and pH: Both soil Eh and pH play a significant role
in determining the chemical form that selenium takes up in soils. Selenate (Se6+) is
the predominant form of selenium (Se) found in well-aerated, alkaline soils. Selenite
(Se4+) is the predominant form of selenium in neutral and acid soils. Due to its
adsorption by clays and iron oxides, selenite is slightly less readily available than other
forms of selenium. The oxidation state of selenium is affected by the pH of the soil
(Figure 1), but the ability of clays and ferric oxide to adsorb selenium is also impacted
5
Selenium and Human Health
by this property [30]. Between pH 3 and 8, there was hardly any change in the amount
of selenite (Se4+) that was adsorbed by Fe2O3. They came to the conclusion that the
pH, and not the layer silicate structure, was the factor that governed selenite (Se4+)
adsorption on clay minerals. The effects of pH on the effect on the sequestration of
selenium by plants was also observed. The greatest quantity of selenite (Se4+) is
available to plants when those plants are cultivated on soils with a pH range of acidic
to neutral. As the pH of the soil rises, hydroxyl ions take the place of selenite (Se4+) on
the adsorption sites. This causes selenite (Se4+) to be released into the solution, which
results in an increase in the availability of the element to plants [10]. Soil texture:
Because selenite (Se4+) is absorbed by clays, the proportion of clay in the soil has a
significant bearing on how well plants are able to take it up. Hence, plants are able to
absorb twice as much Se from sandy-textured soil. Organic matter: Selenium is
released and fixed in part by organic materials. Organometallic complexes may offer
significant Se-adsorbing sites, and organic matter fixes the selenium by removing it
from the soil solution. Because organic matter in soil serves as a source of selenium,
plants absorb more of it than they would in inorganic soils.
5. Accumulation and ecological risk assessment
Selenium accumulation was quantified by calculating the index of Se accumulation
(Igeo). The geo-accumulation index was first purposed by Müller [31], to investigate
heavy metals pollution compared with their background concentration in respective
soils [32], it can be defined as follows:
Geo
accumulationIndex Igeo ¼ log 2
CSe
Soil
k CSe
b
!
(1)
Se
In the above equation, CSe
Soil denotes the selenium contents in soils and Cb denotes
the background concentration of selenium in respective soils. Whereas, in background
concentration, the k is constant and its value is 1.5. The quantification of selenium
contamination in soils was classified by geo-accumulation index criteria (Table 1).
The selenium pollution load index (PLI) was calculated as
6
Geo-accumulation index
Pollution load index
Ecological risk index
Igeo
Level
PLI
Level
ER
Level
Igeo < 0
Uncontaminated
PLI ≤ 1
Low level of
pollution
ER < 40
Low potential
ecological risk
0 < Igeo ≤ 1 Uncontaminated to
moderately
contaminated
1 < PLI ≤ 2
Moderate level 40 ≤ ER < 80
of pollution
Moderate
potential
ecological risk
1 < Igeo ≤ 2 Moderately
contaminated
2 < PLI ≤ 5
High level of
pollution
80 ≤ ER < 160
Considerable
potential
ecological risk
2 < Igeo ≤ 3 Moderately to
heavily
contaminated
PLI > 5
Extremely
high level of
pollution
160 ≤ ER < 320
High potential
ecological risk
Distribution of Selenium in Soils and Human Health
DOI: http://dx.doi.org/10.5772/intechopen.110636
Geo-accumulation index
Pollution load index
Ecological risk index
Igeo
PLI
ER
Level
ER ≥ 320
Very high
potential
ecological risk
Level
Level
3 < Igeo ≤ 4 Heavily
contaminated
4 < Igeo ≤ 5 Heavily to extremely
contaminated
Igeo > 5
Extremely
contaminated
Table 1.
Classification criteria for different indices.
CSoil
Pollution Load Index ¼
Cb
(2)
i
where CSe
Soil is the concentration of selenium in any sample x, and Cb is the
background concentration of selenium in soils before accumulation which was calculated for each soil by determining the Se concentration in the deepest horizon. The
criteria for classifying the pollution load index is presented in Table 1.
The potential ecological risk of selenium accumulation to the ecosystem was calculated by the ecological risk index, which was first suggested by Hakanson [33]. The
potential ecological risk index was by the following equation:
Eir ¼ T ir
CSe
Soil
CSe
b
!
(3)
where T ir is the toxic effect of selenium (Se = 10), CSe
Soil the concentration of
Se
selenium in soil samples, Cb is the background concentration of selenium in soils.
Classifying criteria is presented in Table 1.
6. Health risk assessment
The USEPA approach, which has been extensively used around the world, can be
used to assess the health risks associated with heavy metal exposure through food
consumption [34]. By calculating the target hazard quotient (HQ) and the hazard
index (HI) for selenium, the health risks of ingesting Se will be measured. Below are
the equations as follow:
THQ ¼
EF ∗ ED ∗ Cveg ∗ IRveg
BW ∗ AT ∗ RfD
(4)
The recommended daily intake (RfD) is the amount of selenium consumed each
day through plant-based foods that are deemed to be safe over the course of a lifetime.
Depending on the age, sex, and standard tolerable daily intake of Se, the range is 0.02
to 0.075 mg kg1 day1 [35]. EF stands for exposure frequency (365 days per year), ED
for exposure duration (74.68 years), C for food’s selenium content, IR for food’s
7
Selenium and Human Health
ingestion rate, BW for average body weight, and AT for an average duration of noncarcinogen exposure (365 days divided by 74.68 years).
The following equation was used to calculate the hazard index (HI) of consuming
food while simultaneously absorbing multiple heavy metals:
HI ¼
n
X
THQ i
(5)
i
A negative effect is anticipated to be seen by the exposed population when the HQ/
HI values are equal to or higher than 1 [36].
7. Conclusion
Selenium essentiality and toxicity and the narrow range between them made it
very critical to keep an eye on selenium deficiency and toxicity through the food chain
in humans and animals. While most of the selenium in our food is supplied through
soils in our food. Whereas in soils selenium contents depend on soil parent material
inheriting different selenium contents through different minerals in the soils. Besides
the total selenium contents in soils, other factors also play important role in its
availability to plants including, pH, Eh, clay, organic matter, selenium fractions,
species, and competing ions which ultimately play a role in its deficiency and toxicity.
It is necessary to monitor the food grown in different soils for selenium deficiency or
toxicity. While evaluating the degree of toxicity it is necessary to calculate the selenium environmental or ecological risks and health risks associated with high selenium.
In the end, it is necessary to consider the soil properties and other factors which
influence selenium availability.
Acknowledgements
The authors acknowledge (Prof. Dr. Mohammad Saleem Akhtar, Arid Agriculture
University Rawalpindi) for guiding and contributing throughout the research.
Conflict of interest
The authors declare no conflict of interest.
8
Distribution of Selenium in Soils and Human Health
DOI: http://dx.doi.org/10.5772/intechopen.110636
Author details
Muhammad Imran1*, Zhikun Chen1, Ayaz Mehmood2, Shah Rukh3, Wang Weixie1,
Waleed Asghar4 and Farhan Iftikhar5
1 Key Laboratory of Soil Resource and Biotech Application, Shaanxi Academy of
Sciences, Xi’an Botanical Garden of Shaanxi (Institute of Botany Shaanxi Province),
Xi’an, China
2 Department of Agricultural Sciences, University of Haripur, Pakistan
3 National Centre of Excellence in Geology, University of Peshawar, Pakistan
4 Faculty of Life and Environmental Sciences, Department of Environmental
Sciences, University of Yamanashi, Kofu, Japan
5 School of Soil and Water Conservation, Beijing Forestry University, Beijing,
P.R. China
*Address all correspondence to: changwani_baloch2005@yahoo.com
© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of
the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided
the original work is properly cited.
9
Selenium and Human Health
References
[1] Rayman MP. Selenium and human
health. Nutrition Reviews. 2012;379:
1256-1268
[2] Steinbrenner H, Speckmann B,
Pinto A, Sies H. High selenium intake
and increased diabetes risk:
Experimental evidence for interplay
between selenium and carbohydrate
metabolism. Journal of Clinical
Biochemistry & Nutrition. 2010;48(1):
40-45
[3] Yoon JC, Pulgserver P, Chen G,
Donovan J, Wu Z, Rhee J, et al. Control
of hepatic gluconeogenesis through the
transcriptional coactivator PGC-1.
Nature. 2001;413(6852):131-138
[4] Fordyce F. Selenium deficiency and
toxicity in the environment. In:
Selinus O, editor. Essentials of Medical
Geology. Dordrecht: Springer; 2013.
pp. 375-416
[5] N’eve J. Selenium as a ‘nutraceutical’:
How to conciliate physiological and
supra-nutritional effects for an essential
trace element. Current Opinion in
Clinical Nutrition and Metabolic Care.
2002;5(6):659-663
[6] Weekley CM, Harris HH. Which
form is that? The importance of selenium
speciation and metabolism in the
prevention and treatment of disease.
Chemical Society Reviews. 2013;42(23):
8870-8894
[7] Mayland HF, James LF, Panter KE,
Sonderegger JL. Selenium in seleniferous
environments. In: Jacobs LW, editor.
Selenium in Agriculture and
Environments. Madison: Soil Science
Society of America; 1989. pp. 15-50
[8] Imran M, Akhtar MS, Khan KS,
Khalid A, Mehmood A, Rukh S, et al.
10
Total and extractable soil selenium
contents variation within and across the
parent materials. Journal of Biodiversity
and Environmental Sciences. 2016;9:
175-186
[9] Tan J. The Atlas of Endemic Diseases
and their Environments in People’s
Republic of China. Marrickville: Science
Press; 1989. p. 88
[10] Terry N, Zayed AM, deSouza MP.
Tarun AS selenium in higher plants.
Annual Review of Plant Physiology and
Plant Molecular Biology. 2000;51:
401-432
[11] Joy EJM, Broadley MR, Young SD,
Black CR, Chilimba ADC, Ander EL,
et al. Soil type influences crop mineral
composition in Malawi. Science of The
Total Environment. 2015;505:587-595
[12] Dinh QT, Cui Z, Huang J, Tran TAT,
Wang D, Yang W, et al. Selenium
distribution in the Chinese environment
and its relationship with human health:
A review. Environment International.
2018;112:294-309
[13] Ramesh R, Subramanian V, Van-
Grieken R. Heavy metal distribution in
sediments of Krishna river basin, India.
Environmental Geology and Water
Sciences. 1990;15:207-216
[14] Merrill DT, Manzione MA,
Peterson IJ, Parker DS, Chow W,
Hobbs AO. Field evaluation of arsenic
and selenium removal by iron
coprecipitation. Journal - Water
Pollution Control Federation. 1986;58:
18-26
[15] Balistrieri LS, Chao TT. Selenium
adsorption by goethite. Soil Science
Society of America Journal. 1987;51:
1145-1151
Distribution of Selenium in Soils and Human Health
DOI: http://dx.doi.org/10.5772/intechopen.110636
[16] Balistrieri LS, Chao TT. Adsorption
[26] Cutter GA, Bruland KW. The marine
of Se by amorphous iron oxyhydroxide
and manganese dioxide. Geochimica et
Cosmochimica Acta. 1990;54(3):739-751
biogeochemistry of selenium: Areevaluation. Limnology and
Oceanography. 1984;29:1179-1192
[17] Sarquis M, Mickey CD. Selenium.
[27] Sarathchandra SU, Watkinson IH.
Part 1: Its chemistry and occurrence.
Journal of Chemical Education. 1980;57:
886-889
[18] Gissel-Nielsen G, Bisbjerg B. The
uptake of applied selenium from soils by
plants. 2. The utilization of various
selenium compounds. Plant and Soil.
1970;32:382-396
[19] Eisler R. Selenium hazards to fish,
wildlife, and invertebrates: A synoptic
review. U.S. Fish and Wildlife Servo
BioI. Rep. 1985; 85(1.5). U.S. Gov. Print.
Office, Washington, DC
[20] Vokal-Borek H. Selenium, Univ. of
Stockholm Inst. of Physics Rep. Sweden:
Stockholm; 1979. pp. 79-16
[21] Ryden IC, Syers JK, Tillman RW.
Inorganic anion sorption and interactions
with phosphate sorption by hydrous
ferric oxide gel. Journal of Soil Science
and Plant Nutrition;187(38):211-217
[22] Weast RC. Handbook of Chemistry
and Physics. 66th ed. Ceveland, OH: The
Chemical Rubber Co.; 1989
[23] Lakin HW. Selenium accumulation
in soils and its absorption by plants and
animals. Geological Society of America
Bulletin. 1972;83:181-189
Oxidation of elemental selenium to
selenite by Bacillus megaterium. Science
(Washington, DC). 1981;21:600-601
[28] Rosenfeld I, Beath OA. Selenium:
Geobotany, Biochemistry, Toxicity, and
Nutrition. New York: Academic Press;
1964
[29] Tolu J, LeHécho I, Bueno M, Thiry Y,
Potin-Gautier M. Selenium speciation
analysis at trace level in soils. Analytical
Chemistry Acta. 2011;684:126-133
[30] Imran M, Akhtar MS, Mehmood A,
Rukh S, Khan A, Zhikun C, et al. Soil
selenium transformation across different
parent materials in Pothwar uplands of
Pakistan. Arabian Journal of
Geosciences. 2020;13:1098.
DOI: 10.1007/s12517-020-06111-1
[31] Muller G. Schwermetalle in den
sediments des RheinsVeranderungen
seitt 1971. Umschan. 1979;79:778-783
[32] Zhang P, Qin C, Hong X, Kang G,
Qin M, Yang D, et al. Risk assessment
and source analysis of soil heavy metal
pollution from lower reaches of Yellow
River irrigation in China. Science of The
Total Environment. 2018;633:1136-1147
[33] Hakanson L. An ecological risk index
[24] Elrashidi MA, Adriano DC,
Workman SM, Lindsay WL. Chemical
equilibria of selenium in soils: A
theoretical development. Soil Science.
1987;144:141-152
[25] Cutter GA. Selenium in reducing
waters. Science (Washington, DC). 1982;
217:829-831
11
for aquatic pollution control. Sedimentol
Approaches Water Research. 1980;14:
975-1001
[34] Xiao R, Guo D, Ali A, Mi S, Liu T,
Ren C, et al. Accumulation, ecologicalhealth risks assessment, and source
apportionment of heavy metals in paddy
soils: A case study in Hanzhong, Shaanxi,
Selenium and Human Health
China. Environmental Pollution. 2019;
248:349-357
[35] USEPA (United States Environ. Prot.
Agency). Integrated Risk Information
System. 2015. Available from: http://
www.epa.gov/iris
[36] Cao C, Chen XP, Ma ZB, Jia HH,
Wang JJ. Greenhouse cultivation
mitigates metal-ingestion-associated
health risks from vegetables in
wastewater-irrigated agroecosystems.
Science of the Total Environment. 2016;
560:204-211
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Os caminhos da divulgação de conhecimento em terapia ocupacional no Brasil: o papel dos Cadernos de Terapia Ocupacional da UFSCar
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Cadernos de Terapia Ocupacional da UFSCar
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cc-by
| 862
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ISSN 0104-4931
Cad. Ter. Ocup. UFSCar, São Carlos, v. 23, n. 3, p. 449-450, 2015
http://dx.doi.org/10.4322/0104-4931.ctoED2303 ISSN 0104-4931
Cad. Ter. Ocup. UFSCar, São Carlos, v. 23, n. 3, p. 449-450, 2015
http://dx.doi.org/10.4322/0104-4931.ctoED2303 Os caminhos da divulgação de conhecimento
em terapia ocupacional no Brasil: o papel dos
Cadernos de Terapia Ocupacional da UFSCar O periódico Cadernos de Terapia Ocupacional da UFSCar foi gerado em conversas
informais entre os docentes do curso de Terapia Ocupacional da Universidade Federal de
São Carlos (UFSCar), tendo se constituído oficialmente em um projeto editorial em 1989. Vivíamos uma época bastante dinâmica para a terapia ocupacional brasileira e antevíamos
a necessidade desta publicação, que canalizaria a divulgação do conhecimento que
estava sendo produzido pelos profissionais da área. Já contávamos com trabalhos de boa
qualidade, que estavam sendo publicados em revistas de áreas afins, mas precisávamos de
um periódico que pudesse centralizar a produção que estava sendo efetivada. Esta publicação daria suporte também ao grande número de cursos de terapia ocupacional
que estavam se instalando no Brasil no final da década de 1980, os quais sentiam a falta
de material bibliográfico nacional para melhor fundamentar os conteúdos ministrados,
obrigando-os a utilizarem referências prioritariamente estrangeiras. Tomei a frente desta tarefa, liderando a sua edição por quase dez anos e participando
como Editora Associada pelos últimos cinco anos. Foi também no início da década de 1990 que a terapia ocupacional brasileira começou
a formar pesquisadores, mestres e doutores, prioritariamente docentes das universidades
públicas do país, pois eram estas instituições que exigiam e ofereciam melhores condições
para que tal formação se concretizasse. Embora estes profissionais estivessem se capacitando
em programas fora da terapia ocupacional, uma vez que não existiam programas de
pós- graduação stricto sensu na área, no país, esta condição possibilitava ampliar a produção
de conhecimento específico. Foi dentro desse contexto que lançamos o primeiro número dos Cadernos de Terapia
Ocupacional, em 1990. No entanto, muitos desafios ameaçaram a continuidade desta publicação. Apesar de visualizarmos um potencial enorme para a divulgação científica da terapia
ocupacional, os profissionais de nossa área tinham pouca experiência com a publicação de
artigos. Dizia-se que tínhamos uma tradição oral, que transmitíamos nossos conhecimentos
em cursos, palestras e conferências, mas éramos extremamente impermeáveis quando se
tratava de escrever artigos científicos. Isso se refletiu em nossa iniciativa de publicação, que
sofreu, nos primeiros anos de sua existência, com a dificuldade para receber submissões. Outro grande desafio que enfrentamos desde o início foi a escassez de recursos para
a edição desta Revista, que foi inicialmente financiada pelo Departamento de Terapia
Ocupacional da UFSCar e, mais tarde, se tornou possível com recursos da administração
central da Universidade Federal de São Carlos. Cad. Ter. Ocup. UFSCar, São Carlos, v. 23, n. 3, p. 449-450, 2015 Os caminhos da divulgação de conhecimento
em terapia ocupacional no Brasil: o papel dos
Cadernos de Terapia Ocupacional da UFSCar Hoje, além desse apoio financeiro, o periódico Os caminhos da divulgação de conhecimento em terapia ocupacional no Brasil:
o papel dos Cadernos de Terapia Ocupacional da UFSCar 450 recebe também recursos do Conselho Regional de Fisioterapia e Terapia Ocupacional da
Terceira Região, CREFITO-3. A falta de recursos humanos também foi uma realidade e esta se mantém até os dias de
hoje, com um número bastante reduzido de colegas que trabalham para tornar possível
a veiculação deste periódico. A inserção crescente dos profissionais em programas de pós-graduação stricto sensu a
partir dos anos 1990 e a criação do Programa de Pós-Graduação em Terapia Ocupacional
da UFSCar em 2010 aumentaram de forma significativa o número e a qualidade dos
artigos publicados. As exigências para uma revista científica foram sendo acirradas durante este período e,
por maiores que sejam as dificuldades para atendê-las, estes fatores contribuíram para a
profissionalização da Revista, que hoje está em importantes fontes de indexação (LILACS,
CINAHL – EBSCO, ProQuest- Social Services Abstracts, ProQuest- Nursing & Allied
Health Source, CUIDEN, LATINDEX, OTDATABASE, Periódica, DOAJ - Directory
of Open Access Journals, Portal de Periódicos da CAPES e Ulrich’s International Periodical
Directory) e encontra-se classificada na categoria B1 do QUALIS CAPES. A Revista
aumentou a sua periodicidade, com quatro edições anuais, e também cresceu o número
de artigos publicados em cada número. Nestes 25 anos de vida, este instrumento divulgou a profissão dentro e fora do país, e
acrescentou qualidade à sua produção, tornando-se um dos principais periódicos brasileiros
específicos da área. Após estes anos todos participando do processo de criação e de crescimento dos Cadernos
de Terapia Ocupacional da UFSCar, comunico minha saída como Editora Associada
da Revista, com a certeza de que a iniciativa de sua criação foi acertada, não só para a
divulgação do conhecimento em terapia ocupacional, como também como um veículo
que pode estimular e ajudar a alicerçar o pensamento científico e a fundamentação de
nossas práticas. Profa. Dra. Maria Luísa Guillaumon Emmel
Professora Titular do Departamento de Terapia Ocupacional e do Programa de
Pós-graduação em Terapia Ocupacional da Universidade Federal de São Carlos -
UFSCar, São Carlos, SP, Brasil. Profa. Dra. Maria Luísa Guillaumon Emmel
Professora Titular do Departamento de Terapia Ocupacional e do Programa de
Pós-graduação em Terapia Ocupacional da Universidade Federal de São Carlos -
UFSCar, São Carlos, SP, Brasil.
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“Fluctuograms” Reveal the Intermittent Intra-Protein Communication in Subtilisin Carlsberg and Correlate Mechanical Coupling with Co-Evolution
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PLOS computational biology/PLoS computational biology
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cc-by
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Abstract The mechanism of intra-protein communication and allosteric coupling is key to understanding the structure-property
relationship of protein function. For subtilisin Carlsberg, the Ca2+-binding loop is distal to substrate-binding and active sites,
yet the serine protease function depends on Ca2+ binding. The atomic molecular dynamics (MD) simulations of apo and
Ca2+-bound subtilisin show similar structures and there is no direct evidence that subtilisin has alternative conformations. To model the intra-protein communication due to Ca2+ binding, we transform the sequential segments of an atomic MD
trajectory into separate elastic network models to represent anharmonicity and nonlinearity effectively as the temporal and
spatial variation of the mechanical coupling network. In analogy to the spectrogram of sound waves, this transformation is
termed the ‘‘fluctuogram’’ of protein dynamics. We illustrate that the Ca2+-bound and apo states of subtilisin have different
fluctuograms and that intra-protein communication proceeds intermittently both in space and in time. We found that
residues with large mechanical coupling variation due to Ca2+ binding correlate with the reported mutation sites selected
by directed evolution for improving the stability of subtilisin and its activity in a non-aqueous environment. Furthermore,
we utilize the fluctuograms calculated from MD to capture the highly correlated residues in a multiple sequence alignment. We show that in addition to the magnitude, the variance of coupling strength is also an indicative property for the
sequence correlation observed in a statistical coupling analysis. The results of this work illustrate that the mechanical
coupling networks calculated from atomic details can be used to correlate with functionally important mutation sites and
co-evolution. Citation: Silvestre-Ryan J, Lin Y, Chu J-W (2011) ‘‘Fluctuograms’’ Reveal the Intermittent Intra-Protein Communication in Subtilisin Carlsberg and Correlate
Mechanical Coupling with Co-Evolution. PLoS Comput Biol 7(3): e1002023. doi:10.1371/journal.pcbi.1002023 Citation: Silvestre-Ryan J, Lin Y, Chu J-W (2011) ‘‘Fluctuograms’’ Reveal the Intermittent Intra-Protein Comm
Mechanical Coupling with Co-Evolution. PLoS Comput Biol 7(3): e1002023. doi:10.1371/journal.pcbi.1002023 Editor: Ruth Nussinov, National Cancer Institute, United States of America and Tel Aviv University, Israel Received October 14, 2010; Accepted February 13, 2011; Published March 24, 2011 Copyright: 2011 Silvestre-Ryan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract Funding: We acknowledge the financial support from the American Chemical Society Petroleum Research Fund; ACS-PRF-49727-DNI6, the Energy Biosciences
Institute; OO0J04, the DOE Office of the Biomass Program; subcontract ZGB-0-40593-01 from the National Renewable Energy Laboratory, and the College of
Chemistry, University of California, Berkeley. We also thank the computational resources provided by NERSC, which is supported by the Office of Science of the
U.S. Department of Energy under Contract No. DE-AC02-05CH11231. The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E mail: jwchu@berkeley edu Competing Interests: The authors have declared that no competing interests exist. * E-mail: jwchu@berkeley.edu PLoS Computational Biology | www.ploscompbiol.org ‘‘Fluctuograms’’ Reveal the Intermittent Intra-Protein
Communication in Subtilisin Carlsberg and Correlate
Mechanical Coupling with Co-Evolution Jordi Silvestre-Ryan1, Yuchun Lin2, Jhih-Wei Chu2*
1 Department of Bioengineering, University of California, Berkeley, Berkeley, California, United States of America, 2 Department of Chemical and Biomolecular Engineering,
University of California, Berkeley, Berkeley, California, United States of America Jordi Silvestre-Ryan1, Yuchun Lin2, Jhih-Wei Chu2*
1 Department of Bioengineering, University of California, Berkeley, Berkeley, California, United States of America, 2 Department of Chemical and Biomolecular Engineering,
University of California, Berkeley, Berkeley, California, United States of America ordi Silvestre-Ryan1, Yuchun Lin2, Jhih-Wei Chu2* March 2011 | Volume 7 | Issue 3 | e1002023 Results The native structure of subtilisin shown in Figure 1(a) has 17
segments of helices and sheets connected by loops and turns. Subtilisin contains several commonly encountered right-handed
bab motifs and one rarely encountered left-handed bab motif (b2-
a3-b4), for which the b1–b2 loop (Asp32-Asp41) and the b2-a3
loop (Ser49-His63) cross each other as circled in Figure 1(a). We
name the loops and turns of subtilisin based on the structural
elements that they connect; i.e., the b1–b2 loop connects b1 and
b2. In the Ca2+-bound and apo trajectories of subtilisin, the time
evolution of Ca root-mean-square differences (RMSDs) show that
both Ca2+-bound and apo subtilisin remained close to the
reference X-ray structure with RMSDs ,1.5 A˚ (Figure S1). mechanical coupling network in protein structure, their integra-
tion
also
determines
the
ensemble
distribution
of
protein
conformation. Therefore, variation in the mechanical coupling
network of protein structure due to molecular signals is linked to
allosteric coupling as well as the concomitant population shift. Subtilisin Carlsberg is a serine protease widely used in industry and
protein engineering studies [4,5,6,7]. Similar to numerous enzymes
and signaling proteins, the functioning of subtilisin is regulated by
Ca2+. Subtilisin has a strong Ca2+-binding site with a dissociation
constant of ,100 nM and Ca2+ exhibits significant effects on stability
and folding kinetics [23,24]. The fold of an engineered construct
without the Ca2+-binding loop is very close to that of native subtilisin
[25], and there is no direct evidence of alternative structure. Ca2+-
mediated intra-protein communication in subtilisin may thus proceed
via local variation in the mechanical coupling network. We also calculate the RMSFs (root-mean-square fluctuation) of Ca
atoms to quantify their flexibility; values from the first 4 ns trajectory
are shown in Figure 1(c). Residues in loops and turns are more flexible
than those in rigid secondary structures as expected. A clear feature is
that apo subtilisin has higher RMSF’s in the Ca2+-binding loop
(Val71-Leu83) and around Asp41 (highlights in Figure 1(c)). The
negatively charged Asp41 in the b1–b2 loop (Asp32-Asp41) loop
coordinates with Ca2+ if present. The RMSF’s predicted via the Ca-
SC-ENM (SC;sidechain) are also shown in Figure 1(c) to illustrate
that the RMSF’s observed in all-atom MD are preserved at the
coarse-gain scale by using fluctuation-matched force constants. To test this hypothesis, we consider the anharmonicity and
nonlinearity of protein dynamics in an effective manner. Results First, we
recognize that the ensemble distribution of protein structures is
determined by the mechanical coupling between amino acids, and
shifts in the population of protein structures would reflect in the
variation of mechanical coupling network. As the PMF of protein
structure is extremely complex, modeling usually employs simplified
basis functions [21,22]. Here, we use an elastic network model (ENM)
[26,27] to approximate the distribution of protein structures. As the
structural distribution corresponding to an ENM is determined by
model parameters, we adjust the lengths and force constants of elastic
bonds to match the statistics of structural fluctuations collected in a
molecular dynamics (MD) simulation with explicit solvent [28]. The
atomic details encoded in the sequence and protein’s environment
are thus reflected in the values of model parameters. The simplicity of
harmonic potentials allows for the development of robust computa-
tional methods such as fluctuation matching for inverting structural
fluctuations into force constants [28,29], which we employ for all of
the calculations performed in this work. g
y
g
To capture the anharmonicity and nonlinearity sampled in all-
atom MD simulations, in each of the sequential time windows of a
user-specified size, we calculate the bond lengths of a Ca-SC-ENM
as mean distances and the force constants by fluctuation matching
[28]. In analogy to the spectrogram of sound waves, the temporal
evolution of the Ca-SC-ENM is termed the ‘‘fluctuogram’’, which
records the choreography of protein dynamics. The window size
Dt is an adjustable parameter, which specifies the timescale with
which the Hamiltonian of a Ca-SC-ENM is used to approximate
the structural fluctuations of subtilisin. A small Dt gives high time
resolution but force constants are determined with a fewer number
of configurations. A larger Dt gives lower time resolution but the
force constants are determined with more configurations. Another
consideration is that Ca-SC-ENM becomes less representative for
configurations sampled in a longer MD segment, and we limit Dt
to a few ns for fluctuogram calculations. Over 100 ns atomic
trajectories, we employ a window size of 4 ns. Fluctuograms with
Dt = 2 ns or 10 ns are qualitatively similar (results not shown). We To effectively represent the anharmonicity and nonlinearity in
protein
dynamics,
we
compute
separate
ENM’s
from
the
sequential segments of a long MD trajectory to follow the time
evolution of the mechanical coupling network in subtilisin. Author Summary A hallmark of protein molecules is their machine-like
behaviors while carrying out biological functions. At the
molecular level, molecular signals such as binding a metal
ion at an action site can cause long-range effects and alter
protein function. Such phenomena are often referred to as
intra-protein communication or allosteric coupling. Eluci-
dating the underlying mechanisms could lead to novel
discovery of molecular modulators to regulate protein
function in a more specific and effective manner. A long-
standing puzzle is the roles of the anharmonicity and
nonlinearity in protein dynamics. To incorporate these
characters in modeling intra-protein communication, we
devise a ‘‘fluctuogram’’ analysis to record the choreogra-
phy of allosteric coupling in an atomic molecular dynamics
simulation. We show that fluctuogram analysis can bridge
the results of physics-based simulation and sequence
alignment in bioinformatics by capturing the residues that
exhibit high correlation in a multiple sequence alignment. We also show that the fluctuograms calculated from
atomic details have the potential to be applied as a tool to
select mutation sites for modulating protein function. We computed the fluctuograms of Ca2+-bound and apo subtilisin
from 100 ns all-atom MD trajectories in explicit water. The
calculated fluctuograms demonstrate that intra-protein communica-
tion proceeds intermittently both in space and in time. We found that
residues with large mechanical coupling variation due to Ca2+
binding significantly overlap with the gain-of-function mutation sites
reported in the directed evolution studies that aim to enhance the
stability and activity of subtilisin by random mutations and screening
[31,32,33,34,35,36]. Furthermore, we utilize the fluctuograms
calculated from atomic MD simulation to capture the highly
correlated residues in a multiple sequence alignment. In addition to
the strength of mechanical coupling, we show that the variance of
coupling strength is also an indicative property for the high sequence
correlation observed in a statistical coupling analysis [37,38]. Overall,
our results illustrate that the mechanical coupling networks and
fluctuograms calculated from atomic details can be used to correlate
with functionally important mutation sites and co-evolution. The Fluctuogram of Protein Dynamics recognition [30], we refer to the temporal variation of the
mechanical coupling network as ‘‘fluctuogram’’, which records the
choreography of protein dynamics. Introduction induce local conformational changes and affect residues in the
next layer via mechanical coupling [1]. The propagation of
molecular signals may thus follow a sequential (stepwise) path
[11,12,13] and pathways of allosteric coupling may be defined
based on the contacted amino acids observed in a protein structure
[14,15,16,17,18]. The population shift model emphasizes that the
ensemble distribution of protein structures depends on ligand
binding or other forms of molecular signals [2], and the equilibria
between pre-existing conformations would shift as a result
[11,12,13]. The response of structural distribution is often non-
linear, leading to properties such as cooperative binding. It has
been shown in many examples that the population shift model can
be used to predict the thermodynamics of allosteric coupling and
protein stability [19,20]. During protein dynamics, the temporal and spatial couplings
between amino acids are governed by the atomic details encoded in
the sequence and protein’s environment. A critical outcome is that
ligand binding, chemical modification, and changes in solvent
conditions not only alter structures and thermal motions locally:
molecular signals can propagate through the protein matrix and
affect the properties of distal sites [1,2,3]. For subtilisin Carlsberg,
the Ca2+-binding loop in proximity to the N-terminal is distant to
the substrate-binding and active sites, yet the protease function and
stability depends on Ca2+ binding [4,5,6,7]. Allosteric coupling is a
ubiquitous mechanism by which protein functions are regulated and
coordinated in the cell [8,9,10]. Mechanistic understanding at the
molecular level, though, is still under development. The ensemble distribution of protein conformation can be
represented by the potential of mean forces (PMF) of the relevant
degrees of freedom used for structural description, such as the
positions of all heavy atoms and polar hydrogen. Other degrees of
freedom are considered averaged out according to statistical
mechanics [21,22]. The mean forces not only reflect the The classical induced-fit and population shift models highlight
two
essential
features
of
intra-protein
communication:
the
mechanical coupling (interaction energetics) between amino acids
and the ensemble distribution of protein structures [1,2,3]. According to the induced-fit theory, molecular signals at a site March 2011 | Volume 7 | Issue 3 | e1002023 1 The Fluctuogram of Protein Dynamics PLoS Computational Biology | www.ploscompbiol.org Results In
analogy to the spectrogram of sound waves (temporal variation of
spectral density) widely used in the fields of linguistics and speech March 2011 | Volume 7 | Issue 3 | e1002023 2 The Fluctuogram of Protein Dynamics Figure 1. The structure and mechanical coupling network of subtilisin. (a) A ribbon representation of the X-ray structure of subtilisin (PDB
ID: 1OYV). The bound Ca2+ is shown in ball. The secondary structural elements are labeled and the residues of the catalytic triad are listed. A
sequential conformational change that represents a pathway of intra-protein communication induced by Ca2+ binding is shown via orange arrows. (b) Residues exhibit significant mechanical coupling in subtilisin. Residues cover the upper-right half are colored red and those cover the lower-left
are colored green. (c) The root of mean square fluctuation (RMSF) of Ca atoms in A˚ calculated from the first 4 ns of Ca2+-bound (top) and apo (down)
simulations. (d) Contour plot of the difference in inter-residue force constant (kcal/mol/A˚2) between Ca2+-bound and apo simulations. Force constants
are calculated from the first 4 ns of Ca2+-bound and apo simulations. doi:10.1371/journal.pcbi.1002023.g001 Figure 1. The structure and mechanical coupling network of subtilisin. (a) A ribbon representation of the X-ray structure of subtilisin (PDB
ID: 1OYV). The bound Ca2+ is shown in ball. The secondary structural elements are labeled and the residues of the catalytic triad are listed. A
sequential conformational change that represents a pathway of intra-protein communication induced by Ca2+ binding is shown via orange arrows. (b) Residues exhibit significant mechanical coupling in subtilisin. Residues cover the upper-right half are colored red and those cover the lower-left
are colored green. (c) The root of mean square fluctuation (RMSF) of Ca atoms in A˚ calculated from the first 4 ns of Ca2+-bound (top) and apo (down)
simulations. (d) Contour plot of the difference in inter-residue force constant (kcal/mol/A˚2) between Ca2+-bound and apo simulations. Force constants
are calculated from the first 4 ns of Ca2+-bound and apo simulations. doi:10.1371/journal.pcbi.1002023.g001 also overlap the sequential time windows by Dt=2 to better resolve
the transitions around the timescale of Dt. In the following, we
characterize mechanical coupling variation and mechanisms of
intra-protein communication via fluctuograms. CG sites. Fluctuation matching determines the force constants
from the statistics of inter-site distances [28]. Differences in kIJ’s
between Ca2+-bound and apo simulations for the first 4 ns are
shown in Figure 1. Results Many kIJ’s in apo subtilisin are lower than
those in the Ca2+-bound state, even though the structures are close
to the initial X-ray structure and to each other. The Fluctuogram of Protein Dynamics observed. The strong electrostatic coupling between Asp41 and
Ca2+ (Figure 1(b)) results in a very large force constant of
133 kcal/mol/A˚ 2, while the His39-Thr207 coupling in Ca2+-
bound subtilisin has a force constant of 6.5 kcal/mol/A˚ 2. Force
constants between I-I+4 residue pairs in a helices are 2–7 kcal/
mol/A˚ 2. Therefore, a cutoff of 2.5 kcal/mol/A˚ 2 is used to assign
whether a residue pair with sequence difference larger than three
has significant mechanical coupling. The force constants of
covalent linkages along the peptide backbone ( I{J
j
jƒ3) are
significantly larger than those of I{J
j
jw3. observed. The strong electrostatic coupling between Asp41 and
Ca2+ (Figure 1(b)) results in a very large force constant of
133 kcal/mol/A˚ 2, while the His39-Thr207 coupling in Ca2+-
bound subtilisin has a force constant of 6.5 kcal/mol/A˚ 2. Force
constants between I-I+4 residue pairs in a helices are 2–7 kcal/
mol/A˚ 2. Therefore, a cutoff of 2.5 kcal/mol/A˚ 2 is used to assign
whether a residue pair with sequence difference larger than three
has significant mechanical coupling. The force constants of
covalent linkages along the peptide backbone ( I{J
j
jƒ3) are
significantly larger than those of I{J
j
jw3. Subtilisin sites with large mechanical coupling variation often
occur at loops and the connecting regions of rigid secondary
structures, Figure 2(a,b). These sites, however, are highly specific
and not all flexible regions have large mechanical coupling
variation. The 25 most affected residues in subtilisin (within top
10%) due to Ca2+ binding (the time average of Dkt
I’s.20 kcal/
mol/A˚ 2) are listed in Figure 2(a), and their spatial locations are
shown in Figure 2(b) in red. As an example, around Asp75 at the
edge of the Ca2+-binding loop and Asp41 that coordinates Ca2+ if
present, Dkt
I have large and negative values, indicating weaker
mechanical coupling in apo subtilisin. The nearby N-terminal site
(residue 2) shows a similar behavior. In addition to such
anticipated results, it is clear from Figure 2(a,b) that Ca2+-binding
causes mechanical coupling variation not only locally around the
Ca2+-binding loop but also residues that are far away. Through
the mechanical coupling network in subtilisin, the local molecular
signal of Ca2+ binding propagates across the network and causes
variation at distal sites. The Fluctuogram of Protein Dynamics g
y
g
j
j
Representative residues with many instances of significant
mechanical coupling (kIJ.2.5 kcal/mol/A˚ 2) and larger sequence
separation ( I{J
j
j.3) are shown in Figure 1. Following residue
pairs with significant mechanical coupling, the Ca2+ binding loop
(Val71-Leu83) can be linked to distal regions in subtilisin. This
result is based on the statistics of structural fluctuations via
fluctuation matching and affirms that intra-protein communica-
tion can occur through the mechanical coupling network in
subtilisin. An important residue is Asp41, which coordinates with
Ca2+ if present. Asp41 locates at the C-terminal end of the b1–b2
loop (Asp32-Asp41), and Asp32 at the other end is one of the three
catalytic triad residues (Asp32, His63, and Ser220). As k33,95 is
significant, the Ca2+ binding loop can be linked to Leu95 from
Asp41 via Thr33 (Figure 1(b)). The junction at Asp32 in the b1–b2
loop is mechanically coupled to the site around His63, a triad
residues located in the b2-a3 loop (Ser49-His63), which also
couples with the catalytic Ser220 in the a14 helix (Thr219-
Lys236). Molecular signals at Ca2+-binding loop can thus
propagate to the active site from Asp41 through residue pairs
having significant mechanical coupling. In establishing this link,
we also apply the fact that amino acids close in sequence are
mechanically coupled through the backbone [39]. Tertiary
contacts with strong mechanical coupling provide shortcuts to
residues with larger sequence separation. In Figure 1(b), residues
with significant mechanical coupling that cover the upper-right
half of subtilisin are colored in red. Since the stability of subtilisin strongly depends on Ca2+,
residues with large mechanical coupling variation between Ca2+-
bound and apo simulations may be hot spots for modulating
protein stability. To test this hypothesis, we compare the residues
shown in red in Figure 2(a,b) with those identified by random
mutations and screening to have positive effects on activity and
stability. Since subtilisin has been used as a model system for
methodology development in protein engineering [7], many
mutation sites had been identified. For example, in converting
subtilisin E to its thermophilic homolog via directed evolution,
Zhao and Arnold found 9 mutation sites after screening ,50,000
clones [31]. Mutations at these sites (Figure 2(c)) increase subtilisin
lifetime at 60uC .200 times longer than that of the wild type [31]. The Fluctuogram of Protein Dynamics Among the 9 sites identified by Zhao and Arnold, 7 (9, 14, 75, 165,
180, 193, 217) are covered to within 61 in residue number by the
25 sites calculated from atomic MD simulations for having large
mechanical coupling variation (Figure 2(a,b)). The specific amino
acid type of a mutant residue is definitely a key in gaining function
in directed evolution, but here we focus on comparing the location
of mutation sites. In addition to Asp41, the terminals of the Ca2+-binding loop,
Val71 and Leu83, are mechanically coupled to the surrounding
residues. Originated from the ends of the Ca2+-binding loop, the
residue pairs with significant mechanical coupling that cover the
lower-left half of subtilisin are colored in green in Figure 1(b). The
grouping of red and green residues is a structure-based catego-
rization, and does not grant their independence. In fact, red and
green residues meet at the a14 helix (Ser220-Lys236) and the
b8–b9 loop (Gly153-Asp171) and have multiple instances of direct
mechanical coupling. The 7 covered residues are listed as boldfaced fonts in red in
Figure 2(b,c). On average, randomly picking 25 residues only
covers 1–3 out of the 9 residues identified by directed evolution. 10,000 runs of random picking were performed to calculate the
average and variance of covering the reported mutation sites;
using 1000 runs gives quantitatively close results. Among the 9 reported mutation sites, even though residue 160
is not covered, its calculated mechanical coupling variation is
actually quite significant; the average of Dkt
I’s is 15.4 kcal/mol/
A˚ 2. If the residues selected by directed evolution have significant
but not large mechanical coupling variation, i.e., the time average
of Dkt
I’s is between 10–20 kcal/mol/A˚ 2, they are colored orange
in Figure 2(b,c). If the residues selected by directed evolution have
medium or small mechanical coupling variation, i.e., the average
of Dkt
I’s is less than 10 kcal/mol/A˚ 2, they are colored light blue in
Figure 2(b,c). If the residues with large mechanical coupling
variation (red) are not within 61 of any of the reported mutation
sites, they are labeled via a red, unboldfaced font in Figure 2(b). The mechanical coupling network in subtilisin The off-diagonal features in Figure 1(d) are due to tertiary
contacts, and a wide range of the values of force constants are The
mechanical
coupling
between
residues
I
and
J
is
represented by kIJ~ P
i[I,j[J
kij, where i and j are the indices for PLoS Computational Biology | www.ploscompbiol.org March 2011 | Volume 7 | Issue 3 | e1002023 PLoS Computational Biology | www.ploscompbiol.org 3 March 2011 | Volume 7 | Issue 3 | e1002023 PLoS Computational Biology | www.ploscompbiol.org Ca2+-binding modulates the mechanical coupling
network in subtilisin Residues specified by orange fonts: mutation sites listed in (c) with significant but not large mechanical coupling variation due to
Ca2+ binding, i.e., the time average of Dkt
I’s is in between 10–20 kcal/mol/A˚2. Residues specified by light blue fonts: mutation sites listed in (c) with
medium or weak mechanical coupling variation, i.e., the time average of Dkt
I’s is less than 10 kcal/mol/A˚2. (c) Mutation sites reported in protein
engineering literature that can enhance the stability of subtilisin and the activity in a non-aqueous solvent. The residues are colored and boldfaced
according to the criteria described in (b). doi:10.1371/journal.pcbi.1002023.g002 interest in subtilisin engineering. Removing the sequence of the
Ca2+-binding loop in subtilisin BPN’ has been shown to achieve
this objective but at the expense of significantly reduced stability. Strausberg et al [32,33] integrated the reported mutation sites of
subtilisin variants and increased the stability (half-life at 75uC) of
their Ca2+-free construct 15,000 folds by directed evolution. The
17 mutations sites that were involved in achieving this success are
shown in Figure 2(c). Residue sites 9, 165, and 217 agree with the
results in Zhao and Arnold [31], and residue 72 was selected
instead of 75 after removing the Ca2+-binding loop. 9 of the
stabilization mutation sites employed in Strausberg et al [32,33] are
covered by the residues with large mechanical coupling variation
(red boldfaced); other 4 residues have weaker but significant
mechanical coupling variation (orange), Figure 2(b,c). In a
different protein engineering study by directed evolution, most
of the stabilization mutation sites reported in Rollence et al [34] are
also in agreement with those in Zhao and Arnold [31] and
Strausberg et al [32,33], and also are listed in Figure 2(c). In total,
the 25 calculated residue sites with large mechanical coupling
variation cover 14 of the 25 stabilization mutation sites reported in
[31,32,33,34] to within 61. Randomly picking 25 residues can loops or connecting regions between rigid secondary structures,
probably because the mutations therein are more tolerable [40]. In
subtilisin, this trend is also observed but mutation sites in well-
defined
secondary
structures
are
also
identified
as
well,
Figure 2(a,b). Residues with large mechanical coupling variation also tend to
locate at loops and connecting regions as shown in Figure 2(a), but
only specific residues would satisfy a designated selection criterion
such as the average of Dkt
I’s is larger than 20 kcal/mol/A˚ 2. Ca2+-binding modulates the mechanical coupling
network in subtilisin The force constants of elastic bonds provide a direct measure of
the mechanical coupling between amino acids. From the atomic
configurations sampled in time window t, the force constant
between ij sites, kij, is calculated by fluctuation matching [28]. The
mechanical coupling between residues I and J is determined as
kt
IJ~ P
i[I,j[J
kt
ij. The mechanical coupling associated with residue I
is then calculated as kt
I~1
2
P
J
kt
IJ and the difference in kI between
Ca2+-bound and apo simulations in a time window is Dkt
I~
kapo,t
I
{kCa2z,t
I
. The profiles of Dkt
I are shown in Figure 2(a). It can
be seen that Ca2+-mediated interactions make certain regions in
apo subtilisin becoming more flexible and others less. The
compensatory
balance
in
mechanical
coupling
variation
is
discussed in detail in Figure S2 and Text S1. Ca2+ binding is a molecular signal known to affect the stability
of subtilisin. Atomic simulations and fluctuation matching reveal
that it indeed has significant effects on the mechanical coupling
network in subtilisin, Figure 2(a,b). The results of directed
evolution in [31] suggest that most of the identified mutation sites
that demonstrate stabilization effects have high susceptibility in
mechanical coupling. In protein engineering, it is often observed
that the mutant residues survived from random mutation locate at PLoS Computational Biology | www.ploscompbiol.org March 2011 | Volume 7 | Issue 3 | e1002023 4 The Fluctuogram of Protein Dynamics Figure 2. Mechanical coupling variation of subtilisin due to Ca2+ binding. (a) Differences in the force constant of each residue between the
Ca2+-bound and apo simulations of subtilisin as a function of time, Dkt
I~kt,apo
I
{kt,Ca2z
I
(kcal/mol/A˚2). Residues with large mechanical coupling
variation are highlighted in the y-axis. See text for the definition of kt
I’s. (b) The location of the residues highlighted in (a) and (c). Residues specified
by red fonts: residues have large mechanical coupling variation to Ca2+ binding, i.e. the average of Dkt
I’s is larger than 20 kcal/mol/A˚2. Residues
specified by red and boldfaced fonts: residues with large mechanical coupling variation and cover the mutation sites listed in (c) to within 61. Residues specified by red and not boldfaced fonts: residues with large mechanical coupling variation but are not within 61 of any of the mutation
sites listed in (c). PLoS Computational Biology | www.ploscompbiol.org Ca2+-binding modulates the mechanical coupling
network in subtilisin The
mechanical coupling calculated from MD simulations reflects the
sequence-specific thermodynamic interactions between residues. The correlation between the stabilization mutation sites and the
residues with large mechanical coupling variation suggests that
having different thermodynamic interactions with the surrounding
could be an indicative property for a residue to be an effective
mutation site for protein engineering. To further test this theory,
we compare simulation results with other protein engineering
works. The strong Ca2+-dependence of stability and folding kinetics of
subtilisin makes its application as an industrial enzyme difficult;
eliminating Ca2+ dependence has thus been a long-standing March 2011 | Volume 7 | Issue 3 | e1002023 5 The Fluctuogram of Protein Dynamics adding DtSrijTt’s together: DtRt
I~
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
P
j,i[I
DtSrijTt
2
r
. To monitor
conformational changes relevant to mechanical coupling, only
bonds with non-zero kt{Dt=2
ij
or kt
ij are involved in the sum. DtRt
I’s
for Ca2+-bound and apo simulations are shown in Figure 3(a) and
Figure 3(b), respectively. adding DtSrijTt’s together: DtRt
I~
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
P
j,i[I
DtSrijTt
2
r
. To monitor
f
i
l
h
l
h
i
l
li
l adding DtSrijTt’s together: DtRt
I~
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
P
j,i[I
DtSrijTt
2
r
. To monitor
f
i
l
h
l
h
i
l
li
l only
cover
4–8
residues,
supporting
the
theory
that
the
susceptibility of mechanical coupling to functionally important
signals such as Ca2+ binding is an indicative property for a residue
to be an effective mutation site in protein engineering. j,
r
conformational changes relevant to mechanical coupling, only
bonds with non-zero kt{Dt=2
ij
or kt
ij are involved in the sum. DtRt
I’s
for Ca2+-bound and apo simulations are shown in Figure 3(a) and
Figure 3(b), respectively. In addition to stability, the mechanical coupling network in
protein structure also affects conformational flexibility and protein
dynamics. It is thus expected that varying mechanical coupling
network would also alter other functional properties such as
substrate binding and activity. In applying subtilisin as an
industrial enzyme, one desired property is the ability to function
in non-aqueous environments. This property has been shown to
relate to the flexibility and dynamics of protein conformation
[41,42,43]. Ca2+-binding modulates the mechanical coupling
network in subtilisin To
establish a tighter connection between mechanical coupling
variation and local conformational changes, a useful parameter is: Dtk
t
I~
X
j,i[I,kt
ij or kt{1=2Dt
ij
w0
kt
ij{kt{1=2Dt
ij
max kt
ij,kt{1=2Dt
ij
0
B
B
@
1
C
C
A=Nj,i[I: ð1Þ Dtk
t
I~
X
j,i[I,kt
ij or kt{1=2Dt
ij
w0
kt
ij{kt{1=2Dt
ij
max kt
ij,kt{1=2Dt
ij
0
B
B
@
1
C
C
A=Nj,i[I: ð1Þ g
g
[
]
Out of the 28 mutation sites reported in [31,32,33,34,35,36]
that had been shown to enhance the stability and activity of
subtilisin, 16 are covered to within 61 by the 25 residues
calculated
to
have
large
mechanical
coupling
variation,
Figure 2(b,c); randomly picking 25 residues only covers 5–9
residues. The ratio of the number of captured mutation sites to the
number of selected residues, 0.64, also far exceeds the corre-
sponding values achieved via random picking, 0.360.09. These
results indicate that the mechanical coupling networks calculated
from atomic details can be used to correlate with the functionally
important mutation sites selected by directed evolution. In eq.(1), Dtk
t
I is the average of relative differences in force
constants for the bonds that are connected to residue I. Only
bonds with a non-zero kt{1=2Dt
ij
or kt
ij are considered; Nj,i[I is the
number of such ij pairs. Dtk
t
I’s of Ca2+-bound and apo simulations
are shown in Figure 3(c) and Figure 3(d), respectively. Normalizing
Dtkt
I by max kt
ij,kt{1=2Dt
ij
in Dtk
t
I incorporates larger contribu-
tions from the tertiary contacts, and Dtk
t
I’s thus follow DRt
I’s more
closely than Dtkt
I’s. Furthermore, Dtk
t
I’s vary between 61 and
provide a simple metric for estimating the extent of the mechanical
coupling variation of residue I. Since Dtk
t
I’s closely follow the
intermittent features of DtRt
I’s in Figure 3, a tight connection
between conformational change and mechanical coupling varia-
tion is established. Prominent Dk
t
I’s can be observed right before,
after, or around DtRt
I bands. Another feature in Figure 2(a) is that mechanical coupling
variation is intermittent. In the following, we analyze the intrinsic
intermittence in the dynamics of Ca2+-bound and apo subtilisin
and explore its functional relevance. Intermittent conformational changes and mechanical
coupling variation in subtilisin The variation of kI
between consecutive time windows,
Dtkt
I~kt
I{kt{1=2Dt
I
, of apo and Ca2+-bound subtilisin (Figure
S3) shows an intermittent pattern similar to that of Dkt
I’s in
Figure 2(a). Intermittence in Dtkt
I indicates that during protein
dynamics, increases in mechanical coupling strength for a peptide
segment do not last extensively long. As the segment enters a
resting period, reduction in flexibility or mechanical coupling
strength tend to follow, although further increases after the resting
period are observed occasionally as well (Figure S3). Prominent
features in Dtkt
I’s thus alternate among different sites with time. This behavior illustrates that protein structural fluctuations are
highly rectified. In the following, we first establish correspondences
between
conformational
changes
and
mechanical
coupling
variation and characterize the pathways of intra-protein commu-
nication. The fluctuograms shown in Figure 3 record the chorography of
protein dynamics with a time window of 4 ns. The movies of the
equilibrium structures of sequential Ca-SC-ENM’s further illustrate
the intermittence of conformational changes and are provided in
VideoS1 and VideoS2. Fluctuograms using Dt = 2 ns and 10 ns
show qualitatively similar patterns (results not shown). Ca2+-binding modulates the mechanical coupling
network in subtilisin In enhancing the activity of subtilisin E in a solution
with a high concentration of a polar organic solvent by directed
evolution, Chen and Arnold had identified 9 mutation sites that
increase the activity in 60% dimethylformamide to 256 times that
of the wild type [35,36]. These residue sites also shown in
Figure 2(c). Residues 59, 96, and 102 are distinct and the other 6
are in the pool of the stabilization mutation sites reported in
[31,32,33,34]. Residues 96 and 102 are in the b4-a5 loop (93–104)
that involves substrate binding; residue 59 is in the b2-a3 loop (49–
63) that extends from His63 in the catalytic triad. Residue 59 and
96 have large (red boldfaces) and 102 has weaker but significant
mechanical
coupling
variation
(orange),
Figure
2(b,c). The
functional relevance of mechanical coupling variation is thus not
limited to stability. Another residue with large mechanical
coupling variation is 174, which had been shown to modulate
the Ca2+ binding of subtilisin at the weaker binding site [44]. If a peptide segment in subtilisin underwent conformational
changes over a period of time, DtRt
I’s of these residues shows up as
a band. For regions with limited mobility, DtRt
I’s are small. If
mechanically coupled segments underwent correlated conforma-
tional changes, DRt
I bands would appear together or close in time. In Ca2+-bound subtilisin, co-occurring DRt
I bands in b1–b2 loop
(Asp32-Asp41), b2-a3 (Ser49-His63), b4-a5 (Lys93-Ser104), b6-a7
(Met123-Thr132), and b8–b9 (Gly153-Asp171) loops are clear in
Figure 3(a), and a set of collective DtRt
I bands spanning ,20 ns is
highlighted
as
an
example. This
event
corresponds
to
a
sequentially collective conformational change with mechanically
coupled residues; the details are shown in Figure S4. Since the values of force constants for residue pairs close in
sequence ( I{J
j
jv3) are much larger than those of tertiary
contacts, variations of bare DtRt
I’s (Figure S3) tend to under-
represent the mechanical coupling variation between tertiary
contacts and do not show a close correspondence with DtRt
I’s. PLoS Computational Biology | www.ploscompbiol.org Intra-protein communication due to Ca2+ binding The fluctuogram of apo subtilisin (Figure 3(b,d)) records a
choreography that the signal of removing Ca2+ propagates
through the mechanical coupling network and affects active and
substrate-binding sites that are 20–30 A˚ away. Such behavior is
not
seen
in
the
fluctuograms
of
Ca2+-bound
subtilisin
(Figure 3(a,c)), which record a different pattern of choreography. Here, the apo fluctuograms are analyzed in detail; the analyses of
Ca2+-bound fluctuograms are discussed in Figure S4 and Text S1. The change of a bond length in the mechanical coupling
network between time windows is: DtSrijTt~SrijTt{SrijTt{Dt=2. The overall conformational change of residue I is estimated by PLoS Computational Biology | www.ploscompbiol.org March 2011 | Volume 7 | Issue 3 | e1002023 6 The Fluctuogram of Protein Dynamics Figure 3. Changes in the local conformation and mechanical coupling of each residue in subtilisin between neighboring time
windows. (a) Conformational changes in the Ca2+-bound simulation. (b) Conformational changes in the apo simulation. The change in inter-site
distance in A˚ between two neighboring time windows t
1=2Dt and t is D Sr Tt
Sr Tt
Sr Tt{Dt=2 and the local conformational change of Figure 3. Changes in the local conformation and mechanical coupling of each residue in subtilisin between neighboring time
windows. (a) Conformational changes in the Ca2+-bound simulation. (b) Conformational changes in the apo simulation. The change in inter-site
distance in A˚ between two neighboring time windows, t{1=2Dt and t, is DtSrijTt~SrijTt{SrijTt{Dt=2 and the local conformational change of
residue I is defined as DtRt
I~
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
P
j,i[I
DtSrijTt
2
r
. Variation in the mechanical coupling of each residue between neighboring time windows for (c) the
Ca2+ simulation and (d) the apo simulation. Mechanical coupling variation of residue I between two neighboring time windows, t{1=2Dt and t, is
defined as Dtk
t
I~
P
j,i[I,kt
ij or kt{1=2Dt
ij
w0
kt
ij{kt{1=2Dt
ij
ð
Þ
max kt
ij,kt{1=2Dt
ij
ð
Þ
0
@
1
A=Nj,i[I. Nj,i[I is the number of ij pairs associate with residue I and with at least one of kt{1=2Dt
ij
or
kt
ij has positive value. The time window Dt for calculating DtRt
Iand Dtk
t
I is 4 ns. doi:10.1371/journal.pcbi.1002023.g003 Figure 3. Changes in the local conformation and mechanical coupling of each residue in subtilisin between neighboring time
windows. (a) Conformational changes in the Ca2+-bound simulation. (b) Conformational changes in the apo simulation. The Fluctuogram of Protein Dynamics In apo subtilisin, the absence of Ca2+ caused prominent bands
in DtRt
I and Dtk
t
I in the Ca2+-binding loop (Val71-Leu83) as
highlighted in Figure 3(b,d). Since Asp41 in the b1–b2 loop
(Asp32-Asp41) loop tightly coordinates with Ca2+ if present, the
absence of Ca2+-mediated interactions affects the mechanical
coupling of this loop, and the b1–b2 loop in apo subtilisin has
larger intermittent bands, as highlighted in Figure 3(b,d). Despite
that the force constants at this region show large differences
between Ca2+-bound and apo subtilisin (Figure 2(a)), differences in
intrinsic mechanic coupling variation are also clear. It is obvious
from Figure 3(b,d) that DtRt
I and Dtk
t
I bands in the b2-a3 loop
occur close in time with those in the b1–b2 loop: Ca2+-mediated
changes continue to affect the b2-a3 loop through mechanical
coupling. residues surviving from random mutagenesis and screening tend
to have large mechanical coupling variation due to molecular
signals. In theory, if the mechanical coupling network in protein
structure was optimized by evolution to facilitate intra-protein
communication, residue pairs with functionally important me-
chanical coupling would tend to correlate during evolution. To test
this hypothesis, we select residue pairs with distinct patterns of
mechanical coupling from the fluctuograms and compare the
results with those of statistical coupling analysis (SCA). After
collecting a pool of sequences with high similarity and constructing
a multiple sequence alignment, the SCA method developed by
Ranganathan and coworkers [37,38] is used to identify residues
with high sequence correlation. Using subtilisin Carlsberg as the query sequence, we collected
465 sequences for SCA (see methods for details), and the pattern of
sequence conservation is shown in Figure S6. The 2nd–4th
eigenvectors were used to screen the correlation matrix for
statistically significant correlation according to random matrix
theory [38,46]. The 274 residues of subtilisin expanded by the 2nd
and 3rd eigenvectors are shown in Figure S7; on this map, a cutoff
value of 0.07 for the distance to origin is used to select 80 residues
(,30% of the total amino acids) that exhibit high correlation in
sequence variation [38]. The cleaned correlated matrix is shown in
Figure S8. The 80 amino acids can be roughly divided into three
sectors according to their values on the 2nd and 3rd eigenvectors,
and their locations in subtilisin are shown in Figure 4(a). The Fluctuogram of Protein Dynamics Spatial
localization of sectors is rather clear but close separation of
residues in different sectors is also observed. The pattern of sectors
is consistent with several features of the long-range coupling and
complex folding pathways of subtilisin [6,24,47]. For example, the
blue sector contains residues in the Ca2+-binding loop (Val71-
Leu83) and the weaker Ca2+ binding site, and analyzing the
fluctuogram shows that the two Ca2+-binding sites are linked
through the mechanical coupling network. Many red sector
residues are localized in the central a3 and a14 of subtilisin
(Figure 1(a)). The green sector contains residues in b1 (Val26-
Leu31), the b1–b2 loop (Asp32-Asp41), and the b4-a5 loop
(Lys93-Ser104) that mechanically couple with Asp32. At the
junction of Asp32, the fluctuograms of apo and Ca2+-bound
simulations show significant differences in Figure 3. Mechanical coupling also causes b1–b2 (Asp32-Asp41), b2-a3
(Ser49-His63), b4-a5 loop (Lys93-Ser104), and the b6-a7 loop
(Met123-Thr132) to have coincident bands in DtRt
I and Dtk
t
I. The
sequentially collective bands highlighted in Figure 3(b,d) constitute
a pathway of intra-protein communication, which is shown in
Figure S5 and discussed in more detail in Text S1. The co-
occurring
bands
of
these
loops
in
Ca2+-bound
subtilisin,
Figure 3(a,c), are less prominent and have different patterns,
showing that Ca2+-mediated interactions alter the choreography of
protein dynamics. Along a similar line, as the b8–b9 loop (Gly153-Asp171)
mechanically couples with the b6-a7 loop (Met123-Thr132)
(Figure 1(d)), and the signal of Ca2+ binding propagates there
accordingly. A clear difference between the fluctuograms of Ca2+-
bound and apo subtilisin is that apo subtilisin has less prominent
bands in b6-a7 and b8–b9 loops, opposite to the responses in b1–
b2, b2-a3, and b4-a5 loops, Figure 3. Opposite responses of
different loops to Ca2+-mediated interactions is reminiscent of the
compensatory balance in mechanical coupling variation shown in
Figure S2. The b8–b9 loop contains residues of the weaker Ca2+
binding site of subtilisin [44] and is 32 A˚ away from the strong Ca2+
binding site; fluctuogram analysis shows that through mechanical
coupling network, signal at the Ca2+ binding site affects distal sites. Other significant differences in the fluctuograms are that apo
subtilisin has more pronounced DtRt
I and Dtk
t
I bands in the b10–
b11 loop (Phe188-Ala193), the b12–b13 turn (Thr207-Tyr213),
the a14–a15 loop (Lys236-Ala242), and the Phe260 turn (Gly257-
Gly263), see highlights in Figure 3(b,d). The Fluctuogram of Protein Dynamics These sites are also
consistent with the results of Dkt
I’s shown in Figure 2(a). In recent years, significant progress has been made in con-
necting the network of protein structure to allosteric coupling
[13,14,15,16,17,18,48,49,50,51,52]. Many of these studies employ
ENM using contact-based determination of connectivity and
heuristics-based assignment of force constants (homogeneous or
via an assumed functional form) [13,14,15,16,17,18]. Despite the
simplicity, impressive success has been achieved in identifying
important residues for allosteric coupling, which are often robust
to sequence variation [53]. A key observation is that amino acids
with many close contacts with others often have significant impact
on allosteric coupling. Such residues are also considered as hubs
that cause the structural network of protein conformation to have
small-world characters [48,50,51,52]. To select residue pairs from
fluctuograms, we also apply this result developed in previous
works. Together, the fluctuograms calculated from all-atom MD
simulations show that intra-protein communication can proceed
through the mechanical coupling network in protein structure
without a drastic conformational change [11,45]. The results
discussed above establish (a) Ca2+ binding induces significant
changes in the mechanical coupling network of subtilisin despite a
small difference in the overall structure, (b) residues with large
mechanical coupling variation due to Ca2+ binding correlate with
the gain-of-function mutation sites selected via directed evolution,
(c) conformational changes and mechanical coupling variation are
temporally and spatially intermittent, (d) large variations in the
mechanical coupling network often occur at the connecting
regions of secondary structures, and (e) the fluctuograms can be
used to capture the pathways of intra-protein communication. To
further strengthen (e), the sequentially collective conformational
changes associated with the co-occurring bands highlighted in
Figure 3 are discussed in Figure S4, Figure S5, and Text S1. The fluctuogram approach proposed in this work bridges
atomic and CG models of protein allostery by computing the force
constants in Ca-SC-ENM from the structures sampled in all-atom
MD simulations. An important result is that mechanical coupling
between residues varies significantly, highlighting the anharmoni-
city and nonlinearity of protein dynamics. Therefore, both the
strength and variation of mechanical coupling will be used to select
residue pairs. From the kIJ’s calculated from sequential time
windows,
the
average,
kavg
IJ ,
standard
deviation,
kstd
IJ ,
and Intra-protein communication due to Ca2+ binding The change in inter-site
distance in A˚ between two neighboring time windows, t{1=2Dt and t, is DtSrijTt~SrijTt{SrijTt{Dt=2 and the local conformational change of
residue I is defined as DtRt
I~
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
P
j,i[I
DtSrijTt
2
r
. Variation in the mechanical coupling of each residue between neighboring time windows for (c) the
Ca2+ simulation and (d) the apo simulation. Mechanical coupling variation of residue I between two neighboring time windows, t{1=2Dt and t, is
defined as Dtk
t
I~
P
j,i[I,kt
ij or kt{1=2Dt
ij
w0
kt
ij{kt{1=2Dt
ij
ð
Þ
max kt
ij,kt{1=2Dt
ij
ð
Þ
0
@
1
A=Nj,i[I. Nj,i[I is the number of ij pairs associate with residue I and with at least one of kt{1=2Dt
ij
or
kt
ij has positive value. The time window Dt for calculating DtRt
Iand Dtk
t
I is 4 ns. doi:10 1371/journal pcbi 1002023 g003 ij
ij
@
A
kt
ij has positive value. The time window Dt for calculating DtRt
Iand Dtk
t
I is 4 ns. doi:10.1371/journal.pcbi.1002023.g003 PLoS Computational Biology | www.ploscompbiol.org March 2011 | Volume 7 | Issue 3 | e1002023 PLoS Computational Biology | www.ploscompbiol.org Correlate fluctuograms with co-evolution
2 The fluctuograms of Ca2+-bound and apo subtilisin illustrate the
mechanism of intra-protein communication and show that PLoS Computational Biology | www.ploscompbiol.org March 2011 | Volume 7 | Issue 3 | e1002023 March 2011 | Volume 7 | Issue 3 | e1002023 8 The Fluctuogram of Protein Dynamics Figure 4. Sequence correlation in subtilisin. (a) The residues of subtilisin exhibit high correlation in our multiple sequence alignment
determined by a statistical coupling analysis (SCA). Residues with high correlation in sequence variation are divided into three sectors, blue, red, and
green according to the eigenvectors of the correlation matrix of sequence conservation [38]. Several residues that are not covered by the selection
from the Ca2+-bound fluctuogram are highlighted. (b) The residues that satisfy either of the three criteria discussed in the text from the Ca2+-bound
fluctuogram and cover the co-evolved residues shown in (a); color codes are the same as in (a). The parameters of the selection criteria are: nA
cut = 10,
kA
t = 2 5, kB
t = 11, kC
t = 8 0, and f C
t = 0 8 (c) The residues selected from the Ca2+-bound fluctuogram based on the parameters listed in (b) Lime: Figure 4. Sequence correlation in subtilisin. (a) The residues of subtilisin exhibit high correlation in our multiple sequence alignment
determined by a statistical coupling analysis (SCA). Residues with high correlation in sequence variation are divided into three sectors, blue, red, and
green according to the eigenvectors of the correlation matrix of sequence conservation [38]. Several residues that are not covered by the selection
from the Ca2+-bound fluctuogram are highlighted. (b) The residues that satisfy either of the three criteria discussed in the text from the Ca2+-bound
fluctuogram and cover the co-evolved residues shown in (a); color codes are the same as in (a). The parameters of the selection criteria are: nA
cut = 10,
kA
cut = 2.5, kB
cut = 11, kC
cut = 8.0, and f C
cut = 0.8. (c) The residues selected from the Ca2+-bound fluctuogram based on the parameters listed in (b). Lime:
residues that cover the co-evolved residues from SCA. Brown: the co-evolved residues from SCA that are not covered by the residues selected from
the Ca2+-bound fluctuogram. Pink: residues selected from the Ca2+-bound fluctuogram but do not cover any of the co-evolved residues. PLoS Computational Biology | www.ploscompbiol.org The Fluctuogram of Protein Dynamics protein communication: comparison (10,000 rounds; results of 1,000 rounds are quantita-
tively similar). In Figure 5(a,b), the hit rates of random picking
correspond to the NA values of nA
cut = 8; the profiles of other nA
cut
values are quantitatively similar. When kA
cut is small, the hit rates
calculated from fluctuograms are close to the values of random
picking. Since there are 80 highly correlated residues observed in
SCA and a 61 criterion is used for counting a hit, the baseline hit
rate via random picking is 0.62. As shown in Figure 5(a,b),
increasing kA
cut significantly improves the hit rates achieved by apo
and Ca2+-bound fluctuograms, which are progressively higher
than the values of random picking by more than one standard
deviation. As kA
cut increases, NA and the coverage decrease due to
the more stringent selection. The coverages achieved by apo and
Ca2+-bound fluctuograms are shown in Figure 5(c) and Figure 5(d),
respectively. At small NA values, the standard deviation of the hit
rates of random picking also becomes higher. ð2Þ Criterion{A :
kmax
IJ wkA
cut\nkmax
I
wnA
cut :
ð2Þ The total number of such residues is denoted as NA. For each of
the NA residues, if it captures any highly correlated residues
observed in SCA to within 61 in residue number, a hit is counted. The hit rate, RA, is calculated as the total number of hits, NA
hit,
divided by NA, RA~NA
hit=NA. For each of the residues identified
by SCA, we also check if it is covered by any of the NA residues
predicted by the fluctuogram. The total number of covered
residues is NA
cov, and the coverage is defined as CA~NA
cov=NSCA. NSCA is the number of highly correlated residues identified in
SCA. 2 The hit rates calculated from the fluctuograms of apo and Ca2+-
bound subtilisin at different values of nA
cut are shown in Figure 5(a)
and Figure 5(b), respectively. At a given value of NA, the hit rate
achieved by randomly picking residues is also calculated for Figure 5(a,b) illustrate the correlation between mechanical
coupling and co-evolution. The increasing hit rates with kA
cut
plateau around the value of 2.5 kcal/mol/A˚ 2. This result is Figure 5. Correlating the fluctuograms of subtilisin with co-evolution. The calculated hit rates (RA’s) and coverages (CA’s) by using
Criterion-A, (eq.(2)). (a) RA’s from the apo fluctuogram. Correlate fluctuograms with co-evolution
2 (d) The
residues selected from the Ca2+-bound fluctuogram based on the parameters listed in (b). Blue: residues selected by Criterion-A. Red: residues
selected from Criterion-B. Green: residues selected from Criterion-C. See text for the definitions of each criterion. doi:10.1371/journal.pcbi.1002023.g004 Figure 4. Sequence correlation in subtilisin. (a) The residues of subtilisin exhibit high correlation in our multiple sequence alignment
determined by a statistical coupling analysis (SCA). Residues with high correlation in sequence variation are divided into three sectors, blue, red, and
green according to the eigenvectors of the correlation matrix of sequence conservation [38]. Several residues that are not covered by the selection
from the Ca2+-bound fluctuogram are highlighted. (b) The residues that satisfy either of the three criteria discussed in the text from the Ca2+-bound
fluctuogram and cover the co-evolved residues shown in (a); color codes are the same as in (a). The parameters of the selection criteria are: nA
cut = 10,
kA
cut = 2.5, kB
cut = 11, kC
cut = 8.0, and f C
cut = 0.8. (c) The residues selected from the Ca2+-bound fluctuogram based on the parameters listed in (b). Lime:
residues that cover the co-evolved residues from SCA. Brown: the co-evolved residues from SCA that are not covered by the residues selected from
the Ca2+-bound fluctuogram. Pink: residues selected from the Ca2+-bound fluctuogram but do not cover any of the co-evolved residues. (d) The
residues selected from the Ca2+-bound fluctuogram based on the parameters listed in (b). Blue: residues selected by Criterion-A. Red: residues
selected from Criterion-B. Green: residues selected from Criterion-C. See text for the definitions of each criterion. doi:10.1371/journal.pcbi.1002023.g004 maximum observed value, kmax
IJ , are computed to devise selection
criteria. assign whether the mechanical coupling between the IJ pair is
significant. For residue I, the total number of coupled residues with
kmax
IJ wkA
cut is denoted as nkmax
I
. If nkmax
I
is larger than a number cut-
off, nA
cut, then residue I is selected as a residue important for intra- In Criterion-A, we consider residue pars with kmax
IJ
larger than a
cut-off value, kA
cut. A value of 2.5 kcal/mol/A˚ 2 was used earlier to PLoS Computational Biology | www.ploscompbiol.org March 2011 | Volume 7 | Issue 3 | e1002023 9 PLoS Computational Biology | www.ploscompbiol.org The Fluctuogram of Protein Dynamics (b) RA’s from the Ca2+-bound fluctuogram. Hit rates achieved by randomly picking the same
numbers as the selected residues based on nA
cut = 8 are shown for comparison. The profiles correspond to other nA
cut values are quantitatively close. (c) CA’s from the apo fluctuogram. (d) CA’s from the Ca2+-bound fluctuogram. doi:10.1371/journal.pcbi.1002023.g005 Figure 5. Correlating the fluctuograms of subtilisin with co-evolution. The calculated hit rates (RA’s) and coverages (CA’s) by using
Criterion-A, (eq.(2)). (a) RA’s from the apo fluctuogram. (b) RA’s from the Ca2+-bound fluctuogram. Hit rates achieved by randomly picking the same
numbers as the selected residues based on nA
cut = 8 are shown for comparison. The profiles correspond to other nA
cut values are quantitatively close. (c) CA’s from the apo fluctuogram. (d) CA’s from the Ca2+-bound fluctuogram. doi:10.1371/journal.pcbi.1002023.g005 March 2011 | Volume 7 | Issue 3 | e1002023 10 The Fluctuogram of Protein Dynamics consistent with the physics-based selection of the value of 2.5 for
assigning significant mechanical coupling. Overall, the hit rate is
also an increasing function of nA
cut, except for the special cases at
small NA values. This trend is in line with the analyses of protein
structure using network theory that residues with more neighbors
tend to play important roles in allosteric coupling [48,50,51,52]. In
balancing
hit
rate
and
coverage,
using
nA
cut = 10
and
kA
cut = 2.5 kcal/mol/A˚ 2 for Criterion A gives RA = 0.81 and
CA = 0.29 from the apo fluctuogram and RA = 0.87 and CA =
0.46 from the Ca2+-bound fluctuogram. values significantly exceeds the average plus a standard deviation,
Figure 7(b). RC is also an increasing function with kC
cut as expected
from Criterion-A. For Criterion-C, we use kC
cut = 8 kcal/mol/A˚ 2
and f C
cut~0:8 (apo: RC = 0.71 and CC = 0.26; Ca2+-bound:
RB = 0.85 and CB = 0.25). As shown in Figure 2 and Figure 3 and discussed earlier, the
fluctuogram of subtilisin depends on Ca2+ binding. As a result,
different behaviors are observed in calculating hit rates from apo
and Ca2+-bound fluctuograms. Since native subtilisin is function-
ing with Ca2+ and we screened for alignable sequences that
contain the Ca2+-binding loop for SCA, the Ca2+-bound fluc-
tuogram should better represent the required mechanical coupling
network for the proper functioning of subtilisin. The Fluctuogram of Protein Dynamics This theory is
supported by the result that the Ca2+-bound fluctuogram has
better predictive power in capturing the correlated residues from
SCA. Using
nA
cut = 10,
kA
cut = 2.5,
kB
cut = 11,
kC
cut = 8.0,
and
f C
cut = 0.8 to select residues satisfying either criterion, the calculated
hit rates and coverages are R = 0.75/C = 0.5 from the apo and
R = 0.84/C = 0.65 from the Ca2+-bound fluctuogram. In Criterion-B, we consider residue pairs with strong mechan-
ical coupling. For any IJ pairs with I{J
j
jw3, I and J are selected
if: Criterion{B :
kavg
IJ wkB
cut :
ð3Þ ð3Þ The hit rates and coverages calculated from apo and Ca2+-bound
fluctuograms are shown in Figure 6(a,b). Increasing RB with kB
cut is
also observed as in Criterion-A. The hit rates from the Ca2+-
bound fluctuogram have steeper increase with kB
cut and exceed the
values
of random picking more than that from the apo
fluctuogram. The coverage, CB, quickly decreases with kB
cut, and
is not as high as CA
apo, which screens kmax
IJ
instead. For Criterion-B,
we use kB
cut = 11 kcal/mol/A˚ 2 (apo: RB = 0.74 and CB = 0.23;
Ca2+-bound: RB = 0.78 and CB = 0.26). The correlated residues from SCA (Figure 4(a)) covered by the
residue pairs with distinct behaviors of mechanical coupling in the
Ca2+-bound fluctuogram are shown in Figure 4(b) for comparison. Several uncovered residues are highlighted in Figure 4(a) and
many of them are in or near the pool of stabilization mutation sites
shown in Figure 2(b). Therefore, comparing fluctuograms can
provide additional information about co-evolution. The covered
(green), missed (brown), and over-predicted (pink) residues based
on the Ca2+-bound fluctuogram are contrasted in Figure 4(c), and
several over-predicted residues are highlighted. Some of these
residues are in or near the pool of the stabilization mutation sites
shown in Figure 4(b) but are not selected in SCA. This result is
consistent with many observations that thermodynamic coupling is
not limited to co-evolved residues [54,55,56]. In Criterion-C, we probe if the variation in kIJ can capture the
residues with high correlation in a multiple sequence alignment. The Fluctuogram of Protein Dynamics In
addition to limiting the magnitude of kIJ, a cutoff for kstd
IJ is also
used: Criterion{C :
kmax
IJ wkC
cut\kstd
IJ =kavg
IJ wf C
cut :
ð4Þ ð4Þ Here, we employ kmax
IJ
instead of kavg
IJ for the advantage of having
higher coverage. The calculated RC’s and CC’s are shown in
Figure 7. From the apo fluctuogram, RC is not strictly increasing
with f C
cut, and the lead over random picking is only slightly higher
or close to the average value plus standard deviation, Figure 7(a). From the Ca2+-bound fluctuogram, on the other hand, RC is
clearly increasing with f C
cut, and the lead over random-picking The increasing hit rates with the magnitude and variation of
mechanical coupling link physics-based MD simulations with co-
evolution. We devise different criteria to probe the properties of
the mechanical coupling network in protein structure and to select
residues to cover the correlated residues from SCA. Based on the
Ca2+-bound fluctuogram, the covered SCA residues by using
Criterion-A, Criterion-B, and Criterion-C together are colored Figure 6. The calculated hit rates (RB’s) and coverages (CB’s) by using Criterion-B, (eq.(3)). (a) RB’s and CB’s from the apo fluctuogram. Hit rates achieved by randomly picking the same numbers as the selected residues are shown for comparison. (b) RB’s and CB’s from the Ca2+-bound
fluctuogram. doi:10.1371/journal.pcbi.1002023.g006 Figure 6. The calculated hit rates (RB’s) and coverages (CB’s) by using Criterion-B, (eq.(3)). (a) RB’s and CB’s from the apo fluctuogram. Hit rates achieved by randomly picking the same numbers as the selected residues are shown for comparison. (b) RB’s and CB’s from the Ca2+-bound
fluctuogram. doi:10.1371/journal.pcbi.1002023.g006 March 2011 | Volume 7 | Issue 3 | e1002023 PLoS Computational Biology | www.ploscompbiol.org Figure 7. The calculated hit rates (RC’s) and coverages (CC’s) by using Criterion-C, (eq.(4)). (a) RC’s from the apo fluctuogram. (b) RC’s
from the Ca2+-bound fluctuogram. Hit rates achieved by randomly picking the same numbers as the selected residues based on kC
cut = 7 kcal/mol/A˚2
are also shown for comparison. The profiles correspond to other kC
cut values are quantitatively close. (c) CC’s from the apo fluctuogram. (d) CC’s from
the Ca2+-bound fluctuogram. doi:10.1371/journal.pcbi.1002023.g007
The Fluctuogram of Protein Dynamics The Fluctuogram of Protein Dynamics Figure 7. The calculated hit rates (RC’s) and coverages (CC’s) by using Criterion-C, (eq.(4)). (a) RC’s from the apo fluctuogram. (b) RC’s
from the Ca2+-bound fluctuogram. The Fluctuogram of Protein Dynamics Hit rates achieved by randomly picking the same numbers as the selected residues based on kC
cut = 7 kcal/mol/A˚2
are also shown for comparison. The profiles correspond to other kC
cut values are quantitatively close. (c) CC’s from the apo fluctuogram. (d) CC’s from
the Ca2+-bound fluctuogram. doi:10.1371/journal.pcbi.1002023.g007 differently in Figure 4(c) to illustrate that in the pool of residues
with high sequence correlation, alternative behaviors of mechan-
ical coupling are found. variation are identified and shown in Figure 8(a,b), see Methods
for the details of methodology. The all-atom MD simulation of
EG1 in explicit water at 300 K and 1 atm started with the X-ray
structure, PDB ID 1EG1 [57], with the protocol described in
Methods. The system contains 62,610 atoms, with 5256 protein
atoms, 69 counter ions, and 19095 water molecules. A total of
80 ns trajectory was collected for calculating fluctuogram with
Dt = 4 ns. As an independent test of the correlation between mechanical
coupling and co-evolution, we analyze the fluctuograms of a
different enzyme using the same criteria, in particular, the family 7
endoglucanase of the Trichoderma reesei fungus, EG1 [57]. The 371-
residue EG1 hydrolyzes the b-1,4-glycosidic bonds in cellulose for
nutritional utilization. To work against a glucose chain, EG1 has a
tunnel-shape active site, Figure 8(a,b). The segments around the
active site contain multiple secondary structures and connecting
loop and are responsible for binding the glucose chain from the
surface of cellulose. Therefore, the mechanical coupling network
in EG1 needs to carry out non-catalytic activities, and correlating
co-evolved residues via fluctuograms can reveal the functional
relevance of the mechanical coupling network in EG1. The calculated hit rates and coverages using Criterion-A,
Criterion-B, and Criterion-C are shown in Figure 8(c), Figure 8(d),
and Figure 8(e), respectively. The hit rates achieved by random
picking are also shown for comparison. The increasing hit rates
with kA
cut and kB
cut are clear in Figure 8(c,d), and the hit rates
calculated from the fluctuogram exceed the mean values plus
standard deviation of random
picking to a large extent. In
Criterion-C, the increase of hit rate with f C
cut starts at larger values
(Figure 8(e)). PLoS Computational Biology | www.ploscompbiol.org March 2011 | Volume 7 | Issue 3 | e1002023 Discussion Criterion-A and Criterion-B. Similar behavior is also observed in
calculating hit rates from the apo fluctuogram of subtilisin
(Figure 7(a)). Currently, we are investigating the effects substrate
binding on the mechanical coupling network in EG1. Using
nA
cut = 8, kA
cut = 3.5, kB
cut = 17, kC
cut = 8.0, and f C
cut = 0.8, the covered,
missed, and over-predicted residues compared to the co-evolved
ones are shown in Figure 8(a,b). Criterion-A and Criterion-B. Similar behavior is also observed in
calculating hit rates from the apo fluctuogram of subtilisin
(Figure 7(a)). Currently, we are investigating the effects substrate
binding on the mechanical coupling network in EG1. Using
nA
cut = 8, kA
cut = 3.5, kB
cut = 17, kC
cut = 8.0, and f C
cut = 0.8, the covered,
missed, and over-predicted residues compared to the co-evolved
ones are shown in Figure 8(a,b). The
mechanism
of
allosteric
coupling
and
intra-protein
communication is key to understand the structure-property
relationship of protein function. An emergent picture is that
induced-fit and population shift theories provide complementary
pictures and do not exclude each other [11,12,13,17,58]. The
interaction energetics between amino acids that cause induced-fit
and the distribution of protein structures are the two sides of the
same coin, and inverse algorithms such fluctuation matching [28]
or the iterative Yvon-Born-Green [22] methods could be used to
establish the connection. In this work, the fluctuation matching
method is used to convert the configurations sampled in MD
simulation into the bond lengths and force constants in a Ca-SC-
ENM to represent the mechanical coupling network in protein
structure. The correlation between mechanical coupling and co-evolution
in EG1 is clear in Figure 8. Therefore, in both subtilisin and EG1,
the results of analyzing fluctuograms illustrate that the mechanical
coupling networks calculated from atomic details can be used to
correlate with co-evolution. Several noticeable differences between
EG1 and subtilisin, though, can be found. First, residues in EG1
do not have as many neighbors with strong mechanical coupling,
and a lower number nA
cut is thus used for Criterion-A. This result is
consistent with the more globular shape of subtilisin. For
Criterion-B, which screens for residue pairs with strong mechan-
ical coupling on average, hit rates plateau at a larger kB
cut value in
EG1 (17 kcal/mol/A˚ 2) than that in subtilisin (11 kcal/mol/A˚ 2). The Fluctuogram of Protein Dynamics The hit rate calculated from the fluctuogram is higher
than the mean values of random picking but not as much as in Using EG1 as the query sequence, we collected 318 sequences
for SCA, and 82 residues with high correlation in sequence March 2011 | Volume 7 | Issue 3 | e1002023 12 The Fluctuogram of Protein Dynamics Figure 8. Correlating the fluctuograms of EG1 with co-evolution. (a) The highly correlated residues observed in a multiple sequence
alignment and SCA using EG1 as the query sequence and the residues selected from the fluctuogram satisfying either of the three criteria with the
following parameters: nA
cut = 8, kA
cut = 2.5, kB
cut = 17, kC
cut = 8.0, and f C
cut = 0.8. Lime: residues that cover the co-evolved residues from SCA. Brown: co-
evolved residues from SCA that are not covered by the residues selected from the fluctuogram. Pink: residues selected from the fluctuogram but do
not cover any co-evolved residue. (b) the same as (a) but view from a different angle. The calculated hit rates and coverages from the fluctuogram of
EG1 by using (c) Criterion-A, (eq.(2)), (d) Criterion-B, (eq.(3)), and (e) Criterion-C, (eq.(4)). doi:10.1371/journal.pcbi.1002023.g008 Figure 8. Correlating the fluctuograms of EG1 with co-evolution. (a) The highly correlated residues observed in a multiple sequence
alignment and SCA using EG1 as the query sequence and the residues selected from the fluctuogram satisfying either of the three criteria with the
following parameters: nA
cut = 8, kA
cut = 2.5, kB
cut = 17, kC
cut = 8.0, and f C
cut = 0.8. Lime: residues that cover the co-evolved residues from SCA. Brown: co-
evolved residues from SCA that are not covered by the residues selected from the fluctuogram. Pink: residues selected from the fluctuogram but do
not cover any co-evolved residue. (b) the same as (a) but view from a different angle. The calculated hit rates and coverages from the fluctuogram of
EG1 by using (c) Criterion-A, (eq.(2)), (d) Criterion-B, (eq.(3)), and (e) Criterion-C, (eq.(4)). doi:10.1371/journal.pcbi.1002023.g008 PLoS Computational Biology | www.ploscompbiol.org The Fluctuogram of Protein Dynamics The robustness of using fluctuogram to capture residues
with high sequence correlation is further tested with a different
enzyme, EG1, and the results also show that the residues selected
from the proposed criteria capture to a large extent the highly
correlated residues observed in SCA (Figure 8(c–e)). Overall, our
results illustrate that the mechanical coupling networks calculated
from atomic details can be used to correlate with functionally
important mutation sites and co-evolution. The Ca2+-dependent fluctuograms of subtilisin are in line with
several experimental observations. In enhancing subtilisin stability
by directed evolution and site-directed mutagenesis, it was found
that certain mutations that stabilize apo subtilisin would destabilize
the protein in the presence of Ca2+ [6]. Therefore, the mechanism
of thermal inactivation depends on Ca2+ binding, implying that
the mechanical coupling network in subtilisin is Ca2+ dependent. Indeed, our simulations show that apo and Ca2+-bound subtilisin
have different fluctuograms. A mutation site of this type is Phe50
in the b2-a3 loop that shows different behaviors in Figure 3. p
The design of MD simulations with relevant scenarios and the
criteria for selecting residues are the two core elements in using
fluctuograms to study protein function and dynamics. We
demonstrate that calculating fluctuograms as a function of
molecular signal such as Ca2+ binding and comparing the
resulting differences is a useful strategy to map out the residues
that are important to the specific property conveyed by the probed
signal. Therefore, calculating fluctuograms during the relevant
rare events of protein function, such as enzymatic reaction,
substrate binding, local unfolding [59], protein-protein association,
and conformational changes in allostery is likely a useful strategy
to establish the connection between a specific protein function and
the fluctuograms of protein dynamics. This approach can be
pursued by using multiscale computational methods such as
reaction path optimization and free-energy simulations [60,61]
and is currently being explored in our laboratory. For modulating the functional properties of subtilisin via protein
engineering, the strong Ca2+-dependence of stability and folding
suggests that the residues with large mechanical coupling variation
due to Ca2+-binding (Figure 2(a,b)) could be potential hot spots. Since the thermodynamic interactions associated with these
residues are more susceptible, mutation of these residues should
achieve the goal of altering protein stability. As mechanical
coupling also affects conformational flexibility and dynamics,
modulating the mechanical coupling network is also expected to
change other functional properties such as substrate binding and
activity. The Fluctuogram of Protein Dynamics function depends on Ca2+ binding. Furthermore, there is no direct
evidence that subtilisin forms alternative structures. Therefore, the
intra-protein communication in subtilisin is likely related to the
anharmonicity and nonlinearity of protein dynamics. functionally
important
mechanical
coupling
would
tend
to
correlate during evolution. To test this hypothesis, we devise
criteria to select residue pairs from the fluctuograms and compare
with those from a SCA on the results of a multiple sequence
alignment [38]. To test this hypothesis, we transform the sequential segments of
an atomic MD trajectory into separate elastic network models. The anharmonicity and nonlinearity are thus effectively repre-
sented as the temporal and spatial variation of the mechanical
coupling network. In analogy to the spectrogram of sound waves,
the periodic transformations of structural fluctuations into ENMs
are termed the ‘‘fluctuogram\, which records the choreography of
protein dynamics. The fluctuograms of Ca2+-bound and apo
subtilisin illustrate that local conformational changes and me-
chanical coupling variation are spatially and temporally intermit-
tent: large changes at one location do not last long and different
segments alternatively have prominent events between time
windows (Figure 3). The fluctuograms also revealed the pathways
of intra-protein communication. Ca2+-bound and apo subtilisin
have distinct fluctuograms, illustrating that although a drastic
structural change did not occur, Ca2+-mediated interactions
caused significant effects at distal sites through the mechanical
coupling network. Since the fluctuograms are calculated from atomic MD
simulations, sequence specific properties of the mechanical
coupling network are captured. Furthermore, the calculation does
not require the knowledge of specific protein motions [14,53]. We
show in Figure 5–8 that the residues calculated to have distinctive
behaviors of mechanical coupling can capture to a large extent the
residues observed to have high correlation in a multiple sequence
alignment. The results also indicate that the predictive power in
capturing residues with high sequence correlation depends on the
fluctuogram used for calculations. For subtilisin, the Ca2+-bound
simulation is expected to better capture the functionally important
mechanical coupling, since native subtilisin requires Ca2+ to work
and globally alignable sequences with the presence of the Ca2+-
binding loop are used for SCA. Indeed, the Ca2+-bound
fluctuogram gives higher hit rates and coverages than the apo
fluctuogram as shown in Figure 5–7. In addition to the magnitude
of force constants, the variation of coupling strength is also found
to be an indicative property for the sequence correlation observed
in SCA. Discussion As
EG1 is required to bind a polymer substrate already interacting
with other molecules on the solid surface, strong mechanical
strength in protein structure is likely needed for carrying out the
required non-catalytic actives. An important concern is the functional roles of the anharmo-
nicity and nonlinearity in protein dynamics, especially in allosteric
coupling without a drastic structural change. The population of
similar but distinct protein structures may still shift due to
molecular signals [11,58] and harmonic models are not suitable
for describing the concomitant reorganization of the mechanical
coupling network. For subtilisin Carlsberg, the Ca2+-binding loop
is distal to substrate-binding and active sites, yet the serine protease March 2011 | Volume 7 | Issue 3 | e1002023 13 March 2011 | Volume 7 | Issue 3 | e1002023 PLoS Computational Biology | www.ploscompbiol.org March 2011 | Volume 7 | Issue 3 | e1002023 All-atom molecular dynamics simulations The fluctuation
matching approach adjusts kij’s iteratively to reduce the difference
between Sdr2
ijTCG and Sdr2
ijTAA: kmz1
ij
~km
ij {a
1
Sdr2
ijTCG {
1
Sdr2
ijTAA
! :
ð6Þ ð6Þ In eq.(6), m is the step of fluctuation matching iteration, and a is a
numerical constant. Each step requires a NMA on the Ca-SC-
ENM to update force constants. The fluctuations of each bond are
approximated via Gaussian statistics and only non-negative force
constants are used. Starting from an initial distribution of kij
inversely proportional to Sdr2
ijTAA, convergence (root-of-mean-
square difference in force constants between steps ,0.005 kcal/
mol/A˚ 2) is typically achieved within 200 steps. In the example of subtilisin Carlsberg (Figure 1(a)), the 274-
residue serine protease results in a 504-site Ca-SC-ENM (incept in
Figure 1(c)) with a specific site dedicated for Ca2+ (orange ball). All
site pairs that have been within 10 A˚ during the course of 100 ns
all-atom trajectory are included in the pool of elastic bonds for
fluctuation matching. Since the force constants are adjusted
according to eq.(6) to match the statistics of inter-site distances
from all-atom MD, the results of fluctuation matching are not
sensitive
to
the
distance
cutoff
used
for
assigning
initial
connectivity. A cutoff of 10 A˚ provides sufficiently large basis for
capturing inter-site mechanical coupling. Compute the mechanical coupling network in subtilisin
from all-atom MD Without loss of generality, we choose to describe the mechanical
coupling between amino acids via a commonly used coarse-
grained (CG) elastic network model (ENM) [26,27]. In most
applications of ENM, a protein structure is used to define
connectivity with a distance cut-off and a universal force constant
is often assigned to ignore atomic details other than the native
structure [26,27]. Despite its simplicity, homogeneous ENM is
robust
in
predicting
collective
conformational
changes
[14,70,71,72,73,74,75] and the profile of atomic mean square
fluctuations when comparing with crystallographic B-factors
[26,27,75]. More sophisticated schemes for determining force
constants have been developed to improve the prediction of B-
factors [76]. All-atom molecular dynamics simulations y
We obtained the details of subtilisin by MD simulations using
the CHARMM22 all-atom force field and the TIP3P water model
[62,63]. The particle mesh Ewald method [64] was used for
calculating long-range electrostatics. For short-range non-bound
interactions, a cutoff of 14 A˚ was used with a switch function
turned on at 12 A˚ . Starting from an X-ray structure (PDB ID
1OYV) [5], a 100 ns trajectory was collected at 300 K and 1 atm
after minimization (100,000 steps), heating (+4 K/ps over 100 ps
via velocity reassignment), and equilibration (4 ns) steps. During
minimization, heating, and the first 3 ns of equilibration, Ca atoms
were restrained to their positions in the X-ray structure via
harmonic potentials with a force constant of 1 kcal/mol/A˚ 2. No
restraint potentials or external forces were applied in the last ns of
equilibration and production runs. Langevin dynamics with a
damping coefficient of 0.5 ps21 were used to maintain system
temperature at 300 K [65], and the Langevin piston method was
used to maintain pressure at 1 atm [66]. A time step of 2 fs was
used to propagate dynamics simulations during which all covalent
bonds associated hydrogen atoms were constrained at their
equilibrium values defined in the CHARMM parameter. A Cl2
ion was added to neutralize the subtilisin system in the Ca2+-
bound simulation; in the apo simulation, a Na+ ion was added. Apo subtilisin is marginally stable [4,5,6,7]. A total of 6,767 and
6,768 water molecules were used to solvate subtilisin in a truncated
octahedral
unit
cell
for
Ca2+-bound
and
apo
simulations,
respectively. Periodic boundary conditions were applied. All-atom
MD simulations were performed using the NAMD software [67]. Normal mode analysis and other analyses were performed using
the CHARMM software [68]. Figures of protein structures were
prepared via VMD [69]. In eq.(5), I and J are indices for residues; Nr is the total number of
residues in subtilisin. i and j are indices for CG sites; r0
ij and kij are
the length and force constant of the elastic bond between sites i
and j. The vibrational partition function corresponding to the
potential energy function of eq.(5) can be computed via normal
model analysis (NMA), and the predicted variance of each bond,
Sdr2
ijTCG, can be determined at a specified temperature [75,77]. However, the statistics computed from a segment of an all-atom
MD trajectory, Sdr2
ijTAA, may be different. The Fluctuogram of Protein Dynamics with experimental measurements that provide amino-acid level
information, such as by NMR methods, would be also valuable in
validating and improving the selection criteria of fluctuogram
analysis. The force constant between two CG sites designates the strength
of mechanical coupling. Each bond is treated separately and can
have a different value. The potential energy function of the Ca-
SC-ENM is: Methods VENM~ 1
2
X
Nr
I~1
X
J§I
X
i[I
X
j[J
kij rij{r0
ij
2
:
ð5Þ ð5Þ PLoS Computational Biology | www.ploscompbiol.org The Fluctuogram of Protein Dynamics Many mutation sites that enhance the stability of subtilisin
and its activity in an non-aqueous environment had been selected
via
random
mutation
and
screening
[31,32,33,34,35,36],
Figure 2(c), and are employed for testing the proposed connection
between the mechanical coupling network and protein engineering
of subtilisin. In Figure 2(a,b), we show that the residues calculated
to have large mechanical coupling variation correlate with the
reported mutation sites that had been shown to increase the
stability and activity of subtilisin. As presented in Results, the
agreement between the residues with large mechanical coupling
variation and the reported gain-of-function mutation sites far
exceeds that of randomly picking up the same number of residues. Therefore, the mechanical coupling networks calculated from
atomic details can be used to correlate with functionally important
mutation sites and a potential usage of fluctuograms is to identify
the susceptible spots in a mechanical coupling network for protein
engineering. Although the proposed procedure for calculating fluctuograms
is general and can be applied to any set of MD trajectories,
identifying residues important for function relies on the design of
selection criteria. Several criteria based on the statistics of force
constants are proposed heuristically and their abilities to correlate
with co-evolution vary from one protein to another as illustrated
by comparing the results of subtilisin and EG1. Such protein
dependence is not unexpected given the complexity and specificity
of protein sequence, structure, and function. In modeling complex
allosteric protein systems that spans a diverse range, we envision
that the success of applying the fluctuogram approach will not only
depend on the design of relevant MD simulations but also on the
criteria used for selecting function-related residues. Therefore, we
are also developing systematic ways to partition and categorize the
different behaviors of mechanical coupling in fluctuograms to
better map out their connection with specific properties that are
relevant to function. Direct comparison of the predicted residues The
fluctuogram
analysis
illustrates
that
the
mechanical
coupling network in protein structure is tightly coupled to
functional properties such as stability and intra-protein commu-
nication. If the mechanical coupling network specified by sequence
was optimized in addition to the structure, residue pairs with March 2011 | Volume 7 | Issue 3 | e1002023 March 2011 | Volume 7 | Issue 3 | e1002023 14 The Fluctuogram of Protein Dynamics Supporting Information Text S1
Discussion of the sequentially collective conformational
changes shown in Figure S4 and Figure S5. Text S1
Discussion of the sequentially collective conformational
changes shown in Figure S4 and Figure S5. (DOC) Text S1
Discussion of the sequentially collective conformational
changes shown in Figure S4 and Figure S5. (DOC) Figure S1
The root-mean-square difference (RMSD) of the Ca
atoms in Ca2+-bound (black) and apo (red) trajectories of subtilisin
to the X-ray structure (PDB ID: 1OYV). The cross RMSD
between the two simulations at each time frame is also shown in
blue. (EPS) Figure S7
Scatter plot of the 2nd and 3rd eigenvectors. A cutoff
distance of 0.07 from the origin was used to select 80 residues that
tend to co-evolve, which were divided into three clusters: blue, red,
and green. Residues at a distance of 0.07–1.0 from the origin are
colored with a lighter shade. Figure S2
The time evolution of the total force constant, kTOT,
of Ca2+-bound and apo subtilisin. kTOT is the sum of all force
constants between CG sites. The time window Dt for calculating
force constants is 4 ns. (TIF) Figure
S8
The
statistical
coupling
matrix,
calculated
as
described in [33]. Eigenvectors 2–4 were used for matrix cleaning
and the matrix is truncated to the 80 positions appearing in the
cluster analysis. Columns are grouped by cluster (in the order blue,
red, and green). Within each cluster, positions are ordered by their
distance from the origin along the 2nd and 3rd eigenvectors (Figure
S7). (TIF) Figure S3
Mechanical coupling variation in subtilisin due to
Ca2+ binding. Variation in the force constant of each residue
between neighboring time windows for (c) the Ca2+ simulation and
(d) the apo simulation. The time window Dt for calculating force
constants is 4 ns. (PDF) The Fluctuogram of Protein Dynamics The Fluctuogram of Protein Dynamics ($95% similar) identified by BLASTClust [81], the sequences
were re-aligned and the resulting alignment of 465 sequences was
used for conducting the statistical coupling analysis. The statistical
coupling matrix was created as described in Halabi et al. [38] and
eigenvectors 2 and 3 of this matrix were used to assign three
sectors. The cleaned SCA matrix (Figure S8) is used to visualize
the sectors and was generated from eigenvectors 2 through 4. The
endoglucanse SCA was conducted in a similar fashion. Our
Mathematica code is based on the MATLAB code from [38] and
is available upon request. Figure S6
Positional conservation of the multiple sequence
alignment, defined as the relative entropy between the observed
amino acid frequencies f(a) in each column i and the back-
ground frequencies q(a) from all proteins: D(a)
i ~f (a)
i
ln f (a)
i
q(a) z
(1{f (a)
i
) ln 1{f (a)
i
1{q(a) . Following [33], a binary approximation was
applied. Each position is represented as 1 if it contains the most
prevalent amino acid in that column, or 0 otherwise. Columns are
colored based on the clusters shown in Figure S7. (TIF) Author Contributions Conceived and designed the experiments: JSR JWC. Performed the
experiments: JSR YL JWC. Analyzed the data: JSR YL JWC. Contributed
reagents/materials/analysis tools: JSR JWC. Wrote the paper: JSR YL
JWC. Conceived and designed the experiments: JSR JWC. Performed the
experiments: JSR YL JWC. Analyzed the data: JSR YL JWC. Contributed
reagents/materials/analysis tools: JSR JWC. Wrote the paper: JSR YL
JWC. Figure S5
The time course of Ca-Ca distances in A˚ between
selected residue pairs for the Ca2+-bound (top) and apo (bottom)
simulations, (a) d(Asp41-Leu74), (b) d(Asp41-Val80), (c) d(Ala37- Video S1
(MPG) Figure S4
The time course of Ca-Ca distances in A˚ between
selected residue pairs for the Ca2+-bound (top) and apo (bottom)
simulations. The trajectories of d(Val51-Leu95), d(Ser100-Gly127),
and d(Gly127-Tyr166) of the Ca2+-bound simulation illustrate the
sequentially collective conformational change corresponding to the
highlighted band in fluctuogram shown in Figure 3(a,c). In the apo
simulation, such conformational change was not observed. (EPS) Figure S4
The time course of Ca-Ca distances in A˚ between
selected residue pairs for the Ca2+-bound (top) and apo (bottom)
simulations. The trajectories of d(Val51-Leu95), d(Ser100-Gly127),
and d(Gly127-Tyr166) of the Ca2+-bound simulation illustrate the
sequentially collective conformational change corresponding to the
highlighted band in fluctuogram shown in Figure 3(a,c). In the apo
simulation, such conformational change was not observed. (EPS) 7. Wells JA, Estell DA (1988) Subtilisin - An enzyme designed to be engineered.
Trends Biochem Sci 13: 291–297. Multiple sequence alignment and statistical coupling
analysis y
Subtilisin homologs were gathered from the NCBI’s non-
redundant database using GGSEARCH of the FASTA suite [78]
as well as Pfam [79]. Sequences from the Pfam peptidase inhibitor
I9 domain (PF05922) and the subtilase family domain (PF00082)
were combined with the results from GGSEARCH. Since
GGSEARCH returns only globally alignable sequences, both the
mature protein sequence (274 aa) and the sequence including the
N-terminal signal peptide and propeptide (379 aa) were used as
queries. For both the GGSEARCH and Pfam sequences, an initial
alignment was constructed with MAFFT [80], and then truncated
to positions in the mature subtilisin Carlsberg structure (PDB
1OYV) [5]. Sequences were selected from these truncated
alignments based on the number of alignable positions (no more
than 100 gaps) and the presence of the Ca2+-binding loop (at most
one gap at positions 75–79). After removing redundant sequences In our implementation, the sidechain and backbone contribu-
tions are treated separately by using two CG sites per amino acid. To determine the coordinates of CG sites from an atomic
configuration, the Ca positions are used to define backbone sites
and the centers of mass of sidechain atoms are used to define the
sidechain sites; glycine has a single site. The mass of the backbone
site is the total mass of all backbone atoms and the mass of the
sidechain site is the total mass of all sidechain atoms. The resulting
CG model is referred to as Ca-SC-ENM (SC;sidechain). March 2011 | Volume 7 | Issue 3 | e1002023 15 PLoS Computational Biology | www.ploscompbiol.org 13. Cui Q, Karplus M (2008) Allostery and cooperativity revisited. Protein Sci 17:
1295–1307. The Fluctuogram of Protein Dynamics Haas RK, Chu J-W (2009) Decomposition of energy and free energy changes by
following the flow of work along reaction path. J Chem Phys 131: 144105. 30. Johnson K (2003) Acoustic and Auditory Phonetics. Malden: Blackwell
Publishing. 62. Mackerell AD (2004) Empirical force fields for biological macromolecules:
Overview and issues. J Comput Chem 25: 1584–1604. Overview and issues. J Comput Chem 25: 1584–1604. g
31. Zhao H, Arnold F (1999) Directed evolution converts subtilisin E into a
functional equivalent of thermitase. Protein Eng 12: 47–53. 63. Mackerell AD, Feig M, Brooks CL (2004) Extending the treatment of backbone
energetics in protein force fields: limitations of gas-phase quantum mechanics in
reproducing protein conformational distributions in molecular dynamics
simulations. J Comput Chem 25: 1400–1415. 32. Strausberg S, Alexander P, Gallagher D, Gilliland G, Barnett B, et al. (1995)
Directed evolution of a subtilisin with calcium-independent stability. Biotech-
nology 13: 669–673. 64. Darden T, York D, Pederson L (1993) Particle mesh Ewald: an Nlog(N) method
for Ewald sums in large systems. J Chem Phys 98: 10089. gy
33. Strausberg S, Ruan B, Fisher K, Alexander P, Bryan P (2005) Directed
coevolution of stability and catalytic activity in calcium-free subtilisin. Biochemistry 44: 3272–3279. 65. Allen MP, Tildesley DJ (1987) Computer Simulation of Liquids. New York:
Oxford. 34. Rollence ML, Filpula D, Pantoliano MW, Bryan PN (1988) Engineering
thermostability in subtilisin BPN’ by in vitro mutagenesis. CRC Crit Rev
Biotech 8: 217–224. 66. Feller SE, Zhang YH, Pastor RW, Brooks BR (1995) Constant-pressure
molecular-dynamics simulation - The Langevin piston method. J Chem Phys
103: 4613–4621. 35. Chen K, Arnold F (1991) Enzyme engineering for nonaqueous solvents - random
mutagenesis to enhance activity of subtilisin-E in polar organic media. Biotechnology 9: 1073–1077. 67. Phillips JC, Braun R, Wang W, Gumbart J, Tajkhorshid E, et al. (2005) Scalable
molecular dynamics with NAMD. J Comput Chem 26: 1781–1802. 68. Brooks BR, Brooks CL, Mackerell AD, Nilsson L, Petrella RJ, et al. (2009)
CHARMM: The Biomolecular Simulation Program. J Comput Chem 30:
1545–1614. gy
36. Chen K, Arnold F (1993) Tuning the activity of an enzyme for unusual
environments - sequential random mutagenesis of subtilisin-E for catalysis in
dimethylformamide. Proc Natl Acad Sci USA 90: 5618–5622. 69. Humphrey W, Dalke A, Schulten K (1996) VMD: Visual molecular dynamics. J Mol Graphics 14: 33–38. y
37. The Fluctuogram of Protein Dynamics 14. Zheng WJ, Brooks BR, Thirumalai D (2006) Low-frequency normal modes that
describe allosteric transitions in biological nanomachines are robust to sequence
variations. Proc Natl Acad Sci USA 103: 7664–7669. 49. Vendruscolo M, Paci E, Dobson CM, Karplus M (2001) Three key residues form a
critical contact network in a protein folding transition state. Nature 409: 641–645. 50. Vendruscolo M, Dokholyan NV, Paci E, Karplus M (2002) Small-world view of
the amino acids that play a key role in protein folding. Phys Rev E 65: 061910. 15. Chennubhotla C, Bahar I (2006) Markov propagation of allosteric effects in
biomolecular systems: application to GroEL-GroES. Mol Syst Biol 2: 36. p y
y
p
g
y
51. del Sol A, Fujihashi H, Amoros D, Nussinov R (2006) Residues crucial for
maintaining short paths in network communication mediate signaling in
proteins. Mol Syst Biol 2: 2006.0019. y
pp
y
16. Del Sol A, Tsai C-J, Ma B, Nussinov R (2009) The Origin of Allosteric Functional
Modulation: Multiple Pre-existing Pathways. Structure 17: 1042–1050. Modulation: Multiple Pre-existing Pathways. Structure 17: 1042–105 p
g
y
17. Bahar I, Lezon TR, Yang L-W, Eyal E (2010) Global Dynamics of Proteins: 17. Bahar I, Lezon TR, Yang L-W, Eyal E (2010) Global Dynamics of Proteins:
Bridging Between Structure and Function. Annu Rev Biophys 39: 23–42. 52. Ghosh A, Vishveshwara S (2007) A study of communication pathways in
methionyl-tRNA synthetase by molecular dynamics simulations and structure
network analysis. Proc Natl Acad Sci USA 104: 15711–15716. Bridging Between Structure and Function. Annu Rev Biophys 39: g g
p y
18. Sherwood P, Brooks BR, Sansom MS (2008) Multiscale methods for
macromolecular simulations. Curr Opin Struct Biol 18: 630. 53. Zheng W, Brooks BR, Thirumalai D (2009) Allosteric transitions in biological
nanomachines are described by robust normal modes of elastic networks. Curr
Protein Pept Sc 10: 128–132. 19. Hilser VJ, Dowdy D, Oas TG, Freire E (1998) The structural distribution of
cooperative interactions in proteins: Analysis of the native state ensemble. Proc
Natl Acad Sci USA 95: 9903–9908. p
54. Fodor AA, Aldrich RW (2004) On evolutionary conservation of thermodynamic
coupling in proteins. J Biol Chem 279: 19046–19050. 20. Hilser VJ, Garcia-Moreno B, Oas TG, Kapp G, Whitten ST (2006) A statistical
thermodynamic model of the protein ensemble. Chem Rev 106: 1545–1558. 55. Chi CN, Elfstrom L, Shi Y, Snall T, Engstrom A, et al. The Fluctuogram of Protein Dynamics Suel GM, Lockless SW, Wall MA, Ranganathan R (2003) Evolutionarily
conserved networks of residues mediate allosteric communication in proteins. Nat Struct Biol 10: 59–69. 70. Lezon TR, Sali A, Bahar I (2009) Global Motions of the Nuclear Pore Complex:
Insights from Elastic Network Models. Plos Comput Biol 5: e1000496. 38. Halabi N, Rivoire O, Leibler S, Ranganathan R (2009) Protein sectors:
Evolutionary units of three-dimensional structure. Cell 138: 774–786. 71. Ming D, Wall ME (2005) Allostery in a coarse-grained model of protein
dynamics. Phys Rev Lett 95: 198103. 39. Zhou HX (2004) Polymer models of protein stability, folding, and interactions. Biochemistry 43: 2141–2154. 72. Maragakis P, Karplus M (2005) Large amplitude conformational change in
proteins explored with a plastic network model: Adenylate kinase. J Mol Biol
352: 807–822. y
40. Tracewell CA, Arnold FH (2009) Directed enzyme evolution: climbing fitness
peaks one amino acid at a time. Curr Opin Chem Biol 13: 3–9. 73. Chu J-W, Voth GA (2007) Coarse-grained free energy functions for studying
protein conformational changes: A double-well network model. Biophys J 93:
3860–3871. 41. Eppler RK, Komor RS, Huynh J, Dordick JS, Reimer JA, et al. (2006) Water
dynamics and salt-activation of enzymes in organic media: Mechanistic implications
revealed by NMR spectroscopy. Proc Natl Acad Sci USA 103: 5706–5710. revealed by NMR spectroscopy. Proc Natl Acad Sci USA 103: 570 74. Tama F, Valle M, Frank J, Brooks CL (2003) Dynamic reorganization of the
functionally active ribosome explored by normal mode analysis and cryo-
electron microscopy. Proc Natl Acad Sci USA 100: 9319–9323. 42. Eppler RK, Hudson EP, Chase SD, Dordick JS, Reimer JA, et al. (2008)
Biocatalyst activity in nonaqueous environments correlates with centisecond-
range protein motions. Proc Natl Acad Sci USA. y
y
q
range protein motions. Proc Natl Acad Sci USA. 75. Ma JP (2005) Usefulness and limitations of normal mode analysis in modeling
dynamics of biomolecular complexes. Structure 13: 373–380. 43. Hudson EP, Eppler RK, Beaudoin JM, S. DJ, Reimer JA, et al. (2009) Active-
Site Motions and Polarity Enhance Catalytic Turnover of Hydrated Subtilisin
Dissolved in Organic Solvents. J Am Chem Soc 131: 7. 76. Yang L, Song G, Jernigan RL (2009) Protein elastic network models and the
ranges of cooperativity. Proc Natl Acad Sci USA 106: 12347–12352. g
J
44. Pantoliano MW, Whitlow M, Wood JF, Rollence ML, Finzel BC, et al. The Fluctuogram of Protein Dynamics (2008) Reassessing a
sparse energetic network within a single protein domain. Proc Natl Acad Sci
USA 105: 4679–4684. 21. Noid WG, Chu J-W, Ayton GS, Krishna V, Izvekov S, et al. (2008) The
multiscale coarse-graining method. I. A rigorous bridge between atomistic and
coarse-grained models. J Chem Phys 128: 244114. 56. Liu Z, Chen J, Thirumalai D (2009) On the accuracy of inferring energetic
coupling between distant sites in protein families from evolutionary imprints:
Illustrations using lattice model. Proteins 77: 823–831. g
J
y
22. Cho H, Chu J-W (2009) Inversion of Radial Distribution Functions to Pair Forces by
Solving the Yvon-Born-Green Equation Iteratively. J Chem Phys 131: 134107. Solving the Yvon-Born-Green Equation Iteratively. J Chem Phys 131: 23. Lee S, Jang DJ (2000) Cation-binding sites of subtilisin Carlsberg probed with
Eu(III) luminescence. Biophys J 79: 2171–2177. 57. Kleywegt G, Zou J, Divne C, Davies G, Sinning I, et al. (1997) The crystal
structure of the catalytic core domain of endoglucanase I from Trichoderma
reesei at 3.6 angstrom resolution, and a comparison with related enzymes. J Mol
Biol 272: 383–397. (
)
p y
24. Alexander PA, Ruan B, Bryan PN (2001) Cation-dependent stability of subtilisin. Biochemistry 40: 10634–10639. y
25. Gallagher T, Bryan PN, Gilliland GL (1993) Calcium-independent subtilisin by
design. Proteins 16: 205–213. 58. Csermely P, Palotai R, Nussinov R (2010) Induced fit, conformational selection
and independent dynamic segments: an extended view of binding events. Trends
Biochem Sci 35: 539–546. g
26. Tirion MM (1996) Large amplitude elastic motions in proteins from a single-
parameter, atomic analysis. Phys Rev Lett 77: 1905–1908. 59. Brokaw J, Chu J-W (2010) On the Roles of Substrate Binding and Hinge Unfolding in
Conformational Changes of Adenylate Kinase. Biophys J 99: 3420–3429. p
y
y
27. Bahar I, Atilgan AR, Jernigan RL, Erman B (1997) Understanding the recognition
of protein structural classes by amino acid composition. Proteins 29: 172–185. 60. Brokaw JB, Haas KR, Chu J-W (2009) Reaction Path Optimization with
Holonomic Constraints and Kinetic-Energy Potentials. J Chem Theory Comput
5: 2050–2061. 28. Chu J-W, Voth GA (2006) Coarse-grained modeling of the actin filament
derived from atomistic-scale simulations. Biophys J 90: 1572–1582. derived from atomistic-scale simulations. Biophys J 90: 1572–1582 29. Lyman E, Pfaendtner J, Voth GA (2008) Systematic Multiscale Parameterization
of Heterogeneous Elastic Network Models of Proteins. Biophys J 95: 4183–4192. 61. References 8. Smock RG, Gierasch LM (2009) Sending signals dynamically. Science 324:
198–203. 1. Koshland DE (1958) Application of a theory of enzyme specificity to protein
synthesis. Proc Natl Acad Sci USA 44: 98–104. y
2. Monod J, Wyman J, Changeux JP (1965) An nature of allosteric transitions - A
plausible model. J Mol Biol 12: 88–118. 9. Ma B, Nussinov R (2009) Amplification of signaling via cellular allosteric relay
and protein disorder. Proc Natl Acad Sci USA 106: 6887–6888. p
J
3. Yu EW, Koshland DE (2001) Propagating conformational changes over long
(and short) distances in proteins. Proc Natl Acad Sci USA 98: 9517–9520. 10. McNicholl ET, Das R, SilDas S, Taylor SS, Melacini G (2010) Communication
between tandem camp binding domains in the regulatory subunit of protein
kinase A-I alpha as revealed by domain-silencing mutations. J Biol Chem 285:
15523–15537. 4. Kraut J (1977) Serine proteases - Structure and mechanism of catalysis. Annu
Rev Biochem 46: 331–358. 11. Whitley MJ, Lee AL (2009) Frameworks for understanding long-range intra-
protein communication. Curr Protein Pept Sci 10: 116–127. 5. Barrette-Ng IH, Ng KKS, Cherney MM, Pearce G, Ryan CA, et al. (2003)
Structural basis of inhibition revealed by a 1 : 2 complex of the two-headed
tomato inhibitor-II and subtilisin Carlsberg. J Biol Chem 278: 24062–24071. mato inhibitor-II and subtilisin Carlsberg. J Biol Chem 278: 24062–24 12. Tsai C-J, del Sol A, Nussinov R (2009) Protein allostery, signal transmission and
dynamics: a classification scheme of allosteric mechanisms. Mol Biosyst 5:
207–216. 6. Bryan PN (2000) Protein engineering of subtilisin. BBA-Protein Struct M 1543:
203–222. 13. Cui Q, Karplus M (2008) Allostery and cooperativity revisited. Protein Sci 17:
1295–1307. 13. Cui Q, Karplus M (2008) Allostery and cooperativity revisited. Protein Sci 17:
1295–1307. 7. Wells JA, Estell DA (1988) Subtilisin - An enzyme designed to be engineered. Trends Biochem Sci 13: 291–297. PLoS Computational Biology | www.ploscompbiol.org March 2011 | Volume 7 | Issue 3 | e1002023 March 2011 | Volume 7 | Issue 3 | e1002023 PLoS Computational Biology | www.ploscompbiol.org 16 The Fluctuogram of Protein Dynamics (1988)
The engineering of binding-affinity at metal-ion binding-sites for the stabilization
of proteins - Subtilisin as a test case. Biochemistry 27: 8311–8317. 77. Brooks BR, Janezic D, Karplus M (1995) Harmonic-analysis of large systems .1. methodology. J Comput Chem 16: 1522–1542. 78. Pearson WR, Lipman DJ (1988) Improved tools for biological sequence
comparison. Proc Natl Acad Sci USA 85: 2444–2448. 45. Tsai C-J, Del Sol A, Nussinov R (2008) Allostery: Absence of a change in shape
does not imply that allostery is not at play. J Mol Biol 378: 1–11. 79. Finn RD, Mistry J, Tate J, Coggill P, Heger A, et al. (2010) The Pfam protein
families database. Nucleic Acids Res 38: D211–D222. 46. Plerou V, Gopikrishnan P, Rosenow B, Amaral LAN, Guhr T, et al. (2002)
Random matrix approach to cross correlations in financial data. Phys Rev E 65:
066126. 80. Katoh K, Kuma K, Toh H, Miyata T (2005) MAFFT version 5: improvement in
accuracy of multiple sequence alignment. Nucleic Acids Res 33: 511–518. 47. Fisher KE, Ruan B, Alexander PA, Wang L, Bryan PN (2007) Mechanism of the
kinetically-controlled folding reaction of subtilisin. Biochemistry 46: 640–651. 81. Altschul SF, Madden TL, Schaffer AA, Zhang JH, Zhang Z, et al. (1997)
Gapped BLAST and PSI-BLAST: a new generation of protein database search
programs. Nucleic Acids Res 25: 3389–3402. 48. Daily MD, Upadhyaya TJ, Gray JJ (2008) Contact rearrangements form
coupled networks from local motions in allosteric proteins. Proteins 71: 455–466. PLoS Computational Biology | www.ploscompbiol.org March 2011 | Volume 7 | Issue 3 | e1002023 PLoS Computational Biology | www.ploscompbiol.org 17
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Feasibility of Primary Tumor Culture Models and Preclinical Prediction Assays for Head and Neck Cancer: A Narrative Review
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UvA-DARE (Digital Academic Repository) Citation for published version (APA):
Dohmen, A. J. C., Swartz, J. E., Van Den Brekel, M. W. M., Willems, S. M., Spijker, R.,
Neefjes, J., & Zuur, C. L. (2015). Feasibility of primary tumor culture models and preclinical
prediction assays for head and neck cancer: A narrative review. Cancers, 7(3), 1716-1742. https://doi.org/10.3390/cancers7030858 Citation for published version (APA):
Dohmen, A. J. C., Swartz, J. E., Van Den Brekel, M. W. M., Willems, S. M., Spijker, R.,
Neefjes, J., & Zuur, C. L. (2015). Feasibility of primary tumor culture models and preclinical
prediction assays for head and neck cancer: A narrative review. Cancers, 7(3), 1716-1742.
https://doi.org/10.3390/cancers7030858 General rights General rights
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will be contacted as soon as possible. Cancers 2015, 7, 1716-1742; doi:10.3390/cancers7030858
OPEN ACCESS
cancers
ISSN 2072-6694
www.mdpi.com/journal/cancers
Review
Feasibility of Primary Tumor Culture Models and
Preclinical Prediction Assays for Head and Neck Cancer:
A Narrative Review
Amy J. C. Dohmen 1,2,*, Justin E. Swartz 3, Michiel W. M. Van Den Brekel 1, Stefan M. Willems 4,
René Spijker 5,6, Jacques Neefjes 2 and Charlotte L. Zuur 1
1 Department of Head and Neck Surgery and Oncology, The Netherlands Cancer Institute—Antoni van
Leeuwenhoek, Plesmanlaan 121, Amsterdam 1066 CX, The Netherlands;
E-Mails: m.vd.brekel@nki.nl (M.W.M.V.D.B.); c.zuur@nki.nl (C.L.Z.)
2 Department of Cell Biology, the Netherlands Cancer Institute—Antoni van Leeuwenhoek,
Plesmanlaan 121, Amsterdam 1066 CX, The Netherlands; E-Mail: j.neefjes@nki.nl
3 Department of Otorhinolaryngology—Head and Neck Surgery, University Medical Center Utrecht,
Heidelberglaan 100, Utrecht 3508 GA, The Netherlands; E-Mail: j.e.swartz@umcutrecht.nl
4 Department of Pathology, University Medical Center Utrecht, Heidelberglaan 100, Utrecht 3508 GA,
The Netherlands; E-Mail: s.m.willems-4@umcutrecht.nl
5 Medical library, Academic Medical Center, Amsterdam 1100 DE, The Netherlands;
E-Mail: r.spijker@amc.uva.nl
6 Dutch Cochrane Centre, Julius Center for Health Sciences and Primary Care, University Medical
Center Utrecht, Heidelberglaan 100, Utrecht 3508 GA, The Netherlands
* Author to whom correspondence should be addressed; E-Mail: a.dohmen@nki.nl;
Tel.: +31-20-512-2550; Fax: +31-20-512-2554.
Academic Editor: Maie A. St. John
Received: 2 July 2015 / Accepted: 20 August 2015 / Published: 28 August 2015
Abstract: Primary human tumor culture models allow for individualized drug sensitivity
testing and are therefore a promising technique to achieve personalized treatment for cancer
patients. This would especially be of interest for patients with advanced stage head and neck Cancers 2015, 7, 1716-1742; doi:10.3390/cancers7030858 OPEN ACCESS
cancers
ISSN 2072-6694
www.mdpi.com/journal/cancers Feasibility of Primary Tumor Culture Models and
Preclinical Prediction Assays for Head and Neck Cancer:
A Narrative Review Amy J. C. Dohmen 1,2,*, Justin E. Swartz 3, Michiel W. M. Van Den Brekel 1, Stefan M. Willems 4,
René Spijker 5,6, Jacques Neefjes 2 and Charlotte L. Zuur 1 Amy J. C. Dohmen 1,2,*, Justin E. Swartz 3, Michiel W. M. Van Den Brekel 1, Stefan
René Spijker 5,6, Jacques Neefjes 2 and Charlotte L. Zuur 1 Received: 2 July 2015 / Accepted: 20 August 2015 / Published: 28 August 2015 Disclaimer/Complaints regulations
f UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl)
will be contacted as soon as possible. Download date:24 Oct 2024 Amy J. C. Dohmen 1,2,*, Justin E. Swartz 3, Michiel W. M. Van Den Brekel 1, Stefan M. Willems 4,
René Spijker 5,6, Jacques Neefjes 2 and Charlotte L. Zuur 1 1 Department of Head and Neck Surgery and Oncology, The Netherlands Cancer Institute—Antoni van
Leeuwenhoek, Plesmanlaan 121, Amsterdam 1066 CX, The Netherlands;
E-Mails: m.vd.brekel@nki.nl (M.W.M.V.D.B.); c.zuur@nki.nl (C.L.Z.)
2 Department of Cell Biology, the Netherlands Cancer Institute—Antoni van Leeuwenhoek,
Plesmanlaan 121, Amsterdam 1066 CX, The Netherlands; E-Mail: j.neefjes@nki.nl
3 Department of Otorhinolaryngology—Head and Neck Surgery, University Medical Center Utrecht,
Heidelberglaan 100, Utrecht 3508 GA, The Netherlands; E-Mail: j.e.swartz@umcutrecht.nl
4 Department of Pathology, University Medical Center Utrecht, Heidelberglaan 100, Utrecht 3508 GA,
The Netherlands; E-Mail: s.m.willems-4@umcutrecht.nl 1 Department of Head and Neck Surgery and Oncology, The Netherlands Cancer Institute—Antoni van
Leeuwenhoek, Plesmanlaan 121, Amsterdam 1066 CX, The Netherlands;
E-Mails: m.vd.brekel@nki.nl (M.W.M.V.D.B.); c.zuur@nki.nl (C.L.Z.)
2 Department of Cell Biology, the Netherlands Cancer Institute—Antoni van Leeuwenhoek,
Plesmanlaan 121, Amsterdam 1066 CX, The Netherlands; E-Mail: j.neefjes@nki.nl
3 Department of Otorhinolaryngology—Head and Neck Surgery, University Medical Center Utrecht,
Heidelberglaan 100, Utrecht 3508 GA, The Netherlands; E-Mail: j.e.swartz@umcutrecht.nl
4 Department of Pathology, University Medical Center Utrecht, Heidelberglaan 100, Utrecht 3508 GA,
The Netherlands; E-Mail: s.m.willems-4@umcutrecht.nl 5 Medical library, Academic Medical Center, Amsterdam 1100 DE, The Netherlands;
E-Mail: r.spijker@amc.uva.nl 5 Medical library, Academic Medical Center, Amsterdam 1100 DE, The Netherlands; 6 Dutch Cochrane Centre, Julius Center for Health Sciences and Primary Care, University 6 Dutch Cochrane Centre, Julius Center for Health Sciences and Primary Care, University Medical
Center Utrecht, Heidelberglaan 100, Utrecht 3508 GA, The Netherlands Center Utrecht, Heidelberglaan 100, Utrecht 3508 GA, The Netherlands * Author to whom correspondence should be addressed; E-Mail: a.dohmen@nki.nl;
Tel.: +31-20-512-2550; Fax: +31-20-512-2554. Academic Editor: Maie A. St. John Academic Editor: Maie A. St. John Received: 2 July 2015 / Accepted: 20 August 2015 / Published: 28 August 2015 Abstract: Primary human tumor culture models allow for individualized drug sensitivity
testing and are therefore a promising technique to achieve personalized treatment for cancer
patients. This would especially be of interest for patients with advanced stage head and neck
cancer. They are extensively treated with surgery, usually in combination with high-dose
cisplatin chemoradiation. However, adding cisplatin to radiotherapy is associated with an
increase in severe acute toxicity, while conferring only a minor overall survival benefit. Hence, there is a strong need for a preclinical model to identify patients that will respond to
the intended treatment regimen and to test novel drugs. One of such models is the technique 1717 Cancers 2015, 7 of culturing primary human tumor tissue. 1. Introduction Seventy percent of all patients with head and neck squamous cell carcinoma (HNSCC) present
with advanced stage disease and are characterized by an overall 5-year survival rate of approximately
35%–60% in the case of surgical treatment with or without chemotherapy (CT) and radiotherapy
(RT) [1–3]. From around 1980 onward the addition of high-dose cisplatin to RT (CCRT) has become
the routine treatment for locally advanced disease [4]. Nevertheless, a meta-analysis of randomized
trials in 2009 indicated that there is only a moderate absolute overall survival benefit of 6.5% at
5 years when adding chemotherapy to loco-regional treatment [5]. A subgroup of head and neck cancer
(HNC) patients with HPV-positive oropharynx carcinomas usually shows better prognosis following
CCRT [6]. A similar analysis in laryngeal cancer patients also described no survival benefit from the
addition of chemotherapy to radiotherapy [7]. Moreover, the addition of high-dose cisplatin to RT is
accompanied with a substantial increase in grade three or worse toxicity of 52% to 89% [8]. A more
personalized patient selection for this treatment should improve the quality-of-life of the non-responding
patient population. More effective and less toxic targeted therapies have not (yet) penetrated in the treatment of patients
with HNC. In recent years, only cetuximab has been registered as a radiosensitizer to improve treatment
for advanced HNSCC. Literature, however, shows inconclusive results for survival benefit of this
treatment compared to CCRT [9,10]. Unfortunately, this leaves CCRT the mainstream of therapy with
rather variable individual clinical outcome. It therefore remains a major challenge in HNSCC to develop
novel drugs for improved survival and to reveal patients prior to therapy that will actually benefit from
the intended treatment regimen. Consequently, there is a strong need for a preclinical model to identify
those tumors of patients that will respond to a particular treatment. One of such models is the technique
of culturing primary tumor tissue and testing drugs prior to treatment. In order for a culture model to be
feasible as a preclinical treatment prediction tool, it should be a short-term culture technique, resembling
the patient’s tumor as closely as possible and it should be low in costs. Xenograft mouse models can be
used to assess therapy response as well. However, they are in fact long-term assays in which the patient’s
tumor cells adjust to the murine environment, leading to genetic drift of the tumor cells. Amy J. C. Dohmen 1,2,*, Justin E. Swartz 3, Michiel W. M. Van Den Brekel 1, Stefan M. Willems 4,
René Spijker 5,6, Jacques Neefjes 2 and Charlotte L. Zuur 1 This review discusses the feasibility and success
rate of existing primary head and neck tumor culturing techniques and their corresponding
chemo- and radiosensitivity assays. A comprehensive literature search was performed and
success factors for culturing in vitro are debated, together with the actual value of these
models as preclinical prediction assay for individual patients. With this review, we aim to
fill a gap in the understanding of primary culture models from head and neck tumors, with
potential importance for other tumor types as well. Keywords: head neck cancer; primary cell cultures; chemosensitivity; radiosensitivity;
personalized therapy Keywords: head neck cancer; primary cell cultures; chemosensitivity; radiosensitivity;
personalized therapy Cancers 2015, 7
Cancers 2015, 7 Cancers 2015, 7
Cancers 2015, 7 percentages of previously described fresh primary HNSCC culturing techniques and their preclinical
chemo- and radiosensitivity assays. success percentages of previously described fresh primary HNSCC culturing techniques and their
preclinical chemo- and radiosensitivity assays. 1. Introduction These models are
not optimal, expensive and difficult for multiple drug testing. For these reasons, we excluded xenograft
mouse models from our literature search. With this review, we aim to study feasibility and success 17183 3.1. HNSCC Cell Lines
3.1. HNSCC Cell Lines The first attempts to establish HNSCC monolayer cell lines were performed in the 1950’s on a variety
of tumors (sarcoma, leukemia, Hodgkin, myeloma, kidney, breast, lung, pharynx, larynx, rectum,
melanoma and ovary) [11]. From the 1980s onward, several groups, including those of Carey and
Grenman [12], Rheinwald and Beckett [13], and Easty [14], were among the earliest to be able to
establish HNSCC cell lines, achieving success rates of around 30% [12]. Nowadays, a myriad of HNSCC
cell lines are available for in vitro experiments, as summarized by Carey in 1994 [12], Sacks in 1996 [15]
and Lin in 2007 [16]. Also, tumor cell lines from particular patient cases are available, such as very
young patients or patients with Fanconi anemia-associated HNSCC [17,18]. It is not exactly known why
certain tumors may be cultured indefinitely, while others cannot, although culture success rates have
improved by taking biopsies under aseptic conditions from non-necrotic and uninfected tumor areas. The first attempts to establish HNSCC monolayer cell lines were performed in the 1950’s on a
variety of tumors (sarcoma, leukemia, Hodgkin, myeloma, kidney, breast, lung, pharynx, larynx, rectum,
melanoma and ovary) [11]. From the 1980s onward, several groups, including those of Carey and
Grenman [12], Rheinwald and Beckett [13], and Easty [14], were among the earliest to be able to
establish HNSCC cell lines, achieving success rates of around 30% [12]. Nowadays, a myriad of HNSCC
cell lines are available for in vitro experiments, as summarized by Carey in 1994 [12], Sacks in 1996 [15]
and Lin in 2007 [16]. Also, tumor cell lines from particular patient cases are available, such as very
young patients or patients with Fanconi anemia-associated HNSCC [17,18]. It is not exactly known why
certain tumors may be cultured indefinitely, while others cannot, although culture success rates have
improved by taking biopsies under aseptic conditions from non-necrotic and uninfected tumor areas. In all studies, HNSCC cell lines were established through the “explant technique”, described
extensively in 1994 by Carey [12]. In this technique, fresh tumor specimens were mechanically minced
into fragments. Samples may be further dispersed enzymatically using trypsin, DNase, collagenase or a
combination thereof [19]. The cell suspension was then placed into a rich culture medium, such as
DMEM or RPMI-1640 with additional fetal bovine serum (FBS) and transferred to petri-dishes or culture
flasks [20–22]. 2. Materials and Methods
2. Materials and Methods A narrative review was performed via a systematic literature search in Pubmed searching for primary
HNSCC tumor culturing techniques (research Question 1) and their in vitro sensitivity assays with
clinical correlation (research Question 2) (Supplementary Materials). We screened titles and abstracts
of the identified literature using preformulated criteria (Figure 1a,b). Thereafter, a full text screen of the
selected articles was done. Included were studies that described any technique for culturing fresh primary
tumor tissue of HNSCC patients, except for techniques involving only cultures using xenografts models. The search includes papers using cell lines. Only papers considering primary tumor tissue to establish
fresh cell lines were included. Studies describing the use of purchased or already established cell lines,
while not reporting the technique of its establishment, were excluded. Also included were fresh HNSCC
culture studies regarding in vitro versus in vivo chemosensitivity or radiosensitivity assays. Additionally,
references of the included studies were screened and added to the literature list when relevant. Final
selection was based on consensus of all authors. A narrative review was performed via a systematic literature search in Pubmed searching for primary
HNSCC tumor culturing techniques (research Question 1) and their in vitro sensitivity assays with
clinical correlation (research Question 2) (Supplementary Materials). We screened titles and abstracts of
the identified literature using preformulated criteria (Figure 1a,b). Thereafter, a full text screen of the
selected articles was done. Included were studies that described any technique for culturing fresh primary
tumor tissue of HNSCC patients, except for techniques involving only cultures using xenografts models. The search includes papers using cell lines. Only papers considering primary tumor tissue to establish
fresh cell lines were included. Studies describing the use of purchased or already established cell lines,
while not reporting the technique of its establishment, were excluded. Also included were fresh HNSCC
culture studies regarding in vitro versus in vivo chemosensitivity or radiosensitivity assays. Additionally,
references of the included studies were screened and added to the literature list when relevant. Final selection
was based on consensus of all authors. selection was based on consensus of all authors. was based on consensus of all authors. Figure 1. Cont. Figure 1. Cont. Figure 1. Cont. Figure 1. Cont. 17194 Cancers 2015, 7
Cancers 2015, 7 Figure 1. 2. Materials and Methods
2. Materials and Methods Flow diagram of the systematic review process for the (a) search of various
culturing techniques used in head and neck cancer; (b) search for chemo- and radiosensitivity
assays. Figure 1. Flow diagram of the systematic review process for the (a) search of
various culturing techniques used in head and neck cancer; (b) search for chemo- and
radiosensitivity assays. Figure 1. Flow diagram of the systematic review process for the (a) search of various
culturing techniques used in head and neck cancer; (b) search for chemo- and radiosensitivity
assays
Figure 1. Flow diagram of the systematic review process for the (a) search of
various culturing techniques used in head and neck cancer; (b) search for chemo- and
radiosensitivity assays. 3. Results
3. Results An overview of key publications for fresh primary tumor cell culture of HNSCC is presented in Table 1,
representing the culturing techniques, and Table 2, showing the clinical correlation. An overview of key publications for fresh primary tumor cell culture of HNSCC is presented in
Table 1, representing the culturing techniques, and Table 2, showing the clinical correlation. An overview of key publications for fresh primary tumor cell culture of HNSCC is presented in Table 1,
representing the culturing techniques, and Table 2, showing the clinical correlation. An overview of key publications for fresh primary tumor cell culture of HNSCC is presented in
Table 1, representing the culturing techniques, and Table 2, showing the clinical correlation. 3.2. Single Cell Cultures One essential way of culturing is by starting off with single cell suspensions from tumor biopsies. This is usually done by mechanical and enzymatic digestion. The first part of Table 1 describes studies
using this technique. Cancers 2015, 7 Cancers 2015, 7 the 20th passage (e.g., several months, depending on growth rate), as about 15% of tumor cells initially
show growth but then stop growing or die. Success percentages of 11%–33% have been described for
establishment of cell lines from HNSCC in this fashion [13,23,24]. Recently, Owen et al. described
higher success rates of 50%. They used fluorescence associated cell sorting to separate fibroblasts from
tumor cells. This appeared to be a promising technique to reduce fibroblast overgrowth and to improve
the success rate of cell line establishment [25]. Regarding in vitro to in vivo correlation, no significant difference was found between radiosensitivity
of HNSCC cell lines established from 7 patients with recurrent disease after RT, and cell lines derived
from 13 patients without prior irradiation. Moreover, two patients with unfavorable clinical response
to RT, provided cell lines with good RT response in vitro [26]. However, these preclinical assays did
not consider fresh tumor specimens but cell regrowth from previously established cell lines up to 22
passages, conditions that may have selected cells with reasonable radiosensitivity. Unfortunately, while assays using HNSCC cell lines have been proven essential for experiments
concerning molecular biology, they seem not useful as a preclinical prediction model for the individual
cancer patient. It is critical to establish cells in culture that best resemble the patient’s tumor. This
implies that the selection on the fast growing stable cells, surviving under culture conditions, should
be prevented. Short tissue cultures, where various cells are still present and not out-selected, would be
critical to arrive at patient-relevant culture conditions for the testing of various treatment conditions. 3.1. HNSCC Cell Lines
3.1. HNSCC Cell Lines A combination of antibiotics and antimycotics was added to prevent bacterial or fungal
overgrowth, and fibroblast overgrowth was managed through selective trypsinisation or cell scraping [12]. The cells were then cultured at 37 °C in an air mixture with 5% CO2. When cells grew to confluency,
they were passaged. According to Carey, a cell line may be considered established after the 20th passage
In all studies, HNSCC cell lines were established through the “explant technique”, described
extensively in 1994 by Carey [12]. In this technique, fresh tumor specimens were mechanically minced
into fragments. Samples may be further dispersed enzymatically using trypsin, DNase, collagenase
or a combination thereof [19]. The cell suspension was then placed into a rich culture medium, such
as DMEM or RPMI-1640 with additional fetal bovine serum (FBS) and transferred to petri-dishes or
culture flasks [20–22]. A combination of antibiotics and antimycotics was added to prevent bacterial
or fungal overgrowth, and fibroblast overgrowth was managed through selective trypsinisation or cell
scraping [12]. The cells were then cultured at 37 ˝C in an air mixture with 5% CO2. When cells grew
to confluency, they were passaged. According to Carey, a cell line may be considered established after 1720 3.2.1. The Cell-Adhesive Matrix (CAM) Assay The cell adhesive matrix (CAM) assay is a monolayer culture system developed by Baker et al., that
uses a fibronectin and fibrinopeptides coated dish for optimized cell adhesion [27]. Cell growth was
stimulated through hormone- and growth factor-supplemented medium. Fresh primary tumor biopsies
(melanoma, sarcoma, lung, colon, ovarian and renal) were mechanically and enzymatically digested and
plated as single cells. After 24 h of incubation, RT or drugs were administered. After 2 weeks the
cultures were fixed for quantification of cell growth and survival. Baker et al. successfully cultured
75%–90% of tumors using this technique. The articles reviewed, reaches culture success rates of 60%,
within 14–21 days of culturing, in studies with a large number of patients (Table 1) [27]. The CAM assay in HNSCC has only been used to assess radiosensitivity. Brock et al. first reported
on radiosensitivity using the CAM-assay in 1990, in which 72 of 121 HNSCC patients were evaluable
(60% success rate) (Table 2) [28,29]. Radiosensitivity was determined by comparing the cell-covered
surface to the total surface of 24-well plates after radiation with 2 Gray (surviving fraction at 2 Gray,
SF2). The SF2 was 0.40 in 12 patients with recurrent disease and 0.30 in 60 patients with local tumor
control (p > 0.05). ** 172
ew of the various culturing techniques from HNSCC tissue. Read-out
Result
Corrected
for stroma
Days
Patient
Succe
(n)
(%)
SF2
SF2 0.33 (0.11–0.91)
No
14
121
60
SF2, alpha
SF2 0.39 (0.37–0.42),
alpha 0.18 (0.13–0.24)
No
14–21
96
60
SF2, alpha
SF2 0.39 (0.37–0.41),
alpha 0.19 (0.14–0.25)
No
14–21
156
60
SF2
SF2 0.41 (0.21–0.88)
-
-
92
-
CE (>20 cells), 3-Th
CE 0.006 (0.001–0.08)
No
10–14
36
64
CE (>20 cells)
CE 0.001–0.19
-
14–21
73
45
E (ě30 cells, ě5 colonies)
CE 0.005
No
7–14
73
49
CE (ě6 colonies)
CE -
No
14–21
158
36
CE (ě30 cells, >50 µm,
>5 colonies)
Growth observation
No
7–14
51
0
CE (>20 cells)
CE 0.002–0.08
No
7–21
19
56/90
PE (>50 cells), SF2
SF2 ˘0.18–0.45. PE 0.6–2.2
No
28–35
4
33
(>50 cells, >60 µm), SF2
SF2 0.36 (0.19–0.88). PE 0.02–0.75
Yes
28
15
-
PE (>60 µm), SF2
SF2 0.50 (0.11–1.00). PE 0.052 (0.005–1.60)
Yes
28
105
70
E (>50 cells/>60 µm), SF2
SF2 0.50 (0.19–1.00). PE 0.043 (0.005–1.03)
Yes
28
105
68
E (>50 cells/>60 µm), SF2
SF2 0.48 (0.10–1.00). . Cont.
Re
Sensitivity
Sensitivit
Sensitivity: >
dependin
Sensitivity: >
Sensitivit
Cytoto
Viability
IL-6, MCP
IL-6, M
PCR, IHC, FA
PCR, IHC, F
blot, x
DRA = histocu
lating efficiency
zole is reduced t 3.2.1. The Cell-Adhesive Matrix (CAM) Assay CE 0.093 (0.002–1.30)
Yes
28
140
74
CE (>50 cells), SF2
SF2 0.40 (0.10–1.00). CE -
Yes
28
156
70
CE (> 16 cells); C100
Yes
4
13
92
CE (> 16 cells); C100
Yes
4
19
89
CE (> 16 cells); C100
Yes
4
13
-
CE (> 16 cells); C100
Yes
4
12
- 1. Cont. Result
Corrected
for stroma
Days
Patient
Success
(n)
(%)
Sensitivity: ě84% IR
Yes
3–15
26
88
Sensitivity: >30% IR
No
2
42
98
Sensitivity: >40%–60% IR,
depending on drug
No
7
19
100
Sensitivity: >40%–60% IR
No
7
49
-
Sensitivity: >50% IR
No
8
57
91
Cytotoxic effect
No
5 h–7
days
12
-
Viability, cytokine
No
10–28
18
90
IL-6, MCP-1, TNF-α *
Yes/No
>7
31
-
IL-6, MCP-1 *
Yes/No
10–28
65
-
PCR, IHC, FACS, xenograft
No
>14
47
6
PCR, IHC, FACS, western
blot, xenograft
No
>14
-
-
HDRA = histoculture drug response assay; IR = inhibition rate; SF2 = surviving
plating efficiency; FACS = fluorescence-activated cell sorting; C100 = complete
azole is reduced to purple formazan in living cells; ** 56% soft-agar, 90% agarose; 1723
he various assays and their chemo- and radiosensitivity correlations. n vitro
reatment
In vivo
treatment
Read-out
Correlation
Outcome correlation
FU
(months)
Single cell cultures after enzyme digest
RT
Post-op RT
SF2
Yes
Local control. SF2: recurrent 0.40 (n = 12), not
yet recurred 0.30 (n = 60). Not significant. 24
RT
70% RT, 30%
post-op RT
SF2, alpha
Yes
Local control: alpha value. Not for survival
15
(1–29)
RT
62% RT, 38%
post-op RT
SF2, alpha
Yes
Local control: alpha value
24
(9–47)
RT
RT
SF2
No
Local control, survival
68
(45–80)
CT
-
CE, 3-Th
-
-
-
CT
-
CE
Yes
Early mortality: CE > 0.02%
-
CT
-
CE
Yes
Stage, N-class and survival: high CE (n = 29)
-
CT
-
CE
No
No correlation positive culture with stage,
N-class, recurrence. 3.2.2. Soft-Agar Clonogenic Assays Clonogenic assays, in which single tumor cells were cultured on agar-coated plates, were first
described by Puck and Marcus on HeLa cervical tumors [33,34]. In 1977, Salmon and Hamburger
utilized an adaptation of this technique as an in vitro clonogenic assay of anticancer drugs on tumor
cells (myeloma, lymphoma, leukemia, lung, ovary, melanoma and neuroblastoma) [35]. Later, it was
used for human pancreatic and colon tumor cells grown in immune-suppressed mice, popularized by
Courtenay and Mills and referred to as the Courtenay-Mills clonogenic assay [36]. The main feature
of this agar method is its selection for stem cells or transformed cells [37,38]. Although agar cultures
also support benign tumors and anchorage-dependent cells, if supplemented with high serum levels or
transforming growth factors, soft-agar is still a broadly accepted method for tumor cell selection based
on their anchorage-independent growth behavior. The successful use of the Courtenay-Mills soft-agar clonogenic assay with biopsies of HNSCC was
first described by Mattox and Von Hoff [39–41], Johns [37] and Schiff [42] (Table 1). Primary HNSCC
samples were washed, minced with scalpels and further disaggregated, as in the “explant” technique
described by Carey [12]. The cell suspensions were placed in culture plates covered with a feeding
layer containing agar, culture medium, FBS and a variety of other nutrients, and then incubated with a
chemotherapeutic drug for one hour [39]. After that, the cells were washed, plated and incubated, along
with untreated controls. After 7–21 days, the cultures can be evaluated for colony formation (clumps
of more than 20–40 cells). Plating efficiency (number of colonies compared to number of plated cells)
was generally low, around 0.005, meaning only 1 in 200 cells will grow out as a colony. Cultures were
regarded successful if six or more colonies form in untreated control plates [37,39–41]. The survival
fraction was calculated from the amount of colonies formed in treated, compared to untreated plates. Survival rates of 30% or less, compared to untreated controls, were considered an in vitro indicator of
chemosensitivity [37,39–41]. We reviewed several studies using soft-agar clonogenic assays, showing
overall success rates of 50% (0%–74%), where colonies of 20 to 50 cells form within a time span of 1–5
weeks [37,39–49]. These studies were done on a reasonably amount of patients (Table 1). More poorly
differentiated tumors had higher overall culture success rates than well-differentiated tumors [41,42]. Cancers 2015, 7 1725 In 1994, Girinsky et al. described the CAM assay in 156 HNSCC biopsies. SF2 data were available
for 76 HNSCC patients [30,31]. SF2 values were not predictive for long-term local control (cut-off 0.50;
66% versus 63%). On the other hand, a significantly higher local control rate (p = 0.04) was obtained
for patients with higher alpha values (which illustrates the rate of cell kill by a single dose of RT; cut-off
0.07 Gy´1; 69% versus 38% at 2 years). The third group to work with the CAM assay was Eschwege et al. [32]. They studied 92 HNSCC
patients with mainly oropharyngeal carcinomas treated with RT and found both SF2 and alpha value not
to be prognostic factors for local control and overall survival. 3.2.1. The Cell-Adhesive Matrix (CAM) Assay No difference in survival for
high (>0.02%) and low (<0.02%) CE
24
-
-
CE
-
-
-
-
-
CE
-
-
-
RT
-
PE, SF2
-
-
-
RT
RT
PE, SF2
-
-
-
RT
-
PE, SF2
No
Overall/tumor SF2 were not correlated with T/N
and stage
-
RT
RT
PE, SF2
No
Overall/tumor SF2 and PE did not predict
local-regional control (n = 38)
42
(16–70)
RT
-
CE, SF2
No
SF2 did not correlate with tumor grade, T/N class
RT
RT/CT/Surgery
CE, SF2
Yes
Tumor SF2 (0.40) prognostic for local control,
not for overall survival. SF2: recurrent 0.53
(n = 14), not yet recurrent 0.38 (n = 70)
25
(7–65)
CT
-
CE, C100
-
-
-
CT
-
CE, C100
-
-
-
CT
-
CE, C100
-
-
-
CT
-
CE, C100
-
-
- Table 2. Cont. tro
ment
In vivo
treatment
Read-out
Correlation
Outcome correlation
FU
(months)
Histocultures
T
CT
3-Th
Yes
Clinical response. PPV 83%, NPV 64%. Sensitivity
71%, specificity 78%
-
T
Surgery/(C)RT/CT
MTT,
DNA
Yes
Clinical response. Chemosensitivity is a significant
prognostic variable for 2 year cause specific survival
30
T
CT and CRT
MTT
Yes
Clinical response. CRT: PPV 87%, NPV 50%. Sensitivity 87%, specificity 50% (patients received
RT, in vitro no RT)
-
CT: PPV 90%, NPV 100%. Sensitivity 100%,
specificity 67%
T
CT then
surgery
MTT
Yes
Clinical response. PPV 77%, NPV 80%. Sensitivity
91%, specificity 57%. > 4
weeks
Significant correlation between cisplatin sensitivity
in vitro (50% cut-off) and clinical response. No
correlation for 5-FU. T
Surgery/(C)RT/CT
MTT
Yes
Clinical response. PPV 69%, NPV 80%. Sensitivity
79%, specificity 71%. 2 weeks
Significant correlation between in vitro
chemosensitivity and clinical response
T
-
IHC
-
-
-
-
IHC
-
-
-
-
ELISA,
IHC
-
-
-
-
ELISA
Yes
Increased IL-6 levels predict recurrence and survival
30
T
-
Differentiation
-
-
-
T
-
Differentiation
-
-
-
ppression of colony formation; HDRA = histoculture drug response assay; MTT = a yellow tetrazole, is reduced to
/irradiation; ELISA = enzyme-linked immuno sorbent assay; CT = chemotherapy; IHC = immunohistochemistry;
e predictive value; CE or PE = cloning or plating efficiency; NPV = negative predictive value. nt
In
treat
C
Surgery
CT an
CT
sur
Surgery
ession of col
radiation; EL
redictive valu 3.2.2. Soft-Agar Clonogenic Assays These authors also did in vivo correlations with this assay (Table 2). However, chemosensitivity
testing was often not possible due to low tumor cell count. Mattox [40] and Johns [37] showed
that a higher cloning efficiency (>0.02% and >0.05%) was associated with a higher likelihood of
recurrence [37] and early mortality [37,40]. However, a follow-up study of 158 attempted fresh HNSCC
cultures did not confirm this correlation [39]. Cobleigh attempted a soft-agar assay on HNSCC in 1984 Cancers 2015, 7 Cancers 2015, 7 1726 as well, with no success [43]. Finally, Schiff tried to culture tumors from 19 HNSCC patients [42]. Samples from nine patients were cultured in agar and 10 in agarose. Culture success was higher in
agarose-cultured samples (56% versus 90%). With respect to radiosensitivity correlations done with this clonogenic assay, Rofstad, in 1987, studied
various tumors (including four head and neck tumors) with a 33% culture success rate [44]. The SF2
differed considerably among individual tumors of the same histological type. In 1995, Stausbøl-Grøn
cultured biopsies of 15 HNSCC patients prior to radiotherapy [45]. In 12 tumor biopsies 2%–33% of
the colonies were tumor and 83%–100% of the colonies were fibroblasts. The overall SF2 correlated
significantly to the fibroblast SF2 but not to tumor cell SF2. In 1999, the same group assessed
radiosensitivity in 105 HNSCC patients. Culture was successful in 70%. Data were described from
38 patients who were treated with radiotherapy [46,47]. The majority of the colonies obtained from the
biopsies were again fibroblast-marker positive. No significant correlations were found between overall
or tumor SF2 and T/N-class and disease stage. Neither tumor cell SF2, overall SF2, nor plating efficiency
predicted the locoregional tumor control probability. Björk-Eriksson determined the intrinsic radiosensitivity of primary HNSCC on data collected over
5 years for 140 patients using a soft-agar clonogenic assay [48]. Care was taken to ensure that
only colonies from malignant cells were scored by morphology and staining. Colonies with a
radius of more than 60 µm (>50 cells) after 4 weeks of culture were quantified. They reached
a culture success rate of 74% (104/140) with a colony-forming efficiency (CFE) of 0.093 and
obtained SF2 data from 63% of the patients with a mean of 0.48 (0.10–1.00). Interestingly, these
authors observed that approximately 0%–10% of cultured colonies were of a non-malignant cell
type. 3.3.1. The Histoculture Drug Response Assay (HDRA) In an effort to preserve the 3-dimensional (3D) histological structure of the tumor, a method
was developed to culture (mouse) breast tumor fragments without further dispersal [57], thereby
maintaining cell heterogeneity and cell-cell interactions [58]. These models became the cornerstone of
the “histoculture drug response assay” (HDRA), further developed by the group of Hoffman for gastric
and colorectal cancers [59,60]. Primary tumor material was minced into fragments of about 0.5 mm diameter and placed on 1 ˆ 1
cm collagen sponge gels in a 24-well plate. One mL of RPMI-1640 medium supplemented with FBS
was added and the plate was incubated. RPMI medium was selected rather than (D) MEM, for better
preservation of phenotypic heterogeneity [61]. For chemosensitivity assessment, drugs were added to the
culture medium and cultured for 7 days. Viability was determined using the MTT assay that measures
metabolic activity by a spectrophotometer. When the inhibition rate (absorbance in treated, compared to
untreated samples) was 50% or more, tumors were regarded as chemosensitive [62]. Robbins and colleagues were the first to describe the HDRA in HNSCC [63]. They investigated
inhibition of tumor proliferation by cisplatin using radioactive 3H-thymidine incorporation in tumor
cells as an endpoint. In a group of 26 patients with HNC (21 SCC, five with other histological types), 23
(88%) specimens were evaluable. The authors described a positive predictive value (PPV) of 83% and a
negative predictive value (NPV) of 64% for partial or complete clinical response in patients treated with
cisplatin chemoradiation. Singh observed a correlation between in vitro chemosensitivity and 2-year
cause-specific survival, for cisplatin, 5-FU and both agents [64]. However, the 41 patients included
endured various treatment modalities including chemotherapy, surgery and RT. Therefore, no conclusion
could be drawn considering chemosensitivity in this study. Another study concerning patients treated for
oral cavity SCC showed a PPV of 87% and a NPV of 50% for sensitivity testing with 5-FU, cisplatin,
adriamycin, bleomycin and docetaxel [65]. In 2007, Hasegawa et al. assessed both primary tumors
and lymph node metastases and found a significant correlation between in vitro cisplatin sensitivity
and clinical response. There was no correlation for 5-FU [62]. Pathak studied a rather homogenous
group of oral cavity SCC patients receiving chemotherapy regimens resulting in comparable predictive
values [66]. Cancers 2015, 7 group has used this assay to test chemotherapy response to several drugs in vitro [56]. However, no
reports have been published reporting a proper correlation between predicted outcome based on the
Flavino assay and the actual patient outcome in the clinic. 3.3. Histocultures Another way of culturing is to leave tumor tissue intact by using only mechanical mincing. This
maintains the normal and (largely) unaffected tumor-tumor environment interactions as occurring in vivo. The second part of Table 1 depicts studies using this technique. 3.2.2. Soft-Agar Clonogenic Assays In 2000, the same group reported on 156 previously untreated HNC patients (70% culture
success rate, 110/156) and evaluated in 54% (84/156) of the patients the prognostic value of SF2
prospectively [49]. Eighty-four patients were mainly treated with neoadjuvant chemotherapy plus
radiotherapy, with or without final surgery. For prognostic analyses, patients were divided in
radioresistant (SF2 > 0.40) and radiosensitive (SF2 < 0.40) tumors. After multivariate analysis, tumor
SF2 was found an independent prognostic factor for local control (p = 0.036), but not for overall survival
(p = 0.20). Dollner used a colony forming assay without soft-agar, in a 96-well plate format. In 2000, they used
monochromatic light sources to avoid flavin-mediated photo-oxidative effects (termed “Flavino-assay”)
especially during chemosensitivity testing. Fresh tumor biopsies were digested and after 3 days of
exposure to various drugs adherent colonies were fixed and counted to determine the IC-50 [50,51]. The overall chemoresponse was dominated by stromal cell multidrug resistance [52–54]. Stromal
cells were resistant to drug combinations in 98% of the experiments, whereas epithelial colonies were
sensitive to cisplatin/5-FU in 16%, to carboplatin/5-FU in 8.3%, to cisplatin/docetaxel in 33% and to
carboplatin/docetaxel in 8.3%. In 2010, the assay was correlated to clinical outcome in 18 cultures
receiving neoadjuvant TPF (docetaxel, cisplatin and 5-FU) prior to RT [55]. Twelve tumors could
be successfully cultured (66.7%) The in vivo tumor response to induction chemotherapy was correctly
predicted by the tumor culture assay in 10 patients (83.3%). However, an in vitro prediction of clinical
tumor response to the complete treatment regimen was disregarded [55]. These data were only published
in a meeting abstract; the full article was not published and thus not fully evaluable. In recent years, this 1727 Cancers 2015, 7 3.3.1. The Histoculture Drug Response Assay (HDRA) The efficacy and utility of the HDRA as a useful predictor for chemotherapy response in patients
is described in a number of studies of various human solid tumors, including gastric and esophageal
cancer [67,68], colorectal cancer [59] and ovarian cancer [69–71]. Cancers 2015, 7 1728 Recently, Gerlach and colleagues described an adaptation of the HDRA [72]. In this assay, HNSCC
fragments of 12 tumors were sliced with a vibratome or tissue chopper and were placed on membranes,
rather than a collagen sponge. Tumor slices were incubated with docetaxel, cisplatin, or no drugs and
cultured for 5 h to 7 days in a flavin-free culture medium. The slices were then fixed, embedded in
paraffin and examined using Ki-67 (a proliferation marker), caspase-3 staining (an apoptosis marker)
and γH2AX (a marker for double-strand DNA breaks). After 7 days of culture, tissue quality was
decreased in some tumor slices. Increased apoptosis was observed in the slices exposed to drug,
compared to controls. In their publication on this culture method, no correlations to clinical outcomes
were done [72]. Recently, more groups have started to generate histocultures of HNSCC to investigate
the effect of existing or novel, more targeted drug-based, therapies, such as the PI3K inhibitor LY294002,
to investigate the effect of molecular signaling in tumor growth [73,74]. Cancers 2015, 7 Cancers 2015, 7 Cancers 2015, 7 formation in nude mice was significantly better for undifferentiated cells. This was later confirmed
by Pozzi et al., who found better tumorigenicity in sphere forming cancer stem cell (CSC)-enriched
cell populations than in unselected tumor cells [82]. To investigate whether HNSCC CSCs can be
expanded in adherent cultures without loss of stem cell properties, Lim et al. tested different plate
coatings [83]. HNSCC-CSCs grew much faster on type IV collagen-coated plates than in suspension. Adherent CSCs expressed stem cell markers, were chemoresistant, produced tumors in mice and showed
less spontaneous apoptotic cell death. Leong et al. described the establishment of three cell lines from primary HNSCC grown as spheroids
or monolayers. They confirmed the improved chemoresistance of spheroids when treated with 5FU,
cisplatin, etoposide or irradiation [84]. Unfortunately, correlation of the ex vivo results with the actual
clinical outcome was not one of the aims of this study. On the other hand, while sphere formation or
sphere formation capability of CSCs, may increase resistance to some drugs, another group has shown
in primary HNSCC spheroid cultures that it is also possible to target these CSCs in particular [85]. Until now, only one group described an “organoid culture assay” of HNSCC [86,87]. Although the
authors did not use fresh primary tumor, they aimed for 3D in vitro tumor growth allowing to form
organized and differentiated structures such as those existing in the organism. After full digestion of
a xenografted HNSCC in mice, single cell suspension droplets were seeded on a bridge-like filter in a
petri-dish. In this model the tissue grows at the air-medium interface, as medium was added just until
the bridge. After 4 weeks solid culture nodules were disaggregated again to assess viability of cells
by Trypan Blue. Pathologic evaluation of the nodules showed histological characteristics similar to the
original human hypopharyngeal carcinoma up to 3 weeks of culturing. After 3 weeks degeneration
was seen. 3.4. Other Assays Various other techniques to establish in vitro cultures of primary HNSCC were reported, but were
only described by a single group and not further popularized. For completeness, these assays are briefly
described below. 3.3.2. Spheroids, Squamospheres and Organoids 3.3.2. Spheroids, Squamospheres and Organoids The spheroid culture technique was developed as well to maintain tumor tissue heterogeneity and a
3D architecture (Table 1) [75]. Spheroids would ideally resemble the growth pattern of solid tumors
in vivo as they are composed of an outer layer of proliferating cells closest to a nutrient and oxygen
supply (capillaries) with inner layers of quiescent and -most central- necrotic cells. This was tested with
a variety of cell lines [76,77]. Technically, they can either be grown from cells obtained from monolayer
cell cultures after trypsinisation or grown from fresh tumor biopsy fragments [16]. In 2000, Heimdal described malignant and benign “fragment spheroids” in a nonadhesive system [78]. HNSCC fragments were cultured on agar-coated culture flasks and after 10–14 days rounded
spheroid-like structures were selected for a 2-week co-culture with autologous monocytes derived from
peripheral blood samples of the patients. Cytokine IL-6 production of the monocytes was significantly
higher in case of direct cell-cell (i.e., tumor-monocyte) contact compared to co-cultures where tumor
cells and monocytes were separated by a semi-permeable membrane. In 2005, Kross used the same
model to study the cytokine secretion, and to describe the number of epithelial cells (cytokeratin
positive), fibroblasts (vimentin-positive) and macrophages (CD68 positive) in both malignant HNSCC
and benign spheroids [79]. In malignant spheroids, the proportion of epithelial cells during spheroid
formation decreases from 28% to 13%. The density of macrophages (2%) and fibroblasts (13%) did not
change. Monocytes secreted more IL-6 when co-cultured with malignant compared to benign spheroids. In 2008 they found increased IL-6 cytokine production in vitro to be predictive for recurrence and
survival (Table 2) [80]. A few years later, Lim et al. described “squamospheres” resulting from culturing mechanically
and enzymatically digested biopsies from 47 HNSCC patients [81]. Single cells were incubated for
2–4 weeks to assess sphere forming ability (self-renewal) and other cancer stem cell hallmarks like
tumor-initiating capabilities and chemoresistance. A distinction was made between undifferentiated
squamospheres (cultured in stem cell medium: serum-free, with N2, B27, EGF and bFGF) and
differentiated squamospheres (medium with 10% FBS, without EGF and bFGF). Overall, the success
rate of spheroid formation was 6%. Single cells from spheres were assessed for anchorage-independent
growth ability as an indicator for cell transformation in vitro; undifferentiated cells that maintained
sphere forming capability sustained and differentiated cells diminished in agar. In agreement, tumor 1729 3.4.2. Tumor Slices Grown in Test-Tubes Elprana et al. described a culture system where human HNSCC fragments, from one patient, floated
freely in test tubes containing medium with or without drugs [90,91]. In vitro, the tumor was sensitive to
cisplatin and 5-fluorouracil. The patient received a combination of these drugs and experienced complete
regression in four months, although long-term outcome was not described. 3.4.4. Micronucleus Assay Champion et al. described an assay that involved establishing a monolayer culture of primary HNSCC
tumors and immunohistochemical staining of these cultures after irradiation to identify micronuclei [96]. These micronuclei may be visible in dividing cells and are considered as DNA fragments that cannot be
incorporated in daughter cells, due to (radiation) damage. The primary endpoint was the correlation
between micronuclei formation and the amount of radiation exposure. After optimizing the assay in cell
lines, primary HNSCC specimens were tested. Unfortunately no correlation between assay outcome and
clinical outcome could be established. 3.4.3. Microdevices Recently, the group of Greenman cultured HNSCC samples in vitro with a microfluidic device. Medium flowed through the device and was collected after drug or irradiation treatment [92–94]. Response to chemotherapy or irradiation is determined by measuring LDH in the effluent. Drug treated
samples showed significantly more LDH release than the control groups. No further reports were
found that correlated the in vitro response to clinical data. This culture technique has been reviewed
by Sivagnanam [95]. 3.4.1. Flow-Cytometric Analysis In 1989, Garozzo presented a different model for short-term culturing of HNSCC in which he
acknowledged an equal contribution of all cell populations in the progression of neoplastic disease,
and referred to Von Hoff stating that HNC are not very likely to grow on agar [88,89]. Surgical HNSCC
specimens were disaggregated into cell suspensions and exposed to various drugs for 24 h. The major
endpoint was the presence of cell cycle blocks, determined by flow cytometry. Patients were treated with
a standardized, undisclosed, regimen of polychemotherapy. Thirteen of the 15 patients showed complete
or partial remission. The assay predicted sensitivity to several of the drugs in 11 of these 13 patients
(PPV 85%). Cancers 2015, 7 Cancers 2015, 7 Cancers 2015, 7 1730 3.4.2. Tumor Slices Grown in Test-Tubes Cancers 2015, 7 Cancers 2015, 7 Cancers 2015, 7 not significantly related to outcome in these studies [28,30–32]. Only the alpha value (initial slope of
radiation curve) had a good clinical correlation with local tumor control in two studies [30,31]. Heppner
and colleagues argued that tumor sensitivity to therapeutic agents in a clonal monolayer culture differ to
that of in vivo-like tissue architectures comprised of heterogeneous cells [106]. Another short-term assay is the soft-agar assay. Von Hoff did a meta-analysis on 54 trials in
1990, using a clonogenic assay, which compared in vitro results to clinical outcome in 2300 cases of
solid tumors, including a relatively small number of HNSCCs [107]. Overall, they found a 69% true
positive rate and a favorable true negative rate of 91%, with a sensitivity and specificity of 79% and
86% respectively, in predicting outcome. We reviewed several studies using soft-agar showing that
plating efficiency of HNSCCs is relatively poor. An explanation may be a rather low subpopulation of
stem cells in HNSCC. Moreover, solid HNSCC in these studies were fully digested, likely leading to
mechanical trauma to the cell. Some authors propose that enzymatic digestion is preferable to maintain
viability and growth potential [108]. In addition, the disruption of intercellular attachments may not
only irreversibly damage tumor specimens, but may also lead to higher chemosensitivity of cells, not
representing the actual in vivo sensitivity [109–113]. For example, this is seen in experiments on mouse
mammary tumor cell lines; Miller found that chemoresistance to melphalan and 5-fluorouracil was up to
a 1000-fold higher in 3D collagen gel structures than in monolayer cell lines [109–111]. Unfortunately,
research concerning clonogenic assays also failed to systematically show predictive value for individual
clinical outcome, probably due to disruption of the tissue. Namely, four studies investigating clinical
correlations involving soft-agar HNSCC colony forming assay, did not find any correlation between
in vitro and in vivo response (Table 2) [39,45–47]. In two chemosensitivity studies, plating efficiency
was associated with tumor stage, N-class and survival [37] and early mortality [40], however not with
therapy response. These studies, nevertheless, describe a low number of tumor cells available. Björk
performed a radiosensitivity colony forming assay where SF2 was a significant prognostic factor for
local control, but not for overall survival [49]. 4. Discussion With this review we aimed to evaluate the most successful in vitro culture technique for HNSCC
and to discover which model has the best correlation with clinical response. As the chemotherapeutic
repertoire increases, a simple and reliable assay to determine the expected patient response becomes
critical in making a correct individualized treatment decision. Monolayer cell line culture is not a proficient method for the use of a preclinical prediction assay. Reasons for this are the long duration of cell line establishment, low culture success rates [12,15] and
senescence, the state in which cells no longer divide [13]. Cell line formation is also accompanied
with genetic changes like upregulation of oncogenes, and consequently worse clinical outcome [97,98]. Probably for all these reasons, a good clinical correlation was never shown [26]. Short-term fresh tumor cultures, however, do not experience clonal evolution of tumor cell
(sub)populations [99]. Worsham, Ragin and Bjerkvig found genetic and molecular cytogenetic
resemblance between HNSCC cultures and the primary tumor in vivo [100–102]. The short duration of
culture increases the evaluability of these assays, as these are not influenced by senescence [103–105]. Although tumor biopsies are fully digested in the short-term CAM assay, the assay is thought to
allow for restored cell-cell contact within the anchored monolayer. It was probably thought that this
anchorage was required to establish the predictive value of SF2 for clinical control, however SF2 was 1731 Cancers 2015, 7 Cancers 2015, 7 interior, its low pH and relative inaccessibility to chemotherapeutic agents may be the reason for the
high predictive values described for in vivo correlations, compared to clonogenic or monolayer assays. In addition, the HDRA needs short-term culturing and will therefore have few genetic alterations when
compared to longer-term cultures. Finally, the tumor microenvironment in the HDRA may be of
importance for a proper clinical correlation, as the presence of tumor-infiltrating lymphocytes seems
to determine clinical outcome in patients with HNSCC [115]. Indeed the HDRA has been confirmed
to be a well feasible culture system for fresh HNSCC tissue, as shown by several other research groups
with a good correlation to clinical response [62–66]. Only Singh correlated it with clinical outcome,
and found that in vitro chemosensitivity was a significant prognostic variable for survival [64]. However,
these studies tested only chemosensitivity in vitro, while patients received chemotherapy often combined
with radiotherapy. To improve predictive values and to optimize the clinical relevance for predicting the
long-term clinical outcome of HNSCC patients, the HDRA model may be tested not only to determine
chemosensitivity, but also radiosensitivity. Furthermore, the clinical follow-up duration or the moment
of endpoint determination in the identified studies was not always described or it was short (2 to 4
weeks) [62,66]. This might give an overestimate of the chemosensitivity. Since 2000, several research groups have focused on growing HNSCC “spheres”, “squamospheres”
and “organoids” [79–81]. As the term suggests, the investigators aimed to establish a 3D arrangement
of tumor cells, forming a sphere or organoid, mimicking solid tumor growth in vivo. Therefore,
the in vitro “3D model” might better mimic drug response in vivo. Heimdal and Kross showed
the potential importance of immune cells in culture prediction assays using this model. Increased
cytokine production in co-culture was significantly higher in direct cell-cell contact between autologous
monocytes and tumor [116], and was found to be predictive for a clinical unfavorable prognosis in
HNSCC [80]. However, overall, the reviewed studies did not succeed to systematically generate the
intended organoid like structures. In addition, growing spheres and organoids as described here is
relatively time-consuming. After 2 weeks of spheroid formation, a prediction assay warrants another
2 weeks of incubation. Within the first weeks a decreasing proportion of epithelial cells was seen [80]
and a degeneration of histological characteristics [86]. Cancers 2015, 7 The use of soft-agar should have the advantage of providing support for solid tumor cells, which
frequently have difficulties in attaching to the surface of culture dishes. Tumor cells then grow as
spherical colonies in agar, while the growth of benign cells such as fibroblast, that require anchorage
to a solid substrate, is thought to be reduced [114]. Several groups investigated the impact of stromal
cell contamination on culture and treatment sensitivity and concluded that most colonies consisted of
fibroblasts. The SF2 is then mainly determined by fibroblast SF2 instead of overall or tumor SF2, and
therefore this may contribute as well in not mimicking the correct response in vivo [45,48–51,54,55]. Overall, the number of weeks to culture and the low percentage of evaluable results make the soft-agar
clonogenic assay less suitable for use in individual clinical decision making in HNSCC. In 1994, while other research groups were exploring cultures of fully digested tumor specimens
(CAM- and soft-agar assays), Robbins et al., adopted the HDRA mode. This short-term,
sponge-supported histoculture of HNSCC tissue fragments does not require enzymatic digest, leaving
cell-cell adhesions, 3D character, as well as the tumor heterogeneity intact [60,63]. All cells, benign
and malignant, are co-cultured together. This method allows for the formation of cell aggregates with
identifiable and distinctive tissue patterns simulating the in vivo tumor [57]. This probably explains
that, for the first time, very high culture success rates were reached (88%–100%). The hypoxic tumor 1732 Acknowledgements This work was kindly supported by the Riki Stichting. Cancers 2015, 7 Cancers 2015, 7 assay for personalized treatment decisions. As it stands, the HDRA technique appears to be the best
model to test and identify novel treatment modalities for HNSCC, which is currently specified by a very
poor prognosis. Author Contributions Amy Dohmen: Principal investigator. Acquired the data, analyzed and interpreted data, layout,
design, and wrote the manuscript; Justin Swartz: Contributed to the collection of literature and writing
of the manuscript; Michiel van den Brekel: Critical input and final approval of the manuscript;
Stefan Willems: Critical input and final approval of the manuscript; René Spijker: contributed to the
design of the search strategy, collection of the literature and final approval of the manuscript; Jacques
Neefjes: Critical input and final approval of the manuscript; Charlotte Zuur: Contributed to writing the
manuscript, critical input and final approval of the manuscript. Conflicts of Interest The authors declare no conflict of interest. 5. Conclusions Within the treatment of HNSCC, there is a strong need for predicting individual clinical outcome
prior to therapy, as the overall patient survival rates are relatively low and reliable biomarkers are
not available. Moreover, there is a need to test novel drugs before introduction into clinical practice. A preclinical model that closely resembles the in vivo situation would be highly valuable. In this
review, we observed that the most successful cultures rates and best correlations to clinical response
were reported with the HDRA technique. The HDRA assay has the benefit of better representing
the tumor and its microenvironment as it does not involve tissue disaggregation, thus maintaining cell
heterogeneity, cell-cell interactions and tissue architecture. However, the correlation to clinical outcome
of the HDRA technique has been reported only on a small group of patients and should be validated in
larger patient cohorts. Within the HDRA technique it is important to correct for stroma cell response. Another outstanding and obvious point is that the clinical treatment should be resembled in vitro as
closely as possible, including irradiation. This will ultimately determine the success of this culture-based 1733 Cancers 2015, 7 Cancers 2015, 7 1734 8. Adelstein, D.J.; Li, Y.; Adams, G.L.; Wagner, H.; Kish, J.A.; Ensley, J.F.; Schuller, D.E.;
Forastiere, A.A. An intergroup phase III comparison of standard radiation therapy and two
schedules of concurrent chemoradiotherapy in patients with unresectable squamous cell head and
neck cancer. J. Clin. Oncol. 2003, 21, 92–98. [CrossRef] [PubMed] 9. Ley, J.; Mehan, P.; Wildes, T.M.; Thorstad, W.; Gay, H.A.; Michel, L.; Nussenbaum, B.;
Trinkaus, K.; Adkins, D. Cisplatin versus cetuximab given concurrently with definitive radiation
therapy for locally advanced head and neck squamous cell carcinoma. Oncology 2013, 85,
290–296. [CrossRef] [PubMed] 10. Caudell, J.J.; Sawrie, S.M.; Spencer, S.A.; Desmond, R.A.; Carroll, W.R.; Peters, G.E.;
Nabell, L.M.; Meredith, R.F.; Bonner, J.A. Locoregionally Advanced Head and Neck Cancer
Treated With Primary Radiotherapy:
A Comparison of the Addition of Cetuximab or
Chemotherapy and the Impact of Protocol Treatment. Int. J. Radiat. Oncol. Biol. Phys. 2008, 71,
676–681. [CrossRef] [PubMed] 11. Wright, J.C.; Cobb, J.P.; Gumport, S.L.; Golomb, F.M.; Safadi, D. Investigation of the relation
between clinical and tissue-culture response to chemotherapeutic agents on human cancer. N. Engl. J. Med. 1957, 257, 1207–1211. [CrossRef] [PubMed] 12. Carey, T.E. Head and Neck Tumor Cell Lines. In Atlas of Human Tumor Cell Lines; Hay, R.J.,
Park, J., Gazdar, A., Eds.; Academic Press, Inc.: San Diego, CA, USA, 1994; pp. 79–120. y,
f
;
y,
Park, J., Gazdar, A., Eds.; Academic Press, Inc.: San Diego, CA, USA, 1994; pp. 79–120. 13. Rheinwald, J.G.; Beckett, M.A. Tumorigenic keratinocyte lines requiring anchorage and fibroblast
support cultured from human squamous cell carcinomas. Cancer Res. 1981, 41, 1657–1663. [PubMed] 14. Easty, D.M.; Easty, G.C.; Carter, R.L.; Monaghan, P.; Butler, L.J. Ten human carcinoma cell lines
derived from squamous carcinomas of the head and neck. Br. J. Cancer 1981, 43, 772–785. [CrossRef] [PubMed] 15. Sacks, P.G. Cell, tissue and organ culture as in vitro models to study the biology of squamous cell
carcinomas of the head and neck. Cancer Metastasis Rev. 1996, 15, 27–51. [CrossRef] [PubMed]
16. Lin, C.J.; Grandis, J.R.; Carey, T.E.; Gollin, S.M.; Whiteside, T.L.; Koch, W.M.; Ferris, R.L.;
Lai S Y Head and neck squamous cell carcinoma cell lines: Established models and rationale for 15. Sacks, P.G. Cell, tissue and organ culture as in vitro models to study the biology of squamous cell
carcinomas of the head and neck. Cancer Metastasis Rev. 1996, 15, 27–51. [CrossRef] [PubMed] 16. References 1. Pulte, D.; Brenner, H. Changes in survival in head and neck cancers in the late 20th and early 21st
century: A period analysis. Oncologist 2010, 15, 994–1001. [CrossRef] [PubMed] 1. Pulte, D.; Brenner, H. Changes in survival in head and neck cancers in the late 20th and early 21st
century: A period analysis. Oncologist 2010, 15, 994–1001. [CrossRef] [PubMed] 2. Surveillance Epidemiology and End Results Program Cancer of the Larynx—SEER Fact Sheets. Available online: http://seer.cancer.gov/statfacts/html/laryn.html (accessed on 21 August 2015). 3. Surveillance Epidemiology and End Results Program Cancer of the Oral Cavity and
Pharynx—SEER Fact Sheets. Available online: http://seer.cancer.gov/statfacts/html/oralcav.html
(accessed on 21 August 2015). 3. Surveillance Epidemiology and End Results Program Cancer of the Oral Cavity and
Pharynx—SEER Fact Sheets. Available online: http://seer.cancer.gov/statfacts/html/oralcav.html
(accessed on 21 August 2015). 4. Al-Sarraf, M. Treatment of locally advanced head and neck cancer: Historical and critical review. Cancer Control 2002, 9, 387–399. [PubMed] 4. Al-Sarraf, M. Treatment of locally advanced head and neck cancer: Historical and critical review. Cancer Control 2002, 9, 387–399. [PubMed] 5. Pignon, J.P.; le Maitre, A.; Maillard, E.; Bourhis, J.; Group, M.-N.C. Meta-analysis of
chemotherapy in head and neck cancer (MACH-NC): An update on 93 randomised trials and
17,346 patients. Radiother. Oncol. 2009, 92, 4–14. [CrossRef] [PubMed] 5. Pignon, J.P.; le Maitre, A.; Maillard, E.; Bourhis, J.; Group, M.-N.C. Meta-analysis of
chemotherapy in head and neck cancer (MACH-NC): An update on 93 randomised trials and
17,346 patients. Radiother. Oncol. 2009, 92, 4–14. [CrossRef] [PubMed] 6. Fakhry, C.; Westra, W.H.; Li, S.; Cmelak, A.; Ridge, J.A.; Pinto, H.; Forastiere, A.; Gillison, M.L. Improved survival of patients with human papillomavirus-positive head and neck squamous cell
carcinoma in a prospective clinical trial. J. Natl. Cancer Inst. 2008, 100, 261–269. [CrossRef]
[PubMed] 6. Fakhry, C.; Westra, W.H.; Li, S.; Cmelak, A.; Ridge, J.A.; Pinto, H.; Forastiere, A.; Gillison, M.L. Improved survival of patients with human papillomavirus-positive head and neck squamous cell
carcinoma in a prospective clinical trial. J. Natl. Cancer Inst. 2008, 100, 261–269. [CrossRef]
[PubMed] 7. Forastiere, A.A.; Zhang, Q.; Weber, R.S.; Maor, M.H.; Goepfert, H.; Pajak, T.F.; Morrison, W.;
Glisson, B.; Trotti, A.; Ridge, J.A.; et al. Long-term results of RTOG 91–11: A comparison
of three nonsurgical treatment strategies to preserve the larynx in patients with locally advanced
larynx cancer. J. Clin. Oncol. 2013, 31, 845–852. [CrossRef] [PubMed] Cancers 2015, 7 Cancers 2015, 7 1735 21. Taitz, A.; Petruzzelli, G.; Pak, A.S.; Wright, M.A.; Matthews, J.P.; Raslan, W.F.; Lozano, Y.;
Young, M.R. Immune parameters of mice bearing human head and neck cancer. Cancer Immunol. Immunother. 1995, 40, 283–291. [CrossRef] [PubMed] 21. Taitz, A.; Petruzzelli, G.; Pak, A.S.; Wright, M.A.; Matthews, J.P.; Raslan, W.F.; Lozano, Y.;
Young, M.R. Immune parameters of mice bearing human head and neck cancer. Cancer Immunol. Immunother. 1995, 40, 283–291. [CrossRef] [PubMed] 22. Ji, Z.W.; Oku, N.; Umeda, M.; Komori, T. Establishment of an oral squamous cell carcinoma cell
line (NOS-1) exhibiting amplification of the erbB-1 oncogene and point mutation of p53 tumor
suppressor gene: Its biological characteristics and animal model of local invasion by orthotopic
transplanta. Oral Oncol. 2001, 37, 386–392. [CrossRef] 23. Kim, S.Y.; Chu, K.C.; Lee, H.R.; Lee, K.S.; Carey, T.E. Establishment and characterization of
nine new head and neck cancer cell lines. Acta Otolaryngol. 1997, 117, 775–784. [CrossRef]
[PubMed] 24. Charoenrat, P.; Rhys-Evans, P.; Eccles, S. Characterization of ten newly-derived human head and
neck squamous carcinoma cell lines with special reference to c-erbB proto-oncogene expression. Anticancer Res. 2001, 21, 1953–1963. 25. Owen, J.H.;
Graham, M.P.;
Chinn, S.B.;
Darr, O.F.;
Chepeha, D.B.;
Wolf, G.T.;
Bradford, C.R.; Carey, T.E.; Prince, M.E.P. Novel method of cell line establishment utilizing
fluorescence-activated cell sorting resulting in 6 new head and neck squamous cell carcinoma
lines. Head Neck 2015, 55, 691–696. [CrossRef] [PubMed] 26. Grénman, R.; Carey, T.E.; McClatchey, K.D.; Wagner, J.G.; Pekkola-Heino, K.; Schwartz, D.R.;
Wolf, G.T.; Lacivita, L.P.; Ho, L.; Baker, S.R.; et al. In vitro radiation resistance among cell lines
established from patients with squamous cell carcinoma of the head and neck. Cancer 1991, 67,
2741–2747. [CrossRef] 27. Baker, F.L.; Spitzer, G.; Ajani, J.A.; Brock, W.A.; Lukeman, J.; Pathak, S.; Tomasovic, B.;
Thielvoldt, D.; Williams, M.; Vines, C.; et al. Drug and radiation sensitivity measurements of
successful primary monolayer culturing of human tumor cells using cell-adhesive matrix and
supplemented medium. Cancer Res. 1986, 46, 1263–1274. [PubMed] 28. Brock, W.A.; Baker, F.L.; Wike, J.L.; Sivon, S.L.; Peters, L.J. Cellular radiosensitivity of primary
head and neck squamous cell carcinomas and local tumor control. Int. J. Radiat. Oncol. Biol. Phys. 1990, 18, 1283–1286. [CrossRef] 29. Brock, W.A.; Baker, F.L.; Peters, L.J. Radiosensitivity of human head and neck squamous cell
carcinomas in primary culture and its potential as a predictive assay of tumor radiocurability. Int. J. Radiat. Biol. 1989, 56, 751–760. Cancers 2015, 7 Lin, C.J.; Grandis, J.R.; Carey, T.E.; Gollin, S.M.; Whiteside, T.L.; Koch, W.M.; Ferris, R.L.;
Lai, S.Y. Head and neck squamous cell carcinoma cell lines: Established models and rationale for
selection. Head Neck 2007, 29, 163–188. [CrossRef] [PubMed] 17. Owen, J.H.; Hauff, S.J.; Tang, A.L.; Graham, M.P.; Czerwinski, M.J.; Kaddoura, M.;
Papagerakis, S.; Bradford, C.R.; Carey, T.E.; Prince, M.E. UM-SCC-103: A Unique Tongue
Cancer Cell Line That Recapitulates the Tumorigenic Stem Cell Population of the Primary Tumor. Ann. Otol. Rhinol. Laryngol. 2014, 123, 662–672. [CrossRef] [PubMed] 18. Van Zeeburg, H.J.T. Generation and Molecular Characterization of Head and Neck Squamous
Cell Lines of Fanconi Anemia Patients. Cancer Res. 2005, 65, 1271–1276. [CrossRef] [PubMed] 19. Bijman, J.T.; Wagener, D.J.; van Rennes, H.; Wessels, J.M.; van den Broek, P. Flow cytometric
evaluation of cell dispersion from human head and neck tumors. Cytometry 1985, 6, 334–341. [CrossRef] [PubMed] 20. Kudo, Y.; Kitajjma, S.; Sato, S.; Miyauchi, M.; Ogawa, I.; Takata, T. Establishment of an oral
squamous cell carcinoma cell line with high invasive and p27 degradation activities from a lymph
node metastasis. Oral Oncol. 2003, 39, 515–520. [CrossRef] Cancers 2015, 7 [CrossRef] [PubMed] 30. Girinsky, T.; Lubin, R.; Pignon, J.P.; Chavaudra, N.; Gazeau, J.; Dubray, B.; Cosset, J.M.;
Socie, G.; Fertil, B. Predictive value of in vitro radiosensitivity parameters in head and neck
cancers and cervical carcinomas: Preliminary correlations with local control and overall survival. Int. J. Radiat. Oncol. Biol. Phys. 1993, 25, 3–7. [CrossRef] 31. Girinsky, T.; Bernheim, A.; Lubin, R.; Tavakoli-Razavi, T.; Baker, F.; Janot, F.; Wibault, P.;
Cosset, J.M.; Duvillard, P.; Duverger, A. In vitro parameters and treatment outcome in head and
neck cancers treated with surgery and/or radiation: Cell characterization and correlations with
local control and overall survival. Int. J. Radiat. Oncol. Biol. Phys. 1994, 30, 789–794. [CrossRef] Cancers 2015, 7 1736 32. Eschwege, F.; Bourhis, J.; Girinski, T.; Lartigau, E.; Guichard, M.; Deblé, D.; Kepta, L.;
Wilson, G.D.; Luboinski, B. Predictive assays of radiation response in patients with head and
neck squamous cell carcinoma: A review of the Institute Gustave Roussy experience. Int. J. Radiat. Oncol. Biol. Phys. 1997, 39, 849–853. [CrossRef] 33. Puck, T.T.; Marcus, P.I. A rapid method for viable cell titration and clone production with hela
cells in tissue culture: The use of x-irradiated cells to supply conditioning factors. Proc. Natl. Acad. Sci. USA 1955, 41, 432–437. [CrossRef] [PubMed] 34. Puck, T.T.; Marcus, P.I.; Cieciura, S.J. Clonal growth of mammalian cells in vitro; growth
characteristics of colonies from single HeLa cells with and without a feeder layer. J. Exp. Med. 1956, 103, 273–283. [CrossRef] [PubMed] 35. Hamburger, A.W.; Salmon, S.E. Primary bioassay of human tumor stem cells. Science 1977, 197,
461–463. [CrossRef] [PubMed] 36. Courtenay,
V.D.;
Mills,
J. An
in vitro
colony
assay
for
human
tumours
grown
in
immune-suppressed mice and treated in vivo with cytotoxic agents. Br. J. Cancer 1978, 37,
261–268. [CrossRef] [PubMed] 37. Johns, M.E. The clonal assay of head and neck tumor cells: Results and clinical correlations. Laryngoscope 1982, 92, 1–26. [CrossRef] [PubMed] 38. Macpherson, I.; Montagnier, L. Agar suspension culture for the selective assay of cells
transformed by polyoma virus. Virology 1964, 23, 291–294. [CrossRef] 39. Mattox, D.E.; von Hoff, D.D.; Clark, G.M.; Aufdemorte, T.B. Factors that influence growth of
head and neck squamous carcinoma in the soft agar cloning assay. Cancer 1984, 53, 1736–1740. [CrossRef] 40. Mattox, D.E.; von Hoff, D.D. In vitro stem cell assay in head and neck squamous carcinoma. Am. J. Surg. 1980, 140, 527–530. [CrossRef] 41. Mattox, D.E.; von Hoff, D.D. Cancers 2015, 7 Cancers 2015, 7 1737 47. Stausbol-Gron, B.; Overgaard, J. Relationship between tumour cell in vitro radiosensitivity and
clinical outcome after curative radiotherapy for squamous cell carcinoma of the head and neck. Radiother. Oncol. 1999, 50, 47–55. [CrossRef] 48. Björk-Eriksson, T.; West, C.M.; Karlsson, E.; Slevin, N.J.; Davidson, S.E.; James, R.D.;
Mercke, C. The in vitro radiosensitivity of human head and neck cancers. Br. J. Cancer 1998,
77, 2371–2375. [CrossRef] [PubMed] 49. Björk-Eriksson, T.; West, C.; Karlsson, E.; Mercke, C. Tumor radiosensitivity (SF2) is a
prognostic factor for local control in head and neck cancers. Int. J. Radiat. Oncol. Biol. Phys. 2000, 46, 13–19. [CrossRef] 50. Dollner, R.; Dietz, A.; Kopun, M.; Helbig, M.; Wallner, F.; Granzow, C. Ex vivo responsiveness
of head and neck squamous cell carcinoma to glufosfamide, a novel alkylating agent. Anticancer
Res. 2004, 24, 2947–2951. [PubMed] 51. Dollner, R.; Granzow, C.; Tschop, K.; Dietz, A. Ex vivo responsiveness of head and neck
squamous cell carcinoma to vinorelbine. Anticancer Res. 2006, 26, 2361–2365. [PubMed] 52. Dollner, R.; Granzow, C.; Helmke, B.M.; Ruess, A.; Schad, A.; Dietz, A. The Impact of Stromal
Cell Contamination on Chemosensitivity Testing of Head and Neck Carcinoma. Anticancer Res. 2004, 24, 325–331. [PubMed] 53. Dollner, R.; Granzow, C.; Neudert, M.; Dietz, A. Ex vivo chemosensitivity of head and neck
carcinoma to cytostatic drug combinations. Anticancer Res. 2006, 26, 1651–1655. [PubMed] 54. Horn, I.-S.; Wichmann, G.; Mozet, C.; Dietz, A.; Dollner, R.; Tschöp, K.; Boehm, A. Heterogeneity of epithelial and stromal cells of head and neck squamous cell carcinomas in ex vivo
chemoresponse. Cancer Chemother. Pharmacol. 2010, 65, 1153–1163. [CrossRef] [PubMed] 55. Dietz, A.; Boehm, A.; Reiche, A.; Mueller, G.; Kruber, P.; Keilholz, U.; Niederwieser, D.;
Wichmann, G. Prediction of outcome of TPF with or without cetuximab induction chemotherapy
in head and neck squamous cell carcinoma ( HNSCC) using the FLAVINO assay. J. Clin. Oncol. 2010, 28, 15s. 56. Wichmann, G.; Körner, C.; Boehm, A.; Mozet, C.; Dietz, A. Stimulation by Monocyte
Chemoattractant Protein-1 Modulates the Ex-vivo Colony Formation by Head and Neck
Squamous Cell Carcinoma Cells. Anticancer Res. 2015, 35, 3917–3924. [PubMed] 57. Leighton, J. A sponge matrix method for tissue culture; formation of organized aggregates of cells
in vitro. J. Natl. Cancer Inst. 1951, 12, 545–561. [PubMed] 58. Sherwin, R.P.; Richters, A.; Yellin, A.E.; Donovan, A.J. Histoculture of human breast cancers. J. Surg. Oncol. 1980, 13, 9–20. [CrossRef] [PubMed] 59. Cancers 2015, 7 Culture of human head and neck cancer stem cells using soft agar. Arch. Otolaryngol. 1980, 106, 672–674. [CrossRef] [PubMed] 42. Schiff, L.J.; Shugar, M.A. Growth of human head and neck squamous cell carcinoma stem cells
in agarose. Cancer 1984, 53, 286–290. [CrossRef] 43. Cobleigh, M.A.; Gallagher, P.A.; Hill, J.H.; Applebaum, E.L.; McGuire, W.P. Growth of human
squamous head and neck cancer in vitro. Am. J. Pathol. 1984, 115, 397–402. [PubMed] 43. Cobleigh, M.A.; Gallagher, P.A.; Hill, J.H.; Applebaum, E.L.; McGuire, W.P. Growth of human
squamous head and neck cancer in vitro. Am. J. Pathol. 1984, 115, 397–402. [PubMed] 44. Rofstad, E.K.; Wahl, A.; Brustad, T. Radiation sensitivity in vitro of cells isolated from human
tumor surgical specimens. Cancer Res. 1987, 47, 106–110. [PubMed] 44. Rofstad, E.K.; Wahl, A.; Brustad, T. Radiation sensitivity in vitro of cells isolated from human
tumor surgical specimens. Cancer Res. 1987, 47, 106–110. [PubMed] 45. Stausbol-Gron, B.; Nielsen, O.S.; Moller Bentzen, S.; Overgaard, J. Selective assessment
of in vitro radiosensitivity of tumour cells and fibroblasts from single tumour biopsies using
immunocytochemical identification of colonies in the soft agar clonogenic assay. Radiother. Oncol. 1995, 37, 87–99. [CrossRef] 46. Stausbol-Gron, B.; Bentzen, S.M.; Jorgensen, K.E.; Nielsen, O.S.; Bundgaard, T.; Overgaard, J. In vitro radiosensitivity of tumour cells and fibroblasts derived from head and neck carcinomas:
Mutual relationship and correlation with clinical data. Br. J. Cancer 1999, 79, 1074–1084. [CrossRef] [PubMed] Cancers 2015, 7 Furukawa, T.; Kubota, T.; Hoffman, R.M. Clinical applications of the histoculture drug response
assay. Clin. Cancer Res. 1995, 1, 305–311. [PubMed] 60. Hoffman, R.M. In vitro sensitivity assays in cancer: A review, analysis, and prognosis. J. Clin. Lab. Anal. 1991, 5, 133–143. [CrossRef] [PubMed] 60. Hoffman, R.M. In vitro sensitivity assays in cancer: A review, analysis, and prognosis. J. Clin. Lab. Anal. 1991, 5, 133–143. [CrossRef] [PubMed] 61. Burford-Mason, A.P.; Dardick, I.; MacKay, A. Collagen gel cultures of normal salivary gland:
Conditions for continued proliferation and maintenance of major cell phenotypes in vitro. Laryngoscope 1994, 104, 335–340. [CrossRef] [PubMed] 61. Burford-Mason, A.P.; Dardick, I.; MacKay, A. Collagen gel cultures of normal salivary gland:
Conditions for continued proliferation and maintenance of major cell phenotypes in vitro. Laryngoscope 1994, 104, 335–340. [CrossRef] [PubMed] Cancers 2015, 7 1738 62. Hasegawa, Y.; Goto, M.; Hanai, N.; Ijichi, K.; Adachi, M.; Terada, A.; Hyodo, I.; Ogawa, T.;
Furukawa, T. Evaluation of optimal drug concentration in histoculture drug response assay in
association with clinical efficacy for head and neck cancer. Oral Oncol. 2007, 43, 749–756. [CrossRef] [PubMed] 63. Robbins,
K.T.;
Connors,
K.M.;
Storniolo,
A.M.;
Hanchett,
C.;
Hoffman,
R.M. Sponge-gel-supported histoculture drug-response assay for head and neck cancer. Correlations
with clinical response to cisplatin. Arch. Otolaryngol. Head Neck Surg. 1994, 120, 288–292. [CrossRef] [PubMed] 64. Singh, B.; Li, R.; Xu, L.; Poluri, A.; Patel, S.; Shaha, A.R.; Pfister, D.; Sherman, E.;
Goberdhan, A.; Hoffman, R.M.; et al. Prediction of survival in patients with head and neck cancer
using the histoculture drug response assay. Head Neck 2002, 24, 437–442. [CrossRef] [PubMed] 65. Ariyoshi, Y.; Shimahara, M.; Tanigawa, N. Study on chemosensitivity of oral squamous cell
carcinomas by histoculture drug response assay. Oral Oncol. 2003, 39, 701–707. [CrossRef] 66. Pathak, K.A.; Juvekar, A.S.; Radhakrishnan, D.K.; Deshpande, M.S.; Pai, V.R.; Chaturvedi, P.;
Pai, P.S.; Chaukar, D.A.; D’Cruz, A.K.; Parikh, P.M. In vitro chemosensitivity profile of oral
squamous cell cancer and its correlation with clinical response to chemotherapy. Indian J. Cancer
2007, 44, 142–146. [CrossRef] [PubMed] 67. Kubota, T.; Sasano, N.; Abe, O.; Nakao, I.; Kawamura, E.; Saito, T.; Endo, M.; Kimura, K.;
Demura, H.; Sasano, H.; et al. Potential of the histoculture drug-response assay to contribute to
cancer patient survival. Clin. Cancer Res. 1995, 1, 1537–1543. [PubMed] 68. Suda, S.; Akiyama, S.; Sekiguchi, H.; Kasai, Y.; Ito, K.; Nakao, A. Cancers 2015, 7 1739 Cancers 2015, 7 73. Peria, M.; Donnadieu, J.; Racz, C.; Ikoli, J.-F.; Galmiche, A.; Chauffert, B.; Page, C. Evaluation
of individual sensitivity of Head and Neck Squamous Cell Carcinoma to cetuximab by short-term
culture of tumor slices. Head Neck 2015. [CrossRef] [PubMed] 74. Freudlsperger, C.; Horn, D.; Weißfuß, S.; Weichert, W.; Weber, K.-J.; Saure, D.; Sharma, S.;
Dyckhoff, G.; Grabe, N.; Plinkert, P.; et al. Phosphorylation of AKT(Ser473) serves as an
independent prognostic marker for radiosensitivity in advanced head and neck squamous cell
carcinoma. Int. J. Cancer 2015, 136, 2775–2785. [CrossRef] [PubMed] 75. Freshney, R.I. Culture of Animal Cells:
A Manual of Basic Technique and Specialized
Applications, 6th ed.; Wiley-Liss: Wilmington, DE, USA, 2010. 76. Freyer, J.P. Role of necrosis in regulating the growth saturation of multicellular spheroids. Cancer
Res. 1988, 48, 2432–2439. [PubMed] 77. Schwachöfer, J.H.;
Acker, H.;
Crooijmans, R.P.;
van Gasteren, J.J.;
Holtermann, G.;
Hoogenhout, J.; Jerusalem, C.R.; Kal, H.B. Oxygen tensions in two human tumor cell lines grown
and irradiated as multicellular spheroids. Anticancer Res. 1991, 11, 273–279. [PubMed] 78. Heimdal, J.H.; Aarstad, H.J.; Olofsson, J. Monocytes secrete interleukin-6 when co-cultured
in vitro with benign or malignant autologous fragment spheroids from squamous cell carcinoma
patients. Scand. J. Immunol. 2000, 51, 271–278. [CrossRef] [PubMed] 79. Kross, K.W.; Heimdal, J.-H.; Olsnes, C.; Olofsson, J.; Aarstad, H.J. Head and neck squamous cell
carcinoma spheroid- and monocyte spheroid-stimulated IL-6 and monocyte chemotactic protein-1
secretion are related to TNM stage, inflammatory state and tumor macrophage density. Acta
Otolaryngol. 2005, 125, 1097–1104. [CrossRef] [PubMed] 80. Kross, K.W.; Heimdal, J.H.; Olsnes, C.; Olofsson, J.; Aarstad, H.J. Co-culture of head and neck
squamous cell carcinoma spheroids with autologous monocytes predicts prognosis. Scand. J. Immunol. 2008, 67, 392–399. [CrossRef] [PubMed] 81. Lim, Y.C.; Oh, S.-Y.; Cha, Y.Y.; Kim, S.-H.; Jin, X.; Kim, H. Cancer stem cell traits in
squamospheres derived from primary head and neck squamous cell carcinomas. Oral Oncol. 2011, 47, 83–91. [CrossRef] [PubMed] 82. Pozzi, V.; Sartini, D.; Rocchetti, R.; Santarelli, A.; Rubini, C.; Morganti, S.; Giuliante, R.;
Calabrese, S.; di Ruscio, G.; Orlando, F.; et al. Identification and Characterization of Cancer
Stem Cells from Head and Neck Squamous Cell Carcinoma Cell Lines. Cell. Physiol. Biochem. 2015, 36, 784–798. [CrossRef] [PubMed] 83. Lim, Y.C.; Oh, S.-Y.; Kim, H. Cellular characteristics of head and neck cancer stem cells in
type IV collagen-coated adherent cultures. Exp. Cell Res. 2012, 318, 1104–1111. [CrossRef]
[PubMed] 84. Cancers 2015, 7 Evaluation of the histoculture
drug response assay as a sensitivity test for anticancer agents. Surg. Today 2002, 32, 477–481. [CrossRef] [PubMed] 69. Ohie, S.; Udagawa, Y.; Kozu, A.; Komuro, Y.; Aoki, D.; Nozawa, S.; Moossa, A.R.;
Hoffman, R.M. Cisplatin sensitivity of ovarian cancer in the histoculture drug response assay
correlates to clinical response to combination chemotherapy with cisplatin, doxorubicin and
cyclophosphamide. Anticancer Res. 2000, 20, 2049–2054. [PubMed] 70. Nakada, S.; Aoki, D.; Ohie, S.; Horiuchi, M.; Suzuki, N.; Kanasugi, M.; Susumu, N.;
Udagawa, Y.; Nozawa, S. Chemosensitivity testing of ovarian cancer using the histoculture drug
response assay: Sensitivity to cisplatin and clinical response. Int. J. Gynecol. Cancer 2005, 15,
445–452. [CrossRef] [PubMed] 71. Jung, P.-S.; Kim, D.-Y.; Kim, M.-B.; Lee, S.-W.; Kim, J.-H.; Kim, Y.-M.; Kim, Y.-T.;
Hoffman, R.M.; Nam, J.-H. Progression-free survival is accurately predicted in patients treated
with chemotherapy for epithelial ovarian cancer by the histoculture drug response assay in a
prospective correlative clinical trial at a single institution. Anticancer Res. 2013, 33, 1029–1034. [PubMed] 72. Gerlach, M.M.; Merz, F.; Wichmann, G.; Kubick, C.; Wittekind, C.; Lordick, F.; Dietz, A.;
Bechmann, I. Slice cultures from head and neck squamous cell carcinoma: A novel test system for
drug susceptibility and mechanisms of resistance. Br. J. Cancer 2014, 110, 479–488. [CrossRef]
[PubMed] Cancers 2015, 7 Cancers 2015, 7 1740 86. Kopf-Maier, P.; Kolon, B. An organoid culture assay (OCA) for determining the drug sensitivity
of human tumors. Int. J. Cancer 1992, 51, 99–107. [CrossRef] [PubMed] 87. Kopf-Maier, P. A new approach for realizing the “antioncogram”. Life Sci. 1992, 50, 1711–1718. [CrossRef] 88. Von Hoff, D.D. “Send this patient’s tumor for culture and sensitivity”. N. Engl. J. Med. 1983,
308, 154–155. [CrossRef] [PubMed] 89. Garozzo, A.; Rossi, M.; Denaro, A.; Allegra, E.; Santangelo, G.; Amato, M.; Tomasello, C. In vitro short-term chemosensitivity test in head and neck tumors. J. Chemother. 1989, 1, 59–63. [PubMed] 90. Elprana, D.; Schwachöfer, J.; Kuijpers, W.; van den Broek, P.; Wagener, D.J. In vitro
chemosensitivity testing of squamous cell carcinoma of the head and neck. A preliminary report. Acta Otolaryngol. 1987, 103, 529–536. [PubMed] 91. Elprana, D.; Schwachöfer, J.; Kuijpers, W.; van den Broek, P.; Wagener, D.J. Cytotoxic drug
sensitivity of squamous cell carcinoma as predicted by an in vitro testing model. Anticancer Res. 1989, 9, 1089–1094. [PubMed] 92. Hattersley, S.M.; Sylvester, D.C.; Dyer, C.E.; Stafford, N.D.; Haswell, S.J.; Greenman, J. A
microfluidic system for testing the responses of head and neck squamous cell carcinoma tissue
biopsies to treatment with chemotherapy drugs. Ann. Biomed. Eng. 2012, 40, 1277–1288. [CrossRef] [PubMed] 93. Sylvester, D.; Hattersley, S.M.; Stafford, N.D.; Haswell, S.J.; Greenman, J. Development of
microfluidic-based analytical methodology for studying the effects of chemotherapy agents on
cancer tissue. Curr. Anal. Chem. 2013, 9, 2–8. [CrossRef] 94. Carr, S.D.; Green, V.L.; Stafford, N.D.; Greenman, J. Analysis of radiation-induced cell death
in head and neck squamous cell carcinoma and rat liver maintained in microfluidic devices. Otolaryngol. Head Neck Surg. 2014, 150, 73–80. [CrossRef] [PubMed] 95. Sivagnanam, V.; Gijs, M.A.M. Exploring living multicellular organisms, organs, and tissues using
microfluidic systems. Chem. Rev. 2013, 113, 3214–3247. [CrossRef] [PubMed] 96. Champion, A.R.; Hanson, J.A.; Venables, S.E.; McGregor, A.D.; Gaffney, C.C. Determination
of radiosensitivity in established and primary squamous cell carcinoma cultures using the
micronucleus assay. Eur. J. Cancer 1997, 33, 453–462. [CrossRef] 97. Liu, C.; Tsao, M.S. Proto-oncogene and growth factor/receptor expression in the establishment
of primary human non-small cell lung carcinoma cell lines. Am. J. Pathol. 1993, 142, 413–23. [PubMed] 98. Köhler, G.; Veelken, H.; Rosenthal, F.; Mackensen, A.; Lindemann, A.; Schaefer, H.E.; Lahn, M. Oncogene and HSP-70 expression in primary tumor cell cultures of renal cell carcinoma compared
to their corresponding cell line. Anticancer Res. Cancers 2015, 7 Leong, H.S.; Chong, F.T.; Sew, P.H.; Lau, D.P.; Wong, B.H.; Teh, B.-T.; Tan, D.S.W.; Iyer, N.G. Targeting Cancer Stem Cell Plasticity Through Modulation of Epidermal Growth Factor and
Insulin-Like Growth Factor Receptor Signaling in Head and Neck Squamous Cell Cancer. Stem
Cells Transl. Med. 2014, 3, 1055–1065. [CrossRef] [PubMed] 85. Sun, S.; Liu, S.; Duan, S.Z.; Zhang, L.; Zhou, H.; Hu, Y.; Zhou, X.; Shi, C.; Zhou, R.; Zhang, Z. Targeting the c-Met/FZD8 Signaling Axis Eliminates Patient-Derived Cancer Stem-like Cells in
Head and Neck Squamous Carcinomas. Cancer Res. 2014, 74, 7546–7559. [CrossRef] [PubMed] Cancers 2015, 7 1741 Cancers 2015, 7 100. Worsham, M.J.; Wolman, S.R.; Carey, T.E.; Zarbo, R.J.; Benninger, M.S.; van Dyke, D.L. Chromosomal aberrations identified in culture of squamous carcinomas are confirmed by
fluorescence in situ hybridisation. Mol. Pathol. 1999, 52, 42–46. [CrossRef] [PubMed] 101. Bjerkvig, R. Spheroid Culture in Cancer Research, 1st ed.; CRC Press: Boca Raton, FL, USA,
1992. 102. Ragin, C.C.; Reshmi, S.C.; Gollin, S.M. Mapping and analysis of HPV16 integration sites in a
head and neck cancer cell line. Int. J. Cancer 2004, 110, 701–709. [CrossRef] [PubMed] 103. Gisselsson, D.; Jonson, T.; Yu, C.; Martins, C.; Mandahl, N.; Wiegant, J.; Jin, Y.; Mertens, F.;
Jin, C. Centrosomal abnormalities, multipolar mitoses, and chromosomal instability in head and
neck tumours with dysfunctional telomeres. Br. J. Cancer 2002, 87, 202–207. [CrossRef]
[PubMed] 104. Leess, F.R.; Bredenkamp, J.K.; Lichtenstein, A.; Mickel, R.A. Lymphokine-activated killing
of autologous and allogeneic short-term cultured head and neck squamous carcinomas. Laryngoscope 1989, 99, 1255–1261. [CrossRef] [PubMed] 105. Jin, Y.; Mertens, F.; Mandahl, N.; Heim, S.; Olegård, C.; Wennerberg, J.; Biörklund, A.;
Mitelman, F. Chromosome abnormalities in eighty-three head and neck squamous cell
carcinomas: Influence of culture conditions on karyotypic pattern. Cancer Res. 1993, 53,
2140–2146. [PubMed] 106. Heppner, G.H.; Miller, B.E. Therapeutic implications of tumor heterogeneity. Semin. Oncol. 1989, 16, 91–105. [PubMed] 107. Von Hoff, D.D. He’s not going to talk about in vitro predictive assays again, is he? J. Natl. Cancer
Inst. 1990, 82, 96–101. [CrossRef] [PubMed] 108. Moscona, A. Cell suspensions from organ rudiments of chick embryos. Exp. Cell. Res. 1952, 3,
535–539. [CrossRef] 109. Miller, B.E.; Miller, F.R.; Heppner, G.H. Assessing tumor drug sensitivity by a new in vitro assay
which preserves tumor heterogeneity and subpopulation interactions. J. Cell. Physiol. Suppl. 1984, 3, 105–116. [CrossRef] [PubMed] 110. Lawler, E.M.; Miller, F.R.; Heppner, G.H. Significance of three-dimensional growth patterns of
mammary tissues in collagen gels. In Vitro 1983, 19, 600–610. [CrossRef] [PubMed] 111. Miller, B.E.; Miller, F.R.; Heppner, G.H. Factors affecting growth and drug sensitivity of mouse
mammary tumor lines in collagen gel cultures. Cancer Res. 1985, 45, 4200–4205. [PubMed] 112. Kobayashi, H.; Man, S.; Graham, C.H.; Kapitain, S.J.; Teicher, B.A.; Kerbel, R.S. Acquired
multicellular-mediated resistance to alkylating agents in cancer. Proc. Natl. Acad. Sci. USA
1993, 90, 3294–3298. [CrossRef] [PubMed] 113. Miller, B.E.; Miller, F.R.; Heppner, G.H. Interactions between tumor subpopulations affecting
their sensitivity to the antineoplastic agents cyclophosphamide and methotrexate. Cancer Res. 1981, 41, 4378–4381. Cancers 2015, 7 1997, 17, 3225–3231. [PubMed] 99. Shapiro, J.R.; Shapiro, W.R. The subpopulations and isolated cell types of freshly resected high
grade human gliomas: Their influence on the tumor’s evolution in vivo and behavior and therapy
in vitro. Cancer Metastasis Rev. 1985, 4, 107–124. [CrossRef] [PubMed] Cancers 2015, 7 [PubMed] 114. Lawton, P.A.; Hodgkiss, R.J.; Eyden, B.P.; Joiner, M.C. Growth of fibroblasts as a potential
confounding factor in soft agar clonogenic assays for tumour cell radiosensitivity. Radiother. Oncol. 1994, 32, 218–225. [CrossRef] Cancers 2015, 7 1742 Cancers 2015, 7 115. Balermpas, P.; Rödel, F.; Rödel, C.; Krause, M.; Linge, A.; Lohaus, F.; Baumann, M.; Tinhofer, I.;
Budach, V.; Gkika, E.; et al. CD8+ tumour-infiltrating lymphocytes in relation to HPV status and
clinical outcome in patients with head and neck cancer after postoperative chemoradiotherapy: A
multicentre study of the German cancer consortium radiation oncology group (DKTK-ROG). Int. J. Cancer 2015. [CrossRef] [PubMed] 116. Heimdal, J.H.; Aarstad, H.J.; Olsnes, C.; Olofsson, J. Human autologous monocytes and
monocyte-derived macrophages in co-culture with carcinoma F-spheroids secrete IL-6 by a
non-CD 14-dependent pathway. Scand. J. Immunol. 2001, 53, 162–170. [CrossRef] [PubMed] © 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
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Water Source of Six Woody Plants in Different Habitats on Desertified Land of Ordos Plateau, Semi-Arid China
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Chapter
Water Source of Six Woody
Plants in Different Habitats on
Desertified Land of Ordos Plateau,
Semi-Arid China Yajuan Zhu Abstract Water and soil erosion and sandy desertification are two mainly land
desertification types on eastern and southern Ordos Plateau, north China. Hippophae rhamnoides, Armeniaca sibirica and Pinus tabuliformis are three woody
plants for soil and water conservation on loess slope. Sabina vulgaris, Artemisia
ordosica and Salix psammophila are three shrubs for sand control on sand dune. Water source of six woody plants were investigated by stable isotope technol-
ogy. The results showed that the δ18O of shallow soil water was similar to that of
rainwater in July and September in two habitats. Both of six woody plants in two
habitats mainly used shallow soil water in May. However, three shrubs on sand
dune mainly used both of shallow and deep soil water in July and September. Three woody plants on loess slope mainly used rainwater or deep soil water in July
and September. Therefore, six woody plants utilized different depths of soil water
or rain water based on their availability in different seasons, which is an adaptive
strategy to the semiarid climate on Ordos Plateau. Keywords: land desertification control, rain water, soil water, stable oxygen isotope,
water source 1. Introduction In semi-arid region, water is the key factor for the survival and succession of
plant community [1]. The use of limited water resource concerns not only plant
survival, but also interspecific interaction and community dynamics. Since there
is generally no stable isotope fractionation during water uptake by root system and
water transportation before arriving leaf, water source of a plant could be identified
by comparing stable isotope of xylem water and potential water sources [2]. The
potential water sources for plant species are shallow soil water recharged by rainwa-
ter, deep soil water recharged by rainwater, snow water or groundwater in semi-
arid region [3–10]. Different life form plants usually used different water source,
which related to their root types. Generally, tree and shrub with deep root system
could use deep soil water or groundwater [6]. Woody species with dimorphic root
system may use different depths of soil water or groundwater simultaneously [9]. And shallow rooted shrub and perennial grass only used shallow or middle soil 1 Deserts and Desertification water [11]. Water source of plant species in semi-arid region was affected by many
environmental factors, like season [6, 7], annual variance of climate [8] and habitat
heterogeneity [12]. g
y
Ordos Plateau is located in the middle reaches of Yellow River, which is the
ecotone between Loess Plateau and Mongolian Plateau, with the total area of
1.30 × 105 km2. The eastern part is hilly gully area with loess hill and valley, the
southern part is Mu Us Sandy Land with fixed and semi-fixed sand dune, and the
northern part is Hobq Desert with moving sand dune [13]. The elevation increases
from 774 m in southeast to 2148 m in northwest [14]. From southeast to northwest,
the annual precipitation decreased from 400 mm to 200 mm; meanwhile, the natu-
ral vegetation varies from forest grassland, grassland, and sandy land to desert [13]. In the last decades, land desertification was severely on Ordos Plateau. From 1994 to
2000, land desertification area increased for 1.90 × 104 km2, focused on west Hobq
Desert and south Mu Us Sandy Land [15]. In these years, many measures was taken
to increase vegetation coverage, decrease sand storm hazard and improve ecological
environment, such as air seeding, fence and afforestation. 1. Introduction g
In eastern Ordos Plateau, the hilly gully area is one of the most severely soil and
water erosion region in the middle reaches of Yellow River, which is sand stone
covered by loess and contributed up to 25% of the total course sediment for the
lower reaches [16]. Both of engineering measure and vegetation restoration was
carried out to control soil and water erosion, such as plant trees and shrubs, includ-
ing Hippophae rhamnoides, Pinus tabuliformis and Armeniaca sibirica. g
pp p
f
Mu Us Sandy Land is one of the four sandy land in China, which located in
central Inner Mongolia, Northern Shaanxi and Northeast Ningxia. The total area
is 4.22 × 104 km2, with the elevation varies from 1000 to 1600 m [17]. The main
natural vegetation are forest steppe, steppe, and shrub sandy land and desert
steppe. Vegetation growth was improved in Mu Us Sandy Land, which were resulted
by climate change and human activity [18]. The dominant species of sand control
are Sabina vulgaris, Artemisia ordosica, Salix psammophila and Caragana intermedia
in shrub sandy land [19]. y
Previous studies in Mu Us Sandy Land showed that S. vulgaris mainly used soil
water within 1.5 m and groundwater, whereas A. ordosica mainly used shallow
soil water within 50 cm [5]. Moreover, A. ordosica mainly used deep soil water
recharged by 65 mm rainstorm in summer, Cynanchum komarovii mainly used
middle rain of 10–20 mm, whereas Stipa bungeana mainly used shallow soil water
recharged by small rain [3]. However, we still do not know the seasonal dynamic of
water source for these dominant shrubs in Mu Us Sandy Land. Moreover, there is
few report about water source of woody species in hilly gully area of Ordos Plateau. Therefore, seasonal dynamic of water source of six woody plants was explored by
stable isotope technology in two different habitats of Ordos Plateau. The purpose
of this study was to understand how these trees and shrubs adapt to the semi-arid
climate by adjusting water source in the growing season. The results would give
theoretical supports for ecological forestry engineering, including Natural Forest
Protection, Grain for Green and Three Norths Shelterbelt Program. 2.1 Three soil and water conservation woody species on loess slope This study was conducted in Soil and Water Conservation Park of Jungar
Banner, Ordos City, Inner Mongolia. The banner has a temperate continental
climate, which mean air temperature varies from 6.2°C to 8.7°C, mean annual 2 Water Source of Six Woody Plants in Different Habitats on Desertified Land of Ordos Plateau…
DOI: http://dx.doi.org/10.5772/intechopen.99658 Water Source of Six Woody Plants in Different Habitats on Desertified Land of Ordos Plateau…
DOI: http://dx.doi.org/10.5772/intechopen.99658 Water Source of Six Woody Plants in Different Habitats on Desertified Land of Ordos Plateau…
DOI: http://dx.doi.org/10.5772/intechopen.99658 precipitation is 400 mm, potential evapotranspiration is 2093 mm and forest free
days are 145 d [20]. The natural vegetation is steppe dominated by Stipa bungeana,
whereas few P. tabuliformis, Juniperus rigida and Platycladus orientalis are distrib-
uted on hill slope. p
Hippophae rhamnoides (sea buckthorn) is a small tree or shrub with the height of
1–5 m. It often inhabits in hill ridge, valley, dry river bed or slope with rock, sandy
loam or loam, which distributes in Hebei, Inner Mongolia, Shanxi, Shaanxi, Gansu,
Qinghai and West Sichuan. This shrub was widely used on Loess Plateau as a soil
and water conservation species [21]. It grows fast, resistant to drought and could fix
nitrogen. g
Pinus tabuliformis (Chinese pine) is an evergreen tree with the height of 25 m
and the diameter at breast height of 1 m. It is the dominant species in coniferous
forest with elevation varies from 100 to 2600 m, which distributes in south Jilin,
Liaoning, Hebei, Henan, Shandong, Shanxi, Inner Mongolia, Shaanxi, Ningxia,
Qinghai and Sichuan. It is used as a sand binding and soil and water conservation
trees in north China [22]. Armeniaca sibirica (wild apricot) is a small tree or shrub with the height of
2–5 m. It mainly inhabits on dry slope, hill steppe or mixed with deciduous forest,
which distributes in Heilongjiang, Jilin, Liaoning, Inner Mongolia, Gansu, Hebei
and Shanxi, and also in East and Southeast Mongolia, Far East and Siberia of Russia
[23]. It is cold-resistant and was used as a soil and water conservation species in
Northeast and North China. In the study site, three woody species was planted in 2013 with density of 1000
plants per hectare. The mean height of H. rhamnoides, P. tabuliformis and A. sibirica
were 2.10, 2.14 and 2.71 m, respectively. 2.2 Precipitation of study site in the growing season of 2018 The total precipitation of Dongsheng District was 385.00 mm in the growing
season of 2018 (Figure 1), which was slightly lower than annual mean precipitation
of 400 mm. The maximal daily precipitation was 52.0 mm and occurred on May 19,
the next value was 48.8 mm on July 19. The monthly precipitation were 32.0, 77.9,
15.5, 118.3, 85.0 and 54.3 mm from April to September. 2.1 Three soil and water conservation woody species on loess slope The main water source of three woody spe-
cies was measured by comparing their xylem water with different water sources, e.g. rainwater and soil water in 10, 25, 50, 75 and 100 cm. Lignified, two years old twigs
of shrubs were collected and the bark was removed with scissor, then the xylem was
sampled. Soil and xylem were placed in 8 mL glass vial, sealed with Parafilm, and
stored in a medical cool box. Water in soil and xylem was vacuum-extracted and their
δ18O value was measured with a Flash 2000HT elemental analyzer and a Finnigan
MAT 253 mass spectrometer. Meteorological data was obtained from Meteorology
Bureau of Dongsheng District, Ordos City, which is 50 km west to the study site. The contribution of different water sources to their total water use was analyzed by
Iso-source 1.3.1 software [24]. The input data of the model were δ18O value of xylem
water and potential water source, e.g. soil water in different depths or rain water. The
results of water use ratio to different sources were expressed as mean ± SD. 2.3 Stable oxygen isotope of xylem water of three woody species, soil water and
rainwater on loess slope On May 12, stable oxygen isotope ratio of H. rhamnoides and P. tabuliformis xylem
water were closer to soil water in 10 cm, whereas stable oxygen isotope ratio of
A. sibirica xylem water was closer to soil water in 10–25 cm. On July 15, stable oxygen
isotope ratio of 10–25 cm soil water was closer to rainwater. Stable oxygen isotope 3 Deserts and Desertification Figure 1. Daily precipitation of study site from April to September of 2018. Figure 1. Daily precipitation of study site from April to September of 2018. Figure 1. Daily precipitation of study site from April to September of 2018. ratio of H. rhamnoides, P. tabuliformis and A. sibirica xylem water were closer to soil
water in 10–100 cm, 25–50 cm and 25–100 cm, respectively; moreover, stable oxygen
isotope ratio of P. tabuliformis and A. sibirica xylem water were also closer to rainwa-
ter. On September 23, stable oxygen isotope ratio of H. rhamnoides xylem water was
closer to soil water in 25–100 cm, whereas stable oxygen isotope ratio of xylem water
of other two woody species were closer to soil water in 10–100 cm (Figure 2). ratio of H. rhamnoides, P. tabuliformis and A. sibirica xylem water were closer to soil
water in 10–100 cm, 25–50 cm and 25–100 cm, respectively; moreover, stable oxygen
isotope ratio of P. tabuliformis and A. sibirica xylem water were also closer to rainwa-
ter. On September 23, stable oxygen isotope ratio of H. rhamnoides xylem water was
closer to soil water in 25–100 cm, whereas stable oxygen isotope ratio of xylem water
of other two woody species were closer to soil water in 10–100 cm (Figure 2). 2.4 Contribution of different depth of soil water and rainwater to the water
source of three woody species on loess slope Iso-source analysis showed that H. rhmnoides mainly used 10 cm soil water on
May 12, which accounted for 88.5% of its total water source. On July 15, it mainly
used 10–25 cm soil water and rainwater, which accounted for 44.6% and 35.4% of
its total water source. On September 23, it mainly used 25 cm and 75–100 cm soil
water, which accounted for 88.9% of its total water source (Table 1). Iso-source analysis showed that P. tabuliformis mainly used 10 cm soil water on
May 12, which accounted for 94.0% of its total water source. On July 15, it mainly
used rainwater, which accounted for 93.7% of its total water source. On September
23, it mainly used 10 cm and 50–75 cm soil water, which accounted for 84.5% of its
total water source (Table 2). Iso-source analysis showed that A. sibirica mainly used 10 cm soil water on May
12, which accounted for 91.6% of its total water source. On July 15, it mainly used
25–100 cm soil water and rainwater, which accounted for 55.9% and 36.8% of its
total water source. On September 23, it evenly used 10–100 cm soil water (Table 3). Three woody species on loess slope selected different water source in the grow-
ing season, which is an adaptive strategy to semi-arid environment. They mainly
used shallow soil water recharged by spring rain. However, there are interspecific
difference of water source in summer. For H. rhmnoides, it mainly used shallow
soil water and rainwater. For P. tabuliformis, it mainly used rainwater. However, for
A. sibirica, it mainly used middle and deep soil water and rainwater. In autumn,
H. rhmnoides mainly used shallow and deep soil water, P. tabuliformis mainly used
shallow and middle soil water. However, A. sibirica evenly used different depths soil
water. The interspecific difference in water source of three woody species is related Iso-source analysis showed that A. sibirica mainly used 10 cm soil water on May
12, which accounted for 91.6% of its total water source. On July 15, it mainly used
25–100 cm soil water and rainwater, which accounted for 55.9% and 36.8% of its
total water source. On September 23, it evenly used 10–100 cm soil water (Table 3). Three woody species on loess slope selected different water source in the grow-
ing season, which is an adaptive strategy to semi-arid environment. Table 2. Table 2. Contribution of different depths of soil water and rainwater to the water source of Pinus tabuliformis in hilly
gully area (%, mean ± SD). Water source
May 12
Jul. 15
Sep. 23
10 cm soil water
91.6 ± 3.3
7.4 ± 6.4
30.4 ± 10.4
25 cm soil water
5.7 ± 4.4
10.3 ± 8.8
27.1 ± 21.1
50 cm soil water
1.2 ± 1.2
21.1 ± 17.6
15.9 ± 12.6
75 cm soil water
0.7 ± 0.9
11.4 ± 9.7
14.2 ± 11.3
100 cm soil water
0.7 ± 0.9
13.1 ± 11.1
12.4 ± 9.9
Rainwater
—
36.8 ± 9.5
—
Table 3. Contribution of different depths of soil water and rainwater to the water source of Armeniaca sibirica in hilly
gully area (%, mean ± SD). Table 2. Contribution of different depths of soil water and rainwater to the water source of Pinus tabuliformis in hilly
gully area (%, mean ± SD). Table 2. Contribution of different depths of soil water and rainwater to the water source of Pinus tabuliformis in hilly
gully area (%, mean ± SD). Table 3. Contribution of different depths of soil water and rainwater to the water source of Armeniaca sibirica in hilly
gully area (%, mean ± SD). Table 3. Contribution of different depths of soil water and rainwater to the water source of Armeniaca sibirica in hilly
gully area (%, mean ± SD). Moreover, the 18-yr Robinia pseudoacacia were sensitive to precipitation variation
and used more deep soil water in a drier year, whereas the 30-yr R. pseudoacacia
always used middle and deep soil water in wetter or drier year in the central region
of Loess Plateau [27]. Therefore, woody species which could use deep soil water as a
reliable water source during drought will have advantage in semi-arid region. 2.4 Contribution of different depth of soil water and rainwater to the water
source of three woody species on loess slope Contribution of different depths of soil water and rainwater to the water source of Pinus tabuliformis in hilly
gully area (%, mean ± SD). Water source
May 12
Jul. 15
Sep. 23
10 cm soil water
88.5 ± 0.7
16.4 ± 13.7
4.6 ± 3.8
25 cm soil water
3.1 ± 2.8
28.2 ± 20.5
17.0 ± 13.3
50 cm soil water
3.0 ± 2.8
6.7 ± 5.6
6.4 ± 5.2
75 cm soil water
1.9 ± 2.2
6.2 ± 5.2
41.4 ± 18.3
100 cm soil water
3.4 ± 2.6
7.1 ± 0.6
30.5 ± 22.8
Rainwater
—
35.4 ± 17.1
—
Table 1. Contribution of different depths of soil water and rainwater to the water source of Hippophae rhamnoides in
hilly gully area (%, mean ± SD). Water source
May 12
Jul. 15
Sep. 23
10 cm soil water
91.6 ± 3.3
7.4 ± 6.4
30.4 ± 10.4
25 cm soil water
5.7 ± 4.4
10.3 ± 8.8
27.1 ± 21.1
50 cm soil water
1.2 ± 1.2
21.1 ± 17.6
15.9 ± 12.6
75 cm soil water
0.7 ± 0.9
11.4 ± 9.7
14.2 ± 11.3
100 cm soil water
0.7 ± 0.9
13.1 ± 11.1
12.4 ± 9.9
Rainwater
—
36.8 ± 9.5
—
Table 3. Contribution of different depths of soil water and rainwater to the water source of Armeniaca sibirica in hilly
gully area (%, mean ± SD). Table 1. Contribution of different depths of soil water and rainwater to the water source of Hippophae rhamnoides in
hilly gully area (%, mean ± SD). 2.4 Contribution of different depth of soil water and rainwater to the water
source of three woody species on loess slope 23
10 cm soil water
94.0 ± 0.9
0.6 ± 0.8
36.8 ± 21.0
25 cm soil water
1.8 ± 2.3
1.2 ± 1.2
7.1 ± 5.3
50 cm soil water
1.6 ± 1.7
1.3 ± 1.2
36.1 ± 21.7
75 cm soil water
1.6 ± 1.7
1.5 ± 1.6
11.6 ± 8.7
100 cm soil water
0.9 ± 1.1
1.6 ± 1.7
8.4 ± 6.3
Rainwater
—
93.7 ± 0.7
—
Table 2. Contribution of different depths of soil water and rainwater to the water source of Pinus tabuliformis in hilly
gully area (%, mean ± SD). Water source
May 12
Jul. 15
Sep. 23
10 cm soil water
88.5 ± 0.7
16.4 ± 13.7
4.6 ± 3.8
25 cm soil water
3.1 ± 2.8
28.2 ± 20.5
17.0 ± 13.3
50 cm soil water
3.0 ± 2.8
6.7 ± 5.6
6.4 ± 5.2
75 cm soil water
1.9 ± 2.2
6.2 ± 5.2
41.4 ± 18.3
100 cm soil water
3.4 ± 2.6
7.1 ± 0.6
30.5 ± 22.8
Rainwater
—
35.4 ± 17.1
—
Table 1. Contribution of different depths of soil water and rainwater to the water source of Hippophae rhamnoides in
hilly gully area (%, mean ± SD). Water source
May 12
Jul. 15
Sep. 23
10 cm soil water
91.6 ± 3.3
7.4 ± 6.4
30.4 ± 10.4 Deserts and Desertification Moreover, the 18-yr Robinia pseudoacacia were sensitive to precipitation variation
and used more deep soil water in a drier year, whereas the 30-yr R. pseudoacacia
always used middle and deep soil water in wetter or drier year in the central region
of Loess Plateau [27]. Therefore, woody species which could use deep soil water as a
reliable water source during drought will have advantage in semi-arid region. 3. Water source of three shrubs in sandy land
3.1 Three sand binding shrubs in Mu Us Sandy Land
Water source
May 12
Jul. 15
Sep. 23
10 cm soil water
94.0 ± 0.9
0.6 ± 0.8
36.8 ± 21.0
25 cm soil water
1.8 ± 2.3
1.2 ± 1.2
7.1 ± 5.3
50 cm soil water
1.6 ± 1.7
1.3 ± 1.2
36.1 ± 21.7
75 cm soil water
1.6 ± 1.7
1.5 ± 1.6
11.6 ± 8.7
100 cm soil water
0.9 ± 1.1
1.6 ± 1.7
8.4 ± 6.3
Rainwater
—
93.7 ± 0.7
—
Table 2. 2.4 Contribution of different depth of soil water and rainwater to the water
source of three woody species on loess slope They mainly
used shallow soil water recharged by spring rain. However, there are interspecific
difference of water source in summer. For H. rhmnoides, it mainly used shallow
soil water and rainwater. For P. tabuliformis, it mainly used rainwater. However, for
A. sibirica, it mainly used middle and deep soil water and rainwater. In autumn,
H. rhmnoides mainly used shallow and deep soil water, P. tabuliformis mainly used
shallow and middle soil water. However, A. sibirica evenly used different depths soil
water. The interspecific difference in water source of three woody species is related 4 Water Source of Six Woody Plants in Different Habitats on Desertified Land of Ordos Plateau…
DOI: http://dx.doi.org/10.5772/intechopen.99658 Water Source of Six Woody Plants in Different Habitats on Desertified Land of Ordos Plateau…
DOI: http://dx.doi.org/10.5772/intechopen.99658 p
g
p
to their life form and root distribution. Other shrubs on Loess Plateau also changed
their water source. For example, Caragana korshinskii and Salix psammophila used
40–80 cm soil water in the drought season. However, they mainly used 0–40 cm
soil water in the rain season [25]. Armeniaca sibirica in the mixed plantation used
more shallow soil water than that in the pure plantation. However, Robinia pseudo-
acacia always used shallow and middle soil water in different plantation types [26]
Figure 2. Stable oxygen isotope ratio of xylem water, soil water and rainwater of Hippophae rhamnoides (A), Pinus
tabuliformis (B) and Armeniaca sibirica (C) in hilly gully area. Dark symbol is soil water, white symbol is
xylem water of three woody species. Figure 2. Stable oxygen isotope ratio of xylem water, soil water and rainwater of Hippophae rhamnoides (A), Pinus
tabuliformis (B) and Armeniaca sibirica (C) in hilly gully area. Dark symbol is soil water, white symbol is
xylem water of three woody species. to their life form and root distribution. Other shrubs on Loess Plateau also changed
their water source. For example, Caragana korshinskii and Salix psammophila used
40–80 cm soil water in the drought season. However, they mainly used 0–40 cm
soil water in the rain season [25]. Armeniaca sibirica in the mixed plantation used
more shallow soil water than that in the pure plantation. However, Robinia pseudo-
acacia always used shallow and middle soil water in different plantation types [26]. 5 Deserts and Desertification
Water source
May 12
Jul. 15
Sep. 3.2 Precipitation in Ordos Ecological Station in the growing season of 2018 The total precipitation of Ordos Ecological Station was 367.00 mm in the
growing season of 2018 (Figure 3), which was slightly higher than the annual
mean precipitation of 350 mm. The maximal daily precipitation was 34.4 mm and
occurred on August 30, the next value was 32.2 mm on July 19, and 31.8 mm on July
16. The monthly precipitation were 25.6, 47.6, 11.4, 144.2, 92.2 and 46.0 mm from
April to September. 3.1 Three sand binding shrubs in Mu Us Sandy Land Artemisia ordosica is a small shrub with mean height of 0.5–1.0 m. It is resistant
to sand burial and was widely used as a good sand-binding plant with air seeding. It
mainly inhabits in moving, semi-fixed and fixed sand dunes in desert and slope in
steppe, which distributes in Inner Mongolia, North Hebei and North Shanxi [29]. Salix psammophila (sand willow) is a shrub with mean height of 3–4 m. It is
resistant to wind and sand burial, which was widely used as a sand-binding and Artemisia ordosica is a small shrub with mean height of 0.5–1.0 m. It is resistant
to sand burial and was widely used as a good sand-binding plant with air seeding. It
mainly inhabits in moving, semi-fixed and fixed sand dunes in desert and slope in
steppe, which distributes in Inner Mongolia, North Hebei and North Shanxi [29]. pp
g
Salix psammophila (sand willow) is a shrub with mean height of 3–4 m. It is
resistant to wind and sand burial, which was widely used as a sand-binding and
afforestation species. It mainly distributes in Shaanxi, Inner Mongolia, Ningxia and
Shanxi [30]. In the study site, the mean height of S. vulgaris, A. ordosica and S. psammophila
were 0.78, 0.92 and 3.33 m, respectively. The semi-shrub Hedysarum fruticosum var. laeve was the associated species in A. ordosica and S. psammophila community. The
main water source of four shrubs was measured by comparing their xylem water
with different water sources, e.g. soil water in 10, 25, 50, 75, 100 and 150 cm for
A. ordosica, whereas soil water in 10, 25, 50, 100, 150 and 200 cm for other two
shrubs. The sample and extraction of soil water and xylem water was shown in 2.1. Groundwater was not sampled because shallow groundwater was depleted after
coal mining in 2012. Deep groundwater table was 70 m at the study site, which is
unavailable for vegetation. Meteorological data was obtained from Ordos Ecological
Station. The contribution of different water sources to their total water use was
analyzed by Iso-source 1.3.1 software [24]. 3.1 Three sand binding shrubs in Mu Us Sandy Land This study was conducted in Ordos Sandy Land Ecological Station, Institute of
Botany, Chinese Academy of Forestry, which located in Ejin Holo Banner, Ordos 6 6 Water Source of Six Woody Plants in Different Habitats on Desertified Land of Ordos Plateau…
DOI: http://dx.doi.org/10.5772/intechopen.99658 Water Source of Six Woody Plants in Different Habitats on Desertified Land of Ordos Plateau…
DOI: http://dx.doi.org/10.5772/intechopen.99658 Water Source of Six Woody Plants in Different Habitats on Desertified Land of Ordos Plateau…
DOI: http://dx.doi.org/10.5772/intechopen.99658 City, Inner Mongolia. The banner has a temperate continental climate, which mean
air temperature varies from 5.0°C to 8.5°C, mean annual precipitation is 350 mm,
potential evapotranspiration is 2300 mm and forest free days are 136 d [28]. The
natural vegetation is sandy land dominated by Sabina vulgaris, Artemisia ordosica,
Salix psammophila and Caragana intermedia, and steppe dominated by Stipa
bungeana and Iris lactea var. chinensis. g
Sabina vulgaris is an evergreen shrub with height of 0.3–1.0 m. It is a drought
resistant species and widely used as sand binding and soil and water conservation
shrub. It mainly inhabits on rocky slope, mixed coniferous and broad-leaf forest or
sand dune, which distributes in Tianshan Mountain, Altai Mountain, Helanshan
Mountain, Inner Mongolia, Northeast Qinghai, Gansu and North Shaanxi [22]. Artemisia ordosica is a small shrub with mean height of 0.5–1.0 m. It is resistant
to sand burial and was widely used as a good sand-binding plant with air seeding. It
mainly inhabits in moving, semi-fixed and fixed sand dunes in desert and slope in
steppe, which distributes in Inner Mongolia, North Hebei and North Shanxi [29]. Salix psammophila (sand willow) is a shrub with mean height of 3–4 m. It is
resistant to wind and sand burial, which was widely used as a sand-binding and
afforestation species. It mainly distributes in Shaanxi, Inner Mongolia, Ningxia and
Shanxi [30] Sabina vulgaris is an evergreen shrub with height of 0.3–1.0 m. It is a drought
resistant species and widely used as sand binding and soil and water conservation
shrub. It mainly inhabits on rocky slope, mixed coniferous and broad-leaf forest or
sand dune, which distributes in Tianshan Mountain, Altai Mountain, Helanshan
Mountain, Inner Mongolia, Northeast Qinghai, Gansu and North Shaanxi [22]. A
i i
d i
i
ll h
b
i h
h i h
f 0 5 1 0
I i
i Mountain, Inner Mongolia, Northeast Qinghai, Gansu and North Shaanxi [22]. 3.3 Stable oxygen isotope of xylem water of four shrubs, soil water and
rainwater in sandy land On May 13, stable oxygen isotope ratio of S. vulgaris xylem water were closer to
soil water in 10–25 cm, stable oxygen isotope ratio of A. ordosica and H. fruticosum
var. laeve xylem water were closer to soil water in 10–150 cm, whereas stable oxy-
gen isotope ratio of S. psammophila and H. fruticosum var. laeve xylem water was
closer to soil water in 10 cm and 50–200 cm. On July 13, stable oxygen isotope ratio
of 10 cm soil water was closer to rainwater on July 11. Stable oxygen isotope ratio
of S. vulgaris was closer to soil water in 25 cm and 100–200 cm, stable oxygen iso-
tope ratio of A. ordosica and H. fruticosum var. laeve xylem water were closer to soil
water in 10 cm and 150 cm, whereas stable oxygen isotope ratio of S. psammophila 7 Deserts and Desertification Figure 3. Daily precipitation at Ordos Ecological Station from April to September of 2018. Figure 3. Figure 3. Daily precipitation at Ordos Ecological Station from April to September of 2018. g
3
Daily precipitation at Ordos Ecological Station from April to September of 2018. and H. fruticosum var. laeve xylem water was closer to soil water in 10–25 cm and
100–200 cm. On September 22, stable oxygen isotope ratio of 10 cm soil water
was closer to rainwater on September 11. Stable oxygen isotope ratio of S. vulgaris
xylem water was closer to soil water in 25–200 cm, stable oxygen isotope ratio of
A. ordosica and H. fruticosum var. laeve xylem water were closer to soil water in
10–150 cm, whereas stable oxygen isotope ratio of S. psammophila and H. frutico-
sum var. laeve xylem water were closer to soil water in 25–200 cm (Figure 4). and H. fruticosum var. laeve xylem water was closer to soil water in 10–25 cm and
100–200 cm. On September 22, stable oxygen isotope ratio of 10 cm soil water
was closer to rainwater on September 11. Stable oxygen isotope ratio of S. vulgaris
xylem water was closer to soil water in 25–200 cm, stable oxygen isotope ratio of
A. ordosica and H. fruticosum var. laeve xylem water were closer to soil water in
10–150 cm, whereas stable oxygen isotope ratio of S. psammophila and H. frutico-
sum var. laeve xylem water were closer to soil water in 25–200 cm (Figure 4). 3.4 Contribution of different depth of soil water to the water source of four
shrubs in sandy land Iso-source analysis showed that S. vulgaris mainly used 25 cm soil water on May
13, which accounted for 78.5% of its total water source. On July 17, it mainly used
10–25 cm and 100–200 cm soil water and rainwater, which accounted for 50.8%
and 40.7% of its total water source. On September 22, it mainly used 10–25 cm
and 100–200 cm soil water, which accounted for 51.2% and 39.8% of its total water
source (Table 4). Iso-source analysis showed that A. ordosica and H. fruticosum var. laeve mainly
used 10 cm soil water on May 13, which accounted for 72.9% and 66.5% of their
total water source, respectively. On July 17, they mainly used 10 cm and 150 cm soil
water, which accounted for 65.8% and 54.9% of their total water source, respec-
tively. On September 22, they mainly used 10–25 cm soil water, which accounted for
46.1% and 49.0% of their total water source, respectively (Table 5). p
y
Iso-source analysis showed that S. psammophila and H. fruticosum var. laeve
mainly used 10–25 cm soil water on May 13, which accounted for 59.0% and 37.9%
of their total water source. On July 17, they mainly used 50–200 cm soil water
and 10–25 cm and 100–200 cm soil water, which accounted for 71.0% and 91.8%
of their total water source, respectively. On September 22, they mainly used
10–100 cm soil water, which accounted for 73.8% and 72.5% of their total water
source, respectively (Table 6). y
Four shrubs in sandy land have a resource-dependent water use strategy, e.g. used different depths of soil water based on their availability in the growing season. Sabina vulgaris mainly used 25 cm shallow soil water in spring; however, it mainly 8 Water Source of Six Woody Plants in Different Habitats on Desertified Land of Ordos Plateau…
DOI: http://dx.doi.org/10.5772/intechopen.99658 Water Source of Six Woody Plants in Different Habitats on Desertified Land of Ordos Plateau…
DOI: http://dx.doi.org/10.5772/intechopen.99658 Water Source of Six Woody Plants in Different Habitats on Desertified Land of Ordos Plateau…
DOI: http://dx.doi.org/10.5772/intechopen.99658 p
g
577
p
99 5 9
Figure 4. Stable oxygen isotope ratio of xylem water, soil water and rainwater of Sabina vulgaris (A), Artemisia
ordosica (B) and Salix psammophila (C) in sandy land. Dark symbol is soil water, white symbol is xylem
water of three shrubs, and gray symbol is xylem water of Hedysarum fruticosum var. laeve. Figure 4. Table 4.
C
b Table 4. Contribution of different depths of soil water and rainwater to the water source of Sabina vulgaris in sandy
land (%, mean ± SD). Figure 4. Figure 4. Stable oxygen isotope ratio of xylem water, soil water and rainwater of Sabina vulgaris (A), Artemisia
ordosica (B) and Salix psammophila (C) in sandy land. Dark symbol is soil water, white symbol is xylem
water of three shrubs, and gray symbol is xylem water of Hedysarum fruticosum var. laeve. Figure 4. Stable oxygen isotope ratio of xylem water, soil water and rainwater of Sabina vulgaris (A), Artemisia
ordosica (B) and Salix psammophila (C) in sandy land. Dark symbol is soil water, white symbol is xylem
water of three shrubs, and gray symbol is xylem water of Hedysarum fruticosum var. laeve. 9 9 Deserts and Desertification Deserts and Desertification
Water source
May 13
Jul. 17
Sep. 22
10 cm soil water
8.9 ± 7.7
38.1 ± 5.5
36.8 ± 5.5
25 cm soil water
78.5 ± 4.9
12.7 ± 10.7
14.4 ± 12.1
50 cm soil water
2.8 ± 2.7
8.4 ± 7.2
9.1 ± 7.8
100 cm soil water
2.9 ± 2.8
11.0 ± 9.4
14.2 ± 12.0
150 cm soil water
3.6 ± 3.3
13.3 ± 11.3
15.4 ± 13.0
200 cm soil water
3.4 ± 3.2
16.4 ± 13.8
10.2 ± 8.8
Table 4. Contribution of different depths of soil water and rainwater to the water source of Sabina vulgaris in sandy
land (%, mean ± SD). Water source
May 13
Jul. 17
Sep. 22
Ao
Hf
Ao
Hf
Ao
Hf
10 cm soil water
72.9 ± 3.1
66.5 ± 3.6
51.9 ± 5.5
36.6 ± 7.0
26.0 ± 7.9
30.0 ± 7.5
25 cm soil water
7.9 ± 6.9
9.7 ± 8.4
9.9 ± 8.5
13.1 ± 11.1
20.1 ± 16.8
19.0 ± 16.0
50 cm soil water
4.4 ± 4.0
5.5 ± 4.9
5.9 ± 5.3
7.9 ± 6.8
12.9 ± 11.0
12.2 ± 10.4
75 cm soil water
5.8 ± 5.1
7.2 ± 6.3
8.4 ± 7.3
11.2 ± 9.5
17.6 ± 14.8
16.7 ± 14.0
100 cm soil
water
4.4 ± 4.0
5.5 ± 4.9
9.8 ± 8.4
13.0 ± 11.0
13.3 ± 11.2
12.5 ± 10.7
150 cm soil
water
4.5 ± 4.1
5.7 ± 5.0
13.9 ± 11.8
18.3 ± 15.4
10.1 ± 8.7
9.6 ± 8.2
Table 5. Contribution of different depths of soil water and rainwater to the water source of Artemisia ordosica (Ao)
and Hedysarum fruticosum var. laeve (Hf) in sandy land (%, mean ± SD). Figure 4. Water source
May 13
Jul. 17
Sep. 22 Water source
May 13
Jul. 17
Sep. 22
10 cm soil water
8.9 ± 7.7
38.1 ± 5.5
36.8 ± 5.5
25 cm soil water
78.5 ± 4.9
12.7 ± 10.7
14.4 ± 12.1
50 cm soil water
2.8 ± 2.7
8.4 ± 7.2
9.1 ± 7.8
100 cm soil water
2.9 ± 2.8
11.0 ± 9.4
14.2 ± 12.0
150 cm soil water
3.6 ± 3.3
13.3 ± 11.3
15.4 ± 13.0
200 cm soil water
3.4 ± 3.2
16.4 ± 13.8
10.2 ± 8.8 Table 4. Contribution of different depths of soil water and rainwater to the water source of Sabina vulgaris in sandy
land (%, mean ± SD). Water source
May 13
Jul. 17
Sep. 22
Ao
Hf
Ao
Hf
Ao
Hf
10 cm soil water
72.9 ± 3.1
66.5 ± 3.6
51.9 ± 5.5
36.6 ± 7.0
26.0 ± 7.9
30.0 ± 7.5
25 cm soil water
7.9 ± 6.9
9.7 ± 8.4
9.9 ± 8.5
13.1 ± 11.1
20.1 ± 16.8
19.0 ± 16.0
50 cm soil water
4.4 ± 4.0
5.5 ± 4.9
5.9 ± 5.3
7.9 ± 6.8
12.9 ± 11.0
12.2 ± 10.4
75 cm soil water
5.8 ± 5.1
7.2 ± 6.3
8.4 ± 7.3
11.2 ± 9.5
17.6 ± 14.8
16.7 ± 14.0
100 cm soil
water
4.4 ± 4.0
5.5 ± 4.9
9.8 ± 8.4
13.0 ± 11.0
13.3 ± 11.2
12.5 ± 10.7
150 cm soil
water
4.5 ± 4.1
5.7 ± 5.0
13.9 ± 11.8
18.3 ± 15.4
10.1 ± 8.7
9.6 ± 8.2
Table 5. Contribution of different depths of soil water and rainwater to the water source of Artemisia ordosica (Ao)
and Hedysarum fruticosum var. laeve (Hf) in sandy land (%, mean ± SD). Water source
May 13
Jul. 17
Sep. Figure 4. 22
Sp
Hf
Sp
Hf
Sp
Hf
10 cm soil water
28.6 ± 18.5
19.5 ± 12.1
15.2 ± 12.8
18.0 ± 14.6
26.4 ± 3.2
22.7 ± 3.4
25 cm soil water
30.4 ± 16.0
18.4 ± 10.5
13.8 ± 11.7
16.6 ± 13.3
16.9 ± 14.2
17.8 ± 14.9
50 cm soil water
10.8 ± 8.8
16.2 ± 13.0
16.9 ± 5.1
8.2 ± 4.3
15.1 ± 12.7
15.8 ± 13.3
100 cm soil water
10.5 ± 8.5
15.8 ± 12.7
20.9 ± 17.4
18.6 ± 14.1
15.4 ± 13.0
16.2 ± 13.6
150 cm soil water
9.6 ± 7.8
14.7 ± 11.7
16.7 ± 14.0
19.4 ± 16.0
13.7 ± 11.6
14.3 ± 12.1
200 cm soil water
10.1 ± 8.3
15.4 ± 12.3
16.5 ± 13.8
19.2 ± 15.8
12.6 ± 10.7
13.2 ± 11.2
Table 6. Contribution of different depths of soil water and rainwater to the water source of and Salix psammophila
(Sp) and Hedysarum fruticosum var. laeve (Hf) in sandy land (%, mean ± SD). Table 5. Table 5. Contribution of different depths of soil water and rainwater to the water source of Artemisia ordosica (Ao)
and Hedysarum fruticosum var. laeve (Hf) in sandy land (%, mean ± SD). Water source
May 13
Jul. 17
Sep. 22
Sp
Hf
Sp
Hf
Sp
Hf
10 cm soil water
28.6 ± 18.5
19.5 ± 12.1
15.2 ± 12.8
18.0 ± 14.6
26.4 ± 3.2
22.7 ± 3.4
25 cm soil water
30.4 ± 16.0
18.4 ± 10.5
13.8 ± 11.7
16.6 ± 13.3
16.9 ± 14.2
17.8 ± 14.9
50 cm soil water
10.8 ± 8.8
16.2 ± 13.0
16.9 ± 5.1
8.2 ± 4.3
15.1 ± 12.7
15.8 ± 13.3
100 cm soil water
10.5 ± 8.5
15.8 ± 12.7
20.9 ± 17.4
18.6 ± 14.1
15.4 ± 13.0
16.2 ± 13.6
150 cm soil water
9.6 ± 7.8
14.7 ± 11.7
16.7 ± 14.0
19.4 ± 16.0
13.7 ± 11.6
14.3 ± 12.1
200 cm soil water
10.1 ± 8.3
15.4 ± 12.3
16.5 ± 13.8
19.2 ± 15.8
12.6 ± 10.7
13.2 ± 11.2
Table 6. Contribution of different depths of soil water and rainwater to the water source of and Salix psammophila
(Sp) and Hedysarum fruticosum var. laeve (Hf) in sandy land (%, mean ± SD). Table 6. Table 6. Contribution of different depths of soil water and rainwater to the water source of and Salix psammophila
(Sp) and Hedysarum fruticosum var. laeve (Hf) in sandy land (%, mean ± SD). Table 6. Contribution of different depths of soil water and rainwater to the water source of and Salix psammophila
(Sp) and Hedysarum fruticosum var. laeve (Hf) in sandy land (%, mean ± SD). used both of 10–25 cm shallow and 100–200 cm deep soil water in summer and
autumn. Artemisia ordosica and the accompany plant H. fruticosum var. laeve always
used the same water source, e.g. 10 cm surface soil water in spring, 10 cm surface
soil water and 150 cm deep soil water in summer, and 10–150 cm soil water in
autumn. Salix psammophila mainly used 10–25 cm shallow soil water whereas
H. fruticosum var. leave mainly used 50–200 cm soil water in spring. However, they
both mainly used 10–25 cm shallow soil water and 100–200 cm deep soil water in
summer, and 25–200 cm soil water in autumn. Soil water recharged by rainwater 10 Water Source of Six Woody Plants in Different Habitats on Desertified Land of Ordos Plateau…
DOI: http://dx.doi.org/10.5772/intechopen.99658 Water Source of Six Woody Plants in Different Habitats on Desertified Land of Ordos Plateau…
DOI: http://dx.doi.org/10.5772/intechopen.99658 Water Source of Six Woody Plants in Different Habitats on Desertified Land of Ordos Plateau…
DOI: http://dx.doi.org/10.5772/intechopen.99658 is the main water source for vegetation in sandy land since shallow groundwater
was depleted by coal mining. However, in the habitat with shallow groundwater
in the sandy land, woody species still could use groundwater. For example, Salix
matsudana and S. vulgaris mainly used deep soil water and groundwater, whereas
A. ordosica mainly used groundwater in Mu Us Sandy Land. The groundwater
table were 1.0 m for S. matsudana in interdune, 1.5 m and 1.3 m for S. vulgaris and
A. ordosica on sand dune, respectively [5]. p
y
The water source of the company shrub H. fruticosum var. leave were similar
to the dominant shrub A. ordosica or S. psammophila, which indicated water com-
petition between them. Water source of shrubs was closely related to their root
type in sandy land, especially fine root. The root length of H. fruticosum var. laeve
was 80 cm, with the biomass concentrated within 40 cm [31]. The distribution
of its root system is partly overlap with two dominant shrubs. Acknowledgements The author thanks Ordos Sandy Land Ecological Station for supporting meteo-
rological data. This study was supported by National Key Research & Development Program
of China (2017YFC05040450202) from Ministry of Science and Technology of the
People’s Republic of China. Table 6. The root depth of
A. ordosica was 200 cm, with the fine root concentrated within 40 cm [32]. The
root system of S. psammophila was as deep as 150 cm, with fine root concentrated
within 50 cm [33]. Similar phenomenon occurred in other sandy land vegetation. For example, Salix gordejevii mainly used soil water, whereas Artemisia halodendron
mainly used 10–150 cm soil water in Horqin Sandy Land [11]. Their root biomass
concentrated within 40 cm in the mixed community [34]. Therefore, the distribu-
tion of deep-rooted species should be arranged appropriately to avoid excessive
water competition in the restoration of degraded vegetation in sandy land. It was
suggested to keep reasonable afforestation density in the ecological engineering in
Mu Us Sandy Land and other area of semi-arid region. 4. Conclusion Soil water recharged by rainwater is the main water source for dominant species
on Ordos Plateau. Six woody species have resource-dependent water use strategy,
which is an adaptive advantage to the semi-arid climate. On loess slope of eastern
Ordos Plateau, H. rhmnoides, P. tabuliformis and A. sibirica selected different water
source based on their availability in the growing season, including shallow soil
water, deep soil water and rainwater. In Sandy Land of southern Ordos Plateau, S. vulgaris, A. ordosica, S. psammophila and the company semi-shrub H. fruticosum var. laeve used different depths of soil water based on their availability in the growing
season. However, there were water competition between the company semi-shrub
and two dominant shrubs. Therefore, it was suggested to avoid vegetation degrada-
tion resulted from excessive water competition by appropriate distribution and
afforestation density in semi-arid region. Conflict of interest The author declares no conflict of interest. 11 Deserts and Desertification
Author details
Yajuan Zhu
Institute of Desertification Studies, Chinese Academy of Forestry, Beijing, China
*Address all correspondence to: zhuyj@caf.ac.cn
© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms
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DOI: http://dx.doi.org/10.5772/intechopen.99658 Water Source of Six Woody Plants in Different Habitats on Desertified Land of Ordos Plateau…
DOI: http://dx.doi.org/10.5772/intechopen.99658 Water Source of Six Woody Plants in Different Habitats on Desertified Land of Ordos Plateau…
DOI: http://dx.doi.org/10.5772/intechopen.99658 [14] Wang R, Yan F, Wang Y. Vegetation
growth status and topographic effects in
the Pisha Sandstone Area of China.
Remote Sensing. 2020;12. DOI: 10.3390/
rs12172759 References Land Research. 2017;34(2):350-355. DOI:10.13866/j.azr.2017.02.15 [15] Bai Z, Cui J, Ding X. Desertification
and its driving factors in the Ordos
Plateau, from 1986 to 2015. Arid Zone
Research. 2020;37(3):749-756. DOI:
10.13866/j.azr.2020.03.24 [5] Ohte N, Koba K, Yoshikawa K,
Sugimoto A, Matsuo N, Kabeya N,
Wang L H. Water utilization of natural
and planted trees in the semiarid desert
of Inner Mongolia, China. Ecological
Applications. 2003;13(2):337-351. [4] Yang H, Auerswald K, Bai Y F, Han X
G. Complementarity in water sources
among dominant species in typical
steppe ecosystems of Inner Mongolia,
China. Plant and Soil. 2011;340
(1/2):303-313. DOI: 10.1007/
S11104-010-0307-4 Land Research. 2017;34(2):350-355.
DOI:10.13866/j.azr.2017.02.15 Land Research. 2017;34(2):350-355. DOI:10.13866/j.azr.2017.02.15 [1] Zhang X S. Principles and optimal
models for development of Maowusu
Sandy Grassland. Acta Phytoecologica
Sinica. 1994;18(1):1-16. [1] Zhang X S. Principles and optimal
models for development of Maowusu
Sandy Grassland. Acta Phytoecologica
Sinica. 1994;18(1):1-16. [9] Zhu Y J, Wang G J, Li R Q . Seasonal
dynamics of water use strategy of two
Salix shrubs in alpine sandy land,
Tibetan Plateau. PLoS One. 2016;11(5):e0156586. DOI:10.1371/
journal.pone.0156586 [9] Zhu Y J, Wang G J, Li R Q . Seasonal
dynamics of water use strategy of two
Salix shrubs in alpine sandy land,
Tibetan Plateau. PLoS One. 2016;11(5):e0156586. DOI:10.1371/
journal.pone.0156586 [2] Lin G. Stable Isotope Ecology. Beijing: Higher Education Press;
2013. 492p. [3] Cheng X L, An S Q , Li B, Chen J Q ,
Lin G H, Liu Y H, Luo Y Q , Liu S R. Summer rain pulse size and rainwater
uptake by three dominant desert plants
in a desertified grassland ecosystem in
northwestern China. Plant Ecology. 2006;184(1):1-12. DOI: 10.1007/
s11258-005-9047-6 [10] Zhu Y, Qi K, Pang Z. Water source
of Salix cheilophila plantation in alpine
sandy land in summer. Journal of
Nanjing Forestry University (Natural
Sciences Edition). 2019;43(1):91-97. DOI: 10.3969/j.issn.1000-2006. 201806036 [4] Yang H, Auerswald K, Bai Y F, Han X
G. Complementarity in water sources
among dominant species in typical
steppe ecosystems of Inner Mongolia,
China. Plant and Soil. 2011;340
(1/2):303-313. DOI: 10.1007/
S11104-010-0307-4 [11] Liu B, Liu Z, Qian J, Alamusa,
Zhang F, Peng X. Water sources of
dominant sand-binding plants in dry
season in southern Horqin Sandy Land,
China. Chinese Journal of Applied
Ecology. 2017;28(7):2093-2101. DOI:
10.13287/j.1001-9332.201707.030 [5] Ohte N, Koba K, Yoshikawa K,
Sugimoto A, Matsuo N, Kabeya N,
Wang L H. Water utilization of natural
and planted trees in the semiarid desert
of Inner Mongolia, China. Ecological
Applications. 2003;13(2):337-351. [12] Song L, Zhu J, Li M, Yu Z. Water
utilization of Pinus sylvestris var. mongolica in a sparse wood grassland in
the semiarid sandy region of Northeast
China. Trees. 2014;28(4):971-982. DOI:
10.1007/s00468-014-1010-5 [12] Song L, Zhu J, Li M, Yu Z. Water
utilization of Pinus sylvestris var. mongolica in a sparse wood grassland in
the semiarid sandy region of Northeast
China. Trees. 2014;28(4):971-982. DOI:
10.1007/s00468-014-1010-5 [13] Chen X, Chen Z, Zhao Y. The
determination of ecotone and the
characteristics of biome on Ordos
Plateau. Acta Phytoecologica Sinica. 1998;22(4):312-318. [13] Chen X, Chen Z, Zhao Y. [31] Zhang L, Wang X, Hong G, Wu Y,
Li Z, Hai L, Wang P, Gao X, Yang J.
Root distribution characteristics of
Hedysarum laeve with different aerial [19] Wang J, Liu L, Jia K, Tian L.
Spatiotemporal variation of vegetation
phenology and its affecting factors in
the Mu Us Sandy Land. Journal of
Desert Research. 2015;35(3):624-631.
DOI: 10.7522/j.issn.1000-694X.2015.
00021 Land Research. 2017;34(2):350-355.
DOI:10.13866/j.azr.2017.02.15 00021 [27] Wang J, Fu B, Jiao L, Lu N, Li J,
Chen W, Wang L. Age-related water use
characteristics of Robinia pseudoacacia
on the Loess Plateau. Agricultural and
Forest Meteorology. 2021;301-
302:108344. DOI: 10.1016/j.agrformet. 2021.108344 [27] Wang J, Fu B, Jiao L, Lu N, Li J,
Chen W, Wang L. Age-related water use
characteristics of Robinia pseudoacacia
on the Loess Plateau. Agricultural and
Forest Meteorology. 2021;301-
302:108344. DOI: 10.1016/j.agrformet. 2021.108344 [20] Liu Z, Zhang J, Zhang E. Model of
returning crop plots to forestry and
grassland in hilly and gully area of
Jungar Banner, Inner Mongolia. Journal
of Desert Research. 2002;22(5):506-509. [20] Liu Z, Zhang J, Zhang E. Model of
returning crop plots to forestry and
grassland in hilly and gully area of
Jungar Banner, Inner Mongolia. Journal
of Desert Research. 2002;22(5):506-509. [28] Ye X H, Liu Z L, Zhang S D, Gao S
Q , Liu G F, Cui Q G, Du J, Huang Z Y,
Cornelissen J H C. Experimental sand
burial and precipitation enhancement
alter plant and soil carbon allocation in a
semi-arid steppe in north China. Science of the total Environment. 2019;651:3099-3106. DOI: 10.1016/j. scitotenv.2018.10.208 [28] Ye X H, Liu Z L, Zhang S D, Gao S
Q , Liu G F, Cui Q G, Du J, Huang Z Y,
Cornelissen J H C. Experimental sand
burial and precipitation enhancement
alter plant and soil carbon allocation in a
semi-arid steppe in north China. Science of the total Environment. 2019;651:3099-3106. DOI: 10.1016/j. scitotenv.2018.10.208 [21] Fang W, Chang C, editors. Flora
Reipublicae Popularis Sinicae, Tomus 52
(2): Dicotyledoneae, Elaeagnaceae,
Lecythidaceae, Lythraceae,
Rhizophoraceae, Sonneratiaceae,
Nyssaceae, Crypteroniaceae,
Alangiaceae, Punicaceae. Beijing:
Science Press; 1978. 542p. [21] Fang W, Chang C, editors. Flora
Reipublicae Popularis Sinicae, Tomus 52
(2): Dicotyledoneae, Elaeagnaceae,
Lecythidaceae, Lythraceae,
Rhizophoraceae, Sonneratiaceae,
Nyssaceae, Crypteroniaceae,
Alangiaceae, Punicaceae. Beijing:
Science Press; 1978. 542p. [29] Ling Y, Ling Y, editors. Flora
Reipublicae Popularis Sinicae, Tomus
76(2): Dicotyledoneae, Compositae (3),
Anthemideae (2). Beijing: Science Press;
1991. 333p. [29] Ling Y, Ling Y, editors. Flora
Reipublicae Popularis Sinicae, Tomus
76(2): Dicotyledoneae, Compositae (3),
Anthemideae (2). Beijing: Science Press;
1991. 333p. [22] Cheng W, Fu L, editors. Flora
Reipublicae Popularis Sinicae, Tomus 7:
Gymnospermae. Beijing: Science Press;
1978. 542p. [30] Wang C, Fang C, editors. Flora
Reipublicae Popularis Sinicae, Tomus
20(2): Angiospermae, Dicotyledoneae,
Salicaceae. Beijing: Science Press;
1984. 423p. [23] Yu T, editor. Flora Reipublicae
Popularis Sinicae, Tomus 38:
Dicotyledoneae, Rosaceae (3),
Prunoideae, Connaraceae. [31] Zhang L, Wang X, Hong G, Wu Y,
Li Z, Hai L, Wang P, Gao X, Yang J.
Root distribution characteristics of
Hedysarum laeve with different aerial Land Research. 2017;34(2):350-355.
DOI:10.13866/j.azr.2017.02.15 The
determination of ecotone and the
characteristics of biome on Ordos
Plateau. Acta Phytoecologica Sinica. 1998;22(4):312-318. [6] Wei Y F, Fang J, Liu S, Zhao X Y, Li S
G. Stable isotopic observation of water
use sources of Pinus sylvestris var. mongolica in Horqin Sandy Land, China. Trees. 2013;27(5):1249-1260. DOI:
10.1007/s00468-013-0873-1 [7] Su H, Li Y G, Liu W, Xu H, Sun O J. Changes in water use with growth in
Ulmus pumila in semiarid sandy land of
northern China. Trees. 2014;28(1):41-
52. DOI: 10.1007/s00468-013-0928-3 [15] Bai Z, Cui J, Ding X. Desertification
and its driving factors in the Ordos
Plateau, from 1986 to 2015. Arid Zone
Research. 2020;37(3):749-756. DOI:
10.13866/j.azr.2020.03.24 [8] Jian J, Jia D, Guo S, Qian L. Water
sources in growing season of Salix
gordejevii in the Otindag Sandy Land
traced by stable D isotope in 2014. Arid 13 Deserts and Desertification coping with too many sources. Oecologia. 2003;136(2):261-269. coping with too many sources. Oecologia. 2003;136(2):261-269. [16] Yao W, Wu Z, Liu H, Xiao P, Yang C. Experimental research on the anti-
erosion and vegetation promotion for
sandstone region in the Yellow River
Basin. Yellow River. 2015;37(1):6-10. [25] Yang G, Wang A, Wang L. Water
source and water use efficiency of two
typical shrubs in different seasons in
Liudaogou Watershed. Acta Botanica
Boreali-Occidentalia Sinica. 2018;38(1):140-149. DOI: 10.7606/j. issn.1000-4025.2018.01.0140 [25] Yang G, Wang A, Wang L. Water
source and water use efficiency of two
typical shrubs in different seasons in
Liudaogou Watershed. Acta Botanica
Boreali-Occidentalia Sinica. 2018;38(1):140-149. DOI: 10.7606/j. issn.1000-4025.2018.01.0140 [17] Cao Y, Pang Y, Jia X. Vegetation
growth in Mu Us sandy land from 2001
to 2016. Bulletin of Soil and Water
Conservation. 2019;39(2):29-37. [18] Chen Y, Dong M. Quantitative
analysis of landscape conditions of the
desertified sandy grassland in Ordos
Plateau. Environmental Science. 2002;23(1):87-91. [18] Chen Y, Dong M. Quantitative
analysis of landscape conditions of the
desertified sandy grassland in Ordos
Plateau. Environmental Science. 2002;23(1):87-91. [26] Wang J, Fu B, Wang L, Lu N, Li J. Water use characteristics of the
common tree species in different
plantation types in the Loess Plateau of
China. Agricultural and Forest
Meteorology. 2020;288-289:108020. DOI: 10.1016/j.agrformet.2020.108020 [19] Wang J, Liu L, Jia K, Tian L. Spatiotemporal variation of vegetation
phenology and its affecting factors in
the Mu Us Sandy Land. Journal of
Desert Research. 2015;35(3):624-631. DOI: 10.7522/j.issn.1000-694X.2015. [27] Wang J, Fu B, Jiao L, Lu N, Li J,
Chen W, Wang L. Age-related water use
characteristics of Robinia pseudoacacia
on the Loess Plateau. Agricultural and
Forest Meteorology. 2021;301-
302:108344. DOI: 10.1016/j.agrformet.
2021.108344 Land Research. 2017;34(2):350-355.
DOI:10.13866/j.azr.2017.02.15 Beijing:
Science Press; 1986. 167p. [23] Yu T, editor. Flora Reipublicae
Popularis Sinicae, Tomus 38:
Dicotyledoneae, Rosaceae (3),
Prunoideae, Connaraceae. Beijing:
Science Press; 1986. 167p. [24] Phillips D L, Gregg J W. Source
partitioning using stable isotopes: [24] Phillips D L, Gregg J W. Source
partitioning using stable isotopes: 14 Water Source of Six Woody Plants in Different Habitats on Desertified Land of Ordos Plateau…
DOI: http://dx.doi.org/10.5772/intechopen.99658 Water Source of Six Woody Plants in Different Habitats on Desertified Land of Ordos Plateau…
DOI: http://dx.doi.org/10.5772/intechopen.99658 seeding years in Mu Us Sandy Land. Chinese Journal of Ecology. 2017;36(1):29-34. DOI:10.1329
2/j.1000-4890.201701.001 seeding years in Mu Us Sandy Land.
Chinese Journal of Ecology.
2017;36(1):29-34. DOI:10.1329
2/j.1000-4890.201701.001 [32] Zhang J, Han H, Lei Y, Yang W, Li Y,
Yang D, Zhao X. Correlations between
distribution characteristics of Artemisia
ordosica root system and soil moisture
under different fixation stage of sand
dune. Journal of Southwest Forestry
University. 2012;32(6):1-5. DOI:10.3969/j.issn.2095-1914.2012. 06.001 [33] Liu J, He X, Bao H, Zhou C. Distribution of fine roots of Salix
psammophila and its relationship with
soil moisture in Mu Us Sandland. Journal of Desert Research. 2010;30(6):1362-1366. [33] Liu J, He X, Bao H, Zhou C. Distribution of fine roots of Salix
psammophila and its relationship with
soil moisture in Mu Us Sandland. Journal of Desert Research. 2010;30(6):1362-1366. [34] Huang G, Zhao X, Zhao Y, Su Y. Root distribution of two typical shrubs
in single or mixture circumstance in
Horqin Sandy Land. Journal of Desert
Research. 2007;27(2):239-243. 15
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EEG-Based Brain-Computer Interfaces Using Motor-Imagery: Techniques and Challenges
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PADFIELD, N., ZABALZA, J., ZHAO, H., MASERO, V. and REN, J. 2019. EEG-based brain-computer interfaces using
motor-imagery: techniques and challenges. Sensors [online], 19(6), article 1423. Available from:
https://doi.org/10.3390/s19061423 PADFIELD, N., ZABALZA, J., ZHAO, H., MASERO, V. and REN, J. 2019. EEG-based brain-computer interfaces using
motor-imagery: techniques and challenges. Sensors [online], 19(6), article 1423. Available from:
https://doi.org/10.3390/s19061423 Received: 30 January 2019; Accepted: 19 March 2019; Published: 22 March 2019 Abstract: Electroencephalography (EEG)-based brain-computer interfaces (BCIs), particularly those
using motor-imagery (MI) data, have the potential to become groundbreaking technologies in both
clinical and entertainment settings. MI data is generated when a subject imagines the movement
of a limb. This paper reviews state-of-the-art signal processing techniques for MI EEG-based BCIs,
with a particular focus on the feature extraction, feature selection and classification techniques used. It also summarizes the main applications of EEG-based BCIs, particularly those based on MI data,
and finally presents a detailed discussion of the most prevalent challenges impeding the development
and commercialization of EEG-based BCIs. Keywords: brain-computer interface (BCI); electroencephalography (EEG); motor-imagery (MI)
Sensors 2019, 19, 1423; doi:10.3390/s19061423 PADFIELD, N., ZABALZA, J., ZHAO, H., MASERO, V. and REN, J. 2019. EEG-based brain-computer interfaces using
motor-imagery: techniques and challenges. Sensors [online], 19(6), article 1423. Available from:
https://doi.org/10.3390/s19061423
© 2019 by the authors. Licensee MDPI, Basel, Switzerland.
EEG-based brain-computer interfaces using
motor-imagery: techniques and challenges.
PADFIELD, N., ZABALZA, J., ZHAO, H., MASERO, V. and REN, J.
2019 PADFIELD, N., ZABALZA, J., ZHAO, H., MASERO, V. and REN, J. 2019. EEG-based brain-computer interfaces using
motor-imagery: techniques and challenges. Sensors [online], 19(6), article 1423. Available from:
https://doi.org/10.3390/s19061423 2019 © 2019 by the authors. Licensee MDPI, Basel, Switzerland. © 2019 by the authors. Licensee MDPI, Basel, Switzerland. © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This document was downloaded from
https://openair.rgu.ac.uk sensors sensors Review EEG-Based Brain-Computer Interfaces Using
Motor-Imagery: Techniques and Challenges p
(
); j
(J
)
2
School of Computer Sciences, Guangdong Polytechnic Normal University, Guangzhou 510665, China
3
The Guangzhou Key Laboratory of Digital Content Processing and Security Technologies,
Guangzhou 510665, China p
(
) j
(J
)
2
School of Computer Sciences, Guangdong Polytechnic Normal University, Guangzhou 510665, China
3
The Guangzhou Key Laboratory of Digital Content Processing and Security Technologies,
Guangzhou 510665, China 2
School of Computer Sciences, Guangdong Polytechnic Normal University, Guangzhou 510665, China
3
The Guangzhou Key Laboratory of Digital Content Processing and Security Technologies,
Guangzhou 510665, China 4
Department of Computer Systems and Telematics Engineering, Universidad de Extremadura,
06007 Badajoz, Spain; vmasero@unex.es 4
Department of Computer Systems and Telematics Engineering, Universidad de Extremadura,
06007 Badajoz, Spain; vmasero@unex.es
5 5
School of Electrical and Power Engineering, Taiyuan University of Technology, Taiyuan 030024, Ch School of Electrical and Power Engineering, Taiyuan University of Technology, Taiyuan 030024, China
Correspondence: zhaohuimin@gpnu edu cn (H Z ); jinchang ren@strath ac uk (J R ) School of Electrical and Power Engineering, Taiyuan University of Technology, Taiyuan 030024, China
Correspondence: zhaohuimin@gpnu.edu.cn (H.Z.); jinchang.ren@strath.ac.uk (J.R.) *
Correspondence: zhaohuimin@gpnu.edu.cn (H.Z.); jinchang.ren@strath.ac.uk (J.R.) *
Correspondence: zhaohuimin@gpnu.edu.cn (H.Z.); jinchang.ren@strath.ac.uk (J.R.) 1. Introduction Since the inception of personal computing in the 1970s, engineers have continuously tried to narrow
the communication gap between humans and computer technology. This process began with the
development of graphical user interfaces (GUIs), computers and mice [1], and has led to ever more
intuitive technologies, particularly with the emergence of computational intelligence. Today, the ultimate
frontier between humans and computers is being bridged through the use of brain-computer interfaces
(BCIs), which enable computers to be intentionally controlled via the monitoring of brain signal activity. Electroencephalography (EEG) equipment is widely used to record brain signals in BCI systems
because it is non-invasive, has high time resolution, potential for mobility in the user and a relatively
low cost [2]. Although a BCI can be designed to use EEG signals in a wide variety of ways for control,
motor imagery (MI) BCIs, in which users imagine movements occurring in their limbs in order to
control the system, have been subject to extensive research [3–7]. This interest is due to their wide
potential for applicability in fields such as neurorehabilitation, neuroprosthetics and gaming, where
the decoding of users’ thoughts of an imagined movement would be invaluable [2,8]. The aim of this paper is to review a wide selection of signal processing techniques used in
MI-based EEG systems, with a particular focus on the state-of-the art regarding feature extraction and
feature selection in such systems. It also discusses some of the challenges and limitations encountered
during the design and implementation of related signal processing techniques. Furthermore, the paper
also summarizes the main applications of EEG-based BCIs and challenges currently faced in the
development and commercialization of such BCI systems. www.mdpi.com/journal/sensors www.mdpi.com/journal/sensors Sensors 2019, 19, 1423 2 of 34 The rest of this paper is organized as follows. Section 2 provides an overview of the fundamental
concepts underlying EEG-based BCIs. Section 3 then introduces the main features of MI EEG-based
BCIs, and Section 4 goes on to discuss in detail different feature extraction, feature selection and
classification techniques utilized in the literature. A case study is presented in Section 5 for evaluating
different time-/frequency-domain approaches in controlling motor movement. Section 6 summarizes
the different applications for EEG-based BCIs, particularly those based on MI EEG. Finally, Section 7
discusses the main challenges hindering the development and commercialization of EEG-based BCIs. 2. Overview of EEG-Based BCIs This section introduces some of the core aspects of EEG-based BCIs. It explains why EEG
is a popular technology for BCIs, along with the generic challenges associated with EEG signal
processing. It then introduces the two main classes of EEG-based BCIs, highlighting factors that
should be considered when choosing between the two approaches and the challenges inherent to each. Finally, the basic signal characteristics of MI EEG data are summarized, with a brief discussion of the
signal processing problems this data can present. EEG signals are typically used for BCIs due to their high time resolution and the relative
ease and cost-effectiveness of brain signal acquisition when compared to other methods such as
functional magnetic resonance imaging (fMRI) and magnetoencephalography (MEG) [2]. They are
also more portable than fMRI or MEG technologies [9]. However, EEG signals pose processing
challenges; since they are non-stationary, they can suffer from external noise and are prone to signal
artefacts [2,10]. Furthermore, EEG signals can be affected by the posture and mood of a subject [11]. For example, an upright posture tended to improve the focus and EEG quality during recording [12],
and high-frequency content was stronger when users were in an upright position, as opposed to lying
down [13]. It was also noted in [12] that postural changes could be used to increase attention in subjects
who felt tired. EEG-based BCIs can be classified into two types: evoked and spontaneous [11], though some
works also refer to them as exogenous and endogenous, respectively [2]. In evoked systems, external
stimulation, such as visual, auditory or sensory stimulation, is required. The stimuli evoke responses
in the brain that are then identified by the BCI system in order to determine the will of the user [14]. In spontaneous BCIs, no external stimulation is required, and control actions are taken based on activity
produced as a result of mental activity [2,11]. Table 1 contains examples of typical EEG systems, with
details on the application, functionality, and number of subjects involved in testing, where the mean
accuracy and information transfer rate were also provided. Table 1. A table containing examples of evoked and spontaneous BCIs. 2. Overview of EEG-Based BCIs Sensors 2019, 19, 1423 3 of 34 Typical evoked EEG systems can be separated into two main categories: those dependent
on visually evoked potentials (VEPs), brain signals generated in response to a visual stimulus,
and event-related potentials (ERPs), brain signals generated in response to sensory or cognitive
events [9,14]. Steady-state visually evoked potentials (SSVEPs) are one of the most widely researched
areas of VEP-based BCIs because they enable relatively accurate and rapid command input [14] whilst
requiring little user training [9]. In such a system, the different options available to a user are displayed
as stimuli flickering at unique frequencies, and the user selects an option by focusing on the associated
stimulus. The performance of such systems is dependent on the number of stimuli [19], the modulation
schemes used, and the hardware used for the stimuli [2,19]. Stawicki et al. [10] conducted a survey of
32 subjects on the usability of an SSVEP-based system, where 66% thought that the system required a
lot of concentration to use, 52% thought that the stimuli were annoying, and only 48% considered that
the system was easy to use. When it comes to ERP systems, those based on P300 waves are landmark technologies [2,9]. P300 waves are distinct EEG events related to the categorization or assessment of an external
stimulus [20]. However, in such systems, the results need be integrated over several stimuli, which
adds to the computational time taken to make decisions, and restricts the maximum throughput of
the system [2]. Possible solutions would be to increase the signal-to-noise-ratio (SNR) [21] or find an
optimum number of stimuli [22]. A common example of a spontaneous BCI is a MI BCI, which requires the user to imagine the
movement of a limb. Such BCIs monitor sensorimotor rhythms (SMRs), which are oscillatory events
in EEG signals originating from brain areas associated with preparation, control and carrying out of
voluntary motion [9,23]. Brain activity recorded via EEG is typically classified into five different types,
depending on the predominant frequency content, f, of the signal, summarized as follows: (i) delta
activity: f < 4 Hz; (ii) theta activity: 4 Hz < f < 7 Hz; (iii) alpha activity: 8 Hz < f < 12 Hz; (iv) beta
activity: 12 < f < 30 Hz; and (v) gamma activity: f > 30 Hz. In the literature, alpha activity recorded
from the sensorimotor region is known as mu activity. 2. Overview of EEG-Based BCIs Type
Class
Example/Application
Display & Function
No of
Subjects
Mean
Accuracy
ITR 1
Evoked
VEP
SSVEP/Speller [10]
Look at one of 30 flickering target stimuli
associated with desired character
32
90.81%
35.78 bpm
ERP
P300/Speller [15]
Focus on the desired letter until it next flashes
15
69.28%
20.91 bpm
Auditory/Speller [16]
Spatial auditory cues were used to aid the use
of an on-screen speller
21
86.1%
5.26 bpm/0.94 char/min
Spontaneous
N/A
Blinks/Virtual keyboard [17]
Choose from 29 characters using eye blinks to
navigate/select
14
N/A
1 char/min
Motor imagery
(MI)/Exoskeleton control [18]
Control an exoskeleton of the upper limbs
using right and left hand MI
4
84.29%
N/A
1 ITR—information transfer rate. The decision between using an evoked or spontaneous system is not always clear, and may
require the consideration of the strengths and weaknesses of each approach. Specifically, while evoked
systems typically have a higher throughput, require less training and sensors, and can be mastered by
a larger number of users when compared to spontaneous systems, they require the user’s gaze to be
fixed on the stimuli and this constant concentration can be exhausting [2,9,10]. Table 1. A table containing examples of evoked and spontaneous BCIs. Type
Class
Example/Application
Display & Function
No of
Subjects
Mean
Accuracy
ITR 1
Evoked
VEP
SSVEP/Speller [10]
Look at one of 30 flickering target stimuli
associated with desired character
32
90.81%
35.78 bpm
ERP
P300/Speller [15]
Focus on the desired letter until it next flashes
15
69.28%
20.91 bpm
Auditory/Speller [16]
Spatial auditory cues were used to aid the use
of an on-screen speller
21
86.1%
5.26 bpm/0.94 char/min
Spontaneous
N/A
Blinks/Virtual keyboard [17]
Choose from 29 characters using eye blinks to
navigate/select
14
N/A
1 char/min
Motor imagery
(MI)/Exoskeleton control [18]
Control an exoskeleton of the upper limbs
using right and left hand MI
4
84.29%
N/A
1 ITR—information transfer rate. Table 1. A table containing examples of evoked and spontaneous BCIs. The decision between using an evoked or spontaneous system is not always clear, and may
require the consideration of the strengths and weaknesses of each approach. Specifically, while evoked
systems typically have a higher throughput, require less training and sensors, and can be mastered by
a larger number of users when compared to spontaneous systems, they require the user’s gaze to be
fixed on the stimuli and this constant concentration can be exhausting [2,9,10]. 2. Overview of EEG-Based BCIs Changes in mu and beta activity within EEG
signals are used to identify the type of motor imagery task being carried out [9,23]. Gamma activity is
reliably used in MI BCIs which use internal electrodes, since gamma signals do not reach the scalp
with high enough integrity to be used for MI task identification when recorded using scalp EEG. When activity in a particular band increases, this is called event-related synchronization (ERS), while a
decrease in a particular band is called event-related desynchronization (ERD) [23]. ERSs and ERDs can
be triggered by motor imagery, motor activity and stimulation of the senses [24,25]. Common classes of
movements for MI EEG systems include: left hand movement, right hand movement, movement of the
feet and movement of the tongue [26–28], since these events have been shown to produce significant
and discriminative changes in the EEG signals relative to background EEG [23]. Movement of the
feet is often classed as a single class, with no distinction between the left and right foot movement
because, as Graimann and Pfurtscheller comment [23], it is impossible to distinguish between left
and right foot motor imagery, or between the movements of particular fingers because the cortical
areas associated with these distinct movements are too small to generate discriminative ERD and ERS
signals. However, Hashimoto and Ushiba illustrated that there is potential for beta activity to be used
to discriminate between the left and right MI [29]. The performance of SMR-BCIs is heavily dependent on the neurophysiological and psychological
state of the user, with the control of SMR activity being found to be challenging for many users [9,23]. Furthermore, there is a general lack of understanding of the relationship between good and poor
performance within BCIs in general, and the neuroanatomic state of a user [30] and BCI performance
could have a significant impact on SMR-based BCIs due to their heavy reliance on users successfully
learning to consciously generate the required signals. More research is required to understand how
these neurological factors affect performance of SMR-BCIs, and how they could possibly be exploited
to improve performance [30]. 4 of 34
could Sensors 2019, 19, 1423
understand how t Due to the complex nature of EEG signals, and the strong relationship of signal quality to the
mental state of the user, recording EEG data for testing and ensuring that datasets are ‘valid’ is a
significant challenge. 2. Overview of EEG-Based BCIs This is particularly true for MI EEG data, which requires significant focus by the
user to generate. In An et al. [31], the performance of a classifier for MI data was analyzed, in which
participants carried out MI tasks for an interval of four seconds. They found that during the first two
seconds of a MI task, the classification accuracy for a given a particular processing system was at its
peak, but for the final two seconds, the classification accuracy decreased. They believed that this was
possibly due to subjects losing concentration on the task, resulting in poor-quality EEG data and poor
classification results. This highlights two issues: firstly, the validity of MI EEG data may be closely
linked with the duration of a task, and thus it would be beneficial to test detectors with data from a
complete MI task. The segments of that data should be taken from the beginning and end of the task in
order to observe how performance varies. Secondly, future research could investigate how the quality
of the EEG data changes during an MI EEG task longer than two seconds, and which kinds of signal
processing approaches cope best with longer MI tasks. Zich et al. [32] used fMRI in order to validate
MI EEG data, and suggest that fMRI can be used in conjunction with EEG technologies to investigate
inter-individual differences in MI data generation. This literature review now focuses on EEG-based
MI BCIs in greater depth. Due to the complex nature of EEG signals, and the strong relationship of signal quality to the
mental state of the user, recording EEG data for testing and ensuring that datasets are ‘valid’ is a
significant challenge. This is particularly true for MI EEG data, which requires significant focus by
the user to generate. In An et al. [31], the performance of a classifier for MI data was analyzed, in
which participants carried out MI tasks for an interval of four seconds. They found that during the
first two seconds of a MI task, the classification accuracy for a given a particular processing system
was at its peak, but for the final two seconds, the classification accuracy decreased. They believed
that this was possibly due to subjects losing concentration on the task, resulting in poor-quality EEG
data and poor classification results. 3. Introduction to MI EEG-Based BCIs
3. Introduction to MI EEG-Based BCIs The aim of this section is to explain why MI EEG signals are used in BCIs, to discuss the inherent
challenges presented by the nature of MI EEG data and to introduce the structure of a typical signal
processing pipeline for MI EEG data. It also discusses generic technical challenges in this field,
including the high dimensionality of multichannel EEG data, the choice between averaged and
single-trial results and the choice of pre-processing approach. The aim of this section is to explain why MI EEG signals are used in BCIs, to discuss the inherent
challenges presented by the nature of MI EEG data and to introduce the structure of a typical signal
processing pipeline for MI EEG data. It also discusses generic technical challenges in this field,
including the high dimensionality of multichannel EEG data, the choice between averaged and
single-trial results and the choice of pre-processing approach. MI
d
d
BCI
b
h
ll
d MI is widespread in BCI systems because it has naturally occurring discriminative properties
and also because signal acquisition is not expensive. Furthermore, MI data in particular can be
used to complement rehabilitation therapy following a stroke. This notwithstanding, the processing
of MI data is challenging, and most processing and classification approaches are complex, with
many approaches suffering from poor classification accuracy since EEG signals are unstable [33,34]. Also, many classifiers fail to consider time-series information [33], even though the inclusion of
such data increases classification accuracy [34]. Also, the fact that the MI data of stroke patients is
significantly altered when compared to healthy subjects creates challenges in the design of BCIs for
post-stroke rehabilitation or therapy [5,35]. MI is widespread in BCI systems because it has naturally occurring discriminative properties
and also because signal acquisition is not expensive. Furthermore, MI data in particular can be used
to complement rehabilitation therapy following a stroke. This notwithstanding, the processing of MI
data is challenging, and most processing and classification approaches are complex, with many
approaches suffering from poor classification accuracy since EEG signals are unstable [33,34]. Also,
many classifiers fail to consider time-series information [33], even though the inclusion of such data
increases classification accuracy [34]. Also, the fact that the MI data of stroke patients is significantly
altered when compared to healthy subjects creates challenges in the design of BCIs for post-stroke
rehabilitation or therapy [5,35]. 2. Overview of EEG-Based BCIs This highlights two issues: firstly, the validity of MI EEG data
may be closely linked with the duration of a task, and thus it would be beneficial to test detectors
with data from a complete MI task. The segments of that data should be taken from the beginning
and end of the task in order to observe how performance varies. Secondly, future research could
investigate how the quality of the EEG data changes during an MI EEG task longer than two seconds,
and which kinds of signal processing approaches cope best with longer MI tasks. Zich et al. [32] used
fMRI in order to validate MI EEG data, and suggest that fMRI can be used in conjunction with EEG
technologies to investigate inter-individual differences in MI data generation. This literature review
now focuses on EEG-based MI BCIs in greater depth. 3.1. Raw EEG Data Numerous EEG-based BCIs use data recorded from multiple EEG channels as opposed to a single
channel [3]. A key problem when using multichannel data is the high computational costs and possibly
poorer performance if feature selection is not used [36]. Future work could involve investigating how data
from different channels can be combined or fused using averaging [37], a voting system [38] or PCA [39]. g
g
g
g y
In some areas of BCI research, particularly ERP-based BCIs, salient EEG signal events are often
identified in data which has been averaged across subjects or trials [40,41]. Although this approach is
widely used in neuroimaging research [41], it has the potential to hide poor performance through the
quotation of averaged results [40]. In fact, many studies now use single-trial data, in which results are
not averaged across trials [3,42]. These kinds of results are important as they enable the analysis of the
variability in performance across trials and can also provide a unique insight into brain activity [40]. Quoting results using single-trial data may also provide a clearer picture of BCI performance in a
practical scenario. 3. Introduction to MI EEG-Based BCIs
3. Introduction to MI EEG-Based BCIs h
h
f
EEG b
d
CI f
I
l
I Figure 1 shows the structure of an EEG-based BCIs for MI applications. In many systems, raw EEG
data is pre-processed to remove noise and artefacts [3,11], though not all systems pre-process data [4]. Features are then extracted from the EEG data and the most salient features for classification may be
selected. Based on the extracted features, the classifier then identifies which motor movement was
imagined by the user. Each section of this diagram will be discussed in greater detail in this paper,
with a special focus on feature extraction and selection techniques. Figure 1 shows the structure of an EEG-based BCIs for MI applications. In many systems, raw
EEG data is pre-processed to remove noise and artefacts [3,11], though not all systems pre-process
data [4]. Features are then extracted from the EEG data and the most salient features for classification
may be selected. Based on the extracted features, the classifier then identifies which motor movement
was imagined by the user. Each section of this diagram will be discussed in greater detail in this
paper, with a special focus on feature extraction and selection techniques. Figure 1. A diagram showing the signal processing carried out in a typical MI EEG-based system. Figure 1. A diagram showing the signal processing carried out in a typical MI EEG-based system. Figure 1. A diagram showing the signal processing carried out in a typical MI EEG-based system. Figure 1. A diagram showing the signal processing carried out in a typical MI EEG-based system. Sensors 2019, 19, 1423 5 of 34 3.3. Feature Extraction, Feature Selection and Classification The extracted features must capture salient signal characteristics which can be used as a
basis for the differentiation between task-specific brain states. Some BCIs involve a process of
feature selection, where only the most discriminant of features in a proposed feature set are
passed to the classifier with the aim of reducing computation time and increasing accuracy [3,5]. Based on the selected features, the classifier identifies the type of mental task being carried out,
and activates the necessary control signals in the BCI system. These control signals could be used,
for example, to control the selection of an icon on a graphical user interface, or the movement of a
neuroprosthesis. Classification approaches used in the literature include linear discriminant analysis
(LDA) [3,4,26,46], support-vector machines (SVMs) [3,4,26,47–51], k-nearest neighbor analysis [3,11,51],
logistic regression [51], quadratic classifiers [52] and recurrent neural networks (RNNs) [28]. Some systems group together the feature extraction, feature selection and classification tasks
within a single signal processing block [53–58]. These systems are based on deep learning and largely
use a convolutional neural network (CNN) structure [53–56,58]. The rest of this paper has a particular focus on feature extraction and selection techniques, as well
as classification approaches. 3.2. Pre-Processing In the literature, different approaches have been used to reduce the effects of noise in EEG signals
with the aim of increasing the accuracy and robustness of BCI systems. Kevric and Subasi [11] argue
that linear de-noising approaches, though effective, smooth out sharp transitions in EEG signals, which
may result in salient signal characteristics being deteriorated, and they propose that nonlinear filtering
techniques such as multiscale principle component analysis (MSPCA) are a better alternative, since
they effectively remove noise but preserve sharp transitions [11,43]. MSPCA has been successfully
used in a classification system for EEG signals associated with epileptic seizures [44] and another study
has successfully merged MSPCA with statistical features for EEG signal processing, with encouraging
results [45]. Kevric and Subasi [11] also improved the classification accuracy, in part, for MSPCA
compared to when other pre-processing techniques were used. 3.4. Hybrid BCIs Using MI-EEG: New Horizons A hybrid BCI is one which combines a BCI system with another kind of interface [59], which
can either be another BCI [60,61] or some other kind of interface [62]. In the case that the hybrid is
a merging of two different BCIs, the two BCIs can both be EEG-based [60], or they can be based on
some other technology used to record brain activity [61]. For example, [60] created a paradigm which
helped to improve the success of users training in an MI EEG system by using SSVEP as a training Sensors 2019, 19, 1423 6 of 34 aid. Please note that in these kinds of systems, the EEG brain signal responses must generally be
largely independent of each other. Conversely, in [61], near-infrared spectroscopy (NIRs) was used in
conjunction with EEG signals to identify and classify MI events. Alternatively, an example of a hybrid
BCI created by combining a BCI with another kind of interface was reported in [62], where a MI EEG
BCI was merged with a sensory interface. In hybrid BCIs, there are two main ways of combining the signals from the different technologies. The first approach involves considering both signals simultaneously in order to identify the MI task
being carried out [59]. For example, in the case of [60], in which the SSVEP-based result and the
MI-EEG-based result were both considered at the point of decision-making in the system. In the
second approach, the signals from the different interfaces are considered sequentially [59], as in the
case of [61], where the NIRs system was used to flag the occurrence of a MI event, and the EEG signals
were used to classify the event. The motivation driving the development of hybrid BCIs is sourced in the desire to create systems
with high levels of user literacy, meaning a wide number of users can gain mastery over the system. This is especially important in systems dependent on EEG MI data, since users can struggle to generate
the required signals, leading to frustration in training and poor mastery of the system. In fact,
combining MI EEG training with SSVEP was found to improve user mastery [60]. The struggles a
user may face in generating the required signals may be due to an inherent inability or due to some
pathology or condition which inhibits the required brain functioning. Hybrid BCIs are an emerging field and still face fundamental challenges. 3.4. Hybrid BCIs Using MI-EEG: New Horizons Chief among these is
choosing the right combinations of signals for a given situation and user [59]. Such a design choice should
be made by factoring the abilities and limitations of the user, the environment the system is intended for,
portability requirements, the overall cost and the control system, such as a prosthetic, being influenced by
the BCI. A second challenge is the decision of how to combine the outputs of the two BCIs, and future work
in the area may involve implementing a BCI using the combined and sequential approaches and evaluating
them based on speed, information transfer rate, computational cost, usability for the user, accuracy and
overall BCI literacy in order to see if there are significant differences in any area, and if so, this information
would be used to decide which implementation is best in a given situation. 4. Feature Extraction, Feature Selection and Classification in MI EEG-Based BCIs A variety of feature extraction, feature selection and classification techniques are discussed in
this section of the paper. The first three subsections discuss the signal processing techniques typically
used for feature extraction, feature selection and classification in systems which use distinct signal
processing techniques for each task. Figure 2 provides a summary of the most salient techniques
discussed in these subsections and Table 2 summarizes different feature extraction, feature selection
and classification approaches used in some notable BCI implementations. These works were chosen
merely to illustrate a wide variety of the different pipeline structures which have been implemented in
the literature. It should be noted that all the works in the Table used the BCI competition III dataset
IVa [63], except for the work by Zhou et al. [28], which used the BCI 2003 competition dataset III [64]. The main differences between the datasets was that dataset IVa covers a three-class MI problem with
left hand, right hand and right foot movements being used, and dataset III covers a two-class problem
involving left or right hand movement. Also, dataset III only contains data from one participant, while
dataset IVa has data from five participants. The final subsection discusses the deep learning-based
approaches, in which the three signal processing steps are completed within a single processing block. This whole section deals with the essential challenge of choice of signal processing techniques. To this end, the techniques described in this section are discussed with frequent references to the
signal processing challenges and design choice problems which are faced when using the particular
techniques for MI EEG processing. Furthermore, the shortcomings and issues associated with
particular techniques are also highlighted, as these must be factored when tackling the challenge
of pipeline design. 7 of 34
7 of 34 Sensors 2019, 19, 1423
Sensors 2019 19 x FOR Figure 2. A diagram summarizing some of the feature extraction, feature selection and classification
techniques used in MI EEG-based BCIs. Figure 2. A diagram summarizing some of the feature extraction, feature selection and classification
techniques used in MI EEG-based BCIs. Figure 2. A diagram summarizing some of the feature extraction, feature selection and classification
techniques used in MI EEG-based BCIs. Figure 2. A diagram summarizing some of the feature extraction, feature selection and classification
techniques used in MI EEG-based BCIs. Table 2. 4. Feature Extraction, Feature Selection and Classification in MI EEG-Based BCIs A comparison of the different combinations BCI structures used in the literature, including
features extracted, feature selection approach if used and classification method. Paper
Feature Extraction
Method 1
Feature Selection
Method 2
Classification
Method 3
Classification
Accuracy 7
Rodríguez-
Bermúdez &
García-Laencina,
2012 [26]
AAR modelling, PSD
LARS/LOO-Press
Criterion
LDA with
regularization
62.2% (AAR), 69.4%
(PSD)
Kevric & Subasi,
2017 [11]
Empirical mode
decomposition, DWT,
WPD 4
Kaiser criterion
k-NN
92.8% (WPD) 6
Zhou et al., 2018
[28]
Envelope analysis with
DWT & Hilbert transform
None
RNN LSTM
classifier
91.43%
Kumar et al.,
2017 [47]
CSP & CSSP 5
None, FBCSP, DFBCSP,
SFBCSP, SBLFB,
DFBCSP-MI 4
SVM
Classification
accuracy was not
quoted. Yu et al., 2014
[65]
CSP
PCA
SVM
76.34%
Baig et al., 2017
[3]
CSP
PSO, simulated
annealing, ABC
optimization, ACO, DE 4
LDA, SVM, k-NN,
naive Bayes,
regression trees 4
90.4% (PSO), 87.44%
(simulated
annealing), 94.48%
(ABC optimization),
84.54% (ACO), 95%
DE 8
1 Associated acronyms: AAR—adaptive autoregressive, PSD—power spectral density, DWT—
discrete wavelet transform, WPD—wavelet packet decomposition, CSP—common spatial pattern,
CSSP—common spatio-spectral pattern. 2 Associated acronyms: FBCSP—filter bank CSP, DFBCSP—
discriminative FBCSP, SFBCSP—selective FBCSP, SBLFB—sparse Bayesian learning FB, DFBCSP—
MI—DFBCSP with mutual information, PCA—principal component analysis, PSO—particle swarm
optimization, ABC—artificial bee colony, ACO—ant colony optimization, DE—differential evolution. 3 Associated acronyms: LDA—linear discriminant analysis, k-NN—k- nearest neighbor, RNN LSTM—
Table 2. A comparison of the different combinations BCI structures used in the literature, including
features extracted, feature selection approach if used and classification method. Paper
Feature Extraction
Method 1
Feature Selection
Method 2
Classification
Method 3
Classification Accuracy 7
Rodríguez-Bermúdez
& García-Laencina,
2012 [26]
AAR modelling,
PSD
LARS/LOO-Press
Criterion
LDA with
regularization
62.2% (AAR), 69.4% (PSD)
Kevric & Subasi,
2017 [11]
Empirical mode
decomposition,
DWT, WPD 4
Kaiser criterion
k-NN
92.8% (WPD) 6
Zhou et al.,
2018 [28]
Envelope analysis
with DWT &
Hilbert transform
None
RNN LSTM
classifier
91.43%
Kumar et al.,
2017 [47]
CSP & CSSP 5
None, FBCSP, DFBCSP,
SFBCSP, SBLFB,
DFBCSP-MI 4
SVM
Classification accuracy was
not quoted. Yu et al., 2014 [65]
CSP
PCA
SVM
76.34%
Baig et al., 2017 [3]
CSP
PSO, simulated
annealing, ABC
optimization, ACO, DE 4
LDA, SVM, k-NN,
naive Bayes,
regression trees 4
90.4% (PSO), 87.44%
(simulated annealing), 94.48%
(ABC optimization), 84.54%
(ACO), 95% DE 8
1 Associated acronyms: AAR—adaptive autoregressive, PSD—power spectral density, DWT—discrete wavelet
transform, WPD—wavelet packet decomposition, CSP—common spatial pattern, CSSP—common spatio-spectral
pattern. 4. Feature Extraction, Feature Selection and Classification in MI EEG-Based BCIs 2 Associated acronyms: FBCSP—filter bank CSP, DFBCSP—discriminative FBCSP, SFBCSP—selective
FBCSP, SBLFB—sparse Bayesian learning FB, DFBCSP—MI—DFBCSP with mutual information, PCA—principal
component analysis, PSO—particle swarm optimization, ABC—artificial bee colony, ACO—ant colony optimization,
DE—differential evolution. 3 Associated acronyms: LDA—linear discriminant analysis, k-NN—k- nearest neighbor,
RNN LSTM—recurrent neural network long-short-term memory, SVM—support vector machine. 4 The comma
between the terms denotes that the methods listed were tested separately. 5 The ‘&’ between the terms denotes
that the feature vector was constructed of both types of features. 6 Mean accuracy only available for the proposed
method, which consisted of the WPD combined with higher-order statistics and multiscale principal component
analysis for noise removal. Preliminary tested found WPD to be superior to empirical mode decomposition and
DWT. 7 Mean classification accuracy except result from Zhou et al., for which best accuracy only was quoted. 8
Averaged across the results for individual subjects. Table 2. A comparison of the different combinations BCI structures used in the literature, including
features extracted feature selection approach if used and classification method
Table 2. A comparison of the different combinations BCI structures used in the literature, including
features extracted, feature selection approach if used and classification method. Table 2. A comparison of the different combinations BCI structures used in the literature, including
features extracted feature selection approach if used and classification method
Table 2. A comparison of the different combinations BCI structures used in the literature, including
features extracted, feature selection approach if used and classification method. 4.2.1. Time-Domain and Frequency-Domain Techniques As a typical time-domain approach, autoregressive (AR) modelling has been used for feature
extraction. In this approach an AR model is fitted to segments of EEG data and the AR coefficients
or spectrum are used as features [11,66]. Adaptive autoregressive (AAR) modelling [26,67,68]
involves fitting an adaptive model to data segments, and in the literature, model parameters
have been estimated using recursive least-squares [69], least mean squares [68] and Kalman filter
approaches [70]. Although the Kalman filter is deemed computationally efficient for analyzing EEG
signals, its performance is affected by signal artefacts. Other alternative time-domain feature extraction
techniques include root-mean-square (RMS) and integrated EEG (IEEG) analysis [71]. Batres-Mendoza et al. [72] proposed a novel approach to time-domain modelling of MI EEG
signals based on quaternions. Quaternions, unlike other time-domain techniques used in MI EEG
modelling, can represent objects within a three-dimensional space in terms of their orientation and
rotation—a property which may be useful when dealing with multichannel EEG data. This technique
was found to be effective in extracting features from EEG data for the classification of MI-EEG [72]. Frequency-domain analysis has also been used to extract features from MI EEG data [4,26,73]. While [26] used the fast Fourier transform (FFT) to obtain the power spectrum, [4] used Welch’s
method. Welch’s method reduces the noise content in the spectrum when compared to the FFT, but has
a lower frequency resolution. Another approach to frequency domain analysis, which did not depend
on Fourier theory, was local characteristic-scale decomposition (LCD) [74]. This approach decomposes
the signal into intrinsic scale components which have characteristic instantaneous frequencies linked
to the characteristics of the original signal. g
g
The spectral analysis for feature extraction is weak as it provides no information relating the
frequency content of the signal to the temporal domain. Similarly, time-domain-based analysis ignores
spectral features which may be of use for classification. 4.2. Feature Extraction Feature extraction is the signal processing step in which discriminative and non-redundant
information is extracted from the EEG data to form a set of features on which classification can be
carried out. The most basic feature extraction techniques use time-domain or frequency-domain
analysis in order to extract features. Time-frequency analysis is a more advanced and sophisticated
feature extraction technique which enables spectral information to be related to the time domain. Finally, analysis in the spatial domain using common spectral patterns is also a prevalent method for
feature extraction. recurrent neural network long
between the terms denotes that
4.1. Data and Recording Protocols p
y
denotes that the feature vector was constructed of both types of features. 6 Mean accuracy only
available for the proposed method, which consisted of the WPD combined with higher-order statistics
and multiscale principal component analysis for noise removal. Preliminary tested found WPD to be
superior to empirical mode decomposition and DWT. 7 Mean classification accuracy except result
f
Zh
t l f
hi h b
t
l
t d 8A
d
th
lt f
i di id
l
Data plays a key role in the training and testing of machine learning systems. It should be noted
that the studies discussed in this section of the paper have used different data sets, which all use
slightly different recording protocols. The main variations in the datasets are: (i) number of motor
imagery tasks considered, with a range between two and four classes possible, (ii) variations in the 8 of 34 Sensors 2019, 19, 1423 number of EEG channels recorded and those used in data processing, (iii) variation in the amount of
time subjects are allowed to rest between MI tasks, (iv) number of trials and sessions carried out with
each subject, (v) number of subjects involved, and (vi) whether an open access or private database was
used. These factors should be kept in mind when comparing studies or when applying techniques
similar to the literature on new data. Table 2 aimed to show results using various techniques, which
were largely generated using the similar data, except for one study. 4.2.2. Time-Frequency Domain Techniques Time-frequency analysis is powerful since it enables spectral information about an EEG signal to be
related to the temporal domain, which is advantageous for BCI technologies since spectral brain activity
varies during a period of use of the system as different tasks are carried out [23]. Approaches used for
MI EEG analysis include the short-time Fourier transform (STFT) [55], the wavelet transform (WT) [75]
and the discrete wavelet transform (DWT) [76]. Decomposition methods such as the WT and the
DWT are powerful since different EEG signal frequency bands contain different information about MI
actions [11,23], and they can be used to decompose a signal in multiresolution and multiscale [77–79]. The DWT and WT are competent in deriving dynamic features, which is particularly important Sensors 2019, 19, 1423 9 of 34 in EEG signals since they are non-stationary, non-linear and non-Gaussian [11]. In [80], the DWT
coefficients of the frequency bands of interest were extracted as features, and similarly wavelet packet
decomposition (WPD) was used to break-down the EEG signals into low frequency and high frequency
components, and the coefficients associated with the frequency bands of interest were then extracted
as features. By combining the DWT and AR modelling [76], feature sets are constructed based on
wavelet coefficient statistics and 6th order AR coefficients. Kevric and Subasi [11] conducted a detailed study comparing the performance of decomposition
techniques which took higher-order statistics as input features. While first- and second-order statistics
have been widely used in biomedical applications [79], they restrict the analysis which can be
carried out on nonlinear aspects of the signal. Higher-order statistics enable the representation
of signal features when signal behavior diverges from the ideal stationary, linear and Gaussian model,
something which lends higher-order statistics an advantage over time-frequency approaches [11,79]. This study is one of very few which gives an in-depth comparison of signal decomposition techniques
combined with higher-order statistics for the classification of BCI signals [11]. They compared the
performance of three different decomposition methods: empirical mode decomposition, DWT and
WPD. These decomposition methods were used to create various sub-band signal components from
which 6 features were calculated from the decomposition coefficients, including higher-order statistics
in the form of skewness and kurtosis. k-nearest neighbor (k-NN) analysis was used for classification,
with the k parameter set to 7. MSPCA was used in pre-processing for noise removal. 4.2.2. Time-Frequency Domain Techniques Kevric and
Subasi found that the use of MCSPA and higher-order statistics in feature extraction improved the
classification accuracy when compared to approaches that did not use this combination of techniques,
and the highest classification accuracy obtained was 92.8%. Furthermore, they found that a higher
resolution could be obtained with the WPD coefficients when compared to the DWT coefficients and
that the modelling limitations of wavelets were mitigated by using higher-order statistics. Kevric and
Subasi also suggest that the technique could have an application in stroke rehabilitation technologies. Typical classifiers used for classification, including LDA, SVM, k-NN and logistic regression do
not factor the time-series data present in EEG signals, even though factoring this aspect of the signals
can improve classification accuracy, since EEG signals are nonstationary [34]. RNNs are commonly
used to exploit the time-series nature of signals, but these networks can suffer from gradient vanishing
or gradient explosion during training, and they also have an inherent bias towards new data [28]. To minimize these issues, Zhou et al. [28] used a long-term short-term memory (LSTM) RNN classifier,
an approach also used in other studies [81–83]. In [28], envelope analysis [84] is also used to extract features from the EEG data. This approach
has been used in other works [7,85], as bioelectrical signals naturally exhibit amplitude modulation. Zhou et al. [28] merged the Hilbert transform (HT) and the DWT in order to extract features which
are related to the amplitude and frequency modulation present in EEG signals. In the first step of
the algorithm, the EEG data is decomposed via the DWT, and afterwards, the wavelet envelope of
the decomposed sub-bands was obtained via the HT. The wavelet envelope contained time-series
data which was fed into the LSTM classifier. Thus, this method used both envelope information and
time-series information, and achieved a high classification accuracy. 4.2.3. Common Spatial Patterns Common spatial pattern (CSP) is one of the most common feature extraction methods used in
MI EEG classification [6,47,50,86]. CSP is a spatial filtering method used to transform EEG data into a
new space where the variance of one of the classes is maximized while the variance of the other class
is minimized. It is a strong technique for MI EEG processing since different frequency bands of the
signal contain different information, and CSP enables the extraction of this information from particular
frequency bands. However, pure CSP analysis is not adequate for high-performance MI classification
because different subjects exhibit activity in different frequency bands and the optimal frequency band
is subject-specific. This means that a wide band of frequencies, typically between 4 Hz and 40 Hz, Sensors 2019, 19, 1423 10 of 34 must be used for MI classification, leading to the inclusion of redundant data being processed [47]. The literature has suggested that optimization of filter band selection could improve the classification
accuracy of MI EEG BCIs [48,50,87,88]. However, locating the optimal sub-band using pure CSP is
time-consuming [89,90]. There are also various alterations to the CSP method which aim to improve its feature extraction
capabilities. The common spatio-spectral pattern approach (CSSP) integrates a finite impulse response
(FIR) filter into the CSP algorithm, which was observed to improve performance relative to pure
CSP [42]. Common sparse spatio-spectral patterns (CSSSP) [90] is a more refined technique which aims
to find spectral patterns which are common across channels, as opposed to the individual spectral
patterns in each channel. In sub-band common spatial pattern (SBCSP) [46], EEG is first filtered at
different sub-bands, and then CSP features are calculated for each of the bands. LDA is then used
to decrease the dimensionality of the sub-bands. This was found to obtain improved classification
accuracy when compared to CSP, CSSP and CSSSP [46]. Oikonomou et al. [4] compared the performance of MI classification when using CSP and power
spectral density (PSD) for feature extraction, where LDA and SVM were used for classification. The PSD
approach was found to outperform the CSP approach for classification of left and right MI tasks. This is
possibly because of the high dimensionality feature set obtained via PSD analysis [4]. When using
the CSP features, the LDA and SVM classifiers performed similarly. However, SVM was found to
significantly outperform LDA when the PSD features were used [4]. 1 The performance of the feature selection method can only be truly compared quantitatively to other methods
when they were tested with the same data, feature vector and classifier. Thus, although the classification accuracies
are listed, true comparisons can only be made when the references associated with the selection methods in the first
column are the same. 2 Paper did not quote classification accuracy. 4.3.2. Filter Bank Selection This feature selection approach is specific to systems which use CSP and CSSP for feature
extraction. Previously, in Section 4.2.3, methods to improve the feature extraction capabilities of
traditional CSP analysis were discussed. However, none of the methods discussed thus far exploit
the intrinsic link between the frequency bands and CSP features [47]. Filter bank CSP (FBCSP) [48]
addresses this by estimating the mutual information contained in the CSP features from the various
sub-bands. By choosing those which are most discriminant, selected features are fed into an SVM
for classification. Although the system outperformed the SBCSP approach, it utilized multiple
sub-bands, leading to a hefty computational cost [47]. To decrease the computational demands
of FBCSP, discriminant filter bank CSP (DFBCSP) [88,92] was developed, which considers various
overlapping frequency bands, and uses the Fisher ratio to analyze the band power of each sub-band
in order to identify the most discriminant sub-bands. This analysis is performed on a single channel
of EEG data (C3, C4 or Cz). Sparse filter bank CSP (SFBCSP) [93] also uses multiple frequency
bands, but aims to optimize sparse patterns, and features are selected using a supervised technique. In another technique, SBLFB [50], Bayesian learning is employed to select CSP features from multiple
EEG sub-bands before feeding them into a SVM classifier. This has resulted in improved performance
when compared to state-of-the-art techniques. Kumar et al. [47] noted that the performance of MI classification depends on the selection of the
frequency sub-bands used for feature extraction. They aimed to solve the frequency-band selection
problem by building on the DFBCSP approach. Instead of using single-channel data as proposed in the
DFBCSP approaches reviewed, data from all available channels was used for the extraction of both CSP
and CSSP features from various overlapping sub-bands. Furthermore, Kumar et al. introduced a novel
frequency band covering 7-30 Hz. Using the extracted features, mutual information for the bands is
then calculated, and the most discriminative filter banks are chosen to be forwarded to the next signal
processing stage. In this stage, LDA is used to reduce the dimensionality of the features extracted
from each filter bank. Afterwards, the LDA results are joined and fed into an SVM for classification. 4.3. Feature Selection Three main feature selection techniques are discussed in this section: (i) principal component
analysis (PCA), (ii) filter bank selection, and (iii) evolutionary algorithms (EAs). Table 3 summarizes
the different approaches used, providing information on the mathematical nature of the approaches,
average classification accuracy obtained when applying them in the EEG processing pipeline and
additional comments about these methods. Table 3. A summary of the different feature selection techniques discussed in this subsection. Table 3. A summary of the different feature selection techniques discussed in this subsection. Method
Type
Mean Classification Accuracy 1
Comments
Principal component
analysis (PCA) [65]
Statistical
76.34%
Assumes components with the highest variance have
the most information. Filter Bank Selection [47]
Various
N/A 2
Used only for frequency band selection with CSP [47]
Particle-Swarm
Optimization (PSO) [3]
Metaheuristic
90.4%
Strong Directional search and population-based search
with exploration and exploitation [91]. Simulated Annealing [3]
Probabilistic
87.44%
Aims to find the global maximum through a random
search. [3]
Artificial Bee-Colony
(ABC) Optimization [3]
Metaheuristic
94.48%
Searches regions of the solution space in turn in order
to find the fittest individual in each region. [91]
Ant Colony
Optimization (ACO) [3]
Metaheuristic
84.54%
Uses common concepts of directional and
population-based search but introduces search space
marking. [91]
Differential Evolution
(DE) [3]
Metaheuristic
95%
Similar to GAs, with a strong capability of
convergence. [3]
Firefly Algorithm [74]
Metaheuristic
70.2%
Can get stuck in local minima, [74] introduced a
learning algorithm to prevent this. Genetic Algorithm
(GA) [74]
Metaheuristic
59.85%
Slower than a PSO approach [49], [49] found that PSO
was more accurate. 1 The performance of the feature selection method can only be truly compared quantitatively to other methods
when they were tested with the same data, feature vector and classifier. Thus, although the classification accuracies
are listed, true comparisons can only be made when the references associated with the selection methods in the first
column are the same. 2 Paper did not quote classification accuracy. 11 of 34 Sensors 2019, 19, 1423 11 of 34 4.3.1. Principal Component Analysis (PCA) Analysis and dimensionality reduction techniques, including PCA [65] and independent
component analysis (ICA) [7], have also been applied to MI EEG. PCA has been used for dimensionality
reduction and feature selection for improved classification [3]. In some cases [65,75], both PCA and ICA
are utilized with other signal processing techniques for feature extraction; for example, in [75], ICA and
the WT were used in conjunction with each other in order to extract spatial and time-frequency features. 4.3.2. Filter Bank Selection The performance of this novel technique was compared to that of the CSP, CSSP, FBCSP, DFBSCP,
SFBCSP and SBLFB techniques, and was found to have the smallest misclassification rate, and had a
strong overall prediction capability. Provided that the model used works exceptionally well, a future
improvement to the algorithm could be automatic the learning of the parameters for the filter band. 4.3.3. Evolutionary Algorithms A key issue in BCI development is the high dimensionality of data during feature extraction. Typical dimensionality reduction and feature selection methods such as PCA and ICA involve complex
transformations of features leading to substantial computational demands and a larger sized feature set. These methods often result in low classification accuracy even if the variance of the data is acceptable,
possibly because basic feature extraction tends to retain some redundant features. Furthermore, linear
transforms tend to be used to decrease the dimensionality of the feature set [3]. Evolutionary algorithms (EA) may offer a possible solution, by enabling features to be selected
based on optimization of the classification accuracy of the system. They are promising because in
some applications they have been shown to be successful in searching large feature spaces for optimal
solutions [3]. EAs such as particle swarm optimization (PSO) [3,49,94], differential evolution (DE) Sensors 2019, 19, 1423
Sensors 2019, 19, x FOR 12 of 34
12 of 34 optimization [3,95], artificial bee colony (ABC) optimization [3,96], ant colony optimization (ACO) [3],
genetic algorithms (GAs) [49] and the firefly algorithm [74] have been successfully applied for feature
selection and reduction. optimization [3,95], artificial bee colony (ABC) optimization [3,96], ant colony optimization (ACO)
[3], genetic algorithms (GAs) [49] and the firefly algorithm [74] have been successfully applied for
feature selection and reduction. Baig et al. [3] propose a new feature selection approach based on DE optimization, which aims Baig et al. [3] propose a new feature selection approach based on DE optimization, which aims
to decrease computational demands while improving the effectiveness of the feature set by choosing
only relevant features. Figure 3 summarizes the flow of the DE-based feature extraction and feature
selection process. They implemented a hybrid approach where CSP is used to extract features, a DE
algorithm is used to select an optimized subset of features, and only these features are passed onto
the classifier. The system was also tested with different computational methods for feature selection,
namely PSO, simulated annealing, ACO and ABC optimization. Also, the framework was tested with
five different classifiers: LDA, SVM, k-NN, naive Bayes and regression trees. Although the suggested
algorithm was relatively slow in feature extraction, and the use of wrapper techniques further slowed
the system when compared to state-of-the-art approaches, argued that the significant improvement
in accuracy far outweighs the slower computations. 4.3.3. Evolutionary Algorithms Furthermore, it should be noted that the EA
algorithm is only used to find the optimal feature set for a given application, and thus after selection of
the optimal features, the classification problem can be carried out repeatedly using the pre-selected
features. Thus, the computational burden of the EA is only suffered once, during the initial feature
selection phase, after which the problem becomes one of simple classification. Baig et al. [3] propose a new feature selection approach based on DE optimization, which aims
to decrease computational demands while improving the effectiveness of the feature set by choosing
only relevant features. Figure 3 summarizes the flow of the DE-based feature extraction and feature
selection process. They implemented a hybrid approach where CSP is used to extract features, a DE
algorithm is used to select an optimized subset of features, and only these features are passed onto
the classifier. The system was also tested with different computational methods for feature selection,
namely PSO, simulated annealing, ACO and ABC optimization. Also, the framework was tested with
five different classifiers: LDA, SVM, k-NN, naive Bayes and regression trees. Although the suggested
algorithm was relatively slow in feature extraction, and the use of wrapper techniques further slowed
the system when compared to state-of-the-art approaches, argued that the significant improvement
in accuracy far outweighs the slower computations. Furthermore, it should be noted that the EA
algorithm is only used to find the optimal feature set for a given application, and thus after selection
of the optimal features, the classification problem can be carried out repeatedly using the pre-selected
features. Thus, the computational burden of the EA is only suffered once, during the initial feature
selection phase, after which the problem becomes one of simple classification. Zhiping et al. [49] also implemented a PSO-based two-step method for feature selection from MI Zhiping et al. [49] also implemented a PSO-based two-step method for feature selection from
MI EEG data. Firstly, the PSO algorithm was used to choose the classifier parameters and relevant
features extracted from the EEG data. Afterwards, redundant features were excluded from the selected
features via a voting mechanism. This additional voting mechanism was not implemented in any
of the other EAs surveyed. In this application, the feature vector was constructed of time-frequency
features extracted using the stationary wavelet transform (SWT) and a finite-impulse response (FIR)
filter, and an SVM was used for classification. 4.3.3. Evolutionary Algorithms PSO was found to be effective in increasing the speed of
the system as well as reducing the number of redundant features and a stable performance. The results
obtained using PSO were compared to those obtained using a GA and were found to be superior. The GA approach suffers from a slower learning process than PSO, with PSO taking advantage of
gradient information in order to survey trends in order to obtain an appropriate, optimal answer as
opposed to GAs, which search within the data for trends [49]. p
g
[
]
p
p
EEG data. Firstly, the PSO algorithm was used to choose the classifier parameters and relevant
features extracted from the EEG data. Afterwards, redundant features were excluded from the
selected features via a voting mechanism. This additional voting mechanism was not implemented
in any of the other EAs surveyed. In this application, the feature vector was constructed of time-
frequency features extracted using the stationary wavelet transform (SWT) and a finite-impulse
response (FIR) filter, and an SVM was used for classification. PSO was found to be effective in
increasing the speed of the system as well as reducing the number of redundant features and a stable
performance. The results obtained using PSO were compared to those obtained using a GA and were
found to be superior. The GA approach suffers from a slower learning process than PSO, with PSO
taking advantage of gradient information in order to survey trends in order to obtain an appropriate,
optimal answer as opposed to GAs, which search within the data for trends [49]. The firefly algorithm, which bears close similarity to PSO, has also been applied to feature The firefly algorithm, which bears close similarity to PSO, has also been applied to feature
selection when using CSP and LCD features [74]. In [74], combining the firefly approach with a
learning algorithm was proposed in order to prevent the optimization process getting caught in a
local minimum. Although the firefly algorithm has been criticized as being very similar to the PSO
algorithm [91], it resulted in higher classification accuracies when compared to similar pipelines using
both genetic and adaptive weight PSOs. y
g
y
pp
selection when using CSP and LCD features [74]. In [74], combining the firefly approach with a
learning algorithm was proposed in order to prevent the optimization process getting caught in a
local minimum. 4.4. Classification Methods The aim of this subsection is to provide a brief summary of the various classification techniques
used in the literature. SVMs and LDA were observed to be the widely used classifiers in the
literature [3,4,26,46–51,72], with the performance of the SVM classifier found to be superior when
compared to various classifiers such as LDA, k-NN, naive Bayes and regression trees [3,4,47]. The average classification accuracy of the proposed method using the SVM classifier was 96.02%,
which was a 2% improvement in classification accuracy when compared to state-of-the-art results. LDA was also found to outperform naïve Bayes when CSP and PSD features were used [11]. In [97],
it was also found that the SVM classifier with Gaussian kernel outperforms LDA. Baig et al. [3] also
found that SVM and LDA were the best classifiers for DE-based feature extraction, with both obtaining
a classification accuracy of 95% with a deviation of 0.1. y
Both the LDA and SVM approaches may suffer from overfitting; however, these can be mitigated
by applying regularization in LDA and through choice of training scheme in the case of SVMs [98]
(pp. 9, 336). Although both popular, SVMs and LDA are fundamentally different, with the LDA
approach prone to suffer from the curse of dimensionality, something which is absent when using the
SVM approach. Although SVM is popularly used in the literature [3,4,26,47–51,72], logistic regression
has been found to perform on par with SVM in terms of classification accuracy, obtaining an accuracy
of 73.03% compared to 68.97% for SVM. Logistic regression also performed better than k-NN and
artificial neural network (ANNs) approaches [51]. Although SVM and logistic regression are strong
classifiers in MI EEG processing, there is a relationship between the accuracy of the classification,
classifier type and the type of features used. In fact, the classification performance of the k-NN and
ANN approaches can be further improved by using features which are strongly correlated [99]. k-NN approaches were also found to be common in the literature [3,11,72]; however, these are
memory-based approaches, meaning that a full dataset must be stored in memory and processed all at
once. This inevitably increases computational costs when compared to kernel-based methods such
as SVM [98] (p. 292). Furthermore, SVMs may be viewed as more powerful than the k-NN approach
since it constructs an optimization problem which has one global solution which can be calculated in a
straightforward way [98] (p. 225). 4.3.3. Evolutionary Algorithms Although the firefly algorithm has been criticized as being very similar to the PSO
algorithm [91], it resulted in higher classification accuracies when compared to similar pipelines
using both genetic and adaptive weight PSOs. Figure 3. A diagram of the feature extraction and feature selection process proposed in [3]. Figure 3. A diagram of the feature extraction and feature selection process proposed in [3]. ure 3
A diagram of the feature extraction and feature selection process proposed in [3]
Figure 3. A diagram of the feature extraction and feature selection process proposed in [3]. 13 of 34 Sensors 2019, 19, 1423 13 of 34 4.4. Classification Methods Quadratic classifiers have not typically been applied to MI EEG processing. However, a quadratic
classifier was successfully applied to an EEG classification problem involving the detection of epileptic
activity, obtaining an overall classification accuracy of 99% [52]. Future work could investigate the
application of quadratic classifiers to MI EEG classification problems. Computational intelligence methods have also been used for classification. These include deep
learning architectures [5,58,100], as well as RNNs [28], which were previously discussed. Lu et
al. [101] used a deep neural network constructed using restricted Boltzmann machines and obtained
better accuracy than state-of-the-art methods including CSP and FBCSP. Similarly, using a CNN
approach, [58] obtained a better classification performance than a FBCSP approach. Future work may
involve comparing the performance of the deep learning classifiers in [58,100] with SVM and LDA
classifiers. Cheng et al. [5] tried to improve MI classification for data from stroke patients—which
deviate from MI data from healthy patients- by using deep neural networks (DNNs) to select the
best frequency bands from which to generate features in order to improve classification accuracy. They found that features selected from the identified sub-bands gave better classification accuracies
than when selecting features using standard methods. Furthermore, they found that a DNN classifier
was often more accurate than an SVM classifier. Fuzzy classification is another computational intelligence approach used for EEG classification that
has gained popularity because EEG classification is a decision-making problem suited for fuzzy logic. These approaches challenge established approaches to EEG signal processing and classification [101],
which have already been discussed in this paper. Yang et al. [102] proposed an adaptive neuro-fuzzy
interface system (ANFIS) which aimed to classify background EEG recorded from subjects suffering
from electrical status epilepticus slow wave sleep (ESES) syndrome and healthy controls using sample 14 of 34 14 of 34 Sensors 2019, 19, 1423 entropy and permutation to construct the features. The mean accuracy was reported to be 89%. Alternatively, Herman et al. [103] used an interval type-2 fuzzy logic system, which was designed to
accommodate for the non-stationarity inherent to EEG signals. Using 5-fold cross-validation (CV),
a classification accuracy of 71.2% was obtained, with the approach outperforming state-of-the-art
systems. Finally, Jiang et al. [104] used a Taigi-Sugeno-Kang approach, and applied a multiview
learning approach to provide better generalization. Interestingly, Jiang et al. 4.5. The Deep Learning Approach 4.5. The Deep Learning Approach Deep learning can be used to perform the whole pipeline of feature extraction, selection and
classification within a single processing block [53–58]. The architecture most widely used in MI EEG
processing were CNNs [53–56,58], but RNNs [56], stacked auto encoders (SAEs) [55] and deep belief
networks [56] have also been used. Studies have found deep learning to outperform state-of-the-art
techniques [53,55,56], including those using CSP features [53] and SVM classification [53,56]. Often, other architectures are combined with the CNN architecture. For example, in [55], a CNN
was used for feature extraction while a SAE was used for classification, and in [56], a CNN was
used to extract features that were invariant to spectral and spatial changes, whist a LSTM-RNN was
used to extract temporal information. Finally, Dai et al. [106] used a CNN to extract features and a
variational autoencoder (VAE) for classification, and their implementation was found to outperform
the state-of-the art approaches for the databases they tested on. CNNs hold many advantages for MI EEG data processing [53]: raw data can be input to the
system thus removing the need to prior feature extraction and they inherently exploit the hierarchical
nature of certain signals and they perform well using large datasets. However, their disadvantages are
also evident, since the large number of hyper parameters which must be learnt during training can
increase the training time compared to other methods, they can produce incorrect classification results
with great certainty [107], and the features learnt can be difficult to understand in the context of the
original signal. It should be noted that CNNs were adopted in EEG signal processing after first being established
as a tool in image processing [108]. Thus, when using CNNs for the classification of MI EEG, one of
the greatest differences between approaches involves the pre-processing of the input data, which can
mainly be divided into two solutions, i.e., either configuring the EEG data as an image [55,56], or not
configuring the EEG data as an image [53,54,58]. In approaches which convert the EEG data to an image, a time-frequency domain image is
obtained from the data. In [55] this is achieved by segmenting the EEG data with a two-second
interval, where each interval corresponds to a particular MI task being performed. 4.4. Classification Methods used Friedman rank to
evaluate the performance of the detector, which is a metric which was not observed to be widely used
in the literature. The multiview learning approach provided better results, giving a Friedman rank of 1,
as opposed to the system without multiview learning which obtained a rank of 3.65. Fuzzy classifiers
have also been used with CSP features [105]. It is evident that classification techniques based on supervised learning were overwhelmingly favored
in the literature when compared to those based on unsupervised learning. Unsupervised techniques have
been used mainly for feature selection, as discussed previously in Section 4.2.2. However, unsupervised
techniques such as Gaussian mixture models have been used for EEG classification problems outside of MI
EEG processing, such as in [37], and could possibly be applied to MI EEG as well in future work. 4.5. The Deep Learning Approach The STFT is used to
produce a time-frequency image of the task, from which the frequency bands most associated with
MI EEG are extracted. The extracted image is then fed into the deep network. In [106] it aimed to
preserve the channel relationships between the electrodes used in recording by concatenating STFT
time-frequency images generated from each electrode to form a single image. However, the STFT
ignores any relationship that can exist between the time-frequency domain and the spatial domain. In [56], it attempts to preserve these possible relationships by considering short time segments and
extracting from a given segment the information from three salient frequencies. The data obtained Sensors 2019, 19, 1423 15 of 34 from each band was then projected from the 3D space of the electrodes placed on the scalp to the 2D
space of an image, and this projection maintains the spatial relationships between the information
from each electrode. The resulting 2D images obtained for each frequency band are then grouped to
form an image with three color channels. Contrastingly, [53] proposes a technique in which raw data is fed into the CNN, and the first
layers of the network are devoted to extracting spatial and temporal information. This approach leads
to a lower dimensionality input than the image-based approach in [56]. In [53], the CNN was learnt to
use spectral characteristics to discriminate between tasks. In [54], a time-consuming pre-processing
approach was used, in which the data which has the best markers for MI activity, and the best frequency
bands for each subject in the study were selected via visual inspection. Finally, in the pre-processing
step proposed by [58], augmented CSP (ACSP) features are extracted from the data. Recall that one of
the core issues with CSP feature extraction is the selection of the frequency bands for feature extraction,
with many approaches using a wide-band method or a filter-bank method for selection. However, this
can result in the loss of important information, and ACSP aims to solve this issue by covering as many
frequency bands as possible by varying the partitions between the bands. Deep learning holds much potential in MI EEG classification. Future work could involve a heavier
focus on integrating elements of feature selection. For example, the potential of stacked denoising
auto encoders, which has been used to locate robust features [109], could be explored. 4.5. The Deep Learning Approach Also, network
structure and training could incorporate feature selection elements such as in [110–112], where features
which are not strongly discriminative according to some criterion are suppressed by the network. Architectures using statistical tests such as the t-test [113] or chi-squared test [114] to identify the most
discriminative features could also be investigated. Furthermore, heavier research into architectures
aside from CNNs could be carried out, such as into the use of stacked auto encoders for the entire
pipeline processing and evolutionary neural networks, which has been shown to hold potential for
feature extraction and selection [57]. Section 5 now introduces a case study which provides a practical example of an implementation
of an MI EEG data processing pipeline. 5. Case Study A particular case study of EEG data processing is briefly introduced in this section. This case
was presented at the IEEE brain data bank challenge (BDBC) 2017, hosted in Glasgow [115]. A team
representing the University of Strathclyde participated in the challenge achieving the 2nd place award
for their work ‘Evaluation of different time and frequency domain approaches for a two-directional
imaginary motor movement task’, later extended to [116]. In this competition, participants were allowed to work with any EEG data set, being asked to
develop open analysis for novel, creative or informative conclusions. This case study focused on the
feature extraction stage, implementing different techniques and comparing their performance in terms
of (i) classification accuracy and (ii) computation time required for extracting the features. In the following subsections, information about the data set used in the experiments alon
the proposed data processing and results achieved is provided. 5.1. Selected Data Set For this case study, data set number 4 from the Brain/neural computer interaction (BNCI) Horizon
2020 site [117] was selected. BNCI Horizon 2020 is a coordination-and-support action funded within
the European Commission’s Framework Programme 7 [117], with the objective of promoting collaboration
and communication among the main players in the BCI field. Data set number 4 was selected for several reasons, including high impact and citation, as well as
the data being available in MATLAB files, ready for straightforward use in this platform. The data
consists of three bipolar recordings (C3, CZ, C4) corresponding to the image shown in Figure 4a. During the acquisition process, subjects under study were told to imagine left hand movement or right Sensors 2019, 19, 1423 16 of 34 16 of 34 hand movement for four seconds after the cue was initialized, roughly one second after hearing a short
acoustic tone (beep). Imagination of left or right movement depended on an arrow cue shown in a
screen. The time scheme paradigm followed during acquisition is shown in Figure 4b. Sensors 2019, 19, x FOR PEER REVIEW
16 of 34
Sensors 2019, 19, x FOR PEER REVIEW
16 of 34
Each subject participated in two sessions performed on two different days Each session Each subject participated in two sessions performed on two different days. Each session contained
six runs with ten trials each and two MI classes [118]. A total of 120 trials per session were acquired,
leading to 120 repetitions of left MI class and 120 repetitions of right MI class out of the two sessions. This data set also included information related to electrooculography (EOG), but it was not used in the
case study. Further details about this data set can be found in [118]. Each subject participated in two sessions performed on two different days. Each session
contained six runs with ten trials each and two MI classes [118]. A total of 120 trials per session were
acquired, leading to 120 repetitions of left MI class and 120 repetitions of right MI class out of the two
sessions. This data set also included information related to electrooculography (EOG), but it was not
used in the case study. Further details about this data set can be found in [118]. Each subject participated in two sessions performed on two different days. Each session
contained six runs with ten trials each and two MI classes [118]. 5.2. Data Processing Workflow
5.2. Data Processing Workflow
5.2. Data Processing Workflow These were: (i) template matching (TM) [119–121] and
statistical moments (SM) [122–124] in the time-domain, and (ii) average bandpower (A-BP) [125–127],
The raw EEG evaluated was the same for all the cases, where a common infinite impulse
response (IIR) filter (Butterworth) was implemented for pre-processing. Afterwards, different feature
extraction techniques were implemented. These were: (i) template matching (TM) [119–121] and
statistical moments (SM) [122–124] in the time-domain, and (ii) average bandpower (A-BP) [125–127],
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The raw EEG evaluated was the same for all the cases, where a common infinite impulse response
(IIR) filter (Butterworth) was implemented for pre-processing. Afterwards, different feature extraction
techniques were implemented. These were: (i) template matching (TM) [119–121] and statistical moments
(SM) [122–124] in the time-domain, and (ii) average bandpower (A-BP) [25,125,126], selective bandpower
(S-BP) [69,118,127] and fast Fourier transform power spectrum (FFT) [128–130] in the frequency domain. (
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selective bandpower (S-BP) [69,118,128] and fast Fourier transform power spectrum (FFT) [129–131]
in the frequency domain. TM has been used in previous works related to the detection of salient characteristics in EEG
signal processing. For example, [119] used this technique in order to detect transient events within
EEG recordings from infants, [120] used it to identify early indications of seizures in epileptic patients
and [123] used TM to flap VEP events in a BCI application. SM has also been used in the detection of
selective bandpower (S-BP) [69,118,128] and fast Fourier transform power spectrum (FFT) [129–131]
in the frequency domain. TM has been used in previous works related to the detection of salient characteristics in EEG
signal processing. For example, [119] used this technique in order to detect transient events within
EEG recordings from infants, [120] used it to identify early indications of seizures in epileptic patients
and [123] used TM to flap VEP events in a BCI application. SM has also been used in the detection of
EEG
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TM has been used in previous works related to the detection of salient characteristics in EEG
signal processing. 5.2. Data Processing Workflow
5.2. Data Processing Workflow
5.2. Data Processing Workflow The main purpose of the data processing was to compare different feature extraction techniques
under the same conditions. Figure 5 shows the overall methodology followed for all the techniques,
including (i) raw EEG data (C3, CZ, C4), (ii) pre-processing based on filtering, (iii) feature extraction
(main comparative evaluation), (iv) feature selection, and (v) classification. The main purpose of the data processing was to compare different feature extraction techniques
under the same conditions. Figure 5 shows the overall methodology followed for all the techniques,
including (i) raw EEG data (C3, CZ, C4), (ii) pre-processing based on filtering, (iii) feature extraction
(main comparative evaluation), (iv) feature selection, and (v) classification. The main purpose of the data processing was to compare different feature extraction techniques
under the same conditions. Figure 5 shows the overall methodology followed for all the techniques,
including (i) raw EEG data (C3, CZ, C4), (ii) pre-processing based on filtering, (iii) feature extraction
(main comparative evaluation), (iv) feature selection, and (v) classification. Figure 5. A diagram of the methodology proposed by the University of Strathclyde team in the BDBC
2017 hosted in Glasgow, where different feature extraction techniques were compared under the same
Figure 5. A diagram of the methodology proposed by the University of Strathclyde team in the BDBC
2017 hosted in Glasgow, where different feature extraction techniques were compared under the same
conditions. Figure 5. A diagram of the methodology proposed by the University of Strathclyde team in the BDBC
2017 hosted in Glasgow, where different feature extraction techniques were compared under the
same conditions. Figure 5. A diagram of the methodology proposed by the University of Strathclyde team in the BDBC
2017 hosted in Glasgow, where different feature extraction techniques were compared under the same
Figure 5. A diagram of the methodology proposed by the University of Strathclyde team in the BDBC
2017 hosted in Glasgow, where different feature extraction techniques were compared under the same
conditions
Figure 5. A diagram of the methodology proposed by the University of Strathclyde team in the BDBC
2017 hosted in Glasgow, where different feature extraction techniques were compared under the
same conditions. conditions. The raw EEG evaluated was the same for all the cases, where a common infinite impulse
response (IIR) filter (Butterworth) was implemented for pre-processing. Afterwards, different feature
extraction techniques were implemented. 5.1. Selected Data Set A total of 120 trials per session were
acquired, leading to 120 repetitions of left MI class and 120 repetitions of right MI class out of the two
sessions. This data set also included information related to electrooculography (EOG), but it was not
used in the case study. Further details about this data set can be found in [118]. (a)
(b)
Figure 4. This figure includes information about the acquisition of data set number 4 in BNCI Horizon
2020 [117], where (a) shows the electrodes (C3, CZ, C4) placement on the head [118] and (b) shows the
time scheme paradigm [118] followed during data acquisition. Figure 4. This figure includes information about the acquisition of data set number 4 in BNCI Horizon
2020 [117], where (a) shows the electrodes (C3, CZ, C4) placement on the head [118] and (b) shows the
time scheme paradigm [118] followed during data acquisition. (a)
(b)
Figure 4. This figure includes information about the acquisition of data set number 4 in BNCI Horizon
2020 [117], where (a) shows the electrodes (C3, CZ, C4) placement on the head [118] and (b) shows the
time scheme paradigm [118] followed during data acquisition. (a)
(a)
Figure 4 This figure includes info (b)
(b)
d (a)
(a)
Thi fi Figure 4. This figure includes information about the acquisition of data set number 4 in BNCI Horizon
2020 [117], where (a) shows the electrodes (C3, CZ, C4) placement on the head [118] and (b) shows the
time scheme paradigm [118] followed during data acquisition. Figure 4. This figure includes information about the acquisition of data set number 4 in BNCI Horizon
2020 [117], where (a) shows the electrodes (C3, CZ, C4) placement on the head [118] and (b) shows the
time scheme paradigm [118] followed during data acquisition. Figure 4. This figure includes information about the acquisition of data set number 4 in BNCI Horizon
2020 [117], where (a) shows the electrodes (C3, CZ, C4) placement on the head [118] and (b) shows the
time scheme paradigm [118] followed during data acquisition. 5.3. Performance Comparison
5.3. Performance Comparison After implementing and running the data processing workflow for each one of the feature
extraction techniques mentioned, it was possible to compare the classification accuracy and also the
computation time needed in order to extract the features. Figure 6 shows this comparison among TM,
SM, A-BP, S-BP and FFT. After implementing and running the data processing workflow for each one of the feature
extraction techniques mentioned, it was possible to compare the classification accuracy and also the
computation time needed in order to extract the features. Figure 6 shows this comparison among TM,
SM, A-BP, S-BP and FFT. (a)
(b)
Figure 6. Performance comparison among TM, SM, A-BP, S-BP and FFT feature extraction techniques
evaluated under the same conditions, where (a) shows the classification accuracy (%) and (b) shows
the approximated computation time (μs) required to extract the features. Computation time ( s)
Figure 6. Performance comparison among TM, SM, A-BP, S-BP and FFT feature extraction techniques
evaluated under the same conditions, where (a) shows the classification accuracy (%) and (b) shows
the approximated computation time (µs) required to extract the features. (a) (b)
Computation time ( s) (b) (a) Figure 6. Performance comparison among TM, SM, A-BP, S-BP and FFT feature extraction techniques
evaluated under the same conditions, where (a) shows the classification accuracy (%) and (b) shows
the approximated computation time (μs) required to extract the features. Figure 6. Performance comparison among TM, SM, A-BP, S-BP and FFT feature extraction techniques
evaluated under the same conditions, where (a) shows the classification accuracy (%) and (b) shows
the approximated computation time (µs) required to extract the features. Features from all evaluated techniques were able to provide an accuracy close to 70%, where the
difference among them is not especially significant. Additionally, there is not much difference among
time-domain- and frequency-domain-based techniques, achieving 73% (SM) and 72.3% (S-BP)
accuracies, respectively. This fact may highlight the difficulty in processing EEG data, as it seems not
feasible to go further than the given level of accuracy, probably due to the inherent EEG data nature,
including its acquisition process. Features from all evaluated techniques were able to provide an accuracy close to 70%, where
the difference among them is not especially significant. Additionally, there is not much difference
among time-domain- and frequency-domain-based techniques, achieving 73% (SM) and 72.3% (S-BP)
accuracies, respectively. 5.2. Data Processing Workflow
5.2. Data Processing Workflow
5.2. Data Processing Workflow For example, [119] used this technique in order to detect transient events within
EEG recordings from infants, [120] used it to identify early indications of seizures in epileptic patients
and [123] used TM to flap VEP events in a BCI application. SM has also been used in the detection of
EEG markers for epileptic seizures [123,124] and as well as for the classification of signals based on
modulation [122]. Sensors 2019, 19, 1423 17 of 34 17 of 34 A-BP techniques have been used for the classification of left and right MI [125], as well as for a
four-class MI classification problem involving left hand, right hand, feet and tongue MI tasks [25]. They have also been used for the other band-power related EEG classification problems [126]. S-BP has
also been used for MI classification tasks [69], as well as classification of other mental tasks including
math tasks, geometric rotation, visual counting and letter composition, which are all related to the
frequency content of the signal [128]. It has also shown potential to be used in a practical application,
in which EEG signals are processed as subjects move around a virtual environment [118]. Sensors 2019, 19, x FOR PEER REVIEW
17 of 34
They have also been used for the other band-power related EEG classification problems [127]. S-BP
has also been used for MI classification tasks [69], as well as classification of other mental tasks
including math tasks, geometric rotation, visual counting and letter composition, which are all related
to the frequency content of the signal [129]. It has also shown potential to be used in a practical
application, in which EEG signals are processed as subjects move around a virtual environment [118]. The FFT is also a powerful frequency-domain analysis tool used in EEG signal processing [128–130]. For example, it has been used for the identification of emotional state from EEG data [128,129], as well as
for a classification problem involving the identification of Alzheimer’s from EEG data [130]. pp
,
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[
]
The FFT is also a powerful frequency-domain analysis tool used in EEG signal processing [129–
131]. For example, it has been used for the identification of emotional state from EEG data [129,130],
as well as for a classification problem involving the identification of Alzheimer’s from EEG data [131]. 5.2. Data Processing Workflow
5.2. Data Processing Workflow
5.2. Data Processing Workflow After the feature extraction stage, a common feature selection was introduced, being based on
selecting a particular subset of the extracted features for each case. Finally, the selected features were
used to train, validate and test a classification model based on SVM. The classification accuracy of
the SVM is directly dependent on the features extracted and, therefore, is different for each of the
techniques included in the evaluation. p
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[
]
After the feature extraction stage, a common feature selection was introduced, being based on
selecting a particular subset of the extracted features for each case. Finally, the selected features were
used to train, validate and test a classification model based on SVM. The classification accuracy of
the SVM is directly dependent on the features extracted and, therefore, is different for each of the
techniques included in the evaluation. 6.1.1. Replacement and Restoration of CNS These technologies can restore or replace functionality of the CNS which was lost due to illnesses
such as amyotrophic lateral sclerosis (ALS) and locked-in syndrome, as well as people suffering
from paralysis, amputations and loss of CNS functionality due to trauma, such as spinal cord injury. As previously mentioned, the development of such technologies can be challenging due to the altered
brain functionality that patients with such conditions can experience. Currently, many robotic prosthetics depend on myoelectrics, which record electrical signals
in muscles. However, such technologies are expensive and assume that nerve connections are
largely functional, limiting their applicability for fine control of prosthetics and for patients with CNS
injury [131]. BCI-based prosthetics can solve these problems. Müller-Putz and Pfurtschscheller [132]
implemented an SSVEP-based robotic arms system with 4 flickering stimuli, each one representing a
different function for the arm: later movement to the left or right and opening or closing of the hand. The user selected a movement by looking at the associated stimulus. The system was tested on only
4 subjects, and had a classification accuracy of between 44% and 88%. Such an SSVEP-based system
faces the system-specific challenges previously discussed in Section 2. Elstob and Secco [133] propose a low-cost BCI prosthetic arm based on MI, and which has 5 degrees of
freedom of movement, as opposed to two. The hardware used in the system is shown in Figure 7; note that
an EEG diadem was used. Although this looks significantly different from the standard EEG caps used in
clinical research, manufacturers of EEG diadems tend to place electrodes in standard positions according
to the 10–20 system. Elstob and Secco reported an accuracy of between 56% and 100%, depending on the
movements carried out. MI-based systems may be more suitable than SSVEP systems since they are more
intuitive and remove the fatigue associated with looking at the flickering stimuli. However, as previously
discussed, MI data can be difficult to generate in the brain. Müller-Putz et al. [134] also implemented an MI-based robotic arm system, but with 3 degrees of
freedom. They also designed a novel 64-electrode sleeve which can be worn by the user which gives
feedback via electrical pulses as to the movements carried out, in a process known as functional electrical
stimulation (FES). Although the approach is promising, classification accuracies between 37% and 57%
were obtained. 6.1. Biomedical Applications Roadmaps and research associated with BCI technology has overwhelmingly been focused on
medical applications [2,8], with many BCIs intended for the replacement or restoration of central
nervous system (CNS) functionality which was lost due to disease or injury [2]. Other BCIs are focused
on therapy and motor rehabilitation after illness or trauma to the CNS, in diagnostic applications
and finally BCIs are being used in affective computing for biomedical applications. Each of these
applications will be discussed in more detail in the following subsections. As well as empowering
people suffering from mobility issues or facilitating their recovery, these technologies can also reduce
the time and cost of care. A core challenge in the development of such systems is the need to design
accurate technologies which can deal with the possibly atypical brain responses which can be the
result of illnesses such as stroke [5,35]. 5.3. Performance Comparison
5.3. Performance Comparison This fact may highlight the difficulty in processing EEG data, as it seems not
feasible to go further than the given level of accuracy, probably due to the inherent EEG data nature,
including its acquisition process. g
q
p
On the other hand, the computation time required for extracting features can make a difference,
as some techniques such as TM and A-BP took roughly 3 μs, while others needed up to 40 μs (FFT). Therefore, the main finding from this case study, as presented in the BDBC [115], was to highlight
the importance of computational efficiency, where while the accuracy cannot be increased, at least
high-speed real-time BCI can be proposed, with potential introduction of GPU and FPGA
On the other hand, the computation time required for extracting features can make a difference,
as some techniques such as TM and A-BP took roughly 3 µs, while others needed up to 40 µs (FFT). Therefore, the main finding from this case study, as presented in the BDBC [115], was to highlight
the importance of computational efficiency, where while the accuracy cannot be increased, at least
high-speed real-time BCI can be proposed, with potential introduction of GPU and FPGA architectures. Sensors 2019, 19, 1423 18 of 34 18 of 34 6. Applications Numerous applications exist for BCIs, and the design of a BCI depends on the intended
application [5]. Nijholt [8] suggests that there are two main branches of BCI applications: control and
monitor. Control applications are oriented towards manipulating an external device using brain signals
while monitor applications are oriented towards identifying the mental and emotional state of the user
in order to control the environment they are in or the interface they are using. Practical applications of
BCI technologies within and outside the biomedical sphere are discussed in the following subsections,
with some mentions of the challenges that can be incurred in the development of such systems. 6.1.1. Replacement and Restoration of CNS FES can be used to provide feedback and help to restore aspects of CNS functionality 19 of 34
is the Sensors 2019, 19, 1423
functionality in s in some patients. A challenge when designing such innovative systems is the harmonious merging of
system components, in this case the novel EEG system and the FES aspect of the system, a process that can
require numerous incremental system developments. A study [135] into the performance of off-the-shelf
anthropomorphic arms controlled via BCI showed that such systems hold promise. g
g
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p
y
p
the system, a process that can require numerous incremental system developments. A study [136]
into the performance of off-the-shelf anthropomorphic arms controlled via BCI showed that such
systems hold promise. BCI interfaces are also used for wheelchair control. One prototype uses a GUI to list the different BCI interfaces are also used for wheelchair control. One prototype uses a GUI to list the different
movement options available, and P300 signals are processed to identify the intentions of the user. Voznenko et al. [136] used an extended BCI to replace the joystick functionality of a wheelchair. The system enabled the user to choose to control movement using thought, voice or gestures. This was
a novel approach in which the three control systems worked in parallel to form an ‘extended’ BCI
based on the idea that a user can master a BCI system more effectively when there are multiple control
channels. The core challenge in this project was data fusion during decision making when multiple
control signals were received at once. movement options available, and P300 signals are processed to identify the intentions of the user. Voznenko et al. [137] used an extended BCI to replace the joystick functionality of a wheelchair. The
system enabled the user to choose to control movement using thought, voice or gestures. This was a
novel approach in which the three control systems worked in parallel to form an ‘extended’ BCI based
on the idea that a user can master a BCI system more effectively when there are multiple control
channels. The core challenge in this project was data fusion during decision making when multiple
control signals were received at once. Chella et al. [138] propose a teleoperated robot based on a P300 BCI, enabling remote control of Chella et al. 6.1.2. Therapy, Rehabilitation and Assessment
6.1.2. Therapy, Rehabilitation and Assessment Robotic BCIs are not only used in neuroprosthetics, but also in therapeutic applications. Stroke
rehabilitation can be aided by BCI-controlled robotic arms which guide subjects arm movements
[139] and social robots such as that implemented in [140], in which the user imagines motor
movements which are then carried out by the robot. A core challenge in the development of
rehabilitation technologies is the additional refinement which must be done for systems to pass
Robotic BCIs are not only used in neuroprosthetics, but also in therapeutic applications. Stroke rehabilitation can be aided by BCI-controlled robotic arms which guide subjects arm
movements [138] and social robots such as that implemented in [139], in which the user imagines motor
movements which are then carried out by the robot. A core challenge in the development of rehabilitation
technologies is the additional refinement which must be done for systems to pass clinical trials. rehabilitation technologies is the additional refinement which must be done for systems to pass
clinical trials. BCIs are also being applied to virtual reality (VR) for rehabilitative applications. Luu et al. [141]
propose a system which decodes brain activity while subjects walk on a treadmill and provide visual
feedback to the user on their movements through a virtual avatar. Such systems may hold promise
in post-stroke therapy, and future challenges in the area would involve accurate control of the avatar. Other systems are-based totally in virtual worlds, such as that reported in [142], in which users can
control 3D objects within a VR setting via EEG signals. This system is open-source and inexpensive
and, with further development, holds promise for application in stroke rehabilitation as well as
BCIs are also being applied to virtual reality (VR) for rehabilitative applications. Luu et al. [140]
propose a system which decodes brain activity while subjects walk on a treadmill and provide visual
feedback to the user on their movements through a virtual avatar. Such systems may hold promise
in post-stroke therapy, and future challenges in the area would involve accurate control of the avatar. Other systems are-based totally in virtual worlds, such as that reported in [141], in which users can
control 3D objects within a VR setting via EEG signals. This system is open-source and inexpensive
and, with further development, holds promise for application in stroke rehabilitation as well as
entertainment. 6.1.1. Replacement and Restoration of CNS [137] propose a teleoperated robot based on a P300 BCI, enabling remote control
of the movement of a robot in a space via a BCI. They suggest one application for this system could
be an electronic museum guide, which can send video to the user. Teleoperated systems may suffer
from particular challenges when it comes to delays in control signals being sent, particularly when the
signals travel over the Internet. In such circumstances, the tele-operated robot, in particular, must have
control systems which come into play to prevent it from injuring bystanders or damaging property as
it moved through the space. the movement of a robot in a space via a BCI. They suggest one application for this system could be
an electronic museum guide, which can send video to the user. Teleoperated systems may suffer from
particular challenges when it comes to delays in control signals being sent, particularly when the
signals travel over the Internet. In such circumstances, the tele-operated robot, in particular, must
have control systems which come into play to prevent it from injuring bystanders or damaging
property as it moved through the space. (a)
(b)
Figure 7. This figure shows the hardware setup used for a low-cost MI-based EEG system e.g. in [134],
[137] where (a) shows the 3D-printed prosthetic arm which was controlled and (b) shows the EEG
headset used. Figure 7. This figure shows the hardware setup used for a low-cost MI-based EEG system e.g.,
in [133], [136] where (a) shows the 3D-printed prosthetic arm which was controlled and (b) shows the
EEG headset used. (a) (b) (b) (a) Figure 7. This figure shows the hardware setup used for a low-cost MI-based EEG system e.g. in [134],
[137] where (a) shows the 3D-printed prosthetic arm which was controlled and (b) shows the EEG
headset used. Figure 7. This figure shows the hardware setup used for a low-cost MI-based EEG system e.g.,
in [133], [136] where (a) shows the 3D-printed prosthetic arm which was controlled and (b) shows the
EEG headset used. 6.1.3. Affective Computing for Biomedical Applications In affective computing BCIs, users’ mood and psychological state are monitored, possibly in
order to manipulate their surrounding environment to enhance or alter that state. For example,
Ehrlich et al. [144] implemented closed-loop system in which music is synthesized based on the users’
affective state and then played back to them. Such a system could be used to study human emotions
and sensorimotor integration. Affective computing can also be used to help patients with serious
neurological disorders to communicate their emotions to the outside world [145]. 6.1.2. Therapy, Rehabilitation and Assessment
6.1.2. Therapy, Rehabilitation and Assessment The neurofeedback in such systems would need to be adjusted according to the user Sensors 2019, 19, 1423 20 of 34 and the application, particularly when the neurofeedback from patients with CNS illness or injury is
used, as this feedback may not be reliable. Assessment and diagnosis in a clinical setting can also be complemented by the use of BCIs. In [142] a BCI which users used to play serious games was proposed for the assessment of attention of
children with cerebral palsy. Another study [143] investigated EEG features recorded via BCI as an
aid for the diagnosis of schizophrenia. Assessment and diagnosis technologies play a critical role in
patient wellbeing, and their functionality must be heavily refined to ensure they are safe, appropriate
and have industry-standard high-levels of accuracy. 6.2. Non-Biomedical Applications In recent years, the economic potential of BCI technologies has emerged, particularly in the areas
of entertainment, gaming and affective computing [8]. While medical or military applications require
researchers to focus on robustness and high efficiency, technologies aimed at entertainment or lifestyle
require a heavier focus on enjoyment and social aspects [8]. A key challenge in the design of such
systems is how to identify appropriate and interesting systems that would be appealing to commercial
users, as well as ensuring they are robust enough for being marketed to a wide and varied audience. BCIs can also be used to enhance CNS output, and to control the environment the user
experiences [146]. Such applications include assistive technologies in the form of affective control of
a domestic environment which caters for the emotional needs of the individual in the space as well
as in the transport, games and entertainment industries. This close interaction between the BCI and
the user, particularly the influence on a user’s mood, can raise ethical issues, since such technologies
could be used to exploit user emotions to push targeted marketing or political agendas. Thus, a future
challenge would involve outlining regulatory controls on the applications of such technologies, as well
as technological safeguards to prevent abuse. BCIs can also be used in order to forward research by
driving the development of signal processing algorithms, machine learning, artificial intelligence and
hardware interfaces [2,8]. This section investigates some of the prominent non-biomedical applications
of BCIs. Less commonly [147], P300 potentials are used to control the BCIs [148,149]. Contrary to many
systems depending on SSVEP waves, for systems based on P300 potentials, users do not need to be
trained. Also, P300 waves are more robust than SSVEP waves, and can be evoked visually or by audio. 6.2.1. Gaming BCIs
primarily
aimed
at
the
gaming
industry
have
become
a
significant
area
of
research [8,147,150,151]. However, gaming BCIs currently represent a poor replacement for traditional
methods of controlling games [147], and this represents a possible area for development. Some current
technologies depend on evoked potentials, such as the simple SSVEP-based implementation presented
in [152] and the more advanced system in [153], which combined SSVEP data and MI data to control
a version of Tetris. EEG data has also been used to control difficulty level in multiplayer games,
by triggering dynamic difficulty adjustment (DDA), which increases difficulty for strong players
and decreases difficulty for weaker players. In the system, EEG data was used to monitor each
players’ personal excitement level and activate DDA when the players experience a drop in excitement,
to increase engagement [154]. Refining the algorithms that govern the behavior of the game is a 21 of 34 Sensors 2019, 19, 1423 significant problem when building such systems. In recent years, commercial BCI-based systems have
been emerging in the gaming market [155]. significant problem when building such systems. In recent years, commercial BCI-based systems have
been emerging in the gaming market [155]. 7. Challenges and Future Directions Although research into BCI technology has been ongoing for the last 20 years, these technologies
have remained largely confined to a research environment and have yet to infiltrate into clinical and
home settings. This section discusses the main challenges preventing the widespread adoption of BCIs,
which were divided into five categories: (i) challenges faced during the research and development of
BCIs, (ii) challenges which impede commercialization, (iii) flaws in the testing approaches commonly
seen in the literature, (iv) issues encountered during BCI use which may impede their widespread
uptake, and (v) ethical issues. 6.2.3. Art Wadeston et al. [158] identified four different types of BCIs for artistic applications: passive,
selective, direct and collaborative. Passive artistic BCIs require no active input from the user and
merely select which pre-programmed response to output based on the brain activity of the user. For example, in the BMCI piano [159], the passive brain signals of the user determine which pieces of
music are played, as well as the volume and tempo. In selective systems, the user has some restricted
control over the system but are still not having a leading role in the creative output. For example,
in the drawing application proposed by Todd et al. [160], four SSVEP stimuli are used to enable the
user to choose which type of shape is drawn on screen; however, the application decides on where the
shape is positioned and its color. Direct artistic BCIs give users much higher levels of control, typically
enabling them to select options from detailed menus, with options including things such as brush
type and controlling brush stroke movements [161]. Finally, collaborative systems can be controlled by
multiple users at once [160]. A future development for artistic BCIs could be a merging with virtual
reality, in which designers can meet, collaborate and create designs in a virtual space. The design of
artistic BCIs involves understanding the artistic process to ensure that the technology is designed to be
a help rather than a hindrance. 6.2.2. Industry and Transport Industrial robotics is another area of application for EEG-based BCIs [155], and such technologies
can improve safety in the workplace by keeping humans away from dangerous activities. Such systems
could replace tedious button and joystick systems used to train robots in industrial settings, and they
can also be used to monitor when a user is too tired or unwell to operate the machinery and
take appropriate steps to mitigate any danger, such as by stopping a piece of machinery [155]. Similar BCIs which monitor awareness can also be applied to transport, in order to monitor the
fatigue of drivers [156] and monitor to improve the performance of airline pilots [157]. Such systems
are used in critical applications, and poor decisions can be expensive in terms of both the human life
and monetary burdens on the entities involved. Thus, a key issue in such BCIs is to ensure robustness,
reliability and constituent high accuracy despite the fact that EEG data is highly nonlinear and noisy,
and prone to inter and intra-individual changeability. 7.2.1. Technical Barriers to Commercialization To obtain a clear picture of the barriers preventing the commercialization of BCI interfaces,
Vansteensel et al. [163] sent out a questionnaire to over 3500 BCI researchers worldwide, 95% of which
worked in BCIs related to EEG or electromyography (EMG) technologies. Overall, the researchers
surveyed were confident that BCIs, particularly those to replace or enhance brain functionality, could
be commercialized and were realizable within the next 5 to 10 years. The survey indicated that, in the
case of non-invasive BCIs, major technological developments were needed in sensors, overall system
performance and user-friendliness, while in the case of invasive BCIs, there was a need to develop
completely implantable systems, improve system robustness and performance, and clinical trials
would need to be carried out to ensure the safety of such systems. Van Steen and Kristo [2] also suggested research in BCI technology would need to focus
on improving bit rates [164], improving signal processing techniques and exploring classification
approaches. On the macro-scale, they propose formulating novel approaches to overall BCI system
design and the types of control systems used. 7.1. Challenges Faced in Research and Development The research and development of BCI interfaces, particularly those based on MI EEG, are fraught
with signal processing challenges. These include identifying the most effective techniques for feature
extraction and selection, which is challenging due to the highly non-linear, non-stationary and
artefact-prone nature of EEG data. Other challenges include data fusion, particularly how the
data from different EEG channels can be combined in order to reduce data dimensionality and
also possibly improve the classification results. Further investigations are also needed to identify the
best classification techniques for the selected features. Research into features and classifiers should 22 of 34 Sensors 2019, 19, 1423 also focus on identifying the best methods to be used for patients with CNS injury or illness, as the MI
EEG characteristics of such patients can differ from that of healthy individuals. also focus on identifying the best methods to be used for patients with CNS injury or illness, as the MI
EEG characteristics of such patients can differ from that of healthy individuals. Research also needs to investigate more effective training approaches. For a BCI to be used
by a particular subject, a large number of training trials are typically required from that particular
subject, leading to the calibration stage being unacceptably time consuming for a practical system. Thus, studies focused on reducing the calibration time are required. Attempts have been made to
decrease the training time by using the covariance matrices associated with CSP features extracted from
EEG trials to help the decoding of EEG signals [48,55,58]. However, these approaches fail to exploit the
geometry of the covariance matrix, even though this can be used to extract salient information from
EEG data [162]. The geometric properties of the covariance matrices exist in the symmetrical positive
definite (SPD) space, and Singh et al. [162] developed a framework which used SPD characteristics to
reduce calibration time. This framework outperformed other methods previously tested on the IVa
dataset. Other avenues of research to reduce the calibration time may involve developing EEG models
which are more generalizable. Finally, a hallmark challenge in the research and development of BCIs is the design of dependable
systems with a stable performance, that can be used by a wide variety of users, with different mental
states and in different environments. 7.2. Challenges Impeding Commercialization The commercialization of BCIs is impeded by two main obstacles: the first is concerned with
technical barriers which can be solved through the development of more robust, efficient and accurate
signal processing infrastructures, and the second involves adapting lab-based technologies for use in
the wider world. These two issues are discussed in greater depth in this section. 7.2.2. Adapting Lab-Based Technologies for the Wider World Intuitivism is linked to the choice of brain signals used, which should be application-appropriate. For example, MI-based technologies may be highly suited for the control of a prosthetic limb or a
remote robotic arm since the concept of bodily movement would be intuitive in this kind of scenario. However, for the control of a digital radio, an SSVEP-based system with a 4-option menu consisting of
station-up, station-down, volume-up and volume down options may be more intuitive than a MI-based
system with imagined movements being associated with television controls. This sense of intuitivism
should be factored during the earliest decision phase, when choices of technology and BCI type are
being made. General appeal is another factor underlying all aspects of the design process, and covers anything
that may affect the initial impression of the user in relation to the system. The portability of the system
is important: headsets which use Bluetooth or Wi-Fi enable users to move around while still being able
to use the system, as compared to a wired headset which restricts movements. The aesthetic appeal of
the headset and GUIs used would also affect uptake of the technologies. Furthermore, a choice can be
made about the type of electrodes used: whether wet or dry electrodes should be used. For a practical
application, dry electrodes would be more appropriate as they do not involve the hassle of placing
electro-conductive gel between the scalp electrodes, and there is no residue of gel left in the hair after
use. However, there is open debate on whether dry electrodes provide the same quality of signal, with
some research suggesting dry electrodes produce signals which are noisier and more prone to artifacts
than wet electrodes [172], while other research suggests that the signal quality is similar for both
types of electrodes [173]. Recently, water-based electrodes have also been studied. It was observed
in [174] that in subjects with shorter hair, water-based and dry electrodes performed comparably to
gel-based electrodes, and it was suggested that with further refinement of the electrodes for subjects of
varying hair length, water-based and dry electrodes held potential to be used instead of gel electrodes. 7.2.2. Adapting Lab-Based Technologies for the Wider World One of the biggest leaps in commercialization of BCIs is adapting the interfaces used in the lab
for use in the wider world. Although BCIs hold potential to be applied to various areas including
home automation [165,166], prosthetics [2,8,133,134], rehabilitation [138–140], gaming [8,147,150,151],
transport [157,158], education [167], VR [142], artistic computing [159–162], and possibly even
virtual assistants based on affective computing, the leap to creating viable products involves
considering several factors. These factors mainly include: (i) choice of technology, (ii) general appeal,
(iii) intuitivism, (iv) usability and reliability and (v) cost [2,59,168–171]. Each of these factors will
be discussed in more detail in this section. However, when designers make decisions on any of
these factors, it is very important to consider the particular situation and environment in which the
technology may be used. For example, the design requirements may be different when designing an 23 of 34 Sensors 2019, 19, 1423 affordable technology for the control of a television in a home environment when compared to a BCI
system for monitoring the alertness of a pilot in a plane. Similarly, designers must factor in the health
of the users; technologies designed for rehabilitation or restoration of lost CNS functionality will often
have different or additional requirements when compared to technologies intended for healthy users. Thus, during the earliest stages of system design, it is important that system designers carry out an
in-depth analysis of the basic requirements of the system, factoring the environment, situation and
target audience. The choice of technology is the first and arguably the most fundamental step taken when designing
a human-computer interface, particularly one intended for use in practical situations. The choice of
technology involves first considering all the possible hardware options for the interface, including EEG,
NIRs, EMG, EOG and other type of eye-gaze tracking technology, as well as hybrid combinations of these
technologies. At this stage, it may be determined that a BCI is not the best option for the commercial
application in mind, and an alternative type of technology should be considered. When considering a BCI
technology, the choice between an evoked and spontaneous system needs be made, and it is important to
factor the trade-offs between these two technologies, which were discussed in Section 2. The following
discussions are framed assuming that an EEG-based technology was chosen. Intuitivism is an essential element for any system to be commercializable in the long-term. 7.2.2. Adapting Lab-Based Technologies for the Wider World Until there is a more conclusive consensus on the issue, or the technologies available from different
manufacturers become more homogenous in terms of recording capabilities, researchers aiming to
develop practical systems using dry electrodes should study the signal processing capabilities of the
specific EEG systems and electrodes available to them in order to assess whether the signal quality of
appropriate, following constraints suggested in the research [172], and studying in particular the noise
and artefacts produced in the EEG signals, as in [172,173]. To be successful on the market, BCI systems must be highly usable and reliable. Usability covers
design ergonomics, as well as ease-of-use and the time taken for a new user to train on the system-
which should be minimal. The ideal technology would be one which average users can pick up
and learn how to use through intuition or following a short tutorial, similar to when buying a new 24 of 34 Sensors 2019, 19, 1423 mobile. The systems must also be user-friendly and have in-built safeguards to prevent dangerous use. Furthermore, systems need to also be reliable, with users feeling that the technology is dependable
and provides stable results following the initial learning period associated with the new technology. The system should also be reliable when used in the multisensory environments in which it is targeted
to be used, such as a noisy family home, a busy design studio or the changeable atmosphere of
an emergency operating theatre. These multisensory environments may alter the expected brain
signals when compared to controlled lab recordings, and thorough development and testing of such
systems would need to be carried out. The state of the user during use may also need to be factored,
as heartrate and cortisol levels—a hormone associated with stress—are known to interact with EEG
signal response [175,176]. Seo and Lee found a significant positive correlation between increased
power in the beta band—which is associated with MI EEG data—and cortisol level [176]. Finally, cost is another significant obstacle. The average budget of the expected end users should
be factored early on, as well as possible economies of scale, as this will determine the types of recording
equipment and sensors that can be used, as well as the software and any compliance issues related to
the target audience. 7.2.2. Adapting Lab-Based Technologies for the Wider World Although the aim should always be to provide the best trade-off between cost and
performance, the price ranges affordable by students, families, private healthcare companies, the military,
start-ups and large technological corporations, to name but a few possible target audiences, vary just as
widely as their needs. Researchers serious on developing new technologies to target a particular audience
would do well to consider market research prior to beginning the design process of the system. The commercialization of BCIs would also require the establishment of industry standards,
particularly in terms of databases used for benchmarking, EEG recoding equipment and software
applications [177]. International roadmap projects such as BNCI Horizon 2020 [178] have attempted to
improve communication within industry and research, so that such issues can be resolved. 7.3. A Flawed Testing Process 7.3. A Flawed Testing Process To implement these recommended improvements, extensive testing on wide populations is required,
but the testing process in itself is often flawed. In the literature, a wide array of performance measures
have been used to evaluate BCIs, and the lack of a standard approach or single metric to quantify the
general performance of a system means limited comparison can be carried out among the systems in the
literature [179]. Furthermore, the reporting of basic statistics such as accuracy may obscure prominent
issues in the BCI system such as the trade-off between accuracy and speed, and to remedy this, global
performance measures such as the utility metric have been recommended in [179,180]. Flaws in widely adopted testing approaches are not limited to the choice of performance metrics,
but also the testing data used. Many BCIs which are developed to replace or restore CNS functionality
are tested on healthy subjects within a laboratory environment. However, this can lead to unrealistically
positive results if typical end-users of these systems would be patients with functional disabilities
which have resulted from damage to CNS tissue, for example patients with spinal cord injuries,
as BCI performance tends to be poorer with these subjects when compared to healthy controls. Thus, researchers should factor the needs of users with CNS damage during the design and testing of
BCI systems for restoring CNS functionality, as was done in [181]. Although there has been research
into the design of rehabilitation technologies based on MI for patients following a stroke [5,182–184],
and in people with ALS [185], there are a plethora of other illnesses or conditions which can affect
brain and CNS functioning. These may impede the use of EEG-based BCIs by users who could benefit
significantly from them. Conditions which affect the brain or CNS, but have yet to be investigated
in-depth in relation to MI EEG-based BCIs, include mild cognitive impairment, diabetes causing loss
of cognition, specific types of brain trauma, impairments due to disk and hernia problems, locked-in
syndrome, multiple sclerosis, Huntington’s disease and Parkinson’s disease. A comprehensive
database of MI EEG data from such patients, as well as healthy controls, all performing the same tasks,
would provide a wealth of data for research so the robustness of technologies can be tested, and new
solutions found if they fail users with certain conditions. 7.3. A Flawed Testing Process Sensors 2019, 19, 1423 25 of 34 Even data from healthy subjects tends to be recorded in controlled lab environments. Although this data has an important role in the initial evaluation of signal processing techniques,
the development of more robust and commercializable systems would involve testing under stressful
conditions. As previously mentioned, heartrate and cortisol can affect the quality of EEG signals,
in environments outside the lab, both indoors and outdoors, with varying sensory stimulation in the
environment such as noises, movements and smells, and with the subjects sat in different postures. A database of recording data from the same set of subjects performing the same set of MI tasks in all
these different scenarios, as well as in a controlled laboratory setting would be a valuable starting
point in order to find out how the effectiveness of MI data processing techniques can vary between
different scenarios, and how these techniques can be made more robust. Also, for BCIs to be used in
practical applications, more research is needed into how external factors related to the individual’s
lifestyle can affect BCI performance; for example, consumption of sugar-based drinks has been found
to decrease performance of a BCI [186]. 7.4. Issues with BCI Use BCI illiteracy is another impediment to the universal adoption of BCI technologies, particularly
in EEG-based interfaces. Illiteracy occurs when a user is unable to control a BCI because they do not
manage to produce the high-quality brain signals required [177,187]. EEG signal quality, as well as
overall mastery of the BCI, can be improved by using an interactive, co-learning approach which
provides the user with feedback, such as audio or visual feedback, as they use the system [177]. Long-term use of BCIs requires repeated use of particular neural pathways, and more research
needs to be carried out in order to identify the possible health implications or changes in brain
functionality due to this. For example, studies have indicated that long-term use of external actuators
through BCIs has led to restructuring of the brain’s map of the body, with the actuators being perceived
by the brain as an extension to the subject’s body [177,188]. 7.5. Ethical Issues Ethical standards must also be established in order to guide the development of BCI technology
into the future. Such ethics would establish liability in the case of accidents occurring during the use of
controlled apparatus, as well as deal with the appropriate use of bio-signal data and privacy. The BCI
Society aims to meet some of these needs by releasing standards and guidelines associated with ethical
issues [8,177,178]. Author Contributions: The contributions of the co-authors can be summarized as follows: conceptualization, J.R. and H.Z.; methodology/software/validation for case study: J.Z., V.M. and J.R.; formal analysis/investigation, J.R.,
J.Z. and H.Z.; resources, N.P./J.R.; writing—original draft preparation, N.P. and J.Z.; writing—review and editing,
J.R., H.Z. and V.M.; supervision/project administration and funding acquisition, J.R. Funding: This research was funded by the PhD Scholarship Scheme of the University of Strathclyde, National
Natural Science Foundation of China (61672008), Guangdong Provincial Application-oriented Technical Research
and Development Special fund project (2016B010127006), Scientific and Technological Projects of Guangdong
Province (2017A050501039) and Guangdong Key Laboratory of Intellectual Property Big Data (No.2018B030322016)
of China. Conflicts of Interest: The authors declare no conflict of interest. 1.
Soegaard, M.; Dam, R.F. Human Computer Interaction—Brief intro. In The Encyclopedia of Human-Computer
Interaction, 2nd ed.; The Intetraction Design Foundation: Aarhus, Denmark, 2012.
2.
Van Steen, M.; Kristo, G. Contribution to Roadmap. 2015. Available online: https://pdfs.semanticscholar.
org/5cb4/11de3db4941d5c7ecfc19de8af9243fb63d5.pdf (accessed on 28 January 2019).
3.
Baig, M.Z.; Aslam, N.; Shum, H.P.H.; Zhang, L. Differential evolution algorithm as a tool for optimal feature
subset selection in motor imagery EEG. Expert Syst. Appl. 2017, 90, 184–195. [CrossRef] References Brain computer interfaces, a review. Sensors 2012, 12, 1211–1279. [CrossRef] [PubMed] 0. Stawicki, P.; Gembler, F.; Rezeika, A.; Volosyak, I. A novel hybrid mental spelling application based on
tracking and SSVEP-based BCI. Brain Sci. 2017, 7, 35. [CrossRef] [PubMed] 11. Kevric, J.; Subasi, A. Comparison of signal decomposition methods in classification of EEG signals for
motor-imagery BCI system. Biomed. Signal Process. Control 2017, 31, 398–406. [CrossRef] 12. Caldwell, J.A.; Prazinko, B.; Caldwell, J.L. Body posture affects electroencephalographic activity and
psychomotor vigilance task performance in sleep-deprived subjects. Clin. Neurophysiol. 2003, 114, 23–31. [CrossRef] 13. Thibault, R.T.; Lifshitz, M.; Jones, J.M.; Raz, A. Posture alters human resting-state. Cortex 2014, 58, 199–205. [CrossRef] 14. Suefusa, K.; Tanaka, T. A comparison study of visually stimulated brain-computer and eye-tracking interfaces. J. Neural Eng. 2017, 14, 036009. [CrossRef] [PubMed] 5. Speier, W.; Deshpande, A.; Pouratian, N. A method for optimizing EEG electrode number and configura
for signal acquisition in P300 speller systems. Clin. Neurophysiol. 2015, 126, 1171–1177. [CrossRef] [PubM 6. Schreuder, M.; Rost, T.; Tangermann, M. Listen, you are writing! Speeding up online spelling with a dyna
auditory BCI. Front. Neurosci. 2011, 5, 112. [CrossRef] 17. Chambayil, B.; Singla, R.; Jha, R. Virtual keyboard BCI using Eye blinks in EEG. In Proceedings
of the 2010 IEEE 6th International Conference on Wireless and Mobile Computing, Networking and
Communications, Niagara Falls, ON, Canada, 11–13 October 2010. [CrossRef] 18. Tang, Z.; Sun, S.; Zhang, S.; Chen, Y.; Li, C.; Chen, S. A brain-machine interface based on ERD/ERS for an
upper-limb exoskeleton control. Sensors 2016, 16, 2050. [CrossRef] 19. Gembler, F.; Stawicki, P.; Volosyak, I. Autonomous parameter adjustment for SSVEP-based BCIs with a novel
BCI wizard. Front. Neurosci. 2015, 9, 474. [CrossRef] [PubMed] 20. Picton, T. The P300 wave of the human event-related potential. J. Clin. Neurophysiol. 1992, 9, 456–479. [CrossRef] 21. Andersson, P.; Pluim, J.P.; Siero, J.C.; Klein, S.; Viergever, M.A.; Ramsey, N.F. Real-time decoding
responses to visuospatial attention using 7T fMRI. PLoS ONE 2011, 6, e27638. [CrossRef] [PubMed] 22. Schreuder, M.; Höhne, J.; Blankertz, B.; Haufe, S.; Dickhaus, T.; Tangermann, M. Optimizing event-related
potential based brain-computer interfaces:
A systematic evaluation of dynamic stopping methods. J. Neural Eng. 2013, 10, 036025. [CrossRef] [PubMed] 23. Graimann, B.;
Allison, B.;
Pfurtscheller, G. Brain-computer interfaces:
A gentle introduction. In Brain-Computer Interfaces; Springer: Berlin, Germany, 2009; pp. 1–27. 24. Pfurtscheller, G.; Aranibar, A. Event-related cortical desynchronization detected by power measurements of
scalp EEG. References 1. Soegaard, M.; Dam, R.F. Human Computer Interaction—Brief intro. In The Encyclopedia of Human-Computer
Interaction, 2nd ed.; The Intetraction Design Foundation: Aarhus, Denmark, 2012. 1. Soegaard, M.; Dam, R.F. Human Computer Interaction—Brief intro. In The Encyclopedia of Human-Computer
Interaction, 2nd ed.; The Intetraction Design Foundation: Aarhus, Denmark, 2012. 2. Van Steen, M.; Kristo, G. Contribution to Roadmap. 2015. Available online: https://pdfs.semanticscholar. org/5cb4/11de3db4941d5c7ecfc19de8af9243fb63d5.pdf (accessed on 28 January 2019). 3. Baig, M.Z.; Aslam, N.; Shum, H.P.H.; Zhang, L. Differential evolution algorithm as a tool for optimal feature
subset selection in motor imagery EEG. Expert Syst. Appl. 2017, 90, 184–195. [CrossRef] Sensors 2019, 19, 1423 26 of 34 26 of 34 4. Oikonomou, V.P.; Georgiadis, K.; Liaros, G.; Nikolopoulos, S.; Kompatsiaris, I. A Comparison Study on EEG
Signal Processing Techniques Using Motor Imagery EEG Data. In Proceedings of the IEEE 30th International
Symposium on Computer-Based Medical Systems (CBMS), Thessaloniki, Greece, 22–24 June 2017. [CrossRef] 4. Oikonomou, V.P.; Georgiadis, K.; Liaros, G.; Nikolopoulos, S.; Kompatsiaris, I. A Comparison Study on EEG
Signal Processing Techniques Using Motor Imagery EEG Data. In Proceedings of the IEEE 30th International
Symposium on Computer-Based Medical Systems (CBMS), Thessaloniki, Greece, 22–24 June 2017. [CrossRef] 5. Cheng, D.; Liu, Y.; Zhang, L. Exploring Motor Imagery EEG Patterns for Stroke Patients with Deep Neural
Networks. In Proceedings of the IEEE International Conference on Acoustics, Speech and Signal Processing
(ICASSP), Calgary, AB, Canada, 15–20 April 2018. [CrossRef] 6. Kumar, S.; Sharma, R.; Sharma, A.; Tsunoda, T. Decimation Filter with Common Spatial Pattern and Fishers
Discriminant Analysis for Motor Imagery Classification. In Proceedings of the International Joint Conference
on Neural Networks (IJCNN), Vancouver, BC, Canada, 24–29 July 2016. [CrossRef] 7. Guo, X.; Wu, X.; Gong, X.; Zhang, L. Envelope Detection Based on Online ICA Algorithm and its Application
to Motor Imagery Classification. In Proceedings of the 6th International IEEE/EMBS Conference on Neural
Engineering (NER), San Diego, CA, USA, 6–8 November 2013. [CrossRef] 8. Nijholt, A. The Future of Brain-Computer Interfacing (Keynote Paper). In Proceedings of the 5th International
Conference on Informatics, Electronics and Vision (ICIEV), Dhaka, Bangladesh, 13–14 May 2016. [CrossRef]
9. Nicolas-Alonso, L.F.; Gomez-Gil, J. Brain computer interfaces, a review. Sensors 2012, 12, 1211–1279. [CrossRef] [PubMed] 8. Nijholt, A. The Future of Brain-Computer Interfacing (Keynote Paper). In Proceedings of the 5th International
Conference on Informatics, Electronics and Vision (ICIEV), Dhaka, Bangladesh, 13–14 May 2016. [CrossRef] 9. Nicolas-Alonso, L.F.; Gomez-Gil, J. References Electroencephalogr. Clin. Neurophysiol. 1977, 42, 817–826. [CrossRef] 25. Pfurtscheller, G.; Brunner, C.; Schlogl, A.; da Silva, F.H.L. Mu rhythm (de)synchronization and EEG
single-trial classification of different motor imagery tasks. NeuroImage 2006, 31, 153–159. [CrossRef] [PubMed]
26. Rodríguez-Bermúdez, G.; García-Laencina, P. Automatic and adaptive classification of electroencephalographic 25. Pfurtscheller, G.; Brunner, C.; Schlogl, A.; da Silva, F.H.L. Mu rhythm (de)synchronization and EEG
single-trial classification of different motor imagery tasks. NeuroImage 2006, 31, 153–159. [CrossRef] [PubMed] 26. Rodríguez-Bermúdez, G.; García-Laencina, P. Automatic and adaptive classification of electroencephalographic
signals for brain computer interfaces. J. Med. Stems 2012, 36, S51–S63. [CrossRef] Sensors 2019, 19, 1423 27 of 34 27. Schlögl, A.L.F.;
Bischof, H.P.G. Characterization of four-class motor imagery EEG data for the
BCI-competition 2005. J. Neural Eng. 2005, 2, L14. [CrossRef] 28. Zhou, J.; Meng, M.; Gao, Y.; Ma, Y.; Zhang, Q. Classification of Motor Imagery EEG using Wavelet Envelope
Analysis and LSTM Networks. In Proceedings of the Chinese Control and Decision Conference (CCDC),
Shenyang, China, 9–11 June 2018. [CrossRef] 29. Hashimoto, Y.; Ushiba, J. EEG-based classification of imaginary left and right foot movements using beta
rebound. Clin. Neurophysiol. Pract. 2013, 124, 2153–2160. [CrossRef] 30. Grosse-Wentrup, M.S.B. A review of performance variations in SMR-based Brain-Computer interfaces (BCIs). In Brain-Computer Interface Research; Springer: Berlin, Germany, 2013; pp. 39–51. 31. An, X.; Kuang, D.; Guo, X.; Zhao, Y.; He, L. A Deep Learning Method for Classification of EEG Data Based
on Motor Imagery. In Proceedings of the International Conference on Intelligent Computing, Taiyuan, China,
3–6 August 2014. 32. Zich, C.; Debener, S.; Kranczioch, C.; Bleichner, M.G.; Gutberlet, I.; De Vos, M. Real-time EEG feedback
during simultaneous EEG–fMRI identifies the cortical signature of motor imagery. NeuroImage 2015, 114,
438–447. [CrossRef] 33. Marchesotti, S.; Bassolino, M.; Serino, A.; Bleuler, H.; Blanke, O. Quantifying the role of motor imagery in
brain-machine interfaces. Sci. Rep. 2016, 6, 24076. [CrossRef] 34. Pattnaik, S.; Dash, M.; Sabut, S.K. DWT-based feature extraction and classification for motor imaginary
EEG signals. In Proceedings of the International Conference on Systems in Medicine and Biology (ICSMB),
Kharagpur, India, 4–7 January 2016. [CrossRef] 35. Hara, Y. Brain plasticity and rehabilitation in stroke. J. Nippon Med. Sch. 2015, 82, 4–13. [CrossRef] 36. Hsu, W.Y. Enhanced active segment selection for single-trial EEG classification. Clin. EEG Neurosci. 2012, 43,
87–96. [CrossRef] [PubMed] 37. Patti, C.R.; Penzel, T.; Cvetkovic, D. Sleep spindle detection using multivariate Gaussian mixture models. J. Sleep Res. References 2017, 27. [CrossRef] 38. Lawhern, V.; Kerick, S.; Robbins, K.A. Detecting alpha spindle events in EEG time series using adaptive
autoregressive models. BMC Neurosci. 2013, 14, 101. [CrossRef] 9. Lee, H.; Choi, S. PCA+ HMM+ SVM for EEG Pattern Classification. In Proceedings of the Seve
International Symposium on Signal Processing and Its Applications, Paris, France, 1–4 July 2003. Gaspar, C.M.; Rousselet, G.A.; Pernet, C.R. Reliability of ERP and single-trial analyses. NeuroImage 2011, 58
620–629. [CrossRef] 41. Pernet, C.R.; Sajda, P.; Rousselet, G.A. Single-trial analyses: Why bother? Front. Psychol. 2011, 2, 322. [CrossRef] [PubMed] 42. Lemm, S.; Blankertz, B.; Curio, G.; Muller, K. Spatio-spectral filters for improving the classification of single
trial EEG. IEEE Trans. Biomed. Eng. 2005, 52, 1541–1548. [CrossRef] [PubMed] 43. Sornmo, L.; Laguna, P. Bioelectrical Signal Processing in Cardiac and Neurological Applications, 1st ed.; Academic
Press: Cambridge, MA, USA, 2005. 44. Kevric, J.; Subasi, A. The effect of multiscale PCA de-noising in epileptic seizure detection. J. Med. Syst. 2014,
38, 131. [CrossRef] 45. Kevric, J.; Subasi, A. The impact of MSPCA signal de-Noising in real-Time wireless brain computer interface
system. Southeast Eur. J. Soft Comput. 2015, 4. [CrossRef] 46. Novi, Q.; Cuntai, G.; Dat, T.H.; Xue, P. Sub-Band Common Spatial Pattern (SBCSP) for Brain-Computer
Interface. In Proceedings of the 3rd International IEEE/EMBS Conference on Neural Engineering, Kohala
Coast, HI, USA, 2–5 May 2007. [CrossRef] 47. Kumar, S.; Sharma, A.; Tatsuhiko, T. An Improved Discriminative Filter Bank Selection Approach for Motor
Imagery EEG Signal Classification using Mutual Information. In Proceedings of the 16th International
Conference on Bioinformatics (InCoB 2017): Bioinformatics, Shenzhen, China, 20–22 September 2017. [CrossRef] 48. Ang, K.; Chin, Z.Y.; Zhang, H.; Guan, C. Filter Bank Common Spatial Pattern (FBCSP) in Brain-Computer
Interface. In Proceedings of the IEEE International Joint Conference on Neural Networks (IEEE World
Congress on Computational Intelligence), Hong Kong, China, 1–8 June 2008. [CrossRef] Sensors 2019, 19, 1423 28 of 34 28 of 34 49. Hu, Z.; Chen, G.; Chen, C.; Xu, H.; Zhang, J. A new EEG feature selection method for self-paced
brain-computer interface. In Proceedings of the 10th International Conference on Intelligent Systems
Design and Applications, Cairo, Egypt, 29 November–1 December 2010. 50. Zhang, Y.; Wang, Y.; Jin, J.; Wang, X. Sparse Bayesian learning for obtaining sparsity of EEG frequenc
based feature vectors in motor imagery classification. Int. J. Neural Syst. 2017, 27, 1650032. [CrossRe 51. References Ilyas, M.Z.; Saad, P.; Ahmad, M.I.; Ghani, A.R.I. Classification of EEG signals for brain-computer interface
applications: Performance comparison. In Proceedings of the 6th International IEEE/EMBS Conference on
Neural Engineering (NER), Ayer Keroh, Malaysia, 5–6 November 2016. [CrossRef] 52. Gajic, D.; Djurovic, Z.; Gennaro, S.D.; Gustafsson, F. Classification of EEG signals for detection of epileptic
seizures based on wavelets and statistical pattern recognition. Biomed. Eng. 2014, 26. [CrossRef] 53. Schirrmeister, R.T.; Springenberg, J.T.; Fiederer, L.D.J.; Glasstetter, M.; Eggensperger, K.; Tangermann, M.;
Hutter, F.; Burgard, W.; Ball, T. Deep learning with convolutional neural networks for EEG decoding and
visualization. Hum. Brain Mapp. 2017, 38, 5391–5420. [CrossRef] 54. Tang, Z.; Li, C.; Sun, S. Single-trial EEG classification of motor imagery using deep convolutional neural
networks. Optik 2017, 130, 11–18. [CrossRef] 55. Tabar, Y.; Halici, U. A novel deep learning approach for classification of EEG motor imagery signals. J. Neural Eng. 2017, 14, 016003. [CrossRef] [PubMed] 56. Bashivan, P.;
Rish, I.;
Yeasin, M.;
Codella, N. Learning representations from EEG with deep
recurrent-convolutional neural network. arXiv, 2015; arXiv:1511.06448. 57. Zhang, B. Feature selection based on sensitivity using evolutionary neural network. In Proceedings of the
2nd International Conference on Computer Engineering and Technology, Chengdu, China, 16–18 April 2010. [CrossRef] 58. Yang, H.; Sakhavi, S.; Ang, K.K.; Guan, C. On the Use of Convolutional Neural Networks and Augmented
CSP Features for Multi-Class Motor Imagery of EEG Signals Classification. In Proceedings of the 37th Annual
International Conference of the IEEE Engineering in Medicine and Biology Society (EMBC), Milan, Italy,
25–29 August 2015. [CrossRef] g
59. Pfurtscheller, G.; Allison, B.Z.; Bauernfeind, G.; Brunner, C.; Solis Escalante, T.; Scherer, R.; Zander, T.O.;
Mueller-Putz, G.; Neuper, C.; Birbaumer, N. The hybrid BCI. Front. Neurosci. 2010, 4, 3. [CrossRef] [PubMed]
60. Yu, T.; Xiao, J.; Wang, F.; Zhang, R.; Gu, Z.; Cichocki, A.; Li, Y. Enhanced motor imagery training using a 59. Pfurtscheller, G.; Allison, B.Z.; Bauernfeind, G.; Brunner, C.; Solis Escalante, T.; Scherer, R.; Zander, T.O.;
Mueller-Putz, G.; Neuper, C.; Birbaumer, N. The hybrid BCI. Front. Neurosci. 2010, 4, 3. [CrossRef] [PubMed] Mueller-Putz, G.; Neuper, C.; Birbaumer, N. The hybrid BCI. Front. Neurosci. 2010, 4, 3. [CrossRef] [PubMed]
60. Yu, T.; Xiao, J.; Wang, F.; Zhang, R.; Gu, Z.; Cichocki, A.; Li, Y. Enhanced motor imagery training using a
hybrid BCI with feedback. IEEE Trans. Biomed. Eng. 2015, 62, 1706–1717. [CrossRef] [PubMed] 60. References Yu, T.; Xiao, J.; Wang, F.; Zhang, R.; Gu, Z.; Cichocki, A.; Li, Y. Enhanced motor imagery training using a
hybrid BCI with feedback. IEEE Trans. Biomed. Eng. 2015, 62, 1706–1717. [CrossRef] [PubMed] 61. Koo, B.; Lee, H.G.; Nam, Y.; Kang, H.; Koh, C.S.; Shin, H.C.; Choi, S. A hybrid NIRS-EEG system for self-paced
brain computer interface with online motor imagery. J. Neurosci. Methods 2015, 244, 26–32. [CrossRef] 62. Yao, L.; Meng, J.; Zhang, D.; Sheng, X.; Zhu, X. Combining motor imagery with selective sensation toward a
hybrid-modality BCI. IEEE Trans. Biomed. Eng. 2014, 61, 2304–2312. [CrossRef] 63. BBCI. Data Set IVa ‹Motor Imagery, Small Training Sets›. Available online:
http://www.bbci.de/
competition/iii/desc_IVa.html (accessed on 28 February 2019). 64. Blankertz, B.; Müller, K.-R.; Curio, G.; Vaughan, T.M.; Schalk, G.; Wolpaw, J.R.; Schlögl, A.; Neuper, C.;
Pfurtscheller, G.; Hinterberger, T.; et al. The BCI competition 2003. IEEE Trans. Biomed. Eng. 2004, 51,
1044–1051. [CrossRef] 65. Yu, X.; Chum, P.; Sim, K.B. Analysis the effect of PCA for feature reduction in non-stationary EEG based
motor imagery of BCI system. Optik 2014, 125, 1498–1502. [CrossRef] 66. Krusienski, D.J.; McFarland, D.J.; Wolpaw, J.R. An Evaluation of Autoregressive Spectral Estimation
Model Order for Brain-Computer Interface Applications. In Proceedings of the 28th Annual
International Conference of the IEEE Engineering in Medicine and Biology Society, New York, NY, USA,
30 August–3 September 2006. [CrossRef] 67. Pfurtscheller, G.; Neuper, C.; Guger, C.; Harkam, W.; Ramoser, H.; Schlogl, A.; Obermaier, B.; Pregenzer, M. Current trends in Graz brain-computer interface (BCI) research. IEEE Trans. Rehabil. Eng. 2000, 8, 216–219. [CrossRef] 68. Schloegl,
A.;
Lugger,
K.;
Pfurtscheller,
G. Using
Adaptive
Autoregressive
Parameters
for
a
Brain-Computer-Interface Experiment. In Proceedings of the 19th Annual International Conference of
the IEEE Engineering in Medicine and Biology Society, Chicago, IL, USA, 30 October–2 November 1997. [CrossRef] 29 of 34 29 of 34 Sensors 2019, 19, 1423 69. Pfurtscheller, G.; Neuper, C.; Flotzinger, D.; Pregenzer, M. EEG-based discrimination between imagination
of right and left hand movement. Electroencephalogr. Clin. Neurophysiol. 1997, 103, 642–651. [CrossRef] 70. Schlögl, A. The Electroencephalogram and the Adaptive Autoregressive Model: Theory and Applications. Ph.D. Thesis, Technischen Universität Graz, Graz, Austria, April 2000. Schlögl, A. The Electroencephalogram and the Adaptive Autoregressive Model: Theory and Applications g
p
g
p
g
y
p
Ph.D. Thesis, Technischen Universität Graz, Graz, Austria, April 2000. 71. Hamedi, M.; Salleh, S.H.; Noor, A.M.; Mohammad-Rezazadeh, I. Neural Network-based Three-Class Motor
Imagery. References In Proceedings of the 2014 IEEE Region 10 Symposium, Kuala Lumpur, Malaysia, 14–16 April 2014. [CrossRef] 72. Batres-Mendoza, P.; Montoro-Sanjose, C.; Guerra-Hernandez, E.; Almanza-Ojeda, D.; Rostro-Gonzalez, H.;
Romero-Troncoso, R.; Ibarra-Manzano, M. Quaternion-based signal analysis for motor imagery classification
from electroencephalographic signals. Sensors 2016, 16, 336. [CrossRef] 73. Yang, B.; Zhang, A. Power Spectral Entropy Analysis of EEG Signal Based-on BCI. In Proceedings of the
32nd Chinese Control Conference, Xi’an, China, 26–28 July 2013. 74. Liu, A.; Chen, K.; Liu, Q.; Ai, Q.; Xie, Q.; Chen, A. Feature selection for motor imagery EEG classification
based on firefly algorithm and learning automata. Sensors 2017, 17, 2576. [CrossRef] [PubMed] 75. Wang, Y.; Li, X.; Li, H.; Shao, C.; Ying, L.; Wu, S. Feature extraction of motor imagery electroencephalography
based on time-frequency-space domains. J. Biomed. Eng. 2014, 31, 955–961. 76. Xu, B.G.; Song, A.G.; Fei, S.M. Feature extraction and classification of EEG in online brain-computer inter
Acta Electron. Sin. 2011, 39, 1025–1030. 77. Day, T.T. Applying a locally linear embedding algorithm for feature extraction and visualization of MI-EEG. J. Sens. 2016, 2016, 7481946. [CrossRef] 78. Graps, A. An introduction to wavelets. Comput. Sci. Eng. 1995, 2, 50–61. [CrossRef] 79. Kutlu, Y.; Kuntalp, D. Feature extraction for ECG heartbeats using higher order statistics of WPD coefficients. Comput. Methods Programs Biomed. 2012, 105, 257–267. [CrossRef] [PubMed] 80. Unser, M.; Aldroubi, A. A review of wavelets in biomedical applications. Proc. IEEE 1996, 84, 626–638. [CrossRef] 81. Zazo, R.;
Lozano-Diez, A.;
Gonzalez-Dominguez, J.;
Toledano, D.T.;
Gonzalez-Rodriguez, J. Language identification in short utterances using long short-term memory (LSTM) recurrent neural networks. PLoS ONE 2016, 11, e0146917. [CrossRef] [PubMed] 82. How, D.N.T.; Loo, C.K.; Sahari, K.S.M. Behavior recognition for humanoid robots using long short-term
memory. Int. J. Adv. Robot. Syst. 2016, 13. [CrossRef] 83. Zhai, C.; Chen, Z.; Li, J.; Xu, B. Chinese Image Text Recognition with BLSTM-CTC: A Segmentation-Free
Method. In Proceedings of the 7th Chinese Conference on Pattern Recognition (CCPR), Chengdu, China,
5–7 November 2016. [CrossRef] 84. Li, M.; Chen, W.; Zhang, T. Classification of epilepsy EEG signals using DWT-based envelope analysis and
neural network ensemble. Biomed. Signal Process. Control 2017, 31, 357–365. [CrossRef] 85. Clerico, A.; Gupta, R.; Falk, T.H. Mutual Information between Inter-Hemispheric EEG Spectro-Temporal
Patterns: A New Feature for Automated Affect Recognition. In Proceedings of the 7th International 85. Clerico, A.; Gupta, R.; Falk, T.H. Mutual Information between Inter-Hemispheric EEG Spectro-Temporal 85. References An Idea Based on Honey Bee Swarm for Numerical Optimization; Technical Report, Technical Report-tr06;
Computer Engineering Department, Engineering Faculty, Erciyes University: Kayseri, Turkey, 2005. 96. Karaboga, D. An Idea Based on Honey Bee Swarm for Numerical Optimization; Technical Report, Technical Report-tr06;
Computer Engineering Department Engineering Faculty Erciyes University Kayseri Turkey 2005 gineering Faculty, Erciyes University: Kayseri, Turkey, 20 97. Herman, P.; Prasad, G.; McGinnity, T.M.; Coyle, D. Comparative analysis of spectral approaches to feature
extraction for EEG-based motor imagery classification. IEEE Trans. Neural Syst. Rehabil. Eng. 2008, 16,
317–326. [CrossRef] [PubMed] Bishop, C.M. Pattern Recognition and Machine Learning; Springer: New York, NY, USA, 2006; pp. 9, 21, 189
225, 292, 336. 99. Sohaib, A.;
Qureshi, S.;
Hagelbäck, J.;
Hilbom, O.;
Jerˇci´c, P.;
Schmorrow, D.;
Fidopiastis, C. Evaluating Classifiers for Emotion Recognition Using EEG. In Proceedings of the Foundations of Augmented
Cognition, Las Vegas, NV, USA, 21–26 July 2013. 100. Lu, N.; Li, T.; Ren, X.; Miao, H. A deep learning scheme for motor imagery classification based on restricted
boltzmann machines. IEEE Trans. Neural Syst. Rehabil. Eng. 2017, 25, 566–576. [CrossRef] 101. Nauck, D.; Kruse, R. Obtaining interpretable fuzzy classification rules from medical data. Artif. Intell. Med. 1999, 16, 149–169. [CrossRef] 102. Yang, Z.; Wang, Y.; Ouyang, G. Adaptive neuro-fuzzy inference system for classification of background EEG
signals from ESES patients and controls. Sci. World J. 2014, 2014. [CrossRef] 103. Herman, P.A.; Prasad, G.; McGinnity, T.M. Designing an interval type-2 fuzzy logic system for handling
uncertainty effects in brain–computer interface classification of motor imagery induced EEG patterns. IEEE Trans. Fuzzy Syst. 2017, 25, 29–42. [CrossRef] y
y
104. Jiang, Y.; Deng, Z.; Chung, F.L.; Wang, G.; Qian, P.; Choi, K.S. Recognition of epileptic EEG signals using a
novel multiview TSK fuzzy system. IEEE Trans. Fuzzy Syst. 2017, 25, 3–20. [CrossRef] 105. Yoo, B.S.; Kim, J.H. Fuzzy integral-based gaze control of a robotic head for human robot interaction. IEEE Trans. Cybern. 2015, 45, 1769–1783. [CrossRef] 106. Dai, M.; Zheng, D.; Na, R.; Wang, S.; Zhang, S. EEG Classification of Motor Imagery Using a Novel Deep
Learning Framework. Sesnors 2019, 19, 551. [CrossRef] [PubMed] 107. Nguyen, A.; Yosinski, J.; Clune, J. Deep neural networks are easily fooled: High confidence predictions for
unrecognizable images. In Proceedings of the IEEE conference on computer vision and pattern recognition,
Boston, MA, USA, 7–12 June 2015. 108. LeCun, Y.; Bengio, Y. References Clerico, A.; Gupta, R.; Falk, T.H. Mutual Information between Inter-Hemispheric EEG Spectro-Temporal
Patterns: A New Feature for Automated Affect Recognition. In Proceedings of the 7th International
IEEE/EMBS Conference on Neural Engineering (NER), Montpellier, France, 22–24 April 2015. [CrossRef] Patterns: A New Feature for Automated Affect Recognition. In Proceedings of the 7th International
IEEE/EMBS Conference on Neural Engineering (NER), Montpellier, France, 22–24 April 2015. [CrossRef]
86. Yang, B.; Li, H.; Wang, Q.; Zhang, Y. Subject-based feature extraction by using fisher WPD-CSP in
brain–computer interfaces. Comput. Methods Prog. Biomed. 2016, 129, 21–28. [CrossRef] 86. Yang, B.; Li, H.; Wang, Q.; Zhang, Y. Subject-based feature extraction by using fisher WPD-CSP in
brain–computer interfaces. Comput. Methods Prog. Biomed. 2016, 129, 21–28. [CrossRef] 7. Wei, Q.; Wei, Z. Binary particle swarm optimization for frequency band selection in motor imagery ba
brain-computer interfaces. Biomed. Mater. Eng. 2015, 26, S1523–S1532. [CrossRef] [PubMed] 88. Higashi, H.; Tanaka, T. Simultaneous design of FIR filter banks and spatial patterns for EEG signal
classification. IEEE Trans. Biomed. Eng. 2013, 60, 1100–1110. [CrossRef] [PubMed] 89. Wankar, R.V.; Shah, P.; Sutar, R. Feature extraction and selection methods for motor imagery EEG signals: A
review. In Proceedings of the International Conference on Intelligent Computing and Control (I2C2), Tamil
Nadu, India, 23–24 June 2017. [CrossRef] 90. Dornhege, G.; Blankertz, B.; Krauledat, M.L.F.; Curio, G.; Muller, K.R. Combined optimization of spatial and
temporal filters for improving brain-computer interfacing. IEEE Trans. Biomed. Eng. 2006, 53, 2274–2281. [CrossRef] 91. Lones, M.A. Metaheuristics in Nature-Inspired Algorithms. In Proceedings of the Genetic and Evolutionary
Computation Conference’14, Vancouver, BC, Canada, 12–16 July 2014. 30 of 34 30 of 34 Sensors 2019, 19, 1423 92. Thomas, K.P.; Guam, C.; Lau, C.T.; Vinod, A.P.; Keng, K. A new discriminative common spatial pattern
method for motor imagery brain computer interfaces. IEEE Trans. Biomed. Eng. 2009, 56, 2730–2733. [CrossRef] 93. Zhang, Y.; Zhou, G.; Jin, J.; Wang, X.; Cichocki, A. Optimizing spatial patterns with sparse filter ba
motor-imagery based brain-computer interface. J. Neurosci. Methods 2015, 255, 85–91. [CrossRef] 94. Kennedy, J. Particle swarm optimization. In Encyclopedia of Machine Learning; Springer: Berlin, Germany,
2011; pp. 760–766. 95. Qin, A.K.; Huang, V.L.; Suganthan, P.N. Differential evolution algorithm with strategy adaptation for global
numerical optimization. IEEE Trans. Evol. Comput. 2009, 13, 398–417. [CrossRef] 96. Karaboga, D. References Convolutional networks for images, speech, and time series. In The Handbook of Brain
Theory and Neural Networks; MIT Press: Cambridge, MA, USA, 1995; Volume 3361. 109. Al Rahhal, M.M.; Bazi, Y.; AlHichri, H.; Alajlan, N.; Melgani, F.; Yager, R.R. Deep learning approach for
active classification of electrocardiogram signals. Inf. Sci. 2016, 345, 340–354. [CrossRef] 110. Habermann, M.; Fremont, V.; Shiguemori, E.H. Feature selection for hyperspectral images using single-layer
neural networks. In Proceedings of the 8th International Conference of Pattern Recognition Systems
(ICPRS 2017), Madrid, Spain, 11–13 July 2017. 111. Verikas, A.; Bacauskiene, M. Feature selection with neural networks. Pattern Recognit. Lett. 2002, 23,
1323–1335. [CrossRef] 112. Setiono, R.; Liu, H. Neural-Network Feature Selector. IEEE Trans. Neural Netw. 1997, 8, 654–662. [CrossRef] 112. Setiono, R.; Liu, H. Neural-Network Feature Sele 113. Syafiandini, A.F.; Wasito, I.; Yazid, S.; Fitriawan, A.; Amien, M. Multimodal Deep Boltzmann Machines
for Feature Selection on Gene Expression Data. In Proceedings of the 2016 International Conference on
Advanced Computer Science and Information Systems (ICACSIS), Malang, Indonesia, 15–16 October 2016. [CrossRef] Sensors 2019, 19, 1423 31 of 34 31 of 34 114. Ruangkanokmas, P.; Achalakul, T.; Akkarajitsakul, K. Deep belief networks with feature selection for
sentiment classification. In Proceedings of the 7th International Conference on Intelligent Systems, Modelling
and Simulation (ISMS), Bangkok, Thailand, 25–27 January 2016. 15. IEEE Brain (brain.ieee.org). Competitions & Challenges. Available online: https://brain.ieee.o
competitions-challenges/ (accessed on 17 January 2019). 116. Arnin, J.; Kahani, D.; Lakany, H.; Conway, B.A. Evaluation of Different Signal Processing Methods in Time
and Frequency Domain for Brain-Computer Interface Applications. In Proceedings of the 40th Annual
International Conference of the IEEE Engineering in Medicine and Biology Society (EMBC), Honolulu, HI,
USA, 17–21 July 2018. [CrossRef] 117. BNCI Horizon 2020. Data Sets. Available online: http://bnci-horizon-2020.eu/database/data-sets (accessed
on 17 January 2019). 118. Leeb, R.; Lee, F.; Keinrath, C.; Scherer, R.; Bischof, H.; Pfurtscheller, G. Brain-computer communication:
Motivation, aim, and impact of exploring a virtual apartment. IEEE Trans. Neural Syst. Rehabil. Eng. 2007, 15,
473–482. [CrossRef] 119. Aarabi, A.; Kazemi, K.; Grebe, R.; Moghaddam, H.A.; Wallois, F. Detection of EEG transients in neonates
and older children using a system based on dynamic time-warping template matching and spatial dipole
clustering. NeuroImage 2009, 48, 50–62. [CrossRef] 120. Qu, H.; Gotman, J. A patient-specific algorithm for the detection of seizure onset in long-term EEG
monitoring: Possible use as a warning device. IEEE Trans. Biomed. Eng. 1997, 44, 115–122. References [CrossRef]
[PubMed] 121. Bin, G.; Gao, X.; Wang, Y.; Li, Y.; Hong, B.; Gao, S. A high-speed BCI based on code modulation VEP. J. Neural. Eng. 2011, 8. [CrossRef] [PubMed] 122. Soliman, S.S.; Hsue, S.-Z. Signal classification using statistical moments. IEEE Trans. Commun. 1992, 40,
908–916. [CrossRef] 123. Alam, S.M.S.; Bhuiyan, M.I.H. Detection of seizure and epilepsy using higher order statistics in the EMD
domain. IEEE J. Biomed. Health Inform. 2013, 17, 312–318. [CrossRef] 124. Hassan, A.R.; Haque, M.A. Epilepsy and seizure detection using Statistical Features in the Complete
Ensemble Empirical Mode Decomposition Domain. In Proceedings of the TENCON 2015 IEEE Region
10 Conference, Macao, China, 1–4 November 2015. [CrossRef] 125. Pfurtscheller, G.; Neuper, C.; Schlogl, A.; Lugger, K. Separability of EEG signals recorded during right and
left motor imagery using adaptive autoregressive parameters. IEEE Trans. Rehabil. Eng. 1998, 6, 316–325. [CrossRef] 126. Klimesch, K.; Russegger, H.; Doppelmayr, M.; Pachinger, T. A method for the calculation of induced band
power: Implications for the significance of brain oscillations. Electroencephalogr. Clin. Neurophysiol. 1998, 108,
123–130. [CrossRef] 127. Palaniappan, R. Brain Computer Interface Design using Band Powers Extracted during Mental Tasks. In Proceedings of the 2005 2nd International IEEE EMBS Conference on Neural Engineering, Arlington, VA,
USA, 16–19 March 2005. [CrossRef] 128. Ko, K.-E.; Yang, H.-C.; Sim, K.-B. Emotion recognition using EEG signals with relative power values and
Bayesian network. Int. J. Control Autom. Syst. 2009, 7, 865–870. [CrossRef] 29. Ryu, C.S.; Park, S.H.; Kim, S.H. Method for Determining Positive and Negative Emotional State
Electroencephalogram (EEG). U.S. Patent US6,021,346A, 1 February 2000. 130. Lehmann, C.; Koenig, T.; Jelic, V.; Prichep, L.; John, R.E.; Wahlund, L.O.; Dodge, Y.; Dierks, T. Application and
comparison of classification algorithms for recognition of Alzheimer’s disease in electrical brain activity
(EEG). J. Neurosci. Methods 2007, 161, 342–350. [CrossRef] [PubMed] 131. Bright, D.; Nair, A.; Salvekar, D.; Bhisikar, S. EEG-Based Brain Controlled Prosthetic Arm. In Proceedings of
the 2016 Conference on Advances in Signal Processing (CASP), Pune, India, 9–11 June 2016. [CrossRef] 132. Müller-Putz, G.R.; Pfurtscheller, G. Control of an electrical prosthesis with an SSVEP-based BCI. IEEE Trans. Biomed. Eng. 2008, 55, 361–364. [CrossRef] 133. Elstob, D.; Secco, E.L. A low cost EEG based BCI prosthetic using motor imagery. Int. J. Inf. Technol. Converg. Serv. 2016, 6. [CrossRef] 32 of 34 Sensors 2019, 19, 1423 32 of 34 134. References Müller-Putz, G.; Ofner, P.; Schwarz, A.; Pereira, J.; Luzhnica, G.; di Sciascio, C.; Veas, E.; Stein, S.;
Williamson, J.; Murray-Smith, R. Moregrasp: Restoration of Upper Limb Function in Individuals with
High Spinal Cord Injury by Multimodal Neuroprostheses for Interaction in Daily Activities. In Proceedings
of the 7th Graz Brain-Computer Interface Conference, Graz, Austria, 18–22 September 2017. 135. Athanasiou, A.; Xygonakis, I.; Pandria, N.; Kartsidis, P.; Arfaras, G.; Kavazidi, K.R.; Foroglou, N.; Astaras, A.;
Bamidis, P.D. Towards rehabilitation robotics: Off-the-shelf BCI control of anthropomorphic robotic arms. BioMed Res. Int. 2017. [CrossRef] [PubMed] 136. Voznenko, T.I.; Chepin, E.V.; Urvanov, G.A. The Control System Based on Extended BCI for a Robotic
Wheelchair. Procedia Comput. Sci. 2018, 123, 522–527. [CrossRef] 137. Chella, A.; Pagello, E.; Menegatti, E.; Sorbello, R.; Anzalone, S.M.; Cinquegrani, F.; Tonin, L.; Piccione, F. A BCI Teleoperated Museum Robotic Guide. In Proceedings of the International Conference on Complex,
Intelligent and Software Intensive Systems, Fukuoka, Japan, 16–19 March 2009. [CrossRef] 138. Gomez-Rodriguez, M.;
Grosse-Wentrup, M.;
Hill, J.;
Gharabaghi, A.;
Schölkopf, B.;
Peters, J. Towards Brain-Robot Interfaces in Stroke Rehabilitation. In Proceedings of the 2011 IEEE International
Conference on Rehabilitation Robotics, Zurich, Switzerland, 27 June–1 July 2011. [CrossRef] 139. Abiri, R.; Zhao, X.; Heise, G.; Jiang, Y.; Abiri, F. Brain Computer Interface for Gesture Control of a Social
Robot: An Offline Study. In Proceedings of the 2017 Iranian Conference on Electrical Engineering (ICEE),
Tehran, Iran, 2–4 May 2017. [CrossRef] y
140. Luu, T.P.; Nakagome, S.; He, Y.; Contreras-Vidal, J.L. Real-time EEG-based brain-computer interface to a
virtual avatar enhances cortical involvement in human treadmill walking. Sci. Rep. 2017, 7, 8895. [CrossRef] 141. McMahon, M.; Schukat, M. A Low-Cost, Open-Source, BCI-VR Prototype for Real-Time Signal Processing of
EEG to Manipulate 3D VR Objects as a Form of Neurofeedback. In Proceedings of the 29th Irish Signals and
Systems Conference (ISSC), Belfast, Ireland, 21–22 June 2018. 142. Perales, F.A.E. Combining EEG and serious games for attention assessment of children with cerebral palsy. In Converging Clinical and Engineering Research on Neurorehabilitation II; Springer: Cham, Switzerland, 2017;
Volume 15, pp. 395–399. [CrossRef] 143. Shim, M.; Hwang, H.-J.; Kim, D.-W.; Lee, S.-H.; Im, C.-H. Machine-learning-based diagnosis of schizophrenia
using combined sensor-level and source-level EEG features. Schizophr. Res. 2016, 176, 314–319. [CrossRef] 144. Ehrlich, S.; Guan, C.; Cheng, G. A Closed-Loop Brain-Computer Music Interface for Continuous Affective
Interaction. 153. Wang, Z.; Yu, Y.; Xu, M.; Liu, Y.; Yin, E.; Zhou, Z. Towards a hybrid BCI gaming paradigm based on motor
imagery and SSVEP. Int. J. Hum. Comput. Interact. 2018. [CrossRef] References In Proceedings of the 2017 International Conference on Orange Technologies (ICOT), Singapore,
8–10 December 2017. [CrossRef] 145. Placidi, G.; Cinque, L.; Di Giamberardino, P.; Iacoviello, D.; Spezialetti, M. An Affective BCI Driven by
Self-induced Emotions for People with Severe Neurological Disorders. In Proceedings of the International
Conference on Image Analysis and Processing, Catania, Italy, 11–15 September 2017. 146. Ortiz-Rosario, A.; Berrios-Torres, I.; Adeli, H.; Buford, J.A. Combined corticospinal and reticulospinal effects
on upper limb muscles. Neurosci. Lett. 2014, 561, 30–34. [CrossRef] [PubMed] 147. Kerous, B.; Skola, F.; Liarokapis, F. EEG-based BCI and video games: A progress report. Virtual Real. 2017,
22, 119–135. [CrossRef] 148. Edlinger, G.; Güger, C. Social Environments, Mixed Communication and Goal-Oriented Control Application
using a Brain–Computer Interface. In Proceedings of the International Conference on Universal Access in
Human-Computer Interaction, Orlando, FL, USA, 9–14 July 2011. p
y
149. Maby, E.; Perrin, M.; Bertrand, O.; Sanchez, G.; Mattout, J. BCI could make old two-player games even more
fun: A proof of concept with connect four. Adv. Hum.-Comput. Interact. 2012, 2012. [CrossRef] 150. Van de Laar, B.; Reuderink, B.; Bos, D.P.-O.; Heylen, D. Evaluating user experience of actual and imagined
movement in BCI gaming. In Interdisciplinary Advancements in Gaming, Simulations and Virtual Environments:
Emerging Trends; IGI Global: Hershey, PA, USA, 2012; pp. 266–280. [CrossRef] 151. Kawala-Janik, A.; Podpora, M.; Gardecki, A.; Czuczwara, W.; Baranowski, J.; Bauer, W. Game Controller
Based on Biomedical Signals. In Proceedings of the 20th International Conference on Methods and Models
in Automation and Robotics (MMAR), Miedzyzdroje, Poland, 24–27 August 2015. [CrossRef] 152. Martišius, I.; DamaševiIius, R. A prototype SSVEP based real time BCI gaming system. Comput. Intell. Neurosci. 2016, 18. [CrossRef] 153. Wang, Z.; Yu, Y.; Xu, M.; Liu, Y.; Yin, E.; Zhou, Z. Towards a hybrid BCI gaming paradigm based on motor
imagery and SSVEP. Int. J. Hum. Comput. Interact. 2018. [CrossRef] Sensors 2019, 19, 1423 33 of 34 154. Stein, A.; Yotam, Y.; Puzis, R.; Shani, G. EEG-triggered dynamic difficulty adjustment for multiplayer games. Entertain Comput. 2018, 25, 14–25. [CrossRef] 155. Zhang, B.; Wang, J.; Fuhlbrigge, T. A Review of the Commercial Brain-Computer Interface Technology from
Perspective of Industrial Robotics. In Proceedings of the 2010 IEEE International Conference on Automation
and Logistics, Hong Kong and Macau, China, 16–20 August 2010. [CrossRef] 156. Liu, Y.-T.; Wu, S.-L.; Chou, K.-P.; Lin, Y.-Y.; Lu, J.; Zhang, G.; Lin, W.-C.; Lin, C.-T. References Driving Fatigue Prediction
with Pre-Event Electroencephalography (EEG) via a Recurrent Fuzzy Neural Network. In Proceedings of the
2016 IEEE International Conference on Fuzzy Systems (FUZZ-IEEE), Vancouver, BC, Canada, 24–29 July
2016. [CrossRef] 157. Binias, B.; Myszor, D.; Cyran, K. A machine learning approach to the detection of pilot’s reaction to
unexpected events based on EEG signals. Comput. Intell. Neurosci. 2018, 2018. [CrossRef] 158. Wadeson, A.; Nijholt, A.; Nam, C.S. Artistic brain-computer interfaces: State-of-the-art control mechanisms. Brain Comput. Interfaces 2015, 2, 70–75. [CrossRef] 159. Miranda, E.R.; Durrant, S.J.; Anders, T. Towards Brain-Computer Music Interfaces: Progress and Challenges. In Proceedings of the First International Symposium on Applied Sciences on Biomedical and Communication
Technologies, Aalborg, Denmark, 25–28 October 2008. [CrossRef] 160. Todd, D.; McCullagh, P.J.; Mulvenna, M.D.; Lightbody, G. Investigating the Use of Brain-Computer
Interaction to Facilitate Creativity. In Proceedings of the 3rd Augmented Human International Conference,
Megève, France, 8–9 March 2012. [CrossRef] 161. Van De Laar, B.; Brugman, I.; Nijboer, F.; Poel, M.; Nijholt, A. BrainBrush, a Multimodal Application
for Creative Expressivity. In Proceedings of the Sixth International Conference on Advances in
Computer-Human Interactions (ACHI 2013), Nice, France, 24 February–1 March 2013. 162. Singh, A.; Lal, S.; Guesgen, H.W. Reduce Calibration Time in Motor Imagery Using Spatially Regularized
Symmetric Positives-Definite Matrices Based Classification. Sensors 2019, 19, 379. [CrossRef] [PubMed] 163. Vansteensel, M.J.; Kristo, G.; Aarnoutse, E.J.; Ramsey, N.F. The brain-computer interface researcher’s
questionnaire: From research to application. Brain Comput. Interfaces 2017, 4, 236–247. [CrossRef] 164. Speier, W.; Fried, I.; Pouratian, N. Improved P300 speller performance using electrocorticography, spectral
features, and natural language processing. Clin. Neurophysiol. 2013, 124, 1321–1328. [CrossRef] 165. Tavares, N.G.; Gad, R. Steady-state eisual evoked potential-based real-time BCI for smart appliance control. In Cognitive Informatics and Soft Computing; Springer: Singapore, 2019; pp. 795–805. [CrossRef] 166. Bhemjibhaih, D.P.; Sanjay, G.D.; Sreejith, V.; Prakash, B. Brain-Computer Interface Based Home Automation
System for Paralysed People. In Proceedings of the 2018 IEEE Recent Advances in Intelligent Computational
Systems (RAICS), Thiruvananthapuram, India, 6–8 December 2018. [CrossRef] 167. Andujar, M.; Gilbert, J.E. Let’s Learn! Enhancing User’s Engagement Levels through Passive Brain-Computer
Interfaces. In Proceedings of the CHI’13 Extended Abstracts on Human Factors in Computing Systems,
Paris, France, 27 April–2 May 2013. [CrossRef] 168. Veryzer, R.W.; de Mozota, B. The impact of user-oriented design on new product development:
An examination of fundamental relationships. J. Prod. Innov. Manag. 2005, 22, 128–143. [CrossRef] 169. References Lazard, A.J.; Watkins, I.; Mackert, M.S.; Xie, B.; Stephens, K.K.; Shalev, H. Design simplicity influences patient
portal use: The role of aesthetic evaluations for technology acceptance. JAMIA Open 2015, 23, e157–e161. [CrossRef] [PubMed] 170. Mackelprang, A.W.; Habermann, M.; Swink, M. How firm innovativeness and unexpected product reliability
failures affect profitability. JOM 2015, 38, 71–86. [CrossRef] 171. Neumann, P.J.; Weinstein, M.C. The Diffusion of New Technology: Costs and Benefits to Health Care. In The
Changing Economics of Medical Technology; National Academies Press: Washington, DC, USA, 1991. 172. Mathewson, K.E.; Harrison, T.J.; Kizuk, S.A. High and dry? Comparing active dry EEG electrodes to active
and passive wet electrodes. Psychophysiology 2017, 54, 74–82. [CrossRef] [PubMed] 173. Kam, J.W.Y.; Griffin, S.; Shen, A.; Patel, S.; Hinrichs, H.; Deouell, L.Y.; Knight, R.T. Systematic comparison
between a wireless EEG system with dry electrodes and a wired EEG system with wet electrodes. NeuroImage
2019, 184, 119–129. [CrossRef] [PubMed] 34 of 34 Sensors 2019, 19, 1423 174. Mihajlovic, V.; Garcia Molina, G.; Peuscher, J. To what extent can dry and water-based EEG electrodes
replace conductive gel ones? A steady state visual evoked potential brain-computer interface case study. In Proceedings of the ICBE 2011: International Conference on Biomedical Engineering, Venice, Italy,
23–25 November 2011. 175. Jurvsta, F.; van de Borne, P.; Migeotte, P.F.; Dumont, M.; Languart, J.P.; Deguate, J.P.; Linkowski, P. A study of the dynamic interactions between sleep EEG and heart rate variability in healthy young men. Clin. Neurophysiol. 2003, 114, 2146–2155. [CrossRef] 176. Seo, S.H.; Lee, J.T. Stress and EEG. In Convergence and Hybrid Information Technologies; InTech: Vienna, Austria,
2010; pp. 420–422. 177. Burlaka, S.; Gontean, A. Brain-Computer Interface Review. In Proceedings of the 12th IEEE International
Symposium on Electronics and Telecommunications (ISETC), Timisoara, Romania, 27–28 October 2016. 178. BNCI Horizon 2020. Roadmap. Available online: http://bnci-horizon-2020.eu/roadmap (accessed on
21 December 2018). 179. Lee, B.; Liu, C.Y.; Apuzzo, M.L.J. A primer on brain-machine interfaces, concepts, and technology: A
key element in the future of functional neurorestoration. World Neurosurg. 2013, 79, 457–471. [CrossRef]
[PubMed] 180. Chao, Z.C.; Nagsaka, Y.; Fuji, N. Long-term asynchronous decoding of arm motion. Front. Neuroeng. 2010, 3. [CrossRef] 181. Scherer, R.;
Faller, J.;
Friedrich, E.V.C.;
Opisso, E.;
Costa, U.;
Kübler, A.;
Müller-Putz, G.R. Individually adapted imagery improves brain-computer interface performance in end-users with disability. PLoS ONE 2015, 10, e0123727. [CrossRef] 182. References Ang, K.K.; Guan, C.; Chua, K.S.G.; Ang, B.T.; Kuah, C.; Wang, C.; Phua, K.S.; Chin, Z.Y.; Zhang, H. Clinical study of neurorehabilitation in stroke using EEG-based motor imagery brain-computer interface
with robotic feedback. In Proceedings of the 2010 Annual International Conference of the IEEE Engineering
in Medicine and Biology, Buenos Aires, Argentina, 31 August–4 September 2010. [CrossRef] 183. Ang, K.K.; Chua, K.S.G.; Phua, K.S.; Wang, C.; Chin, Z.Y.; Kuah, C.W.K.; Low, G.C. A randomized
controlled trial of EEG-based motor imagery brain-computer interface robotic rehabilitation for stroke. Clin. EEG Neurosci. 2015, 46, 310–320. [CrossRef] [PubMed] 184. Shahid, S.; Sinha, R.K.; Prasad, G. Mu and beta rhythm modulations in motor imagery related post-stroke
EEG: A study under BCI framework for post-stroke rehabilitation. BMC Neurosci. 2010, 11, P127. [CrossRef] 184. Shahid, S.; Sinha, R.K.; Prasad, G. Mu and beta rhythm modulations in motor imagery related post-stroke
EEG: A study under BCI framework for post-stroke rehabilitation. BMC Neurosci. 2010, 11, P127. [CrossRef]
185. Geronimo, A.; Simmons, S.S. Performance predictors of brain–computer interfaces in patients with
hi l
l
l
i
J N
l E
13 026002 [C
R f] ,
;
,
;
,
y
g
y
p
EEG: A study under BCI framework for post-stroke rehabilitation. BMC Neurosci. 2010, 11, P127. [CrossRef]
185. Geronimo, A.; Simmons, S.S. Performance predictors of brain–computer interfaces in patients with
amyotrophic lateral sclerosis. J. Neural Eng. 2016, 13, 026002. [CrossRef] 185. Geronimo, A.; Simmons, S.S. Performance predictors of brain–computer interfaces in patients with
amyotrophic lateral sclerosis. J. Neural Eng. 2016, 13, 026002. [CrossRef] 186. Meng, J.; Mundahl, J.H.; Streitz, T.D.; Maile, K.; Gulachek, N.S.; He, J.; He, B. Effects of soft drinks on resting
state EEG and brain-computer interface performance. IEEE Access 2017, 5. [CrossRef] [PubMed] 186. Meng, J.; Mundahl, J.H.; Streitz, T.D.; Maile, K.; Gulachek, N.S.; He, J.; He, B. Effects of soft drinks on resting
state EEG and brain-computer interface performance. IEEE Access 2017, 5. [CrossRef] [PubMed]
187. Vidaurre, C.; Blankertz, B. Towards a cure for BCI illiteracy. Brain Topogr. 2010, 23, 194–198. [CrossRef]
[P bM d] 187. Vidaurre, C.; Blankertz, B. Towards a cure for BCI illiteracy. Brain Topogr. 2010, 23, 194–198. [CrossRef]
[PubMed] 188. Mariushi, M. Brains activation during manipulation of the myoelectric prosthetic hand: A functional
magnetic reonance imaging study. Neurolmage 2004, 21, 1604–1611. [CrossRef] [PubMed] © 2019 by the authors. Licensee MDPI, Basel, Switzerland. References This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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The effect of relationship quality on individual perceptions of social responsibility in the US
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Frontiers in psychology
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cc-by
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The effect of relationship quality on
individual perceptions of social
responsibility in the US Joseph C. Thornton*
Rubel School of Business, Bellarmine University, Louisville, KY, USA Joseph C. Thornton* Social responsibility (SR) has been of continuing interest in the U.S. and around the
world. Organizations make a wide variety of SR decisions that represent differing
viewpoints. While a number of definitions of SR exist, many of these definitions indicate
that SR decisions may be viewed as existing of various facets, such as legal/regulatory,
financial/economic, ethical, environmental, and voluntary. While drivers of SR have been
proposed, there has been limited research at a micro-level on how individuals perceive
SR activities by the organizations where they work. Based on a prior qualitative study
(Thornton and Byrd, 2013) that found SR decisions are related to several traits and
influenced by relationships, a model was proposed and tested in this research. The
traits found relevant in the qualitative research were conscientiousness, especially in
the sense of being responsible, and self-efficacy. Relationship quality was assessed
based on positive and negative emotional attractors as proposed in intentional change
theory. Perceptions of individuals in management and non-management showed that
relationship quality mediated the effect of conscientiousness and general self-efficacy
on the SR. Because there are multiple facets, the author made use of Carroll’s (1991)
pyramid of SR to identify activities that business owners and managers consider
relevant. The findings indicate that conscientiousness is related to specific SR activities
in the areas of legal/regulatory, ethical and discretionary dimensions while general self-
efficacy is related to financial/economic and legal/regulatory dimensions. The presence
of relationship quality enhanced the effects of both conscientiousness and general self-
efficacy on the various SR dimensions. This suggests that individuals perceived SR
activities along different traits and that enhancing these traits might improve perceptions
of SR decisions. Edited by:
Scott N. Taylor,
Babson College, USA Reviewed by:
Anita Howard,
Case Western Reserve University,
USA Ante Glavas,
Kedge Business School, France Ante Glavas, Kedge Business School, France *Correspondence:
Joseph C. Thornton,
Rubel School of Business, Bellarmine
University, 2001 Newburg Road,
Louisville, KY 40205, USA
jthornton@bellarmine.edu Specialty section:
This article was submitted to
Personality and Social Psychology,
a section of the journal
Frontiers in Psychology Keywords:
social
responsibility,
relationship
quality,
general
self-efficacy,
conscientiousness,
individual
perceptions Received: 10 September 2014
Accepted: 26 May 2015
Published: 10 June 2015
Citation:
Thornton JC (2015) The effect
of relationship quality on individual
perceptions of social responsibility
in the US. Front. Psychol. 6:781. doi: 10.3389/fpsyg.2015.00781 Received: 10 September 2014
Accepted: 26 May 2015
Published: 10 June 2015 Keywords:
social
responsibility,
relationship
quality,
general
self-efficacy,
conscientiousness,
individual
perceptions ORIGINAL RESEARCH
published: 10 June 2015
doi: 10.3389/fpsyg.2015.00781 Edited by:
Scott N. Taylor,
Babson College, USA Citation: Social responsibility (SR) has been the subject of numerous studies (Bowen, 1953; Aguinis and
Glavas, 2012). Most research has focused on large companies and organizations (Williamson et al.,
2006) while research into smaller companies has lagged, especially in the U.S. (Dean et al., 1998). European literature has focused on small and medium enterprises (SMEs) in terms of management
(Jenkins, 2006), resources (Aragón-Correa et al., 2008), and drivers and determinants (Darnall
et al., 2009). A summary of these dissimilarities are as follows. Thornton JC (2015) The effect
of relationship quality on individual
perceptions of social responsibility
in the US. Thornton JC (2015) The effect
of relationship quality on individual
perceptions of social responsibility
in the US. Front. Psychol. 6:781. doi: 10.3389/fpsyg.2015.00781 June 2015 | Volume 6 | Article 781 Frontiers in Psychology | www.frontiersin.org Perceptions of socially responsible actions Thornton Fassin (2008) suggests that small firms do not have the
resources or capabilities of performing SR activities at the same
level as large firms. While Jenkins (2006) indicates that there is
more dependence on the values and traits of the owners and
senior managers in terms of SR resulting in different responses
compared to large firms. A leading driver for small firms is
their employees and families as opposed to external stakeholders
(Murillo and Lozano, 2006; Darnall et al., 2009). in turn may aid the organization or justify its entry into specific
professional societies. Others argue that involvement in SR may
be a way to strategically position the firm (Keim, 1978; Besser and
Miller, 2004). Others claim that it has motivational and marketing
value in that it attracts employees (Rupp et al., 2006; Rodrigo
and Arenas, 2008) or helps the organization in its image in the
community (Bowen, 2000, 2002). y
Using a grounded theory approach, Thornton and Byrd (2013)
found that owners and managers of SMEs used social and
personal values as well as experience in making decisions about
SR. They found relationships often triggered compassionate and
visionary SR responses from the organization toward individuals
or groups both internally or externally. The study also found
that owners and managers who were aware of key stakeholders
and confident were more likely to enact SR. This is supported
by prior research into personal values (Nonis and Swift, 2001),
management attitudes (Marshall et al., 2005), and personality
(Hogan et al., 1996; Moberg, 1999; Giberson et al., 2005) and their
effect on SR decisions. Social Responsibility According to Carroll (1991), every business performs SR activities
that are economic and compliance oriented since these are
required to remain viable. Carroll (1991) argues that ethical
and philanthropic SR dimensions are voluntary in nature. There
is some disagreement with Carroll’s original concept of four
overlapping dimensions (Schwartz and Carroll, 2003) since the
original publication, including that they are poorly defined and
that philanthropic responsibilities are potentially not a true
form of SR. Schwartz and Carroll (2003) note that the four
factor model “remain (s) a leading paradigm of CSR in the
social issues in management field.” Dahlsrud in an evaluation
of 37 definitions of SR found five conceptual dimensions
that occurred with frequencies greater than 50% (stakeholder,
social, economic, voluntariness, and environmental dimensions). This lends support to a multidimensional conceptualization
of SR. Thornton and Byrd (2013) developed a social empathy model
of SR, finding four distinct dimensions of SR that SME owners
and managers found important. The dimensions correspond to
financial/economic, legal/regulatory, ethical, and discretionary. Internally, the focus is on relationships with employees, suppliers
and customers. Externally, the focus is on the community and
local/regional issues. This study focuses on the question of how
individuals perceive SR actions in the presence of relationship
quality. This research also examines whether conscientiousness
and general self-efficacy affect SR equally or differentially. Citation: While there are many definitions for SR (Dahlsrud, 2008), the
author uses a broad definition proposed by Bowen (1953, p. 6)
that businesses have an obligation to society to “. . .pursue those
policies, to make those decisions, or to follow those lines of action
which are desirable in terms of the objectives and values of our
society.” The major issue with a broad definition is in determining
what an organization might consider the most appropriate
policies or objectives that should be addressed. The concept
of saliency, developed by Agle et al. (1999) is an attempt to
provide a way for organizations to determine which issues should
be addressed. They propose that the owners/managers evaluate
each issue based on their perception of power, legitimacy,
and urgency of specific stakeholders. Based on this, power is
seen as the influence of the stakeholder on the organization,
legitimacy is based on the perceived relationship quality and
urgency is the perception of how the stakeholder sees the
issue. Conceptual Framework According to the theory of reasoned action, individuals make
decisions based on two major factors, the beliefs and attitudes
about the behavior and subjective norms (Ajzen and Fishbein,
1980, 2005). Influences of personal beliefs and attitudes of
people relate to their personality, their perception of the
various alternatives available, and anticipation of the outcome
of the action/decision. Subjective norms reflect the opinions of
significant others about the action/decision. It includes the desire
for organizational members to “fit in” and comply with these
norms. Individuals make decisions from among a limited number
of alternatives that they know, learn or experience through
interactions with others. In terms of beliefs, managers and owners
will anticipate congruence of consequences of actions/decisions
with these shared norms and beliefs. Both internal and external
norms affect these forces (Ajzen and Fishbein, 1980, 2005). While
these occur within individuals, the consequences can be for the
entire organization and its strategy (Conley and Williams, 2005;
Cordano et al., 2009). Thornton and Byrd (2013) found, in interviews with business
owners and managers that they want to do the right thing for
their employees, customers, and society at large. In particular,
many organizations want to make a difference or create an
impact on society. Interviews with managers and owners
conducted by Thornton and Byrd (2013) found that ethical
treatment of employees/customers, recycling/cost reduction,
legal compliance, and volunteering/philanthropy
were the
most frequently discussed decisions for SME engagement. These decisions were mapped to Carroll’s four dimensions of
financial/economic (recycling/cost reduction), legal/regulatory
(compliance),
ethical
(employee/customer
treatment),
and
discretionary (volunteering/philanthropic). Individual Traits Thornton and Byrd (2013) found that individual CEOs/Owners
of SMEs are influenced by their own prior success in attempting
new and different things and that prior success led to a sense of
self-efficacy in many arenas, but in particular when addressing
SR. According to Bandura (1982, 1991, 1998), self-efficacy is the
sense that a person believes in their own ability to perform an
activity successfully and suggests that self-efficacy would predict
future activities within the same general realm, such as SR. Penner et al. (1995) found that self-efficacy played a strong role
in why individuals act in a pro-social manner. They suggest that
this may be due to people believing that their actions are effective
in helping others. They also believe that the consequences of their
decisions and actions are their own responsibility. The author
posits that self-efficacy is strengthened by relationship quality
since relationships are often seen as providing legitimacy to SR
related issues and activities. Therefore, the following hypotheses
were developed for this study: If there is a sense of shared purpose or vision, people
feel a common context and direction. They also experience a
positive emotional attractor (PEA) mood state, which is both
psychological and physiological (Schwartz and Carroll, 2003). It
is the this mood state, characterized by positive affect, increasing
intensity of it and neurological activation of the networks, that
enables a person to be more open to new ideas, people and
moral concerns (Boyatzis et al., 2014). Openness to new ideas and
moral concerns can invite SR thoughts and values. The author
proposes that relationship quality creates an opening for broader
thinking about the organization’s purpose and role in society and
the community, resulting in expansion of the mental models of
key stakeholders to be broader than investors do. H1a: Relationship quality will mediate the positive relationship
between self-efficacy and SR. H1b: Relationship quality will mediate the positive relationship
between self-efficacy and legal SR. H1c: Relationship quality will mediate the positive relationship
between self-efficacy and ethical SR. H1d: Relationship quality will mediate the positive relationship
between self-efficacy and Philanthropic/Discretionary SR. According to Roberts et al. (2005) conscientiousness is
often viewed as a broad personality domain made up of
a variety of somewhat similar concepts that are related
including: industriousness, order, self-control, responsibility,
traditionalism, and virtue. Relationship Quality and SR For SR decisions and actions to occur, there have to be norms
and values supporting and even encouraging them. The norms
are expressed through how people act with each other and the
nature of their relationships. Boyatzis (2008) and Schwartz and
Carroll (2003) describe how sustained, desired change emerges In evaluating SR decisions, Campbell (2007) claims that one
reason may be to exploit tax or other group incentives. These June 2015 | Volume 6 | Article 781 Frontiers in Psychology | www.frontiersin.org 2 Perceptions of socially responsible actions Thornton negative affect does not always lead to decreased pro-social
behavior. through a complex system. The emergence of each stage in the
process in organizations appears to be invoked by a tipping point
in the mood of the people in the organization that Boyatzis (2013)
referred to as shifts between a negative and a positive emotional
attractor (see other papers in this special topic for more detailed
explanation of these states and their dynamics). Thornton and
Byrd (2013) found in interviews that owners and managers in
about 1/3 of the cases indicated that they began or increased SR
after a negative emotional event focused them on a particular
issue. The
nature
of
the
relationships
among
those
in
an
organization communicates emotions as well as norms and
values. Positive relationships appear to invoke a more open
rapport that considers not only the needs and interests of others
but the broader community due to the nature of the neural and
hormonal arousal (Boyatzis et al., 2014). In this manner, SV, SC,
and shared OPM might enhance SR decisions and actions. This
has been related to more individualistic ways of thinking about
the world (Boyatzis et al., 2000). While the ultimate decision may rest with upper management,
people in an organization would have to value and perceive the
desirability and justification of the decisions and actions in a
similar manner. This is where the nature and quality of their
relationships become the enabling factors. Each person is pulled
into a mood state by the degree to which they believe their
relationships as having shared compassion (SC), shared vision
(SV), and a shared overall positive mood (OPM). These are
the norms in the relationships that may enhance the likelihood
of seeing the desirability for SR or not, and possibly which
dimension SR is more important to them. Relationship Quality and SR The perception
that relationships in the organization are shared means that
individuals perceive that the image of the ideal or desired future
of the organization is common (Boyatzis, 2013). This shared
relationship means that a degree of trust and caring for each other
in the organization and that a common or shared view of the
future is hopeful and bright. Preparatory Data Analysis The presence of multivariate non-normality was excessive for
use with covariance-based structural equation modeling (SEM),
exceeding the level suggested by Byrne (2010) of less than 7. Thus, further analysis of the data was conducted using partial Social Responsibility Social responsibility behavior was assessed through two separate
scales. One scale developed by Maignan and Ferrell (2000) based
on Carroll’s (1991) four dimensions of SR consists of four scales:
economic, legal, ethical, and philanthropic. The other scale was
developed by Goll and Rasheed (2004) measures discretionary
SR. Each scale used a 5-point Likert scale. According to Maignan
and Ferrell (2000), the composite reliability (CR) of the overall
four dimensional construct was greater than 0.85 and the
Cronbach alpha was 0.94. The discretionary SR scale had good
psychometrics, with an internal consistency of 0.74 in the original
sample (Goll and Rasheed, 2004). H2b: Relationship quality will mediate the positive relationship
between conscientiousness and legal SR. H2c: Relationship quality will mediate the positive relationship
between conscientiousness and ethical SR. H2d: Relationship quality will mediate the positive relationship
between conscientiousness and Philanthropic/Discretionary SR. Individual Traits Conscientiousness is associated with
conformity and self-regulation (Peterson and Seligman, 2004)
leadership and effectiveness (Barrick et al., 1993) and as an
expression of virtue (Aguilera et al., 2007). Conscientiousness has
been used as a predictor of organizational citizenship behavior
at individual (Organ and Ryan, 1995) and organizational
(Taylor et al., 2010) levels. Brief and Motowidlo (1986) note
that conscientiousness, in terms of perseverance, diligence
and putting forth extra effort, is prosocial organizational
behavior. Conscientiousness is also seen as conforming to
values of the organization (Podsakoffet al., 2000) which can
be thought of as individual initiative. Thornton and Byrd
(2013) found evidence that CEOs/managers were concerned Relationship quality has been linked to succession in family
businesses (Overbeke, 2010) as well as longer term financial
success of family businesses (Neff, 2011). Clayton (2009) reported
perceived SV one of the two most significant predictors of
championing behavior in mergers and acquisitions, and SV
was the strongest predictor of the other mediator, autonomous
motivation. A patient’s perception of the quality of their
relationship to the physician was shown to mediate treatment
adherence for Type II Diabetics (Khawaja, 2012). Pittenger
et al. (2012) found that the quality of the relationship between
managers and information technology teams enhanced the
perception of organizational engagement. In addition, Eisenberg
and Miller (1987) argue that when positive emotions are stronger
than negative emotions, there is an increase in pro-social
behavior. According to Brief and Motowidlo (1986), positive
affect has been repeatedly shown to have a positive effect on
pro-social behavior, such as helping others. They also note that June 2015 | Volume 6 | Article 781 Frontiers in Psychology | www.frontiersin.org Frontiers in Psychology | www.frontiersin.org 3 Perceptions of socially responsible actions Thornton with doing the right thing and making responsible decisions. CEOs/managers worked to promote a sense of responsibility in
their employees and an understanding of why it is important
to give back. Based on the results of Thornton and Byrd
(2013) that people want to do the right thing (be virtuous)
be responsible for their actions and persevere, the author
hypothesizes that individuals will perceive economic, legal,
ethical, and philanthropic/discretionary SR as being related to
conscientiousness and that this relationship will be strengthened
by relationship quality. Therefore, the following hypotheses were
developed for this study: scale. This scale is unidimensional according to Chen et al. Materials and Methods The use of self-report data is recommended by Abbott and
Monsen (1979) as having a significant advantage in obtaining
data for SR. Podsakoffand Organ (1986) noted that while
self-reports may be considered soft data, they are useful for
obtaining data related to past behaviors, personality traits,
perceptions, and demographics, although researchers should be
aware of potential issues present in self-report data. Podsakoff
and Organ (1986) noted that one way of increasing reliability
is to make the responses anonymous in nature. Therefore, a
self-administered on-line survey was used to obtain perceived
behaviors, personality traits and demographics of individuals. The survey was designed to avoid collecting any information that
might be used to identify specific organizations or individuals
resulting in a significant amount of anonymity for respondents. The study was IRB exempt at the University where I was doing
my doctoral program, but all human subjects ethical protocols
were followed. Potential control variables selected based on a review of
literature included: job tenure, company tenure, and individual
age. Demographic information collected included: current
job
(management/non-management),
company
ownership
(public/private), and gender. Respondents were asked to identify
if the company was a U.S. or foreign company, and to select their
industry (food or beverage). Individual Traits (2001)
and exhibits good internal consistency and reliability (α = 0.86–
0.90) in prior work. Conscientiousness The conscientiousness scale used was obtained from the
international personality item pool (Goldberg et al., 2006), based
on the work of Saucier (1997). This scale, consists of ten
items (five items reverse scored), was evaluated using a 5-point
Likert scale. It was found to have good reliability and internal
consistency (α = 0.75) according to Saucier (1997). H2a: Relationship quality will mediate the positive relationship
between conscientiousness and SR. Sample The target population for this study includes both publicly and
privately owned organizations in the US food and beverage
industry. This industry was selected based on the industry focus
on customer service and product quality. The data collection
was conducted on-line using Qualtrics, Inc. software. The initial
data were collected from 308 people. One hundred and ten
cases were dropped because the surveys were less than 50%
complete. Twenty cases were from outside the US and dropped
for consistency of the sample and to control for culture. Eleven
cases had more than 500 employees and were dropped to control
for size of organization. Ten cases were missing demographic
information, and eight cases were from non-food or beverage
industries. Relationship Quality To assess the quality of the relationships the positive and negative
emotional attractors (PNEA) survey developed by Boyatzis
and Oliver (2008) was used to measure the three dimensions
of interest: perceived SV, perceived SC, and perceived shared
positive mood (PM). The PNEA scale consists of 20 items
measured using a 5-point Likert scale: SV (eight-items), SC (six
items), and OPM (six items) and has been shown to have good
psychometric properties with Cronbach alphas of 0.94, 0.83, and
0.91, respectively, (Pittenger et al., 2012). The final sample consisted of 149 respondents, with a mean
age of 40.4 years (SD 14.3 years), 84 were female, 81 were in
management positions, representing 11 public companies, and
138 private companies (including non-profits). General Self-Efficacy The general self-efficacy scale used was developed by Chen et al. (2001). It consists of eight items scored using a 5-point Likert June 2015 | Volume 6 | Article 781 Frontiers in Psychology | www.frontiersin.org 4 Perceptions of socially responsible actions Thornton TABLE 1 | Latent factor correlations (n = 149, Cronbach’s α on the diagonal). Variable
Mean
SD
1
2
3
4
5
6
7
Relationship quality
0.035
0.98
0.76
General self-efficacy
4.11
0.82
0.43
0.89
Conscientiousness
3.76
0.92
0.29
0.08
0.80
Discretionary social responsibility (SR)
3.53
1.05
0.43
0.17
0.47
0.89
Ethical SR
3.73
1.03
0.59
0.14
0.41
0.62
0.83
Legal SR
4.00
0.89
0.56
0.31
0.32
0.53
0.55
0.80
Economic SR
3.62
0.98
0.41
0.34
0.34
0.58
0.52
0.58
0.71
FIGURE 1 | Results of structural equation modeling (SEM). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. TABLE 1 | Latent factor correlations (n = 149, Cronbach’s α on the diagonal). 0.71 FIGURE 1 | Results of structural equation modeling (SEM). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. FIGURE 1 | Results of structural equation modeling (SEM). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. FIGURE 1 | Results of structural equation modeling (SEM). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. bias. CMV was assessed using the Lindell and Whitney (2001)
marker variable technique. CMV was non-significant at a 0.05
level of significance. least squares structural equation modeling (PLS-SEM), which is
non-parametric and robust (Ringle et al., 2005; Hair et al., 2010). A confirmatory factor analysis (CFA) of the data was
completed in SmartPLS. When used for CFA, PLS provides
evidence of convergent and discriminant validity of the
measurement model. In particular, the program provides
factor loadings for each measurement and t-statistics for the
significance of the loading to the latent variables. Measurement
variables that had loadings that were not significant were dropped
from the analysis. Structural Analysis The data from this study were analyzed using SmartPLS v2.0
M3 (beta; Ringle et al., 2005). The PLS method provides
standardized betas and R2 values relative to the outer and inner
models, where the outer model represents the measurement
model and the inner model represents the structural model. Bootstrapping determined the level of statistical significance
(t-statistics), AVE, CR, Cronbach alpha (α), communality,
and redundancy in the model. The reliability, validity, betas,
and R2 values indicate good model fit. The fit assessment
was performed using a blindfolding technique as discussed
in Tenenhaus et al. (2005). According to Wold (1982),
the model is run while a selected construct is removed
at specified intervals ranging between 5 and 10, where the
interval is not divisible into the sample size. An omission
factor of seven was selected for this evaluation. This allowed
the creation of cross-validated communalities (Q2) for each
latent variable, where the Q2 values (greater than zero)
indicated that the latent variables were well constructed
and results were relevant and predictive (Tenenhaus et al.,
2005). y
The final measurement model showed good discriminant
validity with no evidence of significant cross loading by measures
on other factors. Convergent validity is demonstrated by average
variance extracted (AVE) values above 0.50 and CR values
that are greater than AVE values, while discriminant validity
is demonstrated by loadings of individual variables only on
the appropriate latent variables. Table 1 provides the construct
correlations, means, SD, and Cronbach alphas. While there
is always some bias related to surveys, allowing individuals a
choice of completing a survey results in fewer external validity
threats. Results The results of the analysis shown in Figure 1, include
standardized betas, p-values, and type of mediation present. Figure 1 shows direct and mediated paths that are significant
based on the hypotheses tested, with the presence of the
mediating variable. Table 2 provides the results of the hypothesis
testing showing hypotheses that are supported, if the support was
direct or mediated, and the type of mediation. Self-efficacy is positively related to economic and legal
SR and fully mediated by relationship quality. Regarding
economic SR, this complements the non-significant findings for
conscientiousness explained above. The stronger relationship of
self-efficacy to economic and legal SR does seem to be a function
of more individualistic and utilitarian nature of economic
perspectives (Boyatzis et al., 2000). Legal SR is often a more
task-oriented perspective, rather than the more philosophical and
larger scale perspectives involved in ethical and discretionary SR. Using Cohen’s f2 (Soper, 2012), the effect size of the regression
coefficients was determined to be moderate for relationship
quality (0.33), and large for economic (0.57), legal (0.52), ethical
(0.61), and discretionary (0.66) SR. The data indicate that a
significant positive correlation exists between conscientiousness,
general self-efficacy, and the various SR dimensions and that
Relationship quality strengthens this effect. The mediation effect
was evaluated following the method of Baron and Kenny and the
effects were assessed using the Sobel test (Baron and Kenny, 1986;
Hayes, 2009; Soper, 2010). g
y
These
findings
suggest
that
regardless
of
individual
dispositions people perceive SR, the quality of one’s relationships
enable multiple aspects of SR. Further research may show how
SR can be motivated through emotional and social contagion. The full mediation of ethical SR is indicative of emotional
involvement through SV pulling the individuals toward a focus
on future opportunities (Goleman et al., 2001; Goleman, 2006). In such settings, relationships with a SV and compassion arouse
intrinsic motivation. Howard (2006) noted that the creation
of positive emotions serves to pull a person toward the ideal
self or their personal vision, shaping the response toward goals
and behaviors that correspond to our intrinsic values and
behaviors. This deflates, somewhat, the argument that ethical
and discretionary SR is only a form of marketing or image
self-interest by the organization. Hypothesis both directly and when partially mediated by relationship quality. Conscientiousness is related to ethical SR and fully mediated
by relationship quality. The link between conscientiousness
and economic SR is not significant. This could be due to the
essential pragmatic nature that would drive economic SR with its
enhanced beliefs in individual effort. This tendency to see things
as individualistic may be at odds with a more socially concerned
sense of duty emerging from conscientiousness. both directly and when partially mediated by relationship quality. Conscientiousness is related to ethical SR and fully mediated
by relationship quality. The link between conscientiousness
and economic SR is not significant. This could be due to the
essential pragmatic nature that would drive economic SR with its
enhanced beliefs in individual effort. This tendency to see things
as individualistic may be at odds with a more socially concerned
sense of duty emerging from conscientiousness. Common Method Bias/Variance Because the data were gathered using a single instrument, the
presence of common method variance (CMV) may be a potential June 2015 | Volume 6 | Article 781 Frontiers in Psychology | www.frontiersin.org 5 Perceptions of socially responsible actions Thornton TABLE 2 | Summary of hypotheses tested. Hypothesis
Direction
Support
Std. β (α)
Mediation
Sobel (α)
H1a: Relationship quality will mediate the positive relationship between
general self-efficacy and economic SR. +
Yes
0.14 (0.003)
Full
2.70 (0.003)
H1b: Relationship quality will mediate the positive relationship between
general self-efficacy and legal SR. +
Yes
0.20 (<0.001)
Full
3.51 (<0.001)
H1c: Relationship quality will mediate the positive relationship between
general self-efficacy and ethical SR. +
No
Not significant
H1d: Relationship quality will mediate the positive relationship between
general self-efficacy and discretionary SR. +
No
Not significant
H2a: Relationship quality will mediate the positive relationship between
Conscientiousness and economic SR. +
No
Not significant
H2b: Relationship quality will mediate the positive relationship between
Conscientiousness and legal SR. +
Yes
0.14 (0.002)
0.17 (0.038)
Partial
Direct
2.80 (0.002)
H2c: Relationship quality will mediate the positive relationship between
Conscientiousness and ethical SR. +
Yes
0.16 (0.002)
Full
2.93 (0.002)
H2d: Relationship quality will mediate the positive relationship between
Conscientiousness and discretionary SR. +
Yes
0.10 (0.003)
0.31 (<0.001)
Partial
Direct
2.74 (0.003) TABLE 2 | Summary of hypotheses tested. References Baron, R. M., and Kenny, D. A. (1986). The moderator–mediator variable
distinction in social psychological research: conceptual, strategic, and statistical
considerations. J. Pers. Soc. Psychol. 51, 1173–1182. doi: 10.1037/0022-
3514.51.6.1173 Abbott, W., and Monsen, R. (1979). On the measurement of corporate
social responsibility: self-reported disclosures as a method of measuring
corporate social involvement. Acad. Manage. J. 22, 501–515. doi: 10.2307/
255740 Barrick, M. R., Mount, M. K., and Strauss, J. P. (1993). Conscientiousness
and performance of sales representatives: test of the mediating effects
of goal setting. J. Appl. Psychol. 78, 715–722. doi: 10.1037/0021-9010. 78.5.715 Agle, B., Mitchell, R., and Sonnenfeld, J. (1999). Who Matters to CEOs? An
investigation
of
stakeholder
attributes
and
salience,
corporate
performance, and CEO values. Acad. Manage. J. 42, 507–525. doi: 10.2307/
256973 Besser, T., and Miller, N. (2004). The risks of enlightened self-interest:
small businesses and support for community. Bus. Soc. 43, 398–425. doi:
10.1177/0007650304271140 Aguilera, R. V., Rupp, D. E., Williams, C. A., and Ganapathi, J. (2007). Putting the
S back in corporate social responsibility: a multilevel theory of social change
in organizations. Acad. Manage. Rev. 32, 836–863. doi: 10.5465/AMR.2007. 25275678 Bowen, F. (2000). Environmental visibility: a trigger of green organizational
response? Business
Strategy
and
the
Environment,
9,
92–107. doi:
10.1002/(SICI)1099-0836(200003/04)9:2<92::AID-BSE230>3.0.CO;2-X Bowen, F. (2002). Does size matter? Organizational slack and visibility as
alternative explanations for environmental. Bus. Soc. 41, 118–124. doi:
10.1177/0007650302041001007 Aguinis, H., and Glavas, A. (2012). What we know and don’t know about corporate
social responsibility a review and research agenda. J. Manage. 38, 932–968. doi:
10.1177/0149206311436079 Bowen, H. (1953). Social Responsibilities of the Businessman. New York, NY:
Harper. Ajzen, I., and Fishbein, M. (1980). A Theory of Reasoned Action. Englewood Cliffs,
NJ: Prentice-Hall. Boyatzis, R. E. (2008). Leadership development from a complexity perspective. Consult. Psychol. J. Pract. Res. 60, 298–313. doi: 10.1037/1065-9293.60. 4.298 Ajzen, I., and Fishbein, M. (2005). “The influence of attitudes on behavior,” in
The Handbook of Attitudes, eds D. Albarracín, B. T. Johnson, and M. P. Zanna
(Mahwah, NJ: Erlbaum), 173–221. Boyatzis, R. E. (2013). “When pulling to the negative emotional attractor is
too much or not enough to inspire or sustain outstanding leadership,” in
The Fulfilling Workplace: The Organization’s Role in Achieving Individual and
Organizational Health, eds R. Burke, C. Cooper, and G. Woods (London: Gower
Publishing), 139–150. Aragón-Correa, J., Hurtado-Torres, N., Sharma, S., and García-Morales, V. (2008). Environmental strategy and performance in small firms: a resource-based
perspective. Implications for Practice and Research Further research is needed to test the linkage between individuals
and organizational SR along specific dimensions, especially
in terms of the effect of positive and negative emotions. In addition, subsequent research should investigate other
personality dispositions, like agreeableness, to determine if they
have an impact of different facets of SR, or even traits like
general mental ability. Given the results of this study, a follow-
up study should examine the differential effects of SV, SC,
and shared PM. It would also be desirable to study composite
views of people in an organization on their perceptions of
each SR dimension and relate this to individual traits and
perceptions. Acknowledgments I am indebted to the two reviewers, whose suggestions helped
to strengthen this paper. I appreciate the comments of Dr. Scott
Taylor, one of the co-editors that also helped to focus the revised
paper. I would also like to thank my colleagues, Michael Luthy,
Ph.D. and J. T. Byrd, Ph.D. for their review and comments on the
paper. Discussion The purpose of this study was to assess selected individual
characteristics and relationship quality in their effect on
individual perceptions of SR decisions along four conceptual
dimensions. The major contribution of the study suggests that
efforts at stimulating increases in SR might focus on fostering
self-efficacy and conscientiousness along with creating higher
quality relationships in terms of SV, compassion and PM
within organizations as opposed to a broad appeal to social
conscience. The findings indicate significant positive direct
relationships of self-efficacy and conscientiousness with different
SR dimensions. These connections are strengthened through
the relationship that individuals perceive to exist within their
organization. Based on the positive aspects of SV, compassion, and PM,
it would appear that various dimensions of SR might be
experienced as intrinsic motivators of the SR of organizations. Dutton et al. (2007) argue that compassion is particularly
important in organizations, since it increases interconnections
between employees leading to greater levels of trust and This study found that conscientiousness, manifested as being
responsible, is positively related to legal and discretionary SR June 2015 | Volume 6 | Article 781 Frontiers in Psychology | www.frontiersin.org 6 Perceptions of socially responsible actions Thornton enthusiasm to acting in a positive manner. Relationship
quality serves to create a linkage between individuals and the
organizations resulting in the organization noticing issues, feeling
with others and finally taking action on those issues. Conclusion The appeal to stimulate more SR along each of the four
dimensions in organizations would be enhanced if people worked
on the degree of SV and compassion in their relationships. Whether a person acts with SR is often attributed to some
individual characteristic, trait, or value. This study examined how
the nature of relationships may alter perceptions of corporate
responses leading to different dimensions of SR activities. In this
sense, the development of better relationships in terms of SV,
compassion, and PM may help promote various forms of SR. As discussed by (Kanov et al., 2004), the sense of SC serves
to link the individual responses within the organization to the
overall response of the organization to the feelings and needs
of others. This has a strong effect on the sense of responsibility
(conscientiousness) that people have when they make decisions
and may lead to more caring organizations. Limitations One limitation of this study is the size and nature of the
population surveyed. The relationships to different dimensions of
SR discovered may be different in large companies, public sector
organizations or non-profits. The use of a single type of data
(self-report) is known to create potential for CMV, which was
addressed in this study using a marker variable technique (Lindell
and Whitney, 2001). References Res. Methods 4, 62–83. doi: 10.1177/109442810141004 Lindell, M. K., and Whitney, D. J. (2001). Accounting for common method
variance in cross-sectional research designs. J. Appl. Psychol. 86, 114–121. doi:
10.1037/0021-9010.86.1.114 Organ. Res. Methods 4, 62–83. doi: 10.1177/109442810141004 Clayton, B. (2009). When Practice and Theory Conflict: Do Financial Incentives
Influence Championing Behaviors in Mergers and Acquisitions? Doctoral thesis,
Weatherhead School of Management, Case Western Reserve University,
Cleveland, OH, 70. Maignan, I., and Ferrell, O. (2000). Measuring corporate citizenship in two
countries: the case of the United States and France. J. Bus. Ethics 23, 283–297. doi: 10.1023/A:1006262325211 Conley, J., and Williams, C. (2005). Engage, embed, and embellish: theory versus
practice in the corporate social responsibility movement. J. Corp. L. 31, 1. doi:
10.2139/ssrn.691521 Marshall, R., Cordano, M., and Silverman, M. (2005). Exploring individual and
institutional drivers of proactive environmentalism in the US wine industry. Bus. Strategy Environ. 14, 92–109. doi: 10.1002/bse.433 Cordano, M., Marshall, R., and Silverman, M. (2009). How do small and medium
enterprises go “green”? A study of environmental management programs in the
US wine industry. J. Bus. Ethics 92, 463–478. doi: 10.1007/s10551-009-0168-z Moberg, D. (1999). The big five and organizational virtue. Bus. Ethics Q. 9, 245–272. doi: 10.2307/3857474 Murillo, D., and Lozano, J. M. (2006). SMEs and CSR: an approach to CSR in their
own words. J. Bus. Ethics 67, 227–240. doi: 10.1007/s10551-006-9181-7 Dahlsrud, A. (2008). How corporate social responsibility is defined: an analysis
of 37 definitions. Corp. Soc. Respons. Environ. Management 15, 1–13. doi:
10.1002/csr.132 Neff, J. (2011). Non-financial Indicators of Family Firm Performance: A Portfolio
Model Approach. Cleveland, OH: Case Western Reserve University. Darnall, N., Henriques, I., and Sadorsky, P. (2009). Adopting proactive
environmental strategy: the influence of stakeholders and firm size. J. Manage. Stud. 47, 1072–1094. doi: 10.1111/j.1467-6486.2009.00873.x pp
y
Nonis, S., and Swift, C. O. (2001). Personal value profiles and ethical
business decisions. J. Educ. Bus. 76, 251–256. doi: 10.1080/08832320109
599644 Dean, T., Brown, R., and Bamford, C. (1998). Differences in large and small
firm responses to environmental context: strategic implications from a
comparative analysis of business formations. Strateg. Manage. J. 19, 709–728. doi: 10.1002/(SICI)1097-0266(199808)19:8<709::AID-SMJ966>3.0.CO;2-9 Organ, D. W., and Ryan, K. (1995). A meta-analytic review of attitudinal and
dispositional predictors of organizational citizenship behavior. Pers. Psychol. 48,
775–802. doi: 10.1111/j.1744-6570.1995.tb01781.x Overbeke, K. K. (2010). The Intersection of the Family and Business: The Father-
Daughter Connection. Cleveland, OH: Case Western Reserve University. References Dutton, J., Lilius, J., and Kanov, J. (2007). “The transformative potential of
compassion at work,” in Handbook of Transformative Cooperation: New Designs
and Dynamics, eds S. Piderit, R. Fry, and D. Cooperrider (Standford, CA:
Standford University Press), 107–126. Penner, L. A., Fritzsche, B. A., Craiger, J. P., and Freifeld, T. R. (1995). Measuring
the prosocial personality. Adv. Pers. Assess. 10, 147–163. Peterson, C., and Seligman, M. E. P. (2004). Character Strengths and Virtues: A
Handbook and Classification. Oxford, MS: Oxford University Press. Eisenberg, N., and Miller, P. A. (1987). The relation of empathy to prosocial
and related behaviors. Psychol. Bull. 101, 91–119. doi: 10.1037/0033-2909. 101.1.91 Pittenger, L., Perelli, S., and Somers, T. (2012). IT professionals: maximizing
engagement
in
the
rapidly
changing
business
environment. Paper
Presented
at
the
Technology
Management
Conference
(ITMC),
2012
IEEE
International
(Dallas,
TX:
IEEE). doi:
10.1177/014920630002
600307 Fassin, Y. (2008). SMEs and the fallacy of formalizing CSR. Bus. Ethics 17, 364–378. doi: 10.1111/j.1467-8608.2008.00540.x Giberson,
T.,
Resick,
C.,
and
Dickson,
M. (2005). Embedding
leader
characteristics: an examination of homogeneity of personality and values
in organizations. J. Appl. Psychol. 90, 1002–1010. doi: 10.1037/0021-9010.90. 5.1002 Podsakoff, P. M., MacKenzie, S. B., Paine, J. B., and Bachrach, D. G. (2000). Organizational citizenship behaviors: a critical review of the theoretical and
empirical literature and suggestions for future research. J. Manage. 26, 513–563. doi: 10.1177/014920638601200408 Goldberg, L. R., Johnson, J. A., Eber, H. W., Hogan, R., Ashton, M. C.,
Cloniger, C. R., et al. (2006). The international personality item pool and the
future of public-domain personality measures. J. Res. Pers. 40, 84–96. doi:
10.1016/j.jrp.2005.08.007 Podsakoff, P., and Organ, D. (1986). Self-reports in organizational research:
problems and prospects. J. Manage. 12, 531–544. Ringle, C. M., Wende, S., and Will, A. (2005). SmartPLS 2.0 (M3) Beta, Hamburg. Available at: http://www.smartpls.de/ Goleman, D. (2006). Working with Emotional Intelligence. New York, NY: Bantam
Dell. Roberts, B. W., Chernyshenko, O. S., Stark, S., and Goldberg, L. R. (2005). The
structure of conscientiousness: an empirical investigation based on seven major
personality questionnaires. Pers. Psychol. 58, 103–139. doi: 10.1007/s10551-007-
9618-7 Goleman, D., Boyatzis, R. E., and McKee, A. (2001). Primal leadership: the hidden
driver of great performance. Harv. Bus. Rev. 79, 42–51. Goll, I., and Rasheed, A. A. (2004). The moderating effect of environmental
munificence and dynamism on the relationship between discretionary
social responsibility and firm performance. J. Bus. Ethics 49, 41–54. doi:
10.1023/B:BUSI.0000013862.14941.4e Rodrigo, P., and Arenas, D. (2008). References J. Environ. Manage. 86, 88–103. doi: 10.1016/j.jenvman.2006. 11.022 Bandura, A. (1982). Self-efficacy mechanism in human agency. Am. Psychol. 37,
122–147. doi: 10.1037/0003-066X.37.2.122 Boyatzis, R. E., Murphy, A. J., and Wheeler, J. V. (2000). Philosophy as a
missing link between values and behavior. Psychol. Rep. 86, 47–64. doi:
10.2466/pr0.2000.86.1.47 Bandura, A. (1991). “Social cognitive theory of moral thought and action,” in
Handbook of Moral Behavior and Development, Vol. 1, eds W. M. Kurtines and
J. L. Gewirtz (Hillsdale, NJ: Erlbaum). Boyatzis, R., and Oliver, W. (2008). PNEA Survey. Cleveland, OH: Case Western
Reserve University. Bandura, A. (ed.). (1998). Self-Efficacy. San Diego, CA: Academic Press. June 2015 | Volume 6 | Article 781 Frontiers in Psychology | www.frontiersin.org Perceptions of socially responsible actions Thornton Howard, A. (2006). Positive and negative emotional attractors and intentional
change. J. Manage. Dev. 25,
657–670. doi:
10.1108/02621710610
678472 Boyatzis, R. E., Rochford, K., and Jack, A. I. (2014). Antagonistic neural networks
underlying differentiated leadership roles. Front. Hum. Neurosci. 8:114. doi:
10.3389/fnhum.2014.00114 Jenkins, H. (2006). Small business champions for corporate social responsibility. J. Bus. Ethics 67, 241–256. doi: 10.1007/s10551-006-9182-6 Brief, A., and Motowidlo, S. (1986). Prosocial organizational behaviors. Acad. Manage. Rev. 11, 710–725. Kanov, J. M., Maitlis, S., Worline, M. C., Dutton, J. E., Frost, P. J., and Lilius, J. M. Kanov, J. M., Maitlis, S., Worline, M. C., Dutton, J. E., Frost, P. J., and Lilius, J. M. (2004). Compassion in organizational life. Am. Behav. Sci. 47, 808–827. doi:
10.1177/0002764203260211 Byrne, B. M. (2010). Structural Equation Modeling with AMOS, 2nd Edn. New York, NY: Routledge. (2004). Compassion in organizational life. Am. Behav. Sci. 47, 808–827. doi:
10.1177/0002764203260211 Campbell, J. (2007). Why would corporations behave in socially responsible ways? An institutional theory of corporate social responsibility. Acad. Manage. Rev. 32, 946–967. doi: 10.5465/AMR.2007.25275684 Keim, G. (1978). Corporate social responsibility: an assessment of the enlightened
self-interest model. Acad. Manage. Rev. 3, 32–39. Khawaja, M. S. (2012). The Mediating Role of Positive and Negative Emotional
Attractors between Psychosocial Correlates of Doctor-Patient Relationship and
Treatment Adherence in Type 2 Diabetes. Ph.D. dissertation, Case Western
Reserve University, Cleveland,OH. Carroll, A. (1991). The pyramid of corporate social responsibility: toward the
moral management of organizational stakeholders. Bus. Horiz. 34, 39–48. doi:
10.1016/0007-6813(91)90005-G Chen, G., Gully, S. M., and Eden, D. (2001). Validation of a new general self-efficacy Chen, G., Gully, S. M., and Eden, D. (2001). Validation of a new general self-efficacy
scale. Organ. References Do employees care about CSR programs? A
typology of employees according to their attitudes. J. Bus. Ethics 83, 265–283. doi: 10.1002/job.380 Hair, J. Jr., Black, W., Babin, B., and Anderson, R. (2010). Multivariate Data
Analysis, 7th Edn. New Jersey: Prentice Hall. Rupp, D., Ganapathi, J., Aguilera, R., and Williams, C. (2006). Employee reactions
to corporate social responsibility: an organizational justice framework. J. Organ. Behav. 27, 537–543. doi: 10.1037/0022-3514.73.6.1296 Hayes, A. F. (2009). Beyond baron and kenny: statistical mediation analysis in the
new millennium. Commun. Monogr. 76, 408–420. doi: 10.1080/036377509033
10360 Saucier, G. (1997). Effects of variable selection on the factor structure of person
descriptors. J. Pers. Soc. Psychol. 73, 1296–1312. doi: 10.5840/beq200313435 Hogan, R., Hogan, J., and Robert, B. (1996). Personality measurement
and employment decisions. Am. Psychol. 51, 469–477. doi: 10.1037/0003-
066X.51.5.469 Schwartz, M. S., and Carroll, A. B. (2003). Corporate social responsibility: a
three-domain approach. Bus. Ethics Q. 13, 503–530. June 2015 | Volume 6 | Article 781 8 Frontiers in Psychology | www.frontiersin.org Perceptions of socially responsible actions Thornton Wold, H. (1982). “Soft modeling: the basic design and some extensions,” in
Systems Under Indirect Observation, Part 2, eds K. G. Joreskog and H. Wold
(Amsterdam: North-Holland). Soper, D. (2010). Sobel Test Calculator for the Significance of Mediation (Software). Available at: http://www.danielsoper.com/statcalc p
p
Soper, D. S. (2012). Effect Size Calculator for Multiple Regression (Online Software). Available at: http://www.danielsoper.com/statcalc Conflict of Interest Statement: The author declares that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest. Taylor, S., Kluemper, D., and Mossholder, K. (2010). Linking personality
to
interpersonal
citizenship
behaviour:
the
moderating
effect
of
empathy. J. Occup. Organ. Psychol. 83, 815–834. doi: 10.1016/j.csda.2004. 03.005 Copyright © 2015 Thornton. This is an open-access article distributed under
the terms of the Creative Commons Attribution License (CC BY). The use,
distribution or reproduction in other forums is permitted, provided the original
author(s) or licensor are credited and that the original publication in this
journal is cited, in accordance with accepted academic practice. No use,
distribution or reproduction is permitted which does not comply with these
terms. Tenenhaus, M., Vinzi, V. E., Chatelin, Y.-M., and Lauro, C. (2005). PLS path
modeling. Comput. Stat. Data Anal. 48, 159–205. Thornton, J. C., and Byrd, J. T. (2013). Social responsibility and the small business. Acad. Entrep. J. 19, 41–75. Frontiers in Psychology | www.frontiersin.org June 2015 | Volume 6 | Article 781 References doi: 10.1007/s10551-006-9187-1 Williamson, D., Lynch-Wood, G., and Ramsay, J. (2006). Drivers of environmental
behaviour in manufacturing SMEs and the implications for CSR. J. Bus. Ethics
67, 317–330. June 2015 | Volume 6 | Article 781 Frontiers in Psychology | www.frontiersin.org
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Scaling of thermal tolerance with body mass and genome size in ectotherms: A comparison between water-and air-breathers
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Downloaded from: http://hdl.handle.net/2066/206113
Download date: 2024-10-24 Version of the following full text: Publisher’s version
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To cite this publication please use the final published version (if applicable). To cite this publication please use the final published version (if applicable). royalsocietypublishing.org/journal/rstb Research
Cite this article: Leiva FP, Calosi P, Verberk
WCEP. 2019 Scaling of thermal tolerance with
body mass and genome size in ectotherms:
a comparison between water- and
air-breathers. Phil. Trans. R. Soc. B 374:
20190035. h
//d d
/
/ b Keywords: Keywords:
cell size, CTmax, CTmin, oxygen limitation,
phylogeny, exposure duration This article is part of the theme issue ‘Physiological diversity, biodiver-
sity patterns and global climate change: testing key hypotheses involving
temperature and oxygen’. Accepted: 31 March 2019 One contribution of 12 to a theme issue
‘Physiological diversity, biodiversity patterns
and global climate change: testing key
hypotheses involving temperature and oxygen’. Subject Areas:
ecology, evolution, physiology
Keywords:
cell size, CTmax, CTmin, oxygen limitation,
phylogeny, exposure duration
Author for correspondence:
Fe´lix P. Leiva
e-mail: f.leiva@science.ru.nl Research Fe´lix P. Leiva1, Piero Calosi2 and Wilco C. E. P. Verberk1 Fe´lix P. Leiva1, Piero Calosi2 and Wilco C. E. P. Verberk1 1Department of Animal Ecology and Physiology, Radboud University Nijmegen, 6500 Nijmegen, The Netherlands
2De´partement de Biologie, Chimie et Ge´ographie, Universite´ du Que´bec a` Rimouski, 300 Alle´e des Ursulines,
Rimouski, Quebec, Canada G5L 3A1 Cite this article: Leiva FP, Calosi P, Verberk
WCEP. 2019 Scaling of thermal tolerance with
body mass and genome size in ectotherms:
a comparison between water- and
air-breathers. Phil. Trans. R. Soc. B 374:
20190035. FPL, 0000-0003-0249-9274; WCEPV, 0000-0002-0691-583X FPL, 0000-0003-0249-9274; WCEPV, 0000-0002-0691-583X FPL, 0000-0003-0249-9274; WCEPV, 0000-0002-0691-583X Global warming appears to favour smaller-bodied organisms, but whether
larger species are also more vulnerable to thermal extremes, as suggested
for past mass-extinction events, is still an open question. Here, we tested
whether interspecific differences in thermal tolerance (heat and cold) of
ectotherm organisms are linked to differences in their body mass and
genome size (as a proxy for cell size). Since the vulnerability of larger,
aquatic taxa to warming has been attributed to the oxygen limitation
hypothesis, we also assessed how body mass and genome size modulate
thermal tolerance in species with contrasting breathing modes, habitats
and life stages. A database with the upper (CTmax) and lower (CTmin) criti-
cal thermal limits and their methodological aspects was assembled
comprising more than 500 species of ectotherms. Our results demonstrate
that thermal tolerance in ectotherms is dependent on body mass and
genome size and these relationships became especially evident in prolonged
experimental trials where energy efficiency gains importance. During
long-term trials, CTmax was impaired in larger-bodied water-breathers, con-
sistent with a role for oxygen limitation. Variation in CTmin was mostly
explained by the combined effects of body mass and genome size and it
was enhanced in larger-celled, air-breathing species during long-term
trials, consistent with a role for depolarization of cell membranes. Our results
also highlight the importance of accounting for phylogeny and exposure
duration. Especially when considering long-term trials, the observed effects
on thermal limits are more in line with the warming-induced reduction in
body mass observed during long-term rearing experiments. http://dx.doi.org/10.1098/rstb.2019.0035 Subject Areas:
ecology, evolution, physiology Subject Areas:
ecology, evolution, physiology 1. Introduction The capacity of organisms to take up and transform resources from their
environment is a key attribute governing growth and reproduction, and sub-
sequently
affecting
population
dynamics,
community
composition
and
ecosystem functioning [1,2]. Such capacity seems to be mainly dictated by
species’ body mass [3]. Macroecological and palaeoecological data show spatial
(e.g. Bergmann’s rule [4,5]) and temporal (Lilliput effect [6]) variation in body
mass, which share a common point related to the environmental temperature:
at warmer, tropical latitudes and during the past mass extinctions, warming
appears to select for smaller-bodied species [5,7–9]. Body size reductions
with warming appear to be stronger in aquatic taxa than in terrestrial taxa Electronic supplementary material is available
online at https://doi.org/10.6084/m9.figshare.c. 4494482. & 2019 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution
License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original
author and source are credited. [5]. In tandem with body size reductions, both aquatic and
terrestrial species are shifting their distribution towards
cooler habitats and their phenology to earlier and hence
cooler conditions [10,11]. One approach that has been taken
to clarify the extent and variation in species redistributions,
and to determine which taxonomic groups are potentially
more vulnerable to the effects of climate change, is that of
comparative studies that analyse thermal tolerance limits
(upper and lower) synthesized from the literature [12–15]. These studies also highlight key differences in thermal
responses between aquatic and terrestrial taxa, likely related
to their breathing mode [16]. The physiological mechanisms
underpinning
size
adjustments
and
thermal
limits
are
actively debated [17–20], but oxygen limitation has been
implicated for both thermal limits [21–23], and size adjust-
ments [24–29] and hypoxia possibly also contributed to
mass extinctions [8,30]. differences
in
cell
size
have
repercussions
for oxygen
uptake at the cellular level. In the same way, a diversity of cel-
lular physiological functions scales with the cell size [45]. Therefore, differences in thermal tolerance between animals
of different body mass may be mechanistically linked to
differences in cell size, rather than body mass. In contrast
to the hypothesized influence of oxygen limitation on heat
tolerance, the evidence for such an influence on cold toler-
ance is rather limited [16], and these limits are thought to
arise from membrane depolarization and subsequent cell
dysfunction due to energy deficits or—in the case of extreme
cold tolerance—the freezing of body fluids [46]. (a) Data search
W
d
l b Larger-bodied species may be more susceptible to oxygen
limitation because of their lower surface area to volume ratio,
which (all else being equal) constrains their capacity to extract
oxygen from theirenvironment and deliver it to their metaboliz-
ing tissues [24,27,32], or because transport distances increase,
which may be especially a problem if oxygen transport is
based on diffusion [28]. If oxygen limitation plays a role in
setting thermal limits, one prediction would be that thermal
limits vary across organisms with distinct capacities to supply
oxygen, including differences between water- and air-breathers,
or between gas exchange systems across life stages. As body
mass is intimately connected to a suite of other traits,
size-dependency of thermal limits may be driven by traits
related to body mass, rather than body mass per se. Forexample,
relative to the larger adults, smaller life stages also may
experience relatively cool early-season conditions, especially
in temperate and polar regions with a clear seasonality, such
that their thermal limits are shifted to lower temperatures, i.e. improved cold tolerance and impaired heat tolerance. In
addition, organisms living in aquatic habitats will experience
different thermal regimes from those living on land [15]. We created a global database of body mass-related traits (body
mass and genome size), life stage (adult, juvenile and larva)
and breathing mode (air-, bimodal- and water-breathers) of
aquatic and terrestrial species belonging to four taxonomic
groups (amphibians, arthropods, fish and reptiles) for which
the critical thermal limits (upper and lower) have been evaluated
using dynamic methods (i.e. CTmax or CTmin, sensu [47]). The
chosen groups comprise taxa for which the determination of
body mass was expected to be straightforward. We started by
retrieving information from articles on body mass and thermal
limits from existing quantitative reviews whose aim has been to
explore global patterns of thermal tolerance in ectotherms
[12,13,15]. We then added information from recently published
references, from January 2015 to October 2018, which were
found by using the following keyword combinations of Boolean
terms through ISI Web of Science: (thermal tolerance OR heat tol-
erance OR cold tolerance OR upper thermal limit OR lower
thermal limit OR thermal range OR CTmax OR CTmin) AND
(body mass OR body size OR length) AND (amphib* OR arthrop*
OR crustacea* OR fish* OR insect* OR reptil*). 1. Introduction Thus, for
cold tolerance, a cellular perspective may be more informa-
tive, although the correlation between cell size and body
mass may result in size-dependency for CTmin. 2 In the present study, we use a global database of lower
(CTmin) and upper (CTmax) critical thermal limits sup-
plemented with information on other biological traits of
ectotherm species and their phylogenetic relationships, to
investigate whether and how the tolerance to high and low
temperatures is modulated by the body mass and genome
size (proxy for cell size) across arthropod and vertebrate
(amphibian, fish and reptile) species have distinct breathing
modes,
life
stages
and
habitats. We
hypothesize
that:
(1) both CTmax and CTmin will be related to the body mass
and genome size of the species, with thermal limits decreasing
with increasing body mass (for CTmax) and with increasing
genome size (for CTmin); (2) both CTmax and CTmin will
differ across breathing modes and a species’ habitat, and
such differences will become more pronounced in large-
bodied organisms or those with larger genomes; and (3)
early life stages will be more susceptible to heat stress than
their adult counterparts, and more resistant to cold stress. By affecting both oxygen demand [31] and the availability
of oxygen in water [32,33], warming is hypothesized to result
in oxygen limitation, which then causes reductions in
thermal limits [22,34] and/or body mass [24,29]. As breath-
ing underwater is more challenging than breathing in air,
this oxygen-based mechanism could explain the divergent
responses observed in air- and water-breathers [25]. While
studies to date hint at a possible size-dependence of thermal
limits, no studies have tested this possibility comprehen-
sively. In fact, most studies have focused on one or a few
species and although these studies often find no effect of
body mass when included as a covariate in analyses, thermal
tolerance limits (heat tolerance rather than cold tolerance) are
more frequently reported to decrease rather than increase
with increasing body mass [35–38]. In an effort to address
this knowledge gap regarding how body mass modulates
the response to the temperature in ectotherms, we took
advantage of the large body of literature and created a data-
base of upper and lower thermal limits supplemented with
biological information of 510 species. (b) Inclusion criteria Before the main analyses, we first performed preliminary
PGLSs in order to determine whether methodological variables
influence thermal limits within this dataset and needed to be
included in the main analyses. For this, we tested whether the
(1) species origin (laboratory or field), or (2) latitude of collection,
or (3) acclimation time in the laboratory and the (4) time necess-
ary to reach the CTmax and CTmin affected these thermal limits. The time was calculated after [58,59], as the relation between
ramping rate (DT, in 8C min21) and the starting temperature
(T0)
for
CTmax
as:
time ¼ [CTmax – T0] DT21,
and
for
CTmin trials as: time ¼ [T0 – CTmin] DT21. Out of these four
methodological variables, only time and/or latitude showed
the highest support and also had significant effects on the ther-
mal limits (for CTmax: latitude and time, and for CTmin: only
latitude) and these two were subsequently included as covariates
in the main models (see electronic supplementary material,
tables S1 and S2 and figure S7). g
For the main analyses, we fitted PGLSs models to each of
CTmax and CTmin, first with body mass (log10-transformed
body mass) as an independent numerical variable, and breathing
mode (air and water), life stage (adult and non-adult) and habitat
(aquatic, intertidal and terrestrial) as categorical variables. We
also ran models that included all possible interactions of these
categorical variables and body mass. In a similar, second set of
models, we used genome size (log10-transformed genome size)
instead of body mass. Since we did not have a reliable estimate
of genome size for all 510 taxa, the models using genome size
were based on a smaller set of species, and hence model perform-
ance cannot be compared directly between models based on
body mass and those based on genome size. All body size data collected in units other than mass were
transformed using appropriate allometric relationships at the
species level [49]; if this was not possible, we moved up to a
higher taxonomic level (e.g. genus or family [50,51]), aiming to
obtain a more representative unit of size for all species in the
database, in this case the body mass in grams (g). As a proxy
of cell size, we collected genome size data (in picograms, pg)
from the Animal Genome Size Database [52] if it was available. (b) Inclusion criteria CTmax and CTmin data established by a dynamic (or ramping)
method were included in our database, which represents the most
common metrics used to assess thermal tolerances in chosen taxa
[48]. To account for methodological variation related to differences
in starting temperatures and heating/cooling rates across species
or studies, we calculated the exposure duration as a single metric
that takes into account how long animals are exposed to thermal
stress during the heating and cooling trials. After having merged
the already published databases with the articles resulting from
our search, all duplicates were removed and each article was
screened and filtered to build our dataset based only on experimen-
tal studies on the basis of three main inclusion criteria: (i) mention of
species name belonging to at least one of the four taxa selected
(amphibians, arthropods, fish and reptiles), (ii) mention of body
mass estimates as mass (wet or dry), width (carapace) or length (car-
apace, fork, intertegular, snout–vent, standard and total), and (iii)
species candidates should be enlisted in the Open Tree of Life
(https://ot39.opentreeoflife.org) for subsequent phylogenetic ana-
lyses (see §2c, Data analyses). Despite the restrictive nature of our
criteria, just in afew cases, multiple articles reported data on thermal
limits for the same species. For this, we prioritized the articles with
the most information available, covering the largest number of
entries in our database. Even so, if there were duplicates per species,
we favoured those studies that (i) give both CTmax and CTmin esti-
mations, over studies reporting only one thermal limit, (ii) mention
the life stage used during the experiments, and (iii) mention meth-
odological information such as cooling/heating rates, starting
temperatures and geographical coordinates of collection. In the
end, all these criteria allowed us to identify 510 species from 174
research articles providing thermal limits and body mass and phy-
logenetic information (electronic supplementary material, figure
S1). For each species, we compiled taxonomic and biological infor-
mation (life-stage, habitat, breathing mode, body mass and
genome size), data on the site where a species was collected (geo-
graphical
coordinates:
latitude
and
longitude,
and
origin:
laboratory or field), methodological information related to the esti-
mation of the thermal limits (starting temperature, heating/
cooling rates and acclimation time) and, finally, the CTmax and
CTmin values. 3. Results We present results of empirical observations on critical ther-
mal limits for 510 (CTmax) and 232 (CTmin) species
(electronic supplementary material, figure S2a,b). For each (a) Data search
W
d
l b Searches were lim-
ited by research area (ecology, evolutionary biology, biodiversity
conservation, environmental sciences, marine freshwater biology,
physiology, entomology, zoology, biology, oceanography, fish-
eries, limnology, environmental studies, behavioural sciences,
toxicology, water resources and multidisciplinary sciences) and
research articles. To supplement our searches, we delved into
the reference list of each paper to identify additional studies Variation in body mass can result from changes in cell
number, cell size or a combination of both [39,40], but usually
larger-bodied species tend to have larger cells, as documen-
ted in arthropods [40,41], fish [42] and birds and mammals
[43]. The theory of optimal cell size [44] highlights how versions
of phylogenetic
generalized
least-squares
(PGLSs)
models. The correlation structure of these models was given by
the potential similarity of species’ traits resulting from the
shared evolutionary history and described by their phylogenetic
signal using the Pagel’s lambda (l) [54]. For this index, a value
closer to 0 indicates non-phylogenetic signal (phylogenetic inde-
pendence between species, or a star phylogeny) while a value
closer to 1 indicates that species’ traits evolved randomly
through evolutionary timescales (Brownian phylogeny) [55]. Such information, available as phylogenetic trees, was accessed
following [56] and pruned to include only species present in
our database. In addition to the estimation of phylogenetic
signal in the model residuals, we tested for phylogenetic signal
both in the dependent variables (i.e. the thermal limits) as well
as in the independent variables of interest included in the main
models following [57] (see electronic supplementary material,
table S11). missed in the initial search and, if necessary, asked corresponding
authors for additional data not provided in the main text or
electronic supplementary material. 3 (b) Inclusion criteria The breathing mode was established on the basis of the species
used in each experiment, through ‘expert judgement’ orconsulting
secondary references if necessary (e.g. [53]). Bimodal-breathers
were classified either as water-breathers (for trials where they
relied on underwater gas exchange) or air-breathers (for trials
where they relied on aerial gas exchange). As most data concerned
adults, with only few data for larvae and juveniles, these two
categories were grouped as non-adults. Data from publications
where CTmax or CTmin was not reported in the text or tables
(i.e. presented only as figures) were extracted using the LibreOffice
extension ‘OOodigitizer v1.2.1’. y
g
For each model, we explored the contribution of covariates
and phylogeny by fitting models that excluded the effects of phy-
logeny (i.e. with l ¼ 0), latitude or exposure duration. Within each
variable and covariate combination, we selected the most infor-
mative model using a multimodel inference approach by means
of the lowest Akaike’s weights (wi), which provide the relative
weight of the evidence towards one of all tested models, and
therefore must add up to 1 [60]. After fitting the models by maxi-
mum likelihood, hypothesis testing was performed in models
with the highest support using an analysis of deviance with a sig-
nificance level of p 0.05. All analyses and figures presented in
this paper were implemented and generated in R version 3.5.1
[61] using the packages ‘AICcmodavg’ [62], ‘APE’ [63], ‘nlme’
[64], ‘phytools’ [65], ‘rotl’ [56] and ‘visreg’ [66]. (c) Data analyses y
All the results presented in the paper, both in the main text and
in the electronic supplementary material were based on linear Table 1. Results for phylogenetic generalized least-squares (PGLSs) models to explain variation in ectotherms’ CTmax (N ¼ 510 species) as a function of log10-
transformed body mass, breathing mode (air and water), life stage (adult and non-adult), habitat (aquatic, intertidal and terrestrial) and their interactions. All models
were assessed using exposure duration (Time) and/or absolute latitude (Lat) of the animal collection as covariates. The number of parameters (k), corrected Akaike’s
information criterion (AICc), difference in AICc with respect to the model with highest support (DAICc) and Akaike’s weight (wi) are given for each model. Pagel’s
lambda (l) denotes correlation structure used (l ¼ 0, star phylogeny, and l ¼ 1, Brownian phylogeny). Bold font indicates model with highest support. 4 species, we also included information on the body mass of
the experimental animals (electronic supplementary material,
figure S2c) used during the tests and their phylogenetic
relationships
(electronic
supplementary
material,
figures
S3–S6). The smallest species (red fire ant, Solenopsis invicta,
0.0000314 g) is separated from the largest (bonefish, Albula
vulpes, 1235.42 g) by 3.93 107 orders of magnitude (or 7.5
on log10-scale). For most of these species, we also included
information on their genome size (electronic supplementary
material, figure S2d), and this ranged from 0.14 pg, for the
aphid Aphidius avenae, to 66.6 pg for the southern torrent
salamander, Rhyacotriton variegatus. Breathing mode was
( )
(
,
p y g y,
,
p y g y)
g
pp
model
k
AICc
DAICc
wi
0. Covariates only
l ¼ 1 þ Lat þ Time
4
3013.58
54.43
0.00
l ¼ 0 þ Lat þ Time
4
3306.34
347.19
0.00
l ¼ 1 þ Lat
3
3040.98
81.83
0.00
l ¼ 1 þ Time
3
3066.19
107.04
0.00
1. Body mass
l ¼ 1 þ Lat þ Time
5
3014.70
55.55
0.00
l ¼ 0 þ Lat þ Time
5
3200.72
241.57
0.00
l ¼ 1 þ Lat
4
3042.19
83.04
0.00
l ¼ 1 þ Time
4
3066.02
106.87
0.00
2. Breathing mode
l ¼ 1 þ Lat þ Time
5
2984.49
25.34
0.00
l ¼ 0 þ Lat þ Time
5
3232.59
273.43
0.00
l ¼ 1 þ Lat
4
3005.53
46.38
0.00
l ¼ 1 þ Time
4
3031.19
72.03
0.00
3. (c) Data analyses Life stage
l ¼ 1 þ Lat þ Time
5
3015.52
56.37
0.00
l ¼ 0 þ Lat þ Time
5
3307.18
348.02
0.00
l ¼ 1 þ Lat
4
3039.31
80.16
0.00
l ¼ 1 þ Time
4
3067.71
108.56
0.00
4. Habitat
l ¼ 1 þ Lat þ Time
6
3007.04
47.89
0.00
l ¼ 0 þ Lat þ Time
6
3246.43
287.27
0.00
l ¼ 1 þ Lat
5
3032.57
73.42
0.00
l ¼ 1 þ Time
5
3063.24
104.09
0.00
5. Body mass Breathing mode
l ¼ 1 þ Lat þ Time
7
2980.31
21.16
0.00
l ¼ 0 þ Lat þ Time
7
3170.76
211.61
0.00
l ¼ 1 þ Lat
6
3002.48
43.33
0.00
l ¼ 1 þ Time
6
3030.55
71.40
0.00
6. Body mass Life stage
l ¼ 1 þ Lat þ Time
7
3004.43
45.27
0.00
l ¼ 0 þ Lat þ Time
7
3190.02
230.87
0.00
l ¼ 1 þ Lat
6
3031.97
72.82
0.00
l ¼ 1 þ Time
6
3056.17
97.02
0.00
7. Body mass Habitat
l ¼ 1 þ Lat þ Time
9
3003.94
44.79
0.00
l ¼ 0 þ Lat þ Time
9
3167.62
208.47
0.00
l ¼ 1 þ Lat
8
3027.88
68.73
0.00
l ¼ 1 þ Time
8
3052.40
93.25
0.00
8. Body mass 3 Breathing mode 3 Time
l 5 1 1 Lat
10
2959.15
0.00
1.00
l ¼ 0 þ Lat
10
3148.75
189.60
0.00
9. Body mass Life stage Time
l ¼ 1 þ Lat
10
2991.95
32.80
0.00
l ¼ 0 þ Lat
10
3189.60
230.44
0.00
10. Body mass Habitat Time
l ¼ 1 þ Lat
14
2976.66
17.51
0.00
l ¼ 0 þ Lat
14
3174.65
215.49
0.00 species, we also included information on the body mass of
the experimental animals (electronic supplementary material,
figure S2c) used during the tests and their phylogenetic
relationships
(electronic
supplementary
material,
figures
S3–S6). The smallest species (red fire ant, Solenopsis invicta,
0.0000314 g) is separated from the largest (bonefish, Albula vulpes, 1235.42 g) by 3.93 107 orders of magnitude (or 7.5
on log10-scale). For most of these species, we also included
information on their genome size (electronic supplementary
material, figure S2d), and this ranged from 0.14 pg, for the
aphid Aphidius avenae, to 66.6 pg for the southern torrent
salamander, Rhyacotriton variegatus. (c) Data analyses phylogenetically structured and may, therefore, obviate the
need to include phylogeny (table 2, model 5 to model 10). represented by 225 and 285 species, corresponding to air- and
water-breathing species, respectively. On the other hand,
most data concerned adults (N ¼ 402), while the remaining
larvae and juveniles were grouped as non-adults (N ¼ 108). In terms of habitat, the majority of species were aquatic
(316 species), or terrestrial (181 species), with only a few
species being intertidal (13 species). Modelled effects of body mass and genome size on both
thermal limits differed according to whether the model
included phylogeny or not. For CTmax, a negative relation-
ship with body mass was most apparent in the model that
did not include phylogeny (l ¼ 0), likely because extreme
values of both CTmax and body mass were phylogenetically
clustered (tables 1 and 3 and figure 1a,b; electronic sup-
plementary material, figures S3 and S4). Effects of both
body mass and genome size on CTmax differed with breath-
ing mode and exposure duration (electronic supplementary
material, tables S3 and S5; see below). For CTmin, the best-
supported models indicated that cold tolerance declined
(i.e. higher CTmin values) with increasing body mass
(table 2 and figure 1c) and with increasing genome size
(table 4 and figure 1d). Effects of body mass on CTmin dif-
fered
with
breathing
mode
and
exposure
duration
(electronic supplementary material, table S4), whereas those
of genome size differed with habitat and exposure duration
(electronic supplementary material, table S6). Both CTmax and CTmin showed a clear phylogenetic
signal (electronic supplementary material, table S11), indicat-
ing that thermal tolerance among the studied species has
been largely conserved across evolutionary lineages. A com-
parison between PGLSs models under a Brownian mode of
evolution
(l ¼ 1)
and
non-phylogenetic
models
(l ¼ 0)
showed, in most cases, that accounting for phylogenetic
relationships
among
the
studied
species
improved
the
model fit for both CTmax (tables 1 and 3) and CTmin
(tables 2 and 4). The two covariates (i.e. absolute latitude
and exposure duration) were always included in the best-sup-
ported model, indicating their importance in explaining
variation in thermal tolerance. For all CTmax models, greater
support and the lowest corrected Akaike information cri-
terion (AICc) were observed when phylogeny was taken
into account (l ¼ 1). (c) Data analyses Breathing mode was Table 2. Results for phylogenetic generalized least-squares (PGLSs) models to explain variation in ectotherms’ CTmin (N ¼ 232 species) as a function of log10-
transformed body mass, breathing mode (air and water), life stage (adult and non-adult), habitat (aquatic, intertidal and terrestrial), exposure duration (Time) and
their interactions. All models were assessed using absolute latitude (Lat) of the animal collection as a covariate. The number of parameters (k), corrected Akaike’s
information criterion (AICc), difference in AICc with respect to the model with highest support (DAICc) and Akaike’s weight (wi) are given for each model. Pagel’s
lambda (l) denotes correlation structure used (l ¼ 0, star phylogeny, and l ¼ 1, Brownian phylogeny). Bold font indicates model with highest support. 5 model
k
AICc
DAICc
wi
0. Covariates only
l ¼ 1 þ Lat
3
1304.72
69.18
0.00
l ¼ 0 þ Lat
3
1399.46
163.92
0.00
1. Body mass
l ¼ 1 þ Lat
4
1306.73
71.19
0.00
l ¼ 0 þ Lat
4
1289.78
54.24
0.00
2. Breathing mode
l ¼ 1 þ Lat
4
1305.39
69.85
0.00
l ¼ 0 þ Lat
4
1384.55
149.01
0.00
3. Life stage
l ¼ 1 þ Lat
4
1306.57
71.03
0.00
l ¼ 0 þ Lat
4
1398.91
163.37
0.00
4. Habitat
l ¼ 1 þ Lat
5
1292.00
56.46
0.00
l ¼ 0 þ Lat
5
1396.35
160.81
0.00
5. Body mass Breathing mode
l ¼ 1 þ Lat
6
1307.00
71.46
0.00
l ¼ 0 þ Lat
6
1273.49
37.95
0.00
6. Body mass Life stage
l ¼ 1 þ Lat
6
1306.84
71.30
0.00
l ¼ 0 þ Lat
6
1285.09
49.55
0.00
7. Body mass Habitat
l ¼ 1 þ Lat
8
1297.78
62.24
0.00
l ¼ 0 þ Lat
8
1274.84
39.30
0.00
8. Body mass 3 Breathing mode 3 Time
l ¼ 1 þ Lat
10
1288.19
52.65
0.00
l 5 0 1 Lat
10
1235.54
0.00
1.00
9. Body mass Life stage Time
l ¼ 1 þ Lat
10
1290.22
54.68
0.00
l ¼ 0 þ Lat
10
1249.66
14.12
0.00
10. Body mass Habitat Time
l ¼ 1 þ Lat
14
1269.39
33.85
0.00
l ¼ 0 þ Lat
14
1264.29
28.75
0.00 phylogenetically structured and may, therefore, obviate the
need to include phylogeny (table 2, model 5 to model 10). (c) Data analyses By contrast, for CTmin, accounting for
the shared evolutionary history of species was less important
for those models that already included body mass as an
explanatory variable, possibly because body mass is strongly Consistent with the expectation that both CTmax and
CTmin differ in species with different breathing modes and
across habitats, our results indicate a generally lower toler-
ance
for
water-breathers
compared
with
air-breathers,
suggesting that water-breathers are more vulnerable to both Table 3. Results for phylogenetic generalized least-squares (PGLSs) models to explain variation in ectotherms’ CTmax (N ¼ 433 species) as a function of log10-
transformed genome size, breathing mode (air and water), life stage (adult and non-adult), habitat (aquatic, intertidal and terrestrial) and their interactions. All
models were assessed using exposure duration (Time) and/or absolute latitude (Lat) of the animal collection as covariates. The number of parameters (k), corrected
Akaike’s information criterion (AICc), difference in AICc with respect to the model with highest support (DAICc) and Akaike’s weight (wi) are given for each model. Pagel’s lambda (l) denotes correlation structure used (l ¼ 0, star phylogeny, and l ¼ 1, Brownian phylogeny). Bold font indicates models with highest support. 6 heat and cold (figure 2a,d). Contrary to our expectation, we
found no differences in thermal limits between different life
stages (figure 2b,e). Intertidal species were shown to be
more tolerant to the effects of cold (figure 2f ). However,
th
lt
h
ld b
i t
t d
ith
ti
i
li ht
f
species for CTmin). Also, this difference for intertidal species
was not present when phylogenetic relationships were not
accounted for (electronic supplementary material, figure
S8). Although breathing mode and habitat strongly covaried
(
t
ti
i
t
b
th
d
t t
t i l
model
k
AICc
DAICc
wi
0. Covariates only
l ¼ 1 þ Lat þ Time
4
2505.59
3.24
0.07
l ¼ 0 þ Lat þ Time
4
2759.27
256.92
0.00
l ¼ 1 þ Lat
3
2521.96
19.60
0.00
l ¼ 1 þ Time
3
2555.30
52.94
0.00
1. Genome size
l ¼ 1 þ Lat þ Time
5
2506.18
3.82
0.05
l ¼ 0 þ Lat þ Time
5
2731.05
228.69
0.00
l ¼ 1 þ Lat
4
2521.94
19.58
0.00
l ¼ 1 þ Time
4
2554.25
51.89
0.00
2. (c) Data analyses Breathing mode
l 5 1 1 Lat 1 Time
5
2504.30
1.95
0.13
l ¼ 0 þ Lat þ Time
5
2644.82
142.46
0.00
l ¼ 1 þ Lat
4
2519.78
17.43
0.00
l ¼ 1 þ Time
4
2551.15
48.79
0.00
3. Life stage
l 5 1 1 Lat 1 Time
5
2503.91
1.55
0.16
l ¼ 0 þ Lat þ Time
5
2758.50
256.14
0.00
l ¼ 1 þ Lat
4
2513.96
11.60
0.00
l ¼ 1 þ Time
4
2551.56
49.20
0.00
4. Habitat
l ¼ 1 þ Lat þ Time
6
2509.48
7.12
0.01
l ¼ 0 þ Lat þ Time
6
2718.37
216.01
0.00
l ¼ 1 þ Lat
5
2525.79
23.43
0.00
l ¼ 1 þ Time
5
2558.64
56.28
0.00
5. Genome size Breathing mode
l ¼ 1 þ Lat þ Time
7
2506.43
4.07
0.05
l ¼ 0 þ Lat þ Time
7
2599.70
97.34
0.00
l ¼ 1 þ Lat
6
2521.26
18.90
0.00
l ¼ 1 þ Time
6
2551.27
48.91
0.00
6. Genome size Life stage
l ¼ 1 þ Lat þ Time
7
2506.65
4.30
0.04
l ¼ 0 þ Lat þ Time
7
2715.88
213.52
0.00
l ¼ 1 þ Lat
6
2515.68
13.32
0.00
l ¼ 1 þ Time
6
2552.49
50.13
0.00
7. Genome size Habitat
l ¼ 1 þ Lat þ Time
9
2511.67
9.31
0.00
l ¼ 0 þ Lat þ Time
9
2669.46
167.10
0.00
l ¼ 1 þ Lat
8
2527.59
25.23
0.00
l ¼ 1 þ Time
8
2553.99
51.64
0.00
8. Genome size 3 Breathing mode 3 Time
l 5 1 1 Lat
10
2502.36
0.00
0.36
l ¼ 0 þ Lat
10
2590.26
87.90
0.00
9. Genome size Life stage Time
l ¼ 1 þ Lat
10
2510.08
7.72
0.01
l ¼ 0 þ Lat
10
2718.49
216.13
0.00
10. Genome size Habitat Time
l ¼ 1 þ Lat
14
2504.72
2.36
0.11
l ¼ 0 þ Lat
14
2669.50
167.14
0.00 heat and cold (figure 2a,d). Contrary to our expectation, we
found no differences in thermal limits between different life
stages (figure 2b,e). Intertidal species were shown to be
more tolerant to the effects of cold (figure 2f ). (c) Data analyses However,
these results should be interpreted with caution in light of
low representation of intertidal species in our analyses (five heat and cold (figure 2a,d). Contrary to our expectation, we
found no differences in thermal limits between different life
stages (figure 2b,e). Intertidal species were shown to be
more tolerant to the effects of cold (figure 2f ). However,
these results should be interpreted with caution in light of
low representation of intertidal species in our analyses (five species for CTmin). Also, this difference for intertidal species
was not present when phylogenetic relationships were not
accounted for (electronic supplementary material, figure
S8). Although breathing mode and habitat strongly covaried
(most aquatic species are water-breathers and most terrestrial
species are air-breathers), variation in CTmax was best Table 4. Results for phylogenetic generalized least-squares (PGLSs) models to explain variation in ectotherms’ CTmin (N ¼ 190 species) as a function of
log10-transformed genome size, breathing mode (air and water), life stage (adult and non-adult), habitat (aquatic, intertidal and terrestrial) and their interactions. All models were assessed using the absolute latitude (Lat) of the animal collection as a covariate. The number of parameters (k), corrected Akaike’s information
criterion (AICc), difference in AICc with respect to the model with highest support (DAICc) and Akaike’s weight (wi) are given for each model. Pagel’s lambda (l)
denotes correlation structure used (l ¼ 0, star phylogeny, and l ¼ 1, Brownian phylogeny). Bold font indicates model with highest support. 7 model
k
AICc
DAICc
wi
0. Covariates only
l ¼ 1 þ Lat
3
1076.21
37.66
0.00
l ¼ 0 þ Lat
3
1157.44
118.88
0.00
1. Genome size
l ¼ 1 þ Lat
4
1074.03
35.47
0.00
l ¼ 0 þ Lat
4
1159.53
120.97
0.00
2. Breathing mode
l ¼ 1 þ Lat
4
1076.83
38.27
0.00
l ¼ 0 þ Lat
4
1150.57
112.01
0.00
3. Life stage
l ¼ 1 þ Lat
4
1077.85
39.29
0.00
l ¼ 0 þ Lat
4
1158.68
120.13
0.00
4. Habitat
l ¼ 1 þ Lat
5
1063.79
25.23
0.00
l ¼ 0 þ Lat
5
1148.50
109.94
0.00
5. Genome size Breathing mode
l ¼ 1 þ Lat
6
1070.58
32.02
0.00
l ¼ 0 þ Lat
6
1144.42
105.87
0.00
6. Genome size Life stage
l ¼ 1 þ Lat
6
1075.65
37.09
0.00
l ¼ 0 þ Lat
6
1154.62
116.06
0.00
7. (c) Data analyses Genome size Habitat
l ¼ 1 þ Lat
8
1046.08
7.53
0.02
l ¼ 0 þ Lat
8
1153.09
114.53
0.00
8. Genome size Breathing mode Time
l ¼ 1 þ Lat
10
1047.97
9.42
0.01
l ¼ 0 þ Lat
10
1136.59
98.03
0.00
9. Genome size Life stage Time
l ¼ 1 þ Lat
10
1045.99
7.43
0.02
l ¼ 0 þ Lat
10
1156.36
117.80
0.00
10. Genome size 3 Habitat 3 Time
l 5 1 1 Lat
14
1038.56
0.00
0.95
l ¼ 0 þ Lat
14
1153.79
115.23
0.00 repeated the analyses on a smaller set of species for which
information on both body mass and genome size was avail-
able. This smaller set included 433 species for CTmax and
190 species for CTmin. These analyses allowed us not only
to compare the contributions of body mass and genome
size but also to test for possible interactions between body
mass and genome size. The results of these analyses were
highly consistent with those presented above, that is,
models with the highest support, for both the CTmax and
CTmin, were those that incorporated body mass, genome
size, breathing mode and exposure duration. Interestingly,
variations in CTmax were mainly driven by those models
that considered body mass instead of genome size (electronic
supplementary material, table S7). On the contrary, for the
CTmin, the model with the highest support (wi ¼ 0.99) was
that which considered the three-way interaction of body
mass and genome size and exposure duration (electronic
supplementary material, table S9). explained by models based on breathing mode (table 1,
model 8), rather than on habitat (table 1, model 10). Variation
in CTmin was best explained by models based on breathing
mode (when including body mass; table 2) and habitat
(when including genome size; table 4). Cold tolerance
declined (i.e. higher CTmin values) with increasing body
mass (figure 1c). More complex models, testing three interactions (body
mass breathing mode exposure duration), showed the
highest
support
to
explain
variations
both
in
CTmax
(tables 1 and 3) and, with some exceptions, in CTmin
(tables 2 and 4). In general, these models indicate that
exposure duration modulates the intensity or even reverses
the direction of the effects of body mass (figure 3) or
genome size (figure 4). (c) Data analyses For water-breathers, larger species
were found to have a lower CTmax in long-term experimen-
tal trials, while the model indicates an opposite effect in
short-term trials (figure 3a,b). For cold tolerance, the three-
way interaction with exposure duration was also important
for models including body mass and genome size. Here,
air-breathers
showed
improved
cold
tolerance
(lower
CTmin values) with increasing genome size, but only for
long-term trials (figure 4d). 4. Discussion Knowing whether the consequences
of global warming are size-dependent is therefore central,
particularly in light of the ongoing global climatic warming. Here, we have taken a comparative approach to shed light on
the relationship between thermal tolerance levels and body
mass and genome size in ectotherms. A challenge in such
large-scale, comparative studies lies in dealing with the
unique evolutionary history of the various species [67], as
well as dealing with differences in methodology across
studies [58,68,69]. Our results show that effects of body
mass and genome size on thermal limits (CTmax and
CTmin) are context-dependent, covarying to some extent
with the evolutionary relationships across species and differ-
ing mainly with breathing mode of species. The methodology
was
also
influential
(see
also
[70]),
as size-dependent
differences in thermal limits were magnified in long-term trials. (a) Do body mass and genome size relate to thermal
limits? Our results indicate that there is not a simple, straightforward
answer as to whether body mass and genome size matter in
defining a species’ thermal tolerance or not. If heat tolerance
limits arise from insufficient oxygen provisioning to meet demand, and if such oxygen limitation is more likely to
occur in larger ectotherms, we would expect heat tolerance
to be more impaired in larger-bodied animals. We found
such a relationship, but only in our analyses that did not
include phylogenetic relationships. Accounting for phylo-
geny appears to be a more parsimonious explanation for
variation in heat tolerance. Still, even when accounting for
phylogenetic relationships, we found size-dependence of
heat tolerance, but this was contingent upon exposure dur-
ation and breathing mode, with impaired heat tolerance
being more apparent in larger, water-breather animals
during longer trials. Owing to the challenge of underwater
gas exchange, water-breathers have been argued to be more
susceptible to oxygen-limited heat tolerance [16,21]. The
timescale is also important here as stress relates to both its
intensity and duration [68]. Heat stress may result in energy
deficits, and while energy can be generated either aerobically
or anaerobically, anaerobic metabolism is much less efficient
and more suitable to deal with acute, short-term energy defi-
cits [71]. 4. Discussion Body mass is of fundamental importance for the ecology of
ectotherms, governing the rates of energy uptake and
energy transformation at the organismal level, with sub-
sequent consequences for species interactions and to the Since different numbers of species were included in our
analyses on body mass and genome size, the performance
of the models cannot be compared directly. We therefore −6
−4
−2
0
2
4
10
20
30
40
50
60
10
20
30
40
50
60
CTmax (°C)
−1.0
−0.5
0
0.5
1.0
1.5
2.0
−6
−4
−2
0
2
4
−1.0
−0.5
0
0.5
1.0
1.5
2.0
−20
−10
0
10
20
−20
−10
0
10
20
log10(mass (g))
log10(genome (pg))
CTmin (°C)
(b)
(a)
(c)
(d)
air-breather
water-breather
Figure 1. Partial residuals plots showing the predicted effects of log10-transformed body mass and log10-transformed genome size in ectotherms’ CTmax (top, red)
and CTmin (bottom, blue). CTmax (a,b) and CTmin plots (c,d) were based on the model with the highest support, shown in electronic supplementary material,
tables S1–S4. Solid lines indicate predictions of models that included all covariates (latitude, time) and phylogenetic relationships, whereas dashed lines indicate
predictions of models that included all covariates, but do not account for phylogenetic relationships (l ¼ 0). For details on model estimates and significance, see
electronic supplementary material, tables S3–S6. (Online version in colour.) 10
20
30
40
50
60
−1.0
−0.5
0
0.5
1.0
1.5
2.0
(b) −6
−4
−2
0
2
4
10
20
30
40
50
60
CTmax (°C)
(a)
air-breather
water-breather (b) (a) (d) −6
−4
−2
0
2
4
−20
−10
0
10
20
log10(mass (g))
CTmin (°C)
(c) −1.0
−0.5
0
0.5
1.0
1.5
2.0
−20
−10
0
10
20
log10(genome (pg))
d) Figure 1. Partial residuals plots showing the predicted effects of log10-transformed body mass and log10-transformed genome size in ectotherms’ CTmax (top, red)
and CTmin (bottom, blue). CTmax (a,b) and CTmin plots (c,d) were based on the model with the highest support, shown in electronic supplementary material,
tables S1–S4. Solid lines indicate predictions of models that included all covariates (latitude, time) and phylogenetic relationships, whereas dashed lines indicate
predictions of models that included all covariates, but do not account for phylogenetic relationships (l ¼ 0). For details on model estimates and significance, see
electronic supplementary material, tables S3–S6. (Online version in colour.) ecosystem functioning. 4. Discussion For details on model estimates and significance, see electronic supplementary material, tables S3–
S6. (Online version in colour.) 10
20
30
40
50
60
adult
non-adult
(b) 10
20
30
40
50
60
aquatic
intertidal
terrestrial
(c) 10
20
30
40
50
60
CTmax (°C)
air
water
(a) 9 (a) adult
non-adult
−20
−10
0
10
20
life stage
(e) ( f ) air
water
−20
−10
0
10
20
breathing mode
CTmin (°C)
(d) (e) aquatic
intertidal
terrestrial
−20
−10
0
10
20
habitat
( f ) life stage Figure 2. Partial residuals plots showing the predicted effects of breathing mode (a,d), life stage (b,e) and habitat (c,f) in ectotherms’ CTmax (top, red) and CTmin
(bottom, blue). CTmax (a–c) and CTmin plots (d–f) were based on the model with the highest support shown in electronic supplementary material, tables S1 and
S3 that included either breathing mode, life stage or habitat. Horizontal solid lines are the predicted medians of the thermal limits within factor level, conditioned
on 2 h of exposure duration (Time) and 458 of absolute latitude. For details on model estimates and significance, see electronic supplementary material, tables S3–
S6. (Online version in colour.) Given these considerations, it is perhaps not surprising to
find the strongest effects of body mass in prolonged trials
on water-breathers. with body mass in juvenile spiders (family Linyphiidae), but
deteriorated with size of adults in Hemiptera (Heterocerus
sp.) and Collembola (Isotoma riparia) [38]. Thus, an oxygen-
based mechanism could play a role in heat tolerance but
appears to be more relevant for water-breathers and on
longer timescales: i.e. exactly those conditions for which a
strong warming-induced reduction in body mass has been
observed [5]. Heat tolerance was lower in water-breathers compared
with air-breathers during prolonged trials when they had
larger body sizes, but also when they had larger cell size. The observed effects of genome size can also be interpreted
from an oxygen perspective, as smaller genome size is
coupled to smaller cell size [75], which can promote a more
efficient diffusion of oxygen towards the mitochondria
owing to increased membrane surface area to cell volume
ratios and shorter diffusion distances [24,44,76,77]. Studies
on flies and isopods have shown that warming-induced size
reductions are more pronounced under hypoxia [29,76,78],
supporting the idea of oxygen shortage setting limits to the
size that an animal can attain. 4. Discussion For fish, it has been suggested that larger species rely
more on anaerobic metabolism when faced with energy def-
icits [72–74] and if this mass scaling generalizes, this could
explain why larger species may be better in coping with
short, acute heat stress, but not with prolonged heat stress. ecosystem functioning. Knowing whether the consequences
of global warming are size-dependent is therefore central,
particularly in light of the ongoing global climatic warming. Here, we have taken a comparative approach to shed light on
the relationship between thermal tolerance levels and body
mass and genome size in ectotherms. A challenge in such
large-scale, comparative studies lies in dealing with the
unique evolutionary history of the various species [67], as
well as dealing with differences in methodology across
studies [58,68,69]. Our results show that effects of body
mass and genome size on thermal limits (CTmax and
CTmin) are context-dependent, covarying to some extent
with the evolutionary relationships across species and differ-
ing mainly with breathing mode of species. The methodology
was
also
influential
(see
also
[70]),
as size-dependent
differences in thermal limits were magnified in long-term trials. (a) Do body mass and genome size relate to thermal
limits? (a) Do body mass and genome size relate to thermal
limits? Our results indicate that there is not a simple, straightforward
answer as to whether body mass and genome size matter in
defining a species’ thermal tolerance or not. If heat tolerance
limits arise from insufficient oxygen provisioning to meet 10
20
30
40
50
60
10
20
30
40
50
60
10
20
30
40
50
60
CTmax (°C)
air
water
adult
non-adult
aquatic
intertidal
terrestrial
air
water
adult
non-adult
aquatic
intertidal
terrestrial
−20
−10
0
10
20
−20
−10
0
10
20
−20
−10
0
10
20
breathing mode
CTmin (°C)
life stage
habitat
(e)
( f )
(b)
(a)
(c)
(d)
Figure 2. Partial residuals plots showing the predicted effects of breathing mode (a,d), life stage (b,e) and habitat (c,f) in ectotherms’ CTmax (top, red) and CTmin
(bottom, blue). CTmax (a–c) and CTmin plots (d–f) were based on the model with the highest support shown in electronic supplementary material, tables S1 and
S3 that included either breathing mode, life stage or habitat. Horizontal solid lines are the predicted medians of the thermal limits within factor level, conditioned
on 2 h of exposure duration (Time) and 458 of absolute latitude. 4. Discussion This idea also implies that
oxygen is unlikely to be limiting in animals that have not yet
approached their maximum species-specific size. As the
body mass used here is that of the experimental species, in
most cases the specimens used in the experiments will not
have represented the upper size classes. This may explain
why phylogeny better explains the variation in CTmax: phylo-
geny is more likely to covary with the maximum size that a
species can attain, than with the size of the individuals used
in the experiments. Indeed, juveniles and larvae, which by
definition are not yet fully developed, both show improved
heat tolerance with increasing body mass, contrasting with
impaired heat tolerance in adults (electronic supplementary
material, figure S9). Along the same lines, in a study looking
at intraspecific variation in body mass, CTmax improved Unlike heat tolerance, cold tolerance has been suggested
to result from depolarization of cell membranes and sub-
sequent
cell
death
[46,79–82],
and
not
from
oxygen
limitation [83]. Our results also suggest that the mechanisms
underpinning CTmin differ from those underpinning CTmax
as the contribution of phylogeny, body mass and genome
size to explaining variation in CTmin differed from that to
explaining variation in CTmax (electronic supplementary
material, tables S3–S6). Models that consider combined
effects of body mass and genome size indicate that this com-
bination better explains variation in CTmin, than variation in
CTmax (see electronic supplementary material, tables S7–
S10). While a small genome size (or small cell size) may
enhance oxygen diffusion, it also entails greater costs in keep-
ing membranes polarized [44,84]. Thus, larger cells may be
more cost efficient and this could explain why larger gen-
omes can improve cold tolerance. The effect of such an
efficiency-based mechanism would likely be more apparent
during prolonged trials, and indeed we found that including
the interaction between genome size, habitat and exposure
duration showed the highest support across all models
(table 4), showing improved cold tolerance in terrestrial long-term trials
short-term trials
air-breather
water-breather
short-term trials
long-term trials
−6
−4
−2
0
2
4
−6
−4
−2
0
2
4
−6
−4
−2
0
2
4
−6
−4
−2
0
2
4
10
20
30
40
50
60
10
20
30
40
50
60
CTmax (°C)
−20
−10
0
10
20
−20
−10
0
10
20
CTmin (°C)
log10(mass (g))
log10(mass (g))
(b)
(a)
(c)
(d)
Figure 3. 4. Discussion First, both the CTmax and CTmin are phylogenetically
structured, displaying high Pagel’s l (electronic supplemen-
tary
material,
table
S11)
and
second,
those
models animals
with
larger
genome
during
prolonged
trials
(figure 4). In line with these findings, results on the larvae
of the pipevine swallowtail Battus philenor (Linnaeus, 1771)
suggested that larger species may have more energy reserves
for maintaining metabolism during chill coma, thus explain-
ing their improved cold tolerance [85]. When coupled to
lower mass-specific metabolism in larger animals, such an
efficiency mechanism would be generally applicable to the
whole size range and not only restricted to the largest size
classes within a species. This may explain why cold tolerance
is most parsimoniously explained by differences in body
mass, since phylogeny is more likely to covary with maxi-
mum size than with the size of the animals used in the
experiment). Interestingly, these patterns for CTmin were
more apparent for air-breathers, perhaps because cold toler-
ance limits in water-breathers are more related to the
freezing of water. incorporating phylogeny generally received greater support
(especially for CTmax) compared with those where the evol-
utionary history of the species was considered independent. Also, the Pagel’s l used in our models (l ¼ 1) is highly
likely to be a representative value of the shared evolution
of species present in our database, since all continuous vari-
ables, both independent (body mass, genome size, exposure
duration and absolute latitude) and dependent (CTmax and
CTmin), showed high phylogenetic signals (all l . 0.7)
(electronic supplementary material, table S11). The influence of phylogeny on thermal limits is also evi-
dent from the contrasting effects of body mass and genome
size between models that considered a Brownian or star phy-
logeny correlation structure (figure 1). This indicates that
body mass and genome size covary with phylogeny, some-
thing that is also evident from the high Pagel’s l value for
body mass and genome size (electronic supplementary
material, table S11). Consequently, incorporating phylogeny
already accounts for much of the variation in thermal toler-
ance, thereby changing the fitted relationship for body
mass and genome size. For CTmax, models that included
phylogeny always showed greater support, suggesting that
the patterns in heat tolerance were more parsimoniously
explained
by
including
evolutionary
history,
possibly
because phylogeny better captures the maximum body size, 4. Discussion Partial residuals plots showing the interactive effects of body mass and breathing mode (brown and blue for air- and water-breathers, respectively) in
ectotherms’ CTmax (top) and CTmin (bottom), for short- (1st quartile) and long-term trials (3rd quartile). Predicted lines were based on models with the highest
support shown in tables 1 and 2 and based on the median of absolute latitude. For details on model estimates and significance, see electronic supplementary
material, tables S3 and S4. (Online version in colour.) 10 short-term trials
air-breather
water-breather
−6
−4
−2
0
2
4
10
20
30
40
50
60
CTmax (°C)
(a) long-term trials
−6
−4
−2
0
2
4
10
20
30
40
50
60
(b) (d) long-term trials
−6
−4
−2
0
2
4
−20
−10
0
10
20
log10(mass (g))
(d) short-term trials
−6
−4
−2
0
2
4
−20
−10
0
10
20
CTmin (°C)
log10(mass (g))
(c) CTmin (°C) Figure 3. Partial residuals plots showing the interactive effects of body mass and breathing mode (brown and blue for air- and water-breathers, respectively) in
ectotherms’ CTmax (top) and CTmin (bottom), for short- (1st quartile) and long-term trials (3rd quartile). Predicted lines were based on models with the highest
support shown in tables 1 and 2 and based on the median of absolute latitude. For details on model estimates and significance, see electronic supplementary
material, tables S3 and S4. (Online version in colour.) animals
with
larger
genome
during
prolonged
trials
(figure 4). In line with these findings, results on the larvae
of the pipevine swallowtail Battus philenor (Linnaeus, 1771)
suggested that larger species may have more energy reserves
for maintaining metabolism during chill coma, thus explain-
ing their improved cold tolerance [85]. When coupled to
lower mass-specific metabolism in larger animals, such an
efficiency mechanism would be generally applicable to the
whole size range and not only restricted to the largest size
classes within a species. This may explain why cold tolerance
is most parsimoniously explained by differences in body
mass, since phylogeny is more likely to covary with maxi-
mum size than with the size of the animals used in the
experiment). Interestingly, these patterns for CTmin were
more apparent for air-breathers, perhaps because cold toler-
ance limits in water-breathers are more related to the
freezing of water. (b) Model fit, phylogenetic correlation structure and
covariates
We found evidence of the influence of phylogeny on two
fronts. covariates We found evidence of the influence of phylogeny on two
fronts. First, both the CTmax and CTmin are phylogenetically
structured, displaying high Pagel’s l (electronic supplemen-
tary
material,
table
S11)
and
second,
those
models short-term trials
long-term trials
long-term trials
short-term trials
air-breather
water-breather
−1.0
−0.5
0
0.5
1.0
1.5
2.0
−1.0
−0.5
0
0.5
1.0
1.5
2.0
−1.0
−0.5
0
0.5
1.0
1.5
2.0
−1.0
−0.5
0
0.5
1.0
1.5
2.0
10
20
30
40
50
60
10
20
30
40
50
60
CTmax (°C)
−20
−10
0
10
20
−20
−10
0
10
20
CTmin (°C)
log10(genome (pg))
log10(genome (pg))
(b)
(a)
(c)
(d)
Figure 4. Partial residuals plots showing the interactive effects of genome size and breathing mode (brown and blue for air- and water-breathers, respectively) in
ectotherms’ CTmax (top) and CTmin (bottom), for short- (1st quartile) and long-term trials (3rd quartile). Predicted lines were based on models with the highest
support shown in tables 3 and 4 and based on the median of absolute latitude. For details on model estimates and significance, see electronic supplementary
material, tables S5 and S6. (Online version in colour.) long-term trials
−1.0
−0.5
0
0.5
1.0
1.5
2.0
10
20
30
40
50
60
(b) 11 short-term trials
air-breather
water-breather
−1.0
−0.5
0
0.5
1.0
1.5
2.0
10
20
30
40
50
60
CTmax (°C)
(a) long-term trials
−1.0
−0.5
0
0.5
1.0
1.5
2.0
−20
−10
0
10
20
log10(genome (pg))
d) (d) short-term trials
−1.0
−0.5
0
0.5
1.0
1.5
2.0
−20
−10
0
10
20
CTmin (°C)
log10(genome (pg))
(c) CTmin (°C) Figure 4. Partial residuals plots showing the interactive effects of genome size and breathing mode (brown and blue for air- and water-breathers, respectively) in
ectotherms’ CTmax (top) and CTmin (bottom), for short- (1st quartile) and long-term trials (3rd quartile). Predicted lines were based on models with the highest
support shown in tables 3 and 4 and based on the median of absolute latitude. For details on model estimates and significance, see electronic supplementary
material, tables S5 and S6. (Online version in colour.) which may be causally related to CTmax (see above). For
CTmin, models that included the species’ body mass as an
independent variable showed greater support when the evol-
ution of the species was assumed as independent (i.e. covariates star
phylogeny), possibly because here the actual body size of
the experimental individuals is causally related to cold toler-
ance (see above). Overall, our results confirm earlier findings
suggesting that evolutionary history matters for thermal tol-
erance [86–89], especially for heat tolerance [86,90] and also
suggest that this applies to the much larger set of ectotherm
species, including insects, crustaceans, fish, amphibians and
reptiles. Our results also point out the importance of includ-
ing mainly methodological aspects as covariates in modelling
(see also [70]). Of the four methodological aspects evaluated
in the preliminary models (absolute latitude, exposure dur-
ation, acclimation time and origin), latitude and exposure
duration
were
consistently
included
in
the
best-fitted
models. The absolute latitude of the site where animals
were collected consistently shifted thermal windows, impair-
ing the heat tolerance and improving the cold tolerance at
higher latitudes (electronic supplementary material, figure
S7). While the effects of latitude were not the focus of our
analyses, these results reinforce the clear patterns of thermal
tolerance across latitudinal gradients documented in the lit-
erature [12] (see also [70]). The exposure duration was also
consistently included in the best-fitted models, with long-
term
trials
consistently
reducing
CTmax
(electronic supplementary material, figure S7). This indicates that meth-
odological
variation
explains
a
significant part
of
the
variation in CTmax and adding exposure duration as covari-
ate may help to reveal more clearly the effects of other factors,
such as that of latitude [68]. Furthermore, the inclusion of
exposure duration as a covariate has direct biological
meaning as tolerance to high-temperature conditions is
time-dependent [68]. References 1. Atkinson D, Hirst AG. 2007 Life histories and body
size. In Body size: the structure and function of
aquatic ecosystems (eds AG Hildrew, DG Raffaelli, R
Edmonds-Brown), pp. 33–54. Cambridge, UK:
Cambridge University Press. aquatic ectotherms? Glob. Ecol. Biogeogr. 28,
64–77. (doi:10.1111/geb.12847) on wild animals and plants. Nature 421, 57–60. (doi:10.1038/nature01333) aquatic ectotherms? Glob. Ecol. Biogeogr. 28,
64–77. (doi:10.1111/geb.12847) on wild animals and plants. Nature 421, 57–60. (doi:10.1038/nature01333) 20. 11. 20. Po¨rtner HO, Bock C, Mark FC. 2017 Oxygen- and
capacity-limited thermal tolerance: bridging ecology
and physiology. J. Exp. Biol. 220, 2685–2696. (doi:10.1242/jeb.134585) 11. Chen I-C, Hill JK, Ohlemu¨ller R, Roy DB, Thomas CD. 2011 Rapid range shifts of species associated with
high levels of climate warming. Science 333,
1024–1026. (doi:10.1126/science.1206432) 2. Enquist BJ, Economo EP, Huxman TE, Allen AP,
Ignace DD, Gillooly JF. 2003 Scaling metabolism
from organisms to ecosystems. Nature 423,
639–642. (doi:10.1038/nature01671) 12. Sunday JM, Bates AE, Dulvy NK. 2011 Global
analysis of thermal tolerance and latitude in
ectotherms. Proc. R. Soc. B 278, 1823–1830. (doi:10.1098/rspb.2010.1295) 21. Verberk WCEP, Bilton DT. 2013 Respiratory control in
aquatic insects dictates their vulnerability to global
warming. Biol. Lett. 9, 20130473. (doi:10.1098/rsbl. 2013.0473) 3. Brown JH, Gillooly JF, Allen AP, Savage VM,
West GB. 2004 Toward a metabolic theory of
ecology. Ecology 85, 1771–1789. (doi:10.1890/03-
9000) 13. Gunderson AR, Stillman JH. 2015 Plasticity in
thermal tolerance has limited potential to buffer
ectotherms from global warming. Proc. R. Soc. B
282, 20150401. (doi:10.1098/rspb.2015.0401) 22. Po¨rtner HO. 2001 Climate change and temperature-
dependent biogeography: oxygen limitation of
thermal tolerance in animals. Naturwissenschaften
88, 137–146. (doi:10.1007/s001140100216) 4. Bergmann C. 1848 U¨ber die Verha¨ltnisse der
Wa¨rmeo¨konomie der Thiere zu ihrer Gro¨sse. Go¨ttinger Studien 1, 595–708 [in German]. 14. Arau´jo MB, Ferri-Ya´n˜ez F, Bozinovic F, Marquet PA,
Valladares F, Chown SL. 2013 Heat freezes niche
evolution. Ecol. Lett. 16, 1206–1219. (doi:10.1111/
ele.12155) 23. Verberk WCEP, Sommer U, Davidson RL, Viant MR. 2013 Anaerobic metabolism at thermal extremes: a
metabolomic test of the oxygen limitation
hypothesis in an aquatic insect. Integr. Comp. Biol. 53, 609–619. (doi:10.1093/icb/ict015) 5. Horne CR, Hirst A, Atkinson D. 2015 Temperature-
size responses match latitudinal-size clines in
arthropods, revealing critical differences between
aquatic and terrestrial species. Ecol. Lett. 18,
327–335. (doi:10.1111/ele.12413) 15. Addo-Bediako A, Chown SL, Gaston KJ. 2000
Thermal tolerance, climatic variability and latitude. Proc. R. Soc. Lond. B 267, 739–745. (doi:10.1098/
rspb.2000.1065) 24. Atkinson D, Morley SA, Hughes RN. 5. Conclusion conducted the statistical analyses and prepared figures with inputs
from W.C.E.P.V. and P.C.; F.P.L. and W.C.E.P.V. wrote the first 5. Conclusion In conclusion, for CTmax we found that support for the oxygen
limitation hypothesis was limited to long-term trials in
larger-bodied water-breathers. For CTmin, we found improved
cold tolerance for air-breather animals with larger genomes,
again when considering long-term trials. Coping with thermal
stress on long timescales requires sustained energy generation. Long-term heat tolerance appears to be enhanced in smaller-
bodied, water-breathing species, possibly as this enables a
higher capacity to generate energy aerobically and efficiently. On the other hand, long-term cold tolerance appears to be
enhanced in species with a larger body mass and cell size (i.e. more energy reserves and lowerenergetic costs), which appeared
especiallyimportantforair-breathers.Incorporatingtheexposure
duration of the experimental trials can reveal body and genome
size-dependence of thermal tolerance, with body size being version of the manuscript with inputs from P.C. All authors contrib-
uted and approved the final version of the manuscript. moreimportantforCTmaxandwater-breathersandgenomesize
being more important for CTmin and air-breathers. Our results
highlight the importance of accounting for phylogeny and
exposure duration. Especially when considering long-term
trials, these effects are more in line with the warming-induced
reduction in body mass observed during long-term rearing
experiments [5] and over past extinctions [8]. Explicitly incorpor-
ating timescale may thus hold the key to resolve discrepancies
between short-term trials, which do not always find evidence
for oxygen limitation, and the results of long-term laboratory
and field studies, which do suggest a role for oxygen limitation. 12 Competing interests. We have no competing interests. p
g
Funding. F.P.L. was supported by CONICYT Becas Chile (no. 72190288) for Doctoral studies at Radboud University Nijmegen. P.C. was supported by an NSERC Discovery Programme Grant
(RGPIN-2015-06500) and the Programme E´tablissement de Nou-
veaux Chercheurs Universitaires of the Fonds de Recherche du
Que´bec - Nature et Technologies (FRQNT) (grant no. 199173); he is
also a member of Quebec Ocean and QCBS FRQNT-funded research
excellence networks. W.C.E.P.V. gratefully acknowledges support
from The Netherlands Organisation for Scientific Research (NWO-
VIDI grant no. 016.161.321). Acknowledgements. We thank D. Sa´nchez-Ferna´ndez, L. Viegas, L.M. Gutie´rrez-Pesquera and A. Barrı´a for kindly providing body mass
data of the experimental animals. We also thank Jennifer Sunday
and two anonymous reviewers for helpful comments and sugges-
tions that greatly improved this manuscript. Data accessibility. Data available from the Dryad Digital Repository:
https://doi.org/10.5061/dryad.878vn25 [91]. rstb
Phil. Trans. R. Soc. B 374: 20190035 Authors’ contributions. F.P.L. extracted the data from the articles; F.P.L. References 2006 From cells
to colonies: at what levels of body organization
does the ‘temperature-size rule’ apply? Evol. Dev. 8,
202–214. (doi:10.1111/j.1525-142X.2006.00090.x) 6. Sallan L, Galimberti AK. 2015 Body-size reduction in
vertebrates following the end-Devonian mass
extinction. Science 350, 812–815. (doi:10.1126/
science.aac7373) 16. Verberk WCEP, Overgaard J, Ern R, Bayley M, Wang
T, Boardman L, Terblanche JS. 2016 Does oxygen
limit thermal tolerance in arthropods? A critical
review of current evidence. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 192, 64–78. (doi:10.1016/j. cbpa.2015.10.020) 25. Verberk WCEP, Atkinson D. 2013 Why polar
gigantism and Palaeozoic gigantism are not
equivalent: effects of oxygen and temperature on
the body size of ectotherms. Funct. Ecol. 27,
1275–1285. (doi:10.1111/1365-2435.12152) 7. Angilletta MJ, Dunham AE. 2003 The temperature-
size rule in ectotherms: simple evolutionary
explanations may not be general. Am. Nat. 162,
332–342. (doi:10.1086/377187) 17. Lefevre S. 2016 Are global warming and ocean
acidification conspiring against marine ectotherms? A meta-analysis of the respiratory effects of
elevated temperature, high CO2 and their
interaction. Conserv. Physiol. 4, cow009. (doi:10. 1093/conphys/cow009) 26. Atkinson D. 1994 Temperature and organism size—
a biological law for ectotherms? Adv. Ecol. Res. 25,
1–58. (doi:10.1016/S0065-2504(08)60212-3) 8. Calosi P, Putnam HM, Twitchett RJ, Vermandele F. 2019 Marine Metazoan modern mass extinction:
improving predictions by integrating fossil, modern,
and physiological data. Annu. Rev. Mar. Sci. 11,
369–390. (doi:10.1146/annurev-marine-010318-
095106) 27. Pauly D. 2010 Gasping fish and panting squids:
oxygen, temperature and the growth of water-
breathing animals. Oldendorf (Luhe), Germany:
International Ecology Institute. 18. Pauly D, Cheung WW. 2018 Sound physiological
knowledge and principles in modeling shrinking of
fishes under climate change. Glob. Change Biol. 24,
13831. (doi:10.1111/gcb.13831) 28. Lane SJ, Shishido CM, Moran AL, Tobalske BW,
Arango CP, Woods HA. 2017 Upper limits to body
size imposed by respiratory–structural trade-offs in
Antarctic pycnogonids. Proc. R. Soc. B 284,
20171779. (doi:10.1098/rspb.2017.1779) 9. Daufresne M, Lengfellner K, Sommer U. 2009
Global warming benefits the small in
aquatic ecosystems. Proc. Natl Acad. Sci. USA
106, 12 788–12 793. (doi:10.1073/pnas. 0902080106) 19. Audzijonyte A, Barneche DR, Baudron AR, Belmaker
J, Clark TD, Marshall CT, Morrongiello JR, van Rijn I. 2018 Is oxygen limitation in warming waters a valid
mechanism to explain decreased body sizes in 29. Hoefnagel KN, Verberk WCEP. 2015 Is the
temperature-size rule mediated by oxygen in 29. Hoefnagel KN, Verberk WCEP. 2015 Is the
temperature-size rule mediated by oxygen in 29. Hoefnagel KN, Verberk WCEP. 1111/j.2041-210X.2011.00169.x) 66. Breheny P, Burchett W. 2017 Visualization of
regression models using visreg. R Journal 9, 56–71. (doi:10.32614/RJ-2017-046) 51. Cane JH. 1987 Estimation of bee size using
intertegular span (Apoidea). J. Kansas Entomol. Soc. 60, 145–147. 67. Spicer JI, Morley SA, Bozinovic F. 2019 Physiological
diversity, biodiversity patterns and global climate
change: testing key hypotheses involving
temperature and oxygen. Phil. Trans. R. Soc. B 374,
20190032. (doi:10.1098/rstb.2019.0032) 37. Peck LS, Clark MS, Morley SA, Massey A, Rossetti H. 2009 Animal temperature limits and ecological
relevance: effects of size, activity and rates of
change. Funct. Ecol. 23, 248–256. (doi:10.1111/j. 1365-2435.2008.01537.x) 52. Gregory TR. 2019 Animal genome size database. See
http://www.genomesize.com/. 53. Rollinson N, Rowe L. 2018 Temperature-dependent
oxygen limitation and the rise of Bergmann’s rule
in species with aquatic respiration. Evolution 72,
977–988. (doi:10.1111/evo.13458) 38. Franken O, Huizinga M, Ellers J, Berg M. 2018
Heated communities: large inter- and intraspecific
variation in heat tolerance across trophic levels of a
soil arthropod community. Oecologia 186,
311–322. (doi:10.1007/s00442-017-4032-z) 68. Rezende EL, Castan˜eda LE, Santos M. 2014
Tolerance landscapes in thermal ecology. Funct. Ecol. 28, 799–809. (doi:10.1111/1365-2435.12268) 54. Pagel M. 1994 Detecting correlated evolution on
phylogenies: a general method for the comparative
analysis of discrete characters. Proc. R. Soc. Lond. B
255, 37–45. (doi:10.1098/rspb.1994.0006) 69. Terblanche JS, Deere JA, Clusella-Trullas S, Janion C,
Chown SL. 2007 Critical thermal limits depend on
methodological context. Proc. R. Soc. B 274,
2935–2943. (doi:10.1098/rspb.2007.0985) 39. Hessen DO, Daufresne M, Leinaas HP. 2013
Temperature-size relations from the cellular-
genomic perspective. Biol. Rev. 88, 476–489. (doi:10.1111/brv.12006) 70. Sunday J et al. 2019 Thermal tolerance patterns
across latitude and elevation. Phil. Trans. R. Soc. B
374, 20190036. (doi:10.1098/rstb.2019.0036) 55. Hernandez CE, Rodrı´guez-Serrano E, Avaria-
Llautureo J, Inostroza-Michael O, Morales-Pallero B,
Boric-Bargetto D, Canales-Aguirre CB, Marquet PA,
Meade A. 2013 Using phylogenetic information and
the comparative method to evaluate hypotheses in
macroecology. Methods Ecol. Evol. 4, 401–415. (doi:10.1111/2041-210X.12033) 40. Partridge L, Barrie B, Fowler K, French V. 1994
Evolution and development of body size and cell
size in Drosophila melanogaster in response to
temperature. Evolution 48, 1269–1276. (doi:10. 1111/j.1558-5646.1994.tb05311.x) 71. Hagerman L. 1998 Physiological flexibility; a
necessity for life in anoxic and sulphidic habitats. Hydrobiologia 375, 241–254. (doi:10.1023/
A:1017033711985) 41. Jeffery NW. 2015 Genome size diversity and
evolution in the Crustacea. PhD thesis: University of
Guelph. See https://atrium.lib.uoguelph.ca/xmlui/
handle/10214/9216. 56. Michonneau F, Brown JW, Winter DJ. References 2015 Is the
temperature-size rule mediated by oxygen in 10. Root TL, Price JT, Hall KR, Schneider SH, Rosenzweig
C, Pounds JA. 2003 Fingerprints of global warming aquatic ectotherms? J. Therm. Biol 54, 56–65. (doi:10.1016/j.jtherbio.2014.12.003) 43. Czarnoleski M, Labecka AM, Dragosz-Kluska D, Pis T,
Pawlik K, Kapustka F, Kilarski WM, Kozłowski J. 2018 Concerted evolution of body mass and cell
size: similar patterns among species of birds
(Galliformes) and mammals (Rodentia). Biol. Open
7, bio-029603. (doi:10.1242/bio.029603) 59. Wang S, Tang J, Hansen JD. 2007 Experimental and
simulation methods of insect thermal death kinetics. In
Heat treatments for postharvest pest control: theoryand
practice (eds J Tang, E Mitcham, S Wang, S Lurie),
pp. 105–132. Oxford, UK: CABI Publishing. 13 30. Penn JL, Deutsch C, Payne JL, Sperling EA. 2018
Temperature-dependent hypoxia explains
biogeography and severity of end-Permian marine
mass extinction. Science 362, eaat1327. (doi:10. 1126/science.aat1327) 60. Burnham KP, Anderson DR. 2003 Model selection
and multimodel inference: a practical information-
theoretic approach. Berlin, Germany: Springer
Science & Business Media. 44. 44. Kozlowski J, Konarzewski M, Gawelczyk AT. 2003
Cell size as a link between noncoding DNA and
metabolic rate scaling. Proc. Natl Acad. Sci. USA
100, 14 080–14 085. (doi:10.1073/pnas. 2334605100) 31. Gillooly JF, Brown JH, West GB, Savage VM, Charnov
EL. 2001 Effects of size and temperature on
metabolic rate. Science 293, 2248–2251. (doi:10. 1126/science.1061967) 61. R Core Team. 2018 R: a language and environment
for statistical computing. Vienna, Austria: R
Foundation for Statistical Computing. See http://
www.R-project.org/. 32. Verberk WCEP, Bilton DT, Calosi P, Spicer JI. 2011
Oxygen supply in aquatic ectotherms: partial
pressure and solubility together explain biodiversity
and size patterns. Ecology 92, 1565–1572. (doi:10. 1890/10-2369.1) 45. Miettinen TP, Bjo¨rklund M. 2017 Mitochondrial
function and cell size: an allometric relationship. Trends Cell Biol. 27, 393–402. (doi:10.1016/j.tcb. 2017.02.006) 62. Mazerolle MJ. 2019 AICcmodavg: model selection
and multimodel inference based on (Q)AIC(c). R
package version v2.2-1. See https://cran.r-project. org/package=AICcmodavg. 46. Overgaard J, MacMillan HA. 2017 The integrative
physiology of insect chill tolerance. Annu. Rev. Physiol. 79, 187–208. (doi:10.1146/annurev-
physiol-022516-034142) 33. Dejours P. 1981 Principles of comparative respiratory
physiology. Amsterdam, The Netherlands: Elsevier
North-Holland Biomedical Press. 63. Paradis E, Claude J, Strimmer K. 2004 APE: analyses
of phylogenetics and evolution in R language. Bioinformatics 20, 289–290. (doi:10.1093/
bioinformatics/btg412) 34. Po¨rtner HO, Knust R. 2007 Climate change affects
marine fishes through the oxygen limitation of
thermal tolerance. Science 315, 95–97. (doi:10. 1126/science.1135471) 47. References Lutterschmidt WI, Hutchison VH. 1997 The critical
thermal maximum: history and critique. Can. J. Zool. 75, 1561–1574. (doi:10.1139/z97-783) 64. Pinheiro J, Bates D, DebRoy S, Sarkar D. 2019 nlme:
linear and nonlinear mixed effects models. R
package version 3.1-140. See https://CRAN.R-
project.org/package=nlme. 48. Bennett JM et al. 2018 GlobTherm, a global
database on thermal tolerances for aquatic and
terrestrial organisms. Scient. Data 5, 180022. (doi:10.1038/sdata.2018.22) 35. Recsetar MS, Zeigler MP, Ward DL, Bonar SA,
Caldwell CA. 2012 Relationship between fish size and
upper thermal tolerance. Trans. Am. Fish. Soc. 141,
1433–1438. (doi:10.1080/00028487.2012.694830) 65. Revell LJ. 2012 phytools: an R package for
phylogenetic comparative biology (and other
things). Methods Ecol. Evol. 3, 217–223. (doi:10. 1111/j.2041-210X.2011.00169.x) 49. Froese R, Pauly D. 2018 FishBase ver. (06/2018). See www.fishbase.org. 36. Verberk WCEP, Leuven RS, van der Velde G, Gabel F. 2018 Thermal limits in native and alien freshwater
peracarid Crustacea: the role of habitat use and
oxygen limitation. Funct. Ecol. 32, 926–936. (doi:10.1111/1365-2435.13050) 50. Tschinkel WR. 2013 The morphometry of Solenopsis
fire ants. PLoS ONE 8, e79559. (doi:10.1371/journal. pone.0079559) 1111/j.2041-210X.2011.00169.x) 2016 rotl: an R
package to interact with the Open Tree of Life data. Methods Ecol. Evol. 7, 1476–1481. (doi:10.1111/
2041-210X.12593) 72. Urbina MA, Glover CN. 2013 Relationship between
fish size and metabolic rate in the oxyconforming
inanga Galaxias maculatus reveals size-dependent
strategies to withstand hypoxia. Physiol. Biochem. Zool. 86, 740–749. (doi:10.1086/673727) 57. Revell LJ. 2010 Phylogenetic signal and linear
regression on species data. Methods Ecol. Evol. 1,
319–329. (doi:10.1111/j.2041-210X.2010.00044.x) 42. Maciak S, Janko K, Kotusz J, Choleva L, Boron´ A,
Juchno D, Kujawa R, Kozłowski J, Konarzewski M. 2011 Standard metabolic rate (SMR) is inversely
related to erythrocyte and genome size in
allopolyploid fish of the Cobitis taenia hybrid
complex. Funct. Ecol. 25, 1072–1078. (doi:10.1111/
j.1365-2435.2011.01870.x) 73. Sloman KA, Wood CM, Scott GR, Wood S, Kajimura
M, Johannsson OE, Almeida-Val VM, Val AL. 2006
Tribute to RG Boutilier: the effect of size on the
physiological and behavioural responses of oscar,
Astronotus ocellatus, to hypoxia. J. Exp. Biol. 209,
1197–1205. (doi:10.1242/jeb.02090) 58. Santos M, Castan˜eda LE, Rezende EL. 2011 Making
sense of heat tolerance estimates in ectotherms:
lessons from Drosophila. Funct. Ecol. 25, 1169–1180. (doi:10.1111/j.1365-2435.2011.01908.x) Petrolisthes cinctipes. J. Exp. Biol. 215, 1824–1836. (doi:10.1242/jeb.069658) 74. Lv X, Xie H, Xia D, Shen C, Li J, Luo Y. 2018 Mass
scaling of the resting and maximum metabolic rates
of the black carp. J. Comp. Physiol. B 188,
591–598. (doi:10.1007/s00360-018-1154-5) 109, 16 228–16 233. (doi:10.1073/pnas. 1207553109) 14 109, 16 228–16 233. (doi:10.1073/pnas. 1207553109) 81. Overgaard J, Sørensen JG, Petersen SO, Loeschcke V,
Holmstrup M. 2005 Changes in membrane lipid
composition following rapid cold hardening in
Drosophila melanogaster. J. Insect. Physiol. 51,
1173–1182. (doi:10.1016/j.jinsphys.2005.06.007) 87. Marshall DJ, Rezende EL, Baharuddin N, Choi F,
Helmuth B. 2015 Thermal tolerance and climate
warming sensitivity in tropical snails. Ecol. Evol. 5,
5905–5919. (doi:10.1002/ece3.1785) 75. Gregory TR. 2001 The bigger the C-value, the larger
the cell: genome size and red blood cell size in
vertebrates. Blood Cells Mol. Dis. 27, 830–843. (doi:10.1006/bcmd.2001.0457) 88. Gutie´rrez-Pesquera LM, Tejedo M, Olalla-Ta´rraga M,
Duarte H, Nicieza A, Sole´ M. 2016 Testing the
climate variability hypothesis in thermal tolerance
limits of tropical and temperate tadpoles. J. Biogeogr. 43, 1166–1178. (doi:10.1111/jbi. 12700) 82. Slotsbo S, Sørensen JG, Holmstrup M, Kostal V,
Kellermann V, Overgaard J. 2015 Tropical to
subpolar gradient in phospholipid composition
suggests adaptive tuning of biological membrane
function in drosophilids. Funct. Ecol. 30, 759–768. (doi:10.1111/1365-2435.12568) 76. 1111/j.2041-210X.2011.00169.x) Czarnoleski M, Cooper BS, Kierat J, Angilletta MJ. 2013
Flies developed small bodies and small cells in warm
and in thermally fluctuating environments. J. Exp. Biol. 216, 2896–2901. (doi:10.1242/jeb.083535) 89. Calosi P, Bilton DT, Spicer JI, Votier SC, Atfield A. 2010 What determines a species’ geographical
range? Thermal biology and latitudinal range size
relationships in European diving beetles
(Coleoptera: Dytiscidae). J. Anim. Ecol. 79,
194–204. (doi:10.1111/j.1365-2656.2009. 01611.x) 77. Szarski H. 1983 Cell size and the concept of
wasteful and frugal evolutionary strategies. J. Theor. Biol. 105, 201–209. (doi:10.1016/S0022-
5193(83)80002-2) 83. Stevens MM, Jackson S, Bester SA, Terblanche JS,
Chown SL. 2010 Oxygen limitation and thermal
tolerance in two terrestrial arthropod species. J. Exp. Biol. 213, 2209–2218. (doi:10.1242/jeb.040170) 78. Frazier MR, Woods HA, Harrison JF. 2001 Interactive
effects of rearing temperature and oxygen on the
development of Drosophila melanogaster. Physiol. Biochem. Zool. 74, 641–650. (doi:10.1086/322172) 84. Hulbert AJ, Else PL. 2005 Membranes and the
setting of energy demand. J. Exp. Biol. 208,
1593–1599. (doi:10.1242/jeb.01482) 90. Diamond SE, Chick LD. 2017 The Janus of
macrophysiology: stronger effects of evolutionary
history, but weaker effects of climate on upper
thermal limits are reversed for lower thermal limits
in ants. Curr. Zool. 64, 223–230. (doi:10.1093/cz/
zox072) 79. Overgaard J, Tomcˇala A, Sørensen JG, Holmstrup M,
Krogh PH, Sˇimek P, Kosˇta´l V. 2008 Effects of
acclimation temperature on thermal tolerance and
membrane phospholipid composition in the fruit fly
Drosophila melanogaster. J. Insect Physiol. 54,
619–629. (doi:10.1016/j.jinsphys.2007.12.011) 85. Fordyce JA, Shapiro AM. 2003 Another perspective
on the slow-growth/high-mortality hypothesis:
chilling effects on swallowtail larvae. Ecology
84, 263–268. (doi:10.1890/0012-9658(2003)
084[0263:APOTSG]2.0.CO;2) 91. Leiva FP, Calosi P, Verberk WCEP. 2019 Data
from: Scaling of thermal tolerance with body mass
and genome size in ectotherms: a comparison
between water- and air-breathers. Dryad Digital
Repository. (doi:10.5061/dryad.878vn25) 86. Kellermann V, Overgaard J, Hoffmann AA, Fløjgaard
C, Svenning J-C, Loeschcke V. 2012 Upper thermal
limits of Drosophila are linked to species
distributions and strongly constrained
phylogenetically. Proc. Natl Acad. Sci. USA 80. Ronges D, Walsh JP, Sinclair BJ, Stillman JH. 2012
Changes in extreme cold tolerance, membrane
composition and cardiac transcriptome during the
first day of thermal acclimation in the porcelain crab
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English
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FOREIGN BODY A LONG TIME EMBEDDED IN THE HAND.
|
Lancet
| 1,902
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public-domain
| 1,653
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FOREIGN BODY A LONG TIME EMBEDDED IN THE
HAND.
BY L. K. HERSCHEL, M.D. VIENNA. BY L. K. HERSCHEL, M.D. VIENNA. THE following short account of a case in which a fragment
of glass remained embedded in the muscles of the hand for
nearly three years may be of interest as showing the useful-
ness of non-interference when no danger can arise from
waiting. g
In March, 1899, the patient, who was engaged in the
analysis of fats in the chemical laboratory of the University
of Jena, unfortunately broke a glass jar while forcing a
stopper into it. The palm of one of his hands was wounded
in a line with the middle finger at a point nearly opposite to
the middle of the metacarpal bone of the thumb. A jet
of blood immediately spurted out as high as the ceiling
and an artery was ligatured by a surgeon. A branch
of the radial nerve was also severed, for numbness at once
appeared on the inside of the thumb and on the outside of
the forefinger. (This still continues to a slight extent.)
At the time of the accident the surgeon who tied the
artery discovered no foreign body, but an x-ray photograph
taken five days afterwards showed a shadow of an abnormal
character. There were, however, no urgent symptoms ;
the wound healed without suppuration, and in order that
the ends of the artery might have time to consolidate the
surgeon advised non-interference. After three weeks, the
appearance of the hand being quite normal, the surgeon
endeavoured to remove the foreign body which from the
x-ray photograph was supposed to be just under the surface,
but the attempt was unsuccessful. All remained quiet until
a few months ago. The patient, who was a violinist, had
given over playing on account of the injury, but on resuming
it he occasionally had painful sensations in the back of his
hand in the triangle between the metacarpal bones of the
thumb and forefinger. In the first week of January, 1902,
he felt considerable pain in this triangle when washing and
drying his hand, and a red spot formed on the back of the
hand almost on a level with the carpo-metacarpal articula-
tion. On Jan. FOREIGN BODY A LONG TIME EMBEDDED IN THE
HAND.
BY L. K. HERSCHEL, M.D. VIENNA. 10th the skin gave way at this spot and a
sharp point protruded ; two days afterwards, as it was coming
more and more to the surface, the patient seized it with a
pair of forceps and drew out a fragment of glass five-eighths
of an inch long. No pain or difficulty was experienced in
doing this and the wound closed without suppuration. adequately recognised. So much, then, for the disadvantages of the double stetho-
scope from the point of view of diagnosis. But there is
another side to the question. Common sense tells us that it
cannot be good for the delicate organ of hearing to be more
or less constantly compressed by the ivory heads and the
unyielding spring of the binaural stethoscope. For it must
not be forgotten that when the instrument is made use of the
pressure of the necessary spring is by no means a negligible
quantity. Without a certain, indeed, a considerable, tension
nothing can be made of the sounds heard within the chest,
and I am strongly of opinion that the minimum pressure
required for this purpose does not remain confined to the
parts on which the ivory heads immediately impinge-i.e.,
to the outer portion of the external meatus-but that it is
transmitted to the rest of the canal and to the mem-
brana tympani. It cannot be denied, therefore,
that
this pressure kept up intermittently for hours, as is the
case with house physicians and others, may be most
injurious to the hearing of the observer. I have known
of extreme tenderness being the result of the
use of
the instrument ; so marked, indeed, was this symptom that in
more than one case experience has taught the sufferer at
once to discontinue the use of the instrument, in order that
wor: e evil might be avoided. The delicate organ of hearing
is not intended to be abused in this manner, and pain and
discomfort in the ear are the first warnings that nature is
being trifled with ; such warnings are not to be neglected
with impunity. impunity. Are there, then, no uses for the binaural stethoscope, or is
the instrument to be altogether condemned
-? FOREIGN BODY A LONG TIME EMBEDDED IN THE
HAND.
BY L. K. HERSCHEL, M.D. VIENNA. It may be said
at once that the use of the double stethoscope should be
altogether discarded were it not for the fact that it is
convenient in illnesses which render it difficult to move the
patient in a manner suitable for examination by the wooden
stethoscope. In many cases of enteric and other fevers, in
pneumonia, and other conditions in which it is undesirable
on account of the feebleness of the patient to move him
more than is absolutely necessary the binaural stethoscope
is often of use in enabling the breath sounds to be doing
Bordighera. A CASE OF SYPHILITIC PNEUMONIA. BY JOHN GEORGE GIBBON, M.D. DUB. The double stetho-
scope should be altogether done away with and abolished off
the face of the earth—.ZMx est Carthago. ace
;
Carthago. Next as concerns the examination of the lungs. Here,
again, there is nothing good to be said as regards the use of
the binaural instrument in its strictly auscultatory aspect. On the contrary, it is easy to point out numerous and grievous
faults. I have noticed that beginners who are learning the
elements of diagnosis of chest disease, and who, alas, are
freely allowed to choose their instrument, never really appre-
ciate
the difference between bronchial,
tubular, and
cavernous breathing. I do not for a moment deny that
this inability may to
a certain extent depend upon
the want of a keen and correct hearing sense; prob-
ably this is so to some extent. But making every allowance
on this account, I maintain that this great fault is due
chiefly to the most foolish and injudicious laxity which
permits to a young and inexperienced student the use of an
untrustworthy and deceitful instrument. This failure to
recognise fine shades and distinctions of sound is practically
always attributed to the defective ear" of the observer. In nine cases out of ten it is nothing of the kind ; on the
contrary, it is the result of a defective system of education. the differentiation of friction sounds and of crepitation is
by no means easy at any time ; when the binaural stethoscope
is made use of such discrimination is absolutely impossible. And the tendency of the instrument is invariably to exaggerate
the pulmonary sounds, whether healthy or morbid, to a
degree which is embarrassing and likely to tend to serious
error in diagnosis. Further, the fact of both ears being
occupied with the examination renders it impossible to appre-
ciate the significance of what is heard by comparison with
the unoccupied ear ;’ this is a very important point and one
not adequately recognised. Wimpole-street, W. 370 370 heard, when this would be possible only with the greatest
difficulty were the wooden instrument made use of. In
babies, again, and in young children, the double instru-
ment may be advantageously employed. But used in this
way the stethoscope in question is merely an accessory, one
to be employed for particular occasions and for special
needs. What I protest against, and in the strongest possible
manner, is the predilection shown for the binaural stetho-
scope by the young student who is allowed to make use of
it from the very commencement of his clinical work, so that
he is totally unable to appreciate the advantages which
appertain to the employment of the old-fashioned instru-
ment. No words can condemn too strongly this adoption
of the binaural instrument which is, I am convinced, the
cause of much of the indifferent diagnosis of chest disease
so often observed at the present time. fault of the latter, not of the auscultator. Again, in the
diffeientiation of mitral murmurs the double stethoscope
leaves much to be desired. I have constantly found
that systolic murmurs have been described as presystolic
when this- instrument has been employed, though the
converse mistake has not occurred. And in the study of
the normal heart sounds I have found the diagnosis of " over
accentuation " or "obscurity," neither of which really existed,
to be constantly made. As regards the heart, therefore, I
look upon the use of the binaural stethoscope as being in
every way most objectionable. As regards the diagnosis of
heart disease there is no middle course. The double stetho-
scope should be altogether done away with and abolished off
the face of the earth—.ZMx est Carthago. fault of the latter, not of the auscultator. Again, in the
diffeientiation of mitral murmurs the double stethoscope
eaves much to be desired. I have constantly found
that systolic murmurs have been described as presystolic
when this- instrument has been employed, though the
converse mistake has not occurred. And in the study of
the normal heart sounds I have found the diagnosis of " over
accentuation " or "obscurity," neither of which really existed,
to be constantly made. As regards the heart, therefore, I
look upon the use of the binaural stethoscope as being in
every way most objectionable. As regards the diagnosis of
heart disease there is no middle course. A CASE OF SYPHILITIC PNEUMONIA.
BY JOHN GEORGE GIBBON, M.D. DUB. THAT the following case was one of syphilitic pneumonia
there can, I think, be little doubt. The course of the tem-
perature was interesting as most of the few authorities to
whose works I have been able to refer mention that little or
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https://openalex.org/W2036513170
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https://zenodo.org/records/1681113/files/article.pdf
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German
| null |
Ueber die Isomerie der sogen. Benzolazocyanessigester
|
Berichte der Deutschen Chemischen Gesellschaft
| 1,905
|
public-domain
| 4,965
|
[
]
2j Kriickebcrg, Joarn. fiir pralrt. Cilern. 46, 57'3: 49, 32, Haller,
Compt. rmd. 106, 1171. Dieee Berichtc 3S, i i 5 [1905]. Iu seiner jiingsten Publicationen iiber diesen Gegenstand hat
(.i iese 1') auf die Existenz eines Emanium X hingewiesen, welches
bei der Fallung des Lanthan-Emaniumoxyds durch Ammoniak in Lo-
sung bleibt. Daher wurde versucht, wie sich das in der zuletzt be-
schriebenen Weise gewonnene , stark emanirende Thorium bei der
A ~iioioniakfallung verhielte. Dabei zeigte sich, dass das so abgeschie-
deiie 'I'horiurnhgdroxyd unverandert stark emanirte, wahrend die Lo-
sung nach deni Abdampfen und Vertreibeu der Amnioniumsalze keinen
crheblich emanirenden Ruckstand liess. Aus dem Vorstehenden ergiebt sich, dass Acti'nium und E m a -
iiiuui nicht i d e n t i s c h s i n d , s o n d e r n in e i n e m g e n e t i s c h e n
Z u s a m m e n h a n g stehen. Emaniurn, wenu wir als sokhes den dein
Lanthan folgenden Stoff bezeichnen, erzeugt das Actinium, den dem
Thorium folgendeu, stark emanirenden Stoff. Der Name Emanium
iat so allerdinge riicht gliicklicli gewahlt, da dieser Stoff gar nicht
emanirt. Passender ware es gewesen das Actinium nach seinern
Emanationsvermiigen zu beneunen. Dergleichen unpassende Trivialnameii wiirden vermieden werden,
wenn die auf deni Gebiete der Radioactivitat thiitigen Fachgenossen
niit der Benennung neu entdeckter Stoffe bis zu einer gr6ndlichen Er-
forschung ihrer Eigenschaften zuriickhalten wiirden. \Venn nicht ge-
iiiigend charakterisirte Stoffe mit Namen reraehen werden, so wirkt
dies spater hiiufig verwirrend, anstatt die Uebersicht zu erleichtern. Xeben dem hier behandelten Fall sei in dieser Hinsicht besonders auf
das Polonium rerwiesen. 397. A. Hantzsch und K. J. Thompson: Ueber die Isomerie
der sogen. Benzolazocyanessigester. 397. A. Hantzsch und K. J. Thompson: Ueber die Isomerie
der sogen. Benzolazocyanessigester. ,Eingcg. am 9. Juni 1905; mitgetbeilt in der Sitzung von Hrn. 17. Ullmann.) ,Eingcg. am 9. Juni 1905; mitgetbeilt in der Sitzung von Hrn. 17. Ullmann.) Die Prodocte der Einwirkung von Diazoniumsalzen auf Natrium-
Cyanrssigester sind bekanntlich zuerst in zwei versehiedenen Modi-
ticarionen erhalten wordena) bei denen es sich nach allen Analogieen
entweder um Striicturieonierie zwischen den1 echten Benzolazocyau-
CN
esigester
Ar N: K CHcCoo R, und dem Hydrazon des Keto-Cyan- Die Prodocte der Einwirkung von Diazoniumsalzen auf Natrium-
Cyanrssigester sind bekanntlich zuerst in zwei versehiedenen Modi-
ticarionen erhalten wordena) bei denen es sich nach allen Analogieen
entweder um Striicturieonierie zwischen den1 echten Benzolazocyau-
CN
esigester, Ar. N: K. CHcCoo R, und dem Hydrazon des Keto-Cyan-
CN
rssipesters, h r . XH. N: Ccco0
Hydrazons handeln konnte. , oder auch urn Stereoisomerie dieses
Sehr auffiillig war aber der von H. W e i s - ,
[
]
2j Kriickebcrg, Joarn. fiir pralrt. Cilern. 46, 57'3: 49, 32, Haller,
Compt. rmd. 106, 1171. Lac h I) angeblich erbrachte Nachweis ron der Existenz eines dritten
und schliesslich sogar eines vierten Iaomeren. Denn wenn W e i s b a c h
in diesen 4 Isomeren je zwei stereoisomere Panre von Hydrazonen
iind Azokorpern rermuthet, so ist schozi die Existenz zweier stereo-
icornerer Azoverbindungen der Fettreihe Ar.N
N. A r
2. ’. CN
ii . CH<E;,
c, HS
N*CH’COzCzH5, Ar.N
N. A r
2. ’. CN
ii . CH<E;,
c, HS
N*CH’COzCzH5, und specie11 dieExistenz eines aliphatischen wgn-Azokorprrs(( (Formel2)
an sich eiu Unicurn, da bisher nur qn-Diszotate, ~,yn-Diazosulfo~ia
uiid syn-Diazocyanide bekannt sind; ferner besitzt aber auch keine
dieser v i u Modificationen n u r eine einzige Eigenschaft der durch ihre
Reactionsflhigkeit SCJ leicht erkennbaren syn-Diazokiirper. Ini Gegen-
theil spricht Weisbaclis Angabe, dass sich (L- uncl $-Form einerseits
uiid j - i i u d 6-Form andererseits wechaelseitig in einander iiberfiihren
lassen, entschieden dagegen, dass zwei voii dieseri vier Formen, und
zwnr nach W e i s b a c h s Ansicht die y- und &Form, stereoisomere
Azokiirper seien. Denn Letztere stehen, wie das Verhalten der stereo-
isomeren Herizolazocyanide (Diazobenzolcyanide) zeigt, in ganz arideren
Eeziehungen zu eiriauder a13 u- und $-, oder ;’- und 8-Benzolazocyan-
essjgester. ‘;I .Jonrn. fur prakt. Chcm, 67, 395. 397. A. Hantzsch und K. J. Thompson: Ueber die Isomerie
der sogen. Benzolazocyanessigester. Da W e i s b n c h diese Thatsache ausser Acht gelassen hat und als ein-
ziges Charakteristicuni tier beiden ron ihin 31s Azokorper angesehenen
dritten oder y- und vierten oder 8-Modification nur ihre rothe, Azo. benzol-ahnliche Farbe und angeblictie Iudifferenz gegen Acetylchlorid
auffijhrt, so hubeu wir diese eigenthiinilichrn Isomeriererhaltnisse ge-
nauer untersiicht; freilich mit einem von W e i s b a c h a Angaben wesent-
lich verschiedenen Resultate, das sich folgendermaassen zusanimenfasaen
lass1 : Von isomer en Benzolazocyarieasigestern existiren nur die langst
bekannten und gut definirten a- und $-Formen. Die Weisbach’schen
7- und 8-Formen sirid dagegen als chemische Individuen zu streichen. Die sogenannte y-Form ist nur eine unreine @-Form, die sogen. 8-Form
eine unreine a Form. Die rothe Farbe der sogenanriten y- und 8-Form,
dir Weisbach veranlasst hatte, in beiden stereoisomere Azokorper
211 rermuthen, beruht auf einer Veriinreinigung durch minimale Mengen
rou Formazylcyanid, das sich bekanntlich in stark alkalischer Losuog
Auf diese wohl in fester
Liisii~g gebundenen Spuren von Formazylcyanid ist auch eine vou
u n s lieu aufgefundene, anscheinend fiir die y- und 6-Form im Gegen-
wtz zor u- und @-Form charakteristische violettrothe Farbenreaction
Cganessigester und Diazosalzen bildet. mit cocceutrirter Schwefels~ure zuriickzufiihren. Die weitere Angabe
W r i s b a c h s , dnss die y- und 8 Icorm, ebenfnlh im Gegensatze zur
a- und $-Form, nls stereoisomere Azokorper durch Acetylchlorid nicht
angegrifen werden sollen, beruht auf eiriem Irrthum; thats~chlich
wwden alle Modificationen gleichxrtig (scliwierig und unvollstiindig)
ace ty 1 i rt. Die Constitution der beiden einzig reellen Isomeren (der i t -
und ,?-Form) als stereoisomerere Hydrazone ergiebt sicli, worauf bereits
die fiii!,eren -4utoren liinwieseu, aus ihrer gi ossen Aehnlichkeit iiii
pliysiknlischeu rind chemisclien Verbalten, sowie nus der Existenz zweier
Acetplderivate welcli’ Letztere eiii und derselben Hydmzon-Strirctur-
fnrrnel, COOR;C:
N . Nhc. Ar rntspreclien, also stereoieomer seiii
niiissen. wie sicli derni auch das eiiie Acefylderirat beim Cnikry-
stallisiien in da3 :tndere umlagert. 397. A. Hantzsch und K. J. Thompson: Ueber die Isomerie
der sogen. Benzolazocyanessigester. C: C(OC2HS)OMe
Ar Nn
Na Ar B Verbind ungen
NC.C.COOR S a k e aus den Hydrazonen: Na Ar Danach leiten sich also die beiden Salze zwar ron einer Azorer-
Itindung, aber doch nicht rom wirklichen Benzolazocyanessigster ab. 397. A. Hantzsch und K. J. Thompson: Ueber die Isomerie
der sogen. Benzolazocyanessigester. CX-, I)ie C o n f i g u r a t i o n sb es tin1 m 11 n g aeist deu beiden Sterenim-
meren folgende speciellen Foimeln zu: a-Derivat
:f-Derirat
NC.C.COOR
KC.C.COOR
N
, S H A r
9
ArNH.N a-Derivat :f-Derirat KC.C.COOR N
, S H A r
9 die eich aus den im experimentellen Theil angefuhrfen Verschiederi-
heiten und namentlich aos der Analogie des i t - Derivats init dem
Hytlrnzon des Dinitrils CN.CX2 HAr. CK und des p’-Dt.rirats mit
deli Hydrazonen der DicarbonsHureester COOR.CN,HAr.COOR mit
eiernlicher Sicherheit ergeben. - Beide Hydrxone bilderi primar aiicL
zwei verschiedene Salzreitien - denn nus der Soddosung der a-Farin
wird beim Arisiiuern wieder die a-Form, aus der Sodalosung der
$-Form auch die $-Form erhnlten - wobei nllerdiiigs lrtztere z . T!i. schon i i i die I*.Forrn iibergegangen ist. Diese durch Carbonat I:ing-
sam erfolgeude Umlagerung der schwiicher sauren $-Form iii die
stPi kere a-Form rollzieht Rich durch Alkalien so rasch, dms, wie be-
h i n t , aus der Natronlijsung lreider Hydrazone durch starke Ssuren
nur die rr-Form gefiillt wild, d i m also durcli Alkali aus den1 $-Salz fnjt
niomentnn das cc-Salz erzaugt wit d. Etwas cornplicirter werderi die Ver-
haltnisse iibrigens noch dadurch , dass liiichst wahrsclieinlich beide
Hydrazone Pseudosauren sind, d:iss also nicht die unveranderten H\--
CNL-C:N.KHAr Salze COOR,C:X.NMeAr
CN,
bilden,son-
COOR-’
drnzone
dern erst nach Verschiebung de3 beweglicheii WasswstoffJ. die eich aus den im experimentellen Theil angefuhrfen Verschiederi-
heiten und namentlich aos der Analogie des i t - Derivats init dem
Hytlrnzon des Dinitrils CN.CX2 HAr. CK und des p’-Dt.rirats mit
deli Hydrazonen der DicarbonsHureester COOR.CN,HAr.COOR mit
eiernlicher Sicherheit ergeben. - Beide Hydrxone bilderi primar aiicL
zwei verschiedene Salzreitien - denn nus der Soddosung der a-Farin
wird beim Arisiiuern wieder die a-Form, aus der Sodalosung der
$-Form auch die $-Form erhnlten - wobei nllerdiiigs lrtztere z . T!i. Diese Verschiebung wird bei beiden stereoisoseren HydrazonGaii
in priricipiell gleicher Weise - durch Wanderling des Wasserstofl.e.;
:III die raunilich benaclibarte Gruppe - erfolgen. uud dn diese 1-1 r- schieden (entweder CN oder COOR) ist, anch zu rerschiedenen
Salzen fiihren: Urspringliche Hydrazone:
('-Verbindungen
B- Verbind ungen
NC.C.COOR
NC.C.COOR
A r 3 H . N
N.NHAr
S a k e aus den Hydrazonen:
n-Salze
8-Salze
Me N : C. COOR
NC . E x p e r i m e n t e lles. D:e Reaction Ton DiazolGsungen auf Cyanessigester sowie auf
xndere alkalilosliche, schwach snure Verbindungen fiihrt bei Anwesen-
heit ron iiberschiissigem Alkali und manchmal auch ron A1k;ilicarbo-
imt in Folge der verseifenden und spaltenden Wirkung des A l -
lidis suf die Ester mehr oder minder leicht zu secundaren Producten,
welche die als Hauptproducte gebildeten gemischten Azok6rper ver-
unreinigen, namlich zri sauren Estern oder zu Forrnazylverbindungen,
z B. rom Cpnessigester zum Formazylcyanid. Man rermeidet diese
Sebenreactionen und erhalt in den meisten Fallen ganz reiue ge-
mischte Azokorper, wenn man das Alkali oder Alkalicarbonat durch
uherecl~dssiges Natriumacetat ersetzt. Man bringt alsdann den in
Alkali loslichen, aber in Acetat natiirlich unloslichen Ester dnrch
Zusatz ron Alkohol in Liisring und fallt nach vollendeter Reaction
rnit Wasser :(us. Durch diese Abiinderung entstehen aus Cyanessig-
wter. Malons5ilireester und Malonitril sofort ganz reine Hydrazone,
oligleich in geringerer Ausbeute als aus alkalischer Losung. Yon den Isomeren aus Cyaneseigester rind Diazobenzol schmilzt
d a s tc-Derivat wie bckannt bei 1250. Dagegen schmilzt das ganz
reine p-Derivat nicht wie K r t i c k e b e r g und W e i s b n c h angeben,
sthon bei 8S0, sondern erst bei S5-Xti0. Die nach den gennnnteu
Autoren hergestellten. schon bei 820 schmelzenden Prapnrate enthal-
ten stets geringe Mengen der in gelben Nadeln krystallisirenden
ci-Form, die sich beim Umkrystallisiren auch der reinen &Form aus
heissc~n Losungsmitteln stets in gcringer hlenge bildet. Dies deutet
d:tr.,uf hin, dass hei hoherer Temperatur die $-Form in die a-Form
wenigatens in Losung theilweise iibergeht oder mit ihr ein Gleich-
gcwicht bildet. Die reiiie $-Form vom Schrnp. 85-86" scheidet sich 147 Bericl te d. D chem. Gesellvchak Jahrg. XXXVIII. 2270 aus warmer Ligroi'ulosung zuerst aus; in der erkaltendeu Xlutterlauge
entstehen dann erst die drircli u-Forni verurireinigten, tiefer schmel-
zenden Krystallr der $-Form. Das angebliche y-Isoniere wurde euerst nach W eisbach's An-
gnbm dnrgestellt : d:ts :tus stark alkalischer Diazol6sung und Cyan-
essigestrr bei tiefer Temperntur gebildetr Robproduct, das zwischeii
GO -75 0 schmolz, lieferte nach der Behandlung init Acetylchlorid liei
105-1 100 (wodurch nach Weisbacli angeblich (c- und $-r)erivat als
Acetylkijrprr entfernt werden), beiiii Umkrystallisiren DUS LigroYn die
fiir dic 7-Form als charakteristisch angegebenen dunkelrothen dicken
Prismen vom Schmp. 84-850. Dieselben losten sich, wie wir fanden. in conccntrirter Schwefelsiiure init riolettrother Farbe, wiihrend a- und
#-Form eine rein gelbe Liisung ergeben. E x p e r i m e n t e lles. Doch waren daneben br-
reits goldgelbe Nadeln vorhanden, .die etwas h6her schmolzen, schon
husserlich der ti-Form sehr ahnelten und sich scliliesslich mit diesw
identificiren liessen. Ferner reranderten die rothen I<rgstallt, voni
Schmp. 8.10 zwar durch wiederholtes Unikrystallisiren aus Ligroin
wrder ihre Farbe noch ihren Schmelzpunkt, noch ihre Scbwefelsiiuw-
reaction - wohl aber liessen sich aus ihnen (wexin rnit etwas
grosseren Mengen operirt wurde) durch ofteres Umkrystallisiren nus
Aether, noch besser aus Alkobol wenigstens theilweise gelbe Prismen
des #-Isomeren vom Schrnp. 85-86" und daneberi auch sehr geringe
Meiigen dvs a-Isomeren gewinnen, wiihrend allerdiogs inimer noch on-
veriinderte rothe Krystalle rnit violetter Schwefelsaure-Reaction auf-
traten. Aebnliche Heobactitungen wurdrn auch bei den Versuchen ge-
mncht, die y-Form in besserer Ausbentt. und iu reiner Form durch Ver-
anderung der Reactionsbedingungen zu erhalten, aljo durch Aenderung der
Ternperatur, der Menge des Alkalis, der Verdiinnung bei der Eiowirkurig
von Diazolosung auf Cyanessigester. Als wichtigates Resultat ergab 5ich
durch zahlreiche Einzelrersuche, dass die sogen. y-Modification sicli niir
aus den i n a1 k a l i s c h e r Losung erzeugrenKuppelungsproducten von Di-
azoverbindung rind Cyanessigrster gewinnen liess, aber niemals eutstnnd,
wenn sich die Reaction bei Anwesenheit ron Natriuniacetat (8. oben)
rollzogen hatte. In letzterem Falle bildeteu sich direct nur gelbe, iii
concentrirter Schwefelsiiure rnit gelber Farbe Ioslichr Producte , die
auch nach der Behandlung mit Acetglchlorid sich ebenso verhielten,
also keiu sogen. y-Derirat ergaben. Andererseits entstanden
:IUS
diesrn Producteii bei Anwesenheit von Soda und nocli deutlicher voii
freiern Alkali direct dunkelgelbe bis rothe Producte, die sich iri
Scliwefelslure roth his violett losten und sich also bereits For der
Bebandlung niit Acetylchlorid Shnlich dein sogenannten ;.-Isoniereii
hi lt 227 1
.~ D n nun in noch starker alkalisclier Liisung und nach lingerer
Einwirkungsdauer beim Einleiten von Kohlendioxyd in die primhre
Liisuug r o r den Reuzolazocyanessigester~i etwas Formazylcyanid aus-
liel. das durch Umkrystallisiren in tief rothen, goldschimmernden Kry-
stallen rom Sclinip. 358--15!3" rein erhalten wurde und schon in Spureti
sich in ScliwefelsBure tief yiolett-Ioste, so ergab sich ails diesen Be-
obachtuiigeri , dass dns sogeii. ;,-Isomere seine rothe Far1.e uud seine
riolette Schwefelshure-Reaction nur einer sehr fest anhafteliden Bei-
niengung oder einer festen Lijsung yon Formazylcyanid verdankt, die
durch blosses Umkrystallisiren aus Ligroi'n kaum beseiligt werdeu
kanrl. Da riun das reine $-Isomere bei 85-86''
und das sogeti. ],-Isomere bei 84" schmilzt, so ist letzteres iiichts anderes ale eine
durch Foruiazylcyanid veruiireinigte $-Form. ’) Derselbe wiinscht auch ausgesprocheu ZII haben, dass er sich, nacl~dt,n~
i!r Ton mir auf die Widersprlche zwischen unseren ond H. Weisbach’s Be-
obachtungen hingewiesen worden war, duich Versuche mit Weisbach’s Ori-
ginalpriparateu, die er friiher nicht selbst gepriift habe, von der Richtigkeit
tinserer Schlussfolgerungen betr. der Nichtesistenz der sogen. y - und S-lllodi-
tication iiberzeugt hat.
Han tzsc ti. g
*) Diese Berichte 24, 1242 [lSSl]. .‘) v. Pechmann, diese Berichte 24, 866 ‘lS911. E x p e r i m e n t e lles. Der Beweis hierfur liegt also erstens darin, dnss die ;,-hIoditica-
tion, wtiin sie wirklicli aus den a- und #-Isomeren durch die Beliand-
lung niit Kohlensiure bri GOu (nach W e i s b a c h ' s Angabe) erzeugt
wiiide, nntiirlich auch aus den reinen, von Formazylcyanid freien und
deslialb in Schwefelsaure gelb liislichen (z- und $- Formen eiitstehen
niiisste, was aber iiicht der Fall ist. Zweitens darin, dass nus Lii-
sungen der reinen $-Form durch Zusatz kleiner Mengen von Forninzyl-
cyaiiid die sogen. ?-Form krystallisirte. Das sogeu. 3-Isoruerr kounte nach W e i s b a c h ' s Angaben iiber-
haupt nicht aus der sogen. 7-Form rnit den ron diesern Autor ange-
gebenen Eigenschaften erhalten werden. Hierbei entstanden zwar nnfangs
ebenfalls rothe Krystalle, die aber nicht, wie angegeben, bpi 118". soudern je nach der Art des Erhitzens bald hijher, bald tiefer und
bisweilen sogar bei 1250, wie das cc-Derivat schmolzen. Da sich dieFe
d-I:orni ebenfalls mit rioletter Farbe in Schwefelsaure 13st und ubrigens
alle wesentlicheu Eigenschafien der (<-Form besass, so ist sie jedenfalls
nichts anderes als eine durcli Formazylcyanid verunreinigte cr-Forni. Ein chemisches Individioni kann sie schon deshalb nicht sein, weil
ihre augebliclie Muttersiibstaiiz, die y-Form kein eolches ist. Nun giebt allerdings W e i s b a c l i als charakteristisch fiir die 1'-
urid die 6-lJorm ihre Indifferenz gegen Acetylchlorid bei etwa 1100 an,
wahrend die beiden llngst bekannten (a- und #-) Isoniereu hierbei
primlr in ein und dasselbe Acetylderivat vom Schmp. 15So verwandelr
werdrn, das leicht in ein isoneres Acetylderivat Yom Schmp. 166'~
iibergeht. ThatsLchlich verhalt sich aber auch die sogen. y- und &Form
gegen Acetylchlorid ganz wie die a- und @-Form. Die Acetylirung er-
folgt iiherhaupt stets so langsani und partiell, dass meiet noch erhebliche
Mengen der nicht acetylirten Muttermbvtanz ueben deli Acetylkiirpern 1.17 1.17 2272 unoerandert bleiben. Ob Weisbach’s Irrthum hierdurch entstanden
ist, lassen wir dabingestellt. Zum Schlusse sei noch erwiihnt, daes
eine Probe der von Weisbach selbst hergestellten r.Form, die wir
dern freundlichen Entgegenkomrneii des Hro. Geheirneu I-lofrzthes
Prof. Dr. E. v. Meyerl) verdauken, sich genau so yerhielt, wie oberi
bescbrieben, narnentlicb auch durch Acetylchlorid nicht intakt blieb. Auch diese Probe gab nach Weisbach’s Angabeu bei der Umlagerung
nicht ein chemiscbes Individuum voni constanten Schrnp. 1 1 8 O , also
nicht die sogen. &Form. unoerandert bleiben. Ob Weisbach’s Irrthum hierdurch entstanden
ist, lassen wir dabingestellt. ’) Derselbe wiinscht auch ausgesprocheu ZII haben, dass er sich, nacl~dt,n
i!r Ton mir auf die Widersprlche zwischen unseren ond H. Weisbach’s Be-
obachtungen hingewiesen worden war, duich Versuche mit Weisbach’s Ori-
ginalpriparateu, die er friiher nicht selbst gepriift habe, von der Richtigkeit
tinserer Schlussfolgerungen betr. der Nichtesistenz der sogen. y - und S-lllodi-
tication iiberzeugt hat.
Han t zsc ti.
*) Diese Berichte 24, 1242 [lSSl].
.‘) v. Pechmann, diese Berichte 24, 866 ‘lS911. E x p e r i m e n t e lles. Sehr gut krystallisirt aber der b r o m i r t e D i g t h y l e s t e r COOC~HS
Ch’? HCdH4 Br. COOCaHS, der ganz analog aus Malonslureathylester
und p-Bromdiazoniumsalz entstrht und nach dem C‘rnkryst.allisiren a03
Ligroi’n in gelblichen Kadelu YOU) Schmp. 76O whalten wird. C13HljOlN?Br. Ber. N 8.2. Gef. N 8.2. Das ebenfalls krystallisirende bronifreie H y d r a z o n d e s D i m e -
t h y 1 e s t e r s , COOCH3. C p i 2 €I Cls Hg. CO OCH3, voni Schmp. 63.5’, das
zuerst v. Pechtnann*) aas Diazornethan uud dern saureii Ester er-
halten hatte, entsteht nach der obigen Methode ebenfalls glatt, also
auf vie1 bequeniere Weise. CllE1204N2. Ber. N 11.9. Gef. N 12.0. CllE1204N2. Ber. N 11.9. Gef. N 12.0. Zn den Angaben v. P e c h m a n n ’ s iet unr berichtigend
ZII cr-
glnzen, dass sich der Ester iu wlssrigen Alkalien nieht nuzersetrt
lost, sondern ziernlich rasch reraeift wird. Denn die gelbe Alkali-
lbsung war durch Kohleusaure nicht rnehr fiillbar und lieferte durch
Salzsaure das Hydrazon der Mesoxalslure rom Zersetzuugspunktv
lG2-163”. Zn den Angaben v. P e c h m a n n ’ s iet unr berichtigend
ZII cr-
glnzen, dass sich der Ester iu wlssrigen Alkalien nieht nuzersetrt
lost, sondern ziernlich rasch reraeift wird. Denn die gelbe Alkali-
lbsung war durch Kohleusaure nicht rnehr fiillbar und lieferte durch
Salzsaure das Hydrazon der Mesoxalslure rom Zersetzuugspunktv
lG2-163”. Das H y d r a z o n d e s D i n i t r i l s , CN.CNaHCp,Hb.CN, das nach
S c h m i d tmann’s Verfahren?) aus Malonitril in alkalischer LBsring in
unreinem Zustaude entsteht, wird ebenfalls nach unserer Methode: also
durch Einwirkung von Malonitril und Diazolosung bei Anwesenheit VOII
Natriumacetat sofort rein, d. i. vorn Zersetzuagspunkt 146-1470
er-
halten. Die aus Benzol umkrystallisirten dunkelgelben, durchsichtigen
Prismen entbalten ‘/z Mol. Krystallbenzol locker gebunden und werden
deshalb an der Luft, nicht aber in riner Benzol-Atrnosphare triibe. CsHo. Ber. 18.8. Gef. I. 18.1, 11. 18.1. Alle diese syrnmetrisch constituirten Verbindungen werden unter
denselben Bedingungen, unter denen die asymrnetrischen Verbindungen
Cir’ . CN2 N Ar. COOR sic!] isomerisiren, nicht urngelagert. Durch Al-
kalien wurden die Ester nur in anderer Weise verandert. als das Di-
nitril. a) Diese Berichte ‘26, llG8 [1S93;.
’) Diese Berichte 2S, S5S [1895]. E x p e r i m e n t e lles. Zum Schlusse sei noch erwiihnt, daes
eine Probe der von Weisbach selbst hergestellten r.Form, die wir
dern freundlichen Entgegenkomrneii des Hro. Geheirneu I-lofrzthes
Prof. Dr. E. v. Meyerl) verdauken, sich genau so yerhielt, wie oberi
bescbrieben, narnentlicb auch durch Acetylchlorid nicht intakt blieb. Auch diese Probe gab nach Weisbach’s Angabeu bei der Umlagerung
nicht ein chemiscbes Individuum voni constanten Schrnp. 1 1 8 O , also
nicht die sogen. &Form. Der Nachweis, dass beide Verbindungen Hydrazone und damit
Stereoisomeie sind, dass also nicht etwa die eine Form ein structur-
isornerer Azokorper ist, wurde znerst indirect dadurch erbracht, dass
ails Diazoniumsalzen und alkaliliislichen , schwach sauren Verbin-
dungen nur dann zwei Isomere hervorgehen, wenn diese der a s p -
rnetrischen Structurforrnel X .CNaHAr .Y entsprechen, dass dagegen
Verbindungen
van symrnetrischer Structur X. CN2 H Ar. X nur in
einer einzigen Form bestehen. Ware von den fraglichen Ison~eren
das Eine ein Hydrazon und das Andere ein Azokorper, so sollten
linter den symnietrischen Verbindongen ebenfalls Isomere X,CH. x. S : N . Ar und x)C:
N . N H Ar gefunden werden. Dass diese Isornerie
nicht auftritt, wiirde durch die folgenden Versuche mit den uachsten
Verwandten der unsymrnetrischen Verbindung CN. CN:, H Ar.COOR
iiiirnlich mit den spmetrischen Verbindungen CO OK..CNaH Ar.COOR
urid CN. CNg H A r . CN bestatigt. s, Der Diathylester COOC1Hg.CN2 €ICs H5.COOC2H5, der nircb
R. M e y e r a ) aus dem Silbersalz der SPure durch Jodiithyl nicht er-
halien werden kann, entsteht zienilich Ieicht aus Diazosalz und >fa-
lonsiureester in wissrig alkoholisclier Losung bei Anwesenheit van
Satriuniacet at, blieb aber auch riach verschiedenen Reinigungsversuchen
iilig. Er gab sich als der DiiithFlester dadurch zu erkennen, dass er
sich durcli ein Mol. Alkali bei gew6hrilicher Temperatur zu dem
Nonoathyle2ter vom Schrnp. 1140 3, verseifen liess; wegen seiner flii3si- ’) Derselbe wiinscht auch ausgesprocheu ZII haben, dass er sich, nacl~dt,n~
i!r Ton mir auf die Widersprlche zwischen unseren ond H. Weisbach’s Be-
obachtungen hingewiesen worden war, duich Versuche mit Weisbach’s Ori-
ginalpriparateu, die er friiher nicht selbst gepriift habe, von der Richtigkeit
tinserer Schlussfolgerungen betr. der Nichtesistenz der sogen. y - und S-lllodi-
tication iiberzeugt hat. Han t zsc ti. Han t zsc ti. geu Heschaffenheit eignete er sich aber nicht zu Urnlngerungsver3uchen. E x p e r i m e n t e lles. Die Ester siod minimal sauer; sie losen sich in wassrigen
Baseti nur schwierig und nur unter rapider Verseifung; echte Salze,
und zwar von roiher Farbe, erzeugen sie nur rnit Natrium. oder Ka-
lium- Alkoholat; aber auch aus diesen alkoholischen Lijsungen werden
sie durch Wasser unrerandert wieder gefallt. Das Nitril verhllt sich
dagegen wie eine ausgesprochene Saure; es lbst sich leicht und ohne
merkliche Verseifung €chon in verdiinnten Alkalien. Wichtig fur die
Frage nach der Natur der isomeren ssymrnetrischen Verbindungen C S . CNzHAr.COOR ist aber die Thatsache, dass auch das Dinitril aus 2374 alkaliscber Losung nicht nur durch starke Sauren (die aus der alka-
lischen Liisuvg von CN.CN2 H Ar.COOR das tc-Isomere ausfallt),
sondern auch durch Kohlensiiure (die aus der Losung des u-Isomeren
das $-Derivat ausfallt), stets unverandert, also nicht isomerisirt erhal-
ten wird. Durch Acetylchlorid erhalt man nus dern Hydrazon des Di-
nitrils, allerdings unter partieller Zersetzurig ein in Platten krystalli-
sirendes Acetylderivat rom Schmp. 16e- 1690, das sich aber, weil
ron symmetrischer Structur, nicht, wie das primare Acetylderivat aus
sogen. Benzolazocyanessigester durch Umkrystallisireri in eine zweite
isomere Form verwandeln liess. Die Verseifung der Dicarbonsiiureester COOR.CN2HAr.CO OR
durch 1 Mol. Alkali fiibrt zu den sauren Estern. So wurde bei der
Verseifuug des Methylesters in wiissrigem Methylalkohol durch 1 Mol. Natron nach mehrstiindigem Stehen durch Salzeaure der von P e c h -
m a n n beschriebene Monomethylester vom Schmp. 1250 gefallt. Dagegen
wurden durch wassrigen Aethylalkohol aus dem Dimethylester stets
tiefer schmelzende Prodncte gebildet, die jedoch nicht Gemische stereo-
isomerer Ester, sondern Gemische von Monomethylester rind Mona-
athylester waren. Es wird namlich durch den Aetbylalkohol aus den
Metbyleatern der Aethylester erzeugt. Dies geht darans hervor, dass
man ans dem Diniethylester wie folgt reinen Monoathylester CO OCa Hs. CN2 HCs HS.COOH erhalt. Zur Losung des Dimethylesters in Aethyl-
dkohol wird 1 Mot. Natron hinzugefagt; die anfangs tiefrothe Liisuug
des Ester-Natriumsalzes wird rasch heller und scheidet nach eiriigeu
Stunden ein gelbliches Salz ab, aus dem nach dem Waschen mit Al-
kohol und hbprrssen durcb Salzsaure der reine Monoathylester rom
Schrnp. 113- 114O erhalten wird. C I I H I V O ~ N . Ber. N 11.9. Gef. N 12.0. Aehnlich wandeln sich bekanntlich nach Pu r d i e rerschiqdene
Methylester Ton fetten Sauren und nach S u d b o r o u g h und Davies')
:ruch solche von aromatischen Sauren durch Aethylalkohol in die
Aethylester um. I) Proc. Chem. Sac. 21, 84. E x p e r i m e n t e lles. Wie das im Wesentlichen gleichartige, schon bekannte Verhalten
der isomeren DBenzolazocyanessigesterc (gegen Alkalien und Acetyl-
chlorid) beide als Hydrazone erkennen lasst, so lasst sich aus den noch
nicht bekannten, bezw. noch nicht geniigend hervorgehobeuen feineren
Unterschieden die Con figuration d e r h eid en i so m e r e n H y d r az o n e
nbleiten. Das a-Derivat ist entschieden saurer; denn es 16st sich in
Soda vie1 rascher uud reichlicher als das $-Derivat. Das a-Derivat
ist damit natiirlich auch die alkalistabile Porm ; das p'-Derivat wandelt
sich daher in Sodaliisung langsam, in NatronlBsuug fast momentan in 2 2 i 5 dns rc-Derivat i i m ; denn nus der Sodalosung des $-Derivats wird durch
etarlie Sauren ein Gemisch ron (c-
und $-Form, nus der Alkalilosung
des jLDerivats die reine cz-Forrn gefallt - wahrend nur durch die
laugsaine Einwirkung der schwachen Kohlensaure aus u- und @-Lo-
>iiog*m das $-Derivat wohl deshalb gefallt wird, weil diese im freien
Zustmde stabilere Form alsdann Zeit findet, sich For dern Uebergang
i i i den festen Zustand zu isornerisiren. Denn diese Verhaltnisse sind
drnen bein, I’henylnitrornethan
gauz Bhnlich: Die Alkaliliisungen des-
Eelhen enthalten die echte Slure, den Aci-Nitrokorper, der auch durch
stnrke Siiureu gefallt wird, - wahrend Kohlensaure auch hier in
Folge der langsameren Fallung den in freiem Zustand stabilen echten
Sitrokorper, die Pseudoeaure, erzeugt. Das stiirker saure u-Hydrazon
scheint auch rascher, ja wahrscheiiilich ausschliesslich direct acetylirt
zu werden, wahrend das $-Derivat wohl nur indirect, d. i. nach Tor-
Ileriger Isomerisation zum cr-Hydrazon angegriffen wird; d e m beide
Hydrazone erzeugen ein und dasselbe, auscheinend der n-Reihe zlige-
Li;rige Acetylderirat, das sich erst beim Uffikrystallisiren in das isornere
$Dei irat unilagert. g
Vergleicht man nun dss Verhalten der beiden isomeren asyrnme-
:rizclien Hydrazone CX.CN:,HAr.COOR gegen Alkalien und gegen
;\cetylchlorid mit deui der beiden nachst verwandten symmetrischen
Hydrazone CX.CK:,HAr.CS und COOR.CN:,HAr.CO@R, so gleicht
clas cf-Derivat den1 IIydrazon des Dinitrils durch seine starke snure
Satur (Alkalistabilitat) und directe Acetylirbarkeit, wahrend das
G-Derivat durch seine griissere Indifferenz dern IJydrazori der Dicar-
hjnsiiureester nilier steht. Dn nun bei der syrnnietrischen Verbindung
dns Cyan stiirker acidificirt als das Carbaethoxyl, so wird das
brwegliche Wasserstoffatom der Gruppe NHAr irn stgrker sauren
irnd :tctiveren tc-Henzolazocyanessigester dern Cyan benachbart sein,
wie v3 irn Dinitril der Fall sein muss; ini indifferenten yJ-Derivat wird
es sich, wie it! ') Diese Berichte 24, 1301 [lSSl]. E x p e r i m e n t e lles. den L)icarbonskureestern, in der Nahe des Carbaethoxyls
befinden - geniiiss den oben nngefiihrten Configurationsforrnelu : CN.C. COOR
6-Derirat =
.. CN. C.COOR
c-Derirnt =
A r S H . ?j
N . N H A r * CN.C. COOR
6-Derirat =
.. CN. C.COOR
c-Derirnt =
A r S H . ?j
N . N H A r * Die Arnrnoiiiakreaciion(( fiihrte bei allen hier besprochenen Hy-
t1r:izorien zu dem Resultnt, dass ausschliesslich die cyanhaltigeri Hj-
drazone, nicht aber die Dic;irbonsaiireester durch trocknes Arnmoiriak
aus indifferenter Idtisung gelitlit werden. Die erhaltenen Ammonsalze
W
~
P
U
jedoch olig uod gabpn ihr Amrnoniak sehr leicht wieder ab. Die u- und $-Form des Benzolazocyanessigesters liesseo sich wegen
ihres leichten wechselseitigen CTebergange3 hierbei nicht deutlich unter-
scheiden, w-ie denn auch das nus ihren oligen Animonsalzen regenerirte 2276 Product ein Gernisch von
(1- und ,3 Form war. - Verschieden sind
jedoch die aus beiden Isomeren hervorgehenden Producte der Verseifuog
in Sodaliirung. Wird das a-Derivat in Sodalosung mehrere Stundpri
auf etwa 600 erwarmt und das noch unveranderte Hydrazon durch
Kohlensaure ausgefallt, so erhalt man im Filtrat durch Salzsaure GI!-
oxylslure-Hydrazon COOH. CHNa H .Ce Hb, gelbe Krystalle 311s Benzol
vom Scbmp. 137O. Das $-Derivat liefert dagegen unter gleichen Be-
dingungen neben unverandertem Ausgangamaterial und Glyoxylsaurr-
Hydrazon auch Mesoxalsaure-Hydrazon COOH.CN2HCs H5. COOH roni
Schmp. 162 -163 '. Somit wird die Haftfestigkeit der Carboxylgruppe
durch die raumliche Nlhe bezw. Ferne der Hpdrazongrnppe ahnlirh
beeinflusst, wie dies bei gewissen stereoisomeren Oximen, 2. B. den
Oximidobernsteinsauren'), schon langst nacbgewiesen wordeu ist. ),
g
g
Scbliesslich rersochten wir, die bei den Hydrazonen CN. CNz I1
A r . COOR auftretende Stereoisomerie auch bei dern zugehiirigen Oxirn
CN.CNOH.COOR nachzuweisen, jedoch ohne Erfolg; es entstand bri
allen Umlagerungsversuchen stets nur die eine, Ilngst bekannte Form. Wahrend also in der Regel stereoisomere Hydrazone trotz der Exir-
tenz stereoisomerer Oxime fehlen, erstere also den selteneren Isomerir-
fall darstellen, ist bier das umgekehrte der Fall: es feblt die Stereo-
iaomerie der Oxirne trotz der Existenz der eiitsprechendeu stereoiso-
mereri Hydrazone. ?) Dicse Berichte 25, 3197 [IS921 iiud Ann. d. Cliem. 2994, 135. 398. A. Lipp und E. Widnmann: Ueber die Einwirkung des
Formaldehyds auf K-Methyl-d*-tetrahydropikolin. 398. A. Lipp und E. Widnmann: Ueber die Einwirkung des
Formaldehyds auf K-Methyl-d*-tetrahydropikolin. [I. Mitt h ei lun g.] (Eingcgangen am 25. Mai 190h.) Vor mehreren .Jahren kam der Einea) von uns zu der Ansicbt,
dnss die Einwirkung von Forrnaldehyd auf N-Methyltetrahydropikolin
in folgender Weise verlaufe: H a C'"-CH
H? C,,
C .CHs
CH:,
CH2
+ CHaO= Ht C,''CH
HzC ...,
C.CH2 CCI2.CJH'
N . CI33
N. CH3 H a C'"-CH
H? C,,
C .CHs
CH:,
CH2
+ CHaO= Ht C,''CH
HzC ...,
C.CH2 CCI2.CJH'
N . CI33
N. CH3 ...,
C.CH
N. CH3 Wegen der Ihnlichen Constitution der genannten Base niit der dr3
Chinii!dins rind Pikolins koiirite mail von rornherein diesen Reactions- ') Diese Berichte 24, 1301 [lSSl]. ?) Dicse Berichte 25, 3197 [IS921 iiud Ann. d. Cliem. 2994, 135.
|
W2913047261.txt
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Limiting the Spectral Diffusion of Nano-Scale Light Emitters using the Purcell effect in a Photonic-Confined Environment
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OPEN
Received: 5 October 2018
Accepted: 10 December 2018
Published: xx xx xxxx
Limiting the Spectral Diffusion of
Nano-Scale Light Emitters using
the Purcell effect in a PhotonicConfined Environment
A. Lyasota, C. Jarlov, A. Rudra, B. Dwir & E. Kapon
Partial suppression of the spectral diffusion of quantum dot (QD) excitons tuned to resonance of a nanophotonic cavity is reported. The suppression is caused by the Purcell enhancement of the QD-exciton
recombination rate, which alters the rate of charging of the solid-state environment by the QD itself.
The effect can be used to spectrally-stabilize solid-state emitters of single photons and other nonclassical states of light.
Electric charging in the environment of nano-scale light emitters can lead to spectral diffusion or intermittency (blinking) of the emission, as observed with nitrogen-vacancy centers in diamond1, molecules2, carbon
nanotubes3, semiconductor nanorods4 and quantum dots5,6 (QDs) embedded in solid-state matrices. Various
aspects7–11 and mechanisms9,12–14 of such spectral dynamics were widely investigated for semiconductor QDs,
partly in an attempt to quench these effects. In particular, engineering of colloidal QD environment15,16 and its
nanostructure17–19 provided stable nano-emitters with suppressed emission intermittency useful for quantum
science and technology applications. While Purcell enhancement of a QD radiation rate through coupling of a
QD transition to a plasmonic or a hybrid mode20–22 strongly quenches non radiative processes including the ones
that lead to emission intermittency, we did not find any report on an effect of modified photonic environment
on the QD spectral diffusion. Here, we report on the suppression of the spectral diffusion of QD-excitons incorporated in photonic cavities. It is shown that, for QD emission tuned to the resonance of the cavity, the Purcell
enhancement of the exciton radiative recombination leads to reduced electrical charging of the QD environment,
which, in turn, reduces the spectral diffusion range. Moreover, the experiments and modeling evidence a QD
environmental self-charging due to carriers trapped in the dot itself, supporting reports on similar QD charging
leading to emission intermittency in open photonic environment23.
Results
Our photonic–confined QD system presented in Fig. 1(a) consists of a single InGaAs/GaAs site-controlled
QD grown in an inverted pyramid, around which a photonic crystal (PhC) membrane cavity was defined (see
Methods). We designed the QD emission wavelength to be close to resonance with the fundamental cavity
mode CM0 and the first excited mode CM1 (Fig. 1(a)). Weak CM-QD coupling was verified by observing the
co-polarization between the QD exciton (X) and the CM line for sufficiently small X-CM detuning24 (Fig. 1(b)).
Such co-polarization is accompanied by reduced X lifetimes, both arising from the Purcell effect25. From the
co-polarization parameters we estimate a Purcell factor of Fp ~ 10 for studied structures (see Methods).
Whereas most structures showed PL spectra stable over acquisition time, several samples exhibited spectral
diffusion of the QD transitions (Fig. 1(c)). We attribute this spectral diffusion mainly to the fabrication-induced
defects around the QDs, which can trap electric charges and induce spectral shifts via the quantum confined Stark
effect and bi-exciton binding energy modifications26. It should be noticed that almost all QDs stopped diffusing
spectrally if exposed to above-barrier excitation during several tens of minutes. We explain this effect by the
quasi-stable states of defects or background impurities that are reached after a long exposure to photoexcitation
and hence high densities of excited electron-hole pairs. For CM-X detuning of at least several meV, as in Fig. 1(c),
we did not observe any effect of the CM on the spectral diffusion.
Laboratory of Physics of Nanostructures, Institute of Physics, Ecole Polytechnique Fédérale de Lausanne (EPFL),
Lausanne, Switzerland. Correspondence and requests for materials should be addressed to A.L. (email: alexey.
lyasota@gmail.com)
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(a)
(c)
Δ
V
Intensity [a.u.]
1
0.5
CM1
0
0
X [µm]
-0.4
1
0.5
0
Δ=0 nm
CM0
1.41
X-
X0
X0
200
150
X-
1.42 1.43 1.44
Energy [eV]
100
0.4
1
H
V
50
0.5
0
1.41
1.416
1.422
Energy [eV]
t=160 s
CM0
X0
Occurrence Intensity
CM0
0
Intensity [a. u.]
(b)
250
|Ex|2+|Ey|2, [a. u.]
Y [µm]
-0.1 0.1 -0.1 0.1
200 nm
Δ=0 nm
H
Time [s]
QD
CM0
1.42
1.43
Energy [eV]
1.44
Figure 1. (a) SEM top view of the fabricated structure and computed near-field intensity patterns of modes
CM0 and CM1. (b) Polarization-resolved photoluminescence (PL) spectra showing exciton-cavity mode
weak coupling (T = 10 K). (c) Temporal-spectral map showing PL spectra acquired sequentially for a structure
exhibiting “free” X spectral diffusion. Excitation power Pexc = 300 µW, T = 10 K. Insets show spectrum acquired
at t = 160 s and occurrence histogram of the neutral excitonic transition X°.
However, for sufficiently small CM-X detuning, we observed spectral diffusion within a restricted energy
range, confined to the vicinity of the CM resonance (Fig. 2(a)). We explain this “spectral trapping” of the otherwise freely diffusing X transition by the combined effects of the enhanced X emission rate γ due to the
CM-induced Purcell enhancement at sufficiently small X-CM detuning δ, and the role of the QD as a primary
source of the charging for the traps in its vicinity (Fig. 2(b,c)). Similar charging mechanism leads to exciton
lifetime-intensity correlations in emission intermittency of colloidal QDs27, recently also reported for InGaAs/
GaAs QDs23. In this scenario, the charging rate of the surrounding traps is proportional to the QD average occu1
pation II with an electron-hole pair. The latter is estimated as Π =
from the steady state solution of the
1 + γ(δ )/P
rate equation dΠ = P(1 − Π) − γ(δ)Π driving the single electron-hole dynamics in the QD at a population rate
dt
P below the X saturation level. The coupling of the X transition with the CM modifies the QD population depending on the X-CM detuning δ = E − ECM, where E and ECM are exciton and CM energies. The Purcell effect on the
X recombination rate reads28–30
(
γ0FP 1 +
γ(δ) =
8
δ2
Δω CM 2
(
QCM
QX
+2 1+
)
QCM
QX
)
2
η2 + γleak
(1)
where γ0 and γleak are the X spontaneous emission rates in the bulk (open photonic environment) and in the PhC
gap, FP is the theoretical Purcell factor, QCM = ω CM and Q X = ω X are the CM and the X quality factors defined
Δω CM
Δω X
through the X and the CM angular frequencies, and η is the effective CM-X spatial overlap25. Thus, the charging
rate of the QD environment and the spectral diffusion of the excitonic transitions are coupled to the X-CM detuning δ = E − ECM (Fig. 2(b,c)). It should be noted that before exciton X0 started spectrally diffusing (Fig. 2(a)), its
emission was almost completely quenched5,6 during the first 36 s due to the high electric field induced by the
environmental charging. This quenching is caused by either QD ionization or by a reduced dipole moment of the
QD transition.
In this picture, the charging level of the surrounding traps is proportional to the QD average occupation with
an electron-hole pair, which depends on the QD pumping rate P and the recombination rate γ of the QD-confined
exciton. The QD recombination rate, in turn, increases with decreasing CM-X energy detuning δ. Thus, the QD
environment is more efficiently charged when the QD optical transition is tuned out of resonance with the CM
(Fig. 2(b)). On the other hand, at resonance the electron-hole recombination rate is enhanced by the cavity
Purcell effect, which reduces the average QD occupation and consequently decreases the charging rate of the
traps (Fig. 2(c)). At large CM-X detuning and high charging levels, the electric field, exerted by the environment,
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Figure 2. (a) Temporal-spectral map for an L3 PhC cavity with Δ = 120 nm showing spectral trapping of the X
by the CM mode (red dashed line). Inset shows a detail of the spectrally diffusing exciton at t = 75 s. Excitation
power Pexc = 100 µW, T = 10 K. (b,c) Schematics of the model showing the QD and charged environment for
high (b) and low (c) charging rates, controlled by the X-CM detuning. Simulated X spectral diffusion: temporal
trace and occurrence histogram without (d) and with (e) the CM. (f) Simulated map of the X occurrence versus
relative CM energy ECM − EX for quantum dot population rate P = 0.25γ0 where EX is unperturbed X energy (see
Methods for details on simulations).
shifts the X-transition provoking spectral diffusion. For small CM-X detuning, the charging level of the environment can be drastically lowered, which reduces the X-level shifts and the consequent spectral diffusion. Here, we
neglected the effect of carrier losses due to traps charging on the average QD occupation. This is because such loss
rate is comparable to that of spectral diffusion, that is, a few Hz, and is at least 7–8 orders of magnitude smaller
than the X recombination rate. The latter varies between 2 GHz to 20 GHz, as can be extracted from the ~2 ns and
~200 ps radiative-limited excitonic lifetime in an open photonic environment and in direct resonance with the
cavity mode (δ = 0), respectively.
Figure 2(d),(e) show the simulated time traces and occurrence histograms of the X-transitions as a function
of the energy shift from the unperturbed (i.e., without energy shift) X energy EX (see Methods for details on the
numerical model). Evidently, the presence of the cavity reduces the range of spectral diffusion of the X-line,
pulling the recombination energy to the vicinity of the CM. At the same time, in the presence of the CM the
occurrence histogram is suppressed at the CM energy, with its peak located at one side of the CM energy. A more
complete description of the occurrence distribution is shown in Fig. 2(f), which displays the simulated spectrum
of the occurrence as a function of the CM energy ECM. The occurrence assumes its maximum at either side of the
CM, a manifestation of the Purcell-enhanced recombination of excitons near the cavity resonance.
Further support to our model is provided by the dependence of the spectral diffusion pattern on the
photo-excitation power Pexc (Fig. 3(a,b)), measured in another device. Increasing the QD pumping rate shifts
the peak occurrence of the X transition to higher energies and produces wider spectral diffusion range, reaching
across the CM resonance. This is brought about by the higher average charging of the environment and resulting
intense electric fields. Remarkably, the measured occurrence of the neutral exciton transition is depleted close
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1
200
0.5
0
(c)
0
CM
Time [s]
P/γ0=0.25
600
400
1
2
3
4
E-EX [meV]
5
CM1
0
0
6
(f)
30 µW
100 µW
8
6 ×10
1
2
3
4
E-EX [meV]
5
6
CM
4
P/γ0=0.09
5
Occurrence
Occurrence
16
1.442 1.443 1.444 1.445
Energy [eV]
200
200
24
200
800
400
(e)
300
100
P/γ0=0.09
600
0
0
400
(d)
CM
CM1
Pexc=100 µW
0
1.442 1.443 1.444 1.445
Energy [eV]
800
600 Δ=180 nm
500
100
Time [s]
(b)
CM1
Intensity [a. u.]
Time [s]
300
Δ=180 nm
Pexc=30 µW
Time [s]
(a)
P/γ0=0.25
4
3
2
1
0
1.442 1.443 1.444 1.445
Energy [eV]
0
0
1
2
3
4
5
E-EX [meV]
6
Figure 3. Measured (a,b) and simulated (c,d) excitonic trajectories for two different photo-excitation powers
and QD pumping rates respectively. Corresponding measured and simulated excitonic occurrences are shown
in (e) and (f). Experimental data in (a) and (b) were acquired at Pexc = 30 µW and Pexc = 100 µW excitation
powers (QD in an L3 PhC cavity with Δ = 180 nm, mode CM1) and temperature T = 10 K. Simulated excitonic
trajectories in (c) and (d) were obtained for quantum dot population rates P = 0.09γ0 and P = 0.25γ0 (See
Methods for details on simulations). Same color scales used in (a) and (b).
to the CM energy, exhibiting a clear dip in the occurrence spectrum for larger excitation powers (Fig. 3(e)).
Our numerical model reproduces this behavior, as reflected in both the simulated spectral diffusion traces
(Fig. 3(c,d)) and spectral occurrence histograms (Fig. 3(f)). In these simulations we used 20 single-electron traps
in the environment, with excitonic shifts uniformly distributed between Emin = −0.06 and Emax = 0.54 meV, and
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corresponding charging and discharging rates lower then fmax = 10 Hz and lmax = 0.1 Hz. It can be seen that the key
parameter controlling the spectral diffusion pattern is the normalized pumping rate P/γ0.
Additional insight into the impact of the photonic environment on the spectral diffusion is obtained by a simple
analytic model for the temporal evolution of the exciton energy E. Assuming that the energy shift E − EX of the excitonic transition is proportional to the total charge accommodated in the QD environment Q (E − EX ~ Q), we can write
dE
Pα
(E max − E ) − β(E − EX )
=
dt
γ (E − ECM ) + P
(2)
where EX and Emax correspond to zero and maximum charge Q, γ(E − ECM) is the exciton emission rate, α and β
are charging and discharging rates (see Methods for details of the analytic model). For simplicity, we ignore the
stochastic nature of the charging process, thus the exciton energy converges to well-defined values at infinite time.
Analysis of the equation permits the determination of “stable” exciton energies, which represent the energies of
highest occurrence during the spectral diffusion process.
Figure 4(a) displays the derivative dE , calculated from (2), for typical parameters of our system (See Methods).
dt
d 2E (E
)
stat
The stationary points Estat satisfying
< 0 are indicated. In an intermediate range of QD population rate P,
dt 2
two points are obtained (Fig. 4(b)), such that the exciton transition most likely occurs above or below the CM
energy. At higher QD population rates, a single stable branch exists, restoring the spectral diffusion to its pattern in
the absence of a CM. As shown in Fig. 4(b), the energy at which spectral diffusion is captured depends on a particular detuning between unperturbed excitonic energy EX and CM energy. We corroborated this result with numerical
simulations as shown in Fig. 4(d,e). Figure 4(d,e) demonstrate that the spectral diffusion ‘capture’ energy, as well as
the range of QD pumping rates at which QD spectral diffusion is limited near ECM, strongly depend on the EX - ECM
detuning. The free spectral diffusion of the QD transition in the absence of a cavity (Fig. 4(c)) persists at higher QD
pumping rates when the CM is tuned closer to the unperturbed excitonic energy EX (Fig. 4(d)), and bunches near
the CM energy for sufficiently low QD population rates (Fig. 4(e)). In all cases, the spectral diffusion in the absence
of CM (Fig. 4(c)) is recovered above a threshold QD population rate, which depends on the ECM - EX value.
Figure 4(f) shows spectral diffusion ranges as a function of Purcell factor Fp and corresponding emission rate
enhancement in resonance γres/γ0 obtained using Eq. (1). Exciton occurrence is limited to the CM side for Fp
above some threshold value Fth
p with a full width at half maximum (FWHM) δE significantly reduced with respect
to the occurrence FWHM δE0 obtained for Fp ≈ 0. Figure 4(g) shows the simulated δE/δE0 for 20 (set 1–2) and 40
(set 3) single-electron charge traps and for various charging and discharging rates: lmax = 0.3 Hz and fmax = 10 Hz
for ‘set 1’, lmax = 0.1 Hz and fmax = 10 Hz for ‘set 2’, and lmax = 0.3 Hz and fmax = 1 Hz for ‘set 3’ (see Methods). δE/δE0
reveals very similar behavior after dropping down for Fp > Fpth (Fig. 4(g)) for all used modeling parameters.
Discussion
We observed the modification of the spectral diffusion of QD excitons tuned to resonance with the modes of a
photonic nano-cavity. The effect is explained by the Purcell enhancement of the QD-exciton lifetime, which, in
turn, reduces the direct charging of the environment by charge released by the QD itself. We note that resonant
excitation of QD excitons may avoid charging of the environment by the photoexcited carriers, but does not
prevent self-charging by charges confined at the QD via the mechanism invoked in our study. Thus, the reported
demonstrations31,32 of emission of indistinguishable photons by selected QD-cavity systems using resonant QD
pumping are not in contradiction with our observations. Similar to the quenching of emission intermittency in
QDs placed in plasmonic nanostructures20,33, the use of confined photonic environments for quenching the spectral diffusion of nano-scale light emitters can be useful for the fabrication of novel photon sources.
Methods
The studied system consists of a single InGaAs/GaAs site-controlled pyramidal QD integrated in an L3 PhC cavity
(Fig. 1(a)) fabricated on a 250 nm thick GaAs suspended membrane. The QD is nominally shifted by Δ = 0 nm,
Δ = 120 nm or Δ = 180 nm from the center of the L3 PhC cavity (placement precision ~20 nm, confirmed by
scanning electron microscopy (SEM)34). We used structures with different quantum dot positions in this study
because of the limited number of samples of the same design. However, we took care to select structures with
both similar QD-CM spectral detuning and similar spectral diffusion behavior. Further details of growth and
fabrication can be found in35–37.
PhC mode modeling. The near field intensity patterns of the CMs shown in Fig. 1 were calculated in the symmetry plane of the PhC slab using a finite-difference method.
Optical measurements. The QDs were optically excited using a continuous wave Ti:sapphire laser emitting at
730 nm wavelength, with the beam focused to a ~1.5 µm wide spot. Photoluminescence (PL) measurements were
carried out with the samples placed in a He-flow optical cryostat using a spectrometer equipped with a charge
coupled device (CCD) detector providing a spectral resolution of 80 µeV (reported excitation powers measured
before the micro-PL setup). Polarization-resolved spectra were obtained using a λ/2 waveplate and a linear polarizer. Time-resolved PL spectra were measured with 1 s dead time between consecutive acquisitions. Importantly,
we avoided any light exposure of the sample from the moment the cooling process started till the PL acquisition.
The reported dynamics of the spectral diffusion were obtained by measuring PL spectra with acquisition time
of ~2 s and 1 s dead time between consecutive acquisitions.
Cooling procedure. We avoided any light exposure of the sample before acquisition of the PL spectra in order
to achieve reproducible charging conditions. Thus, keeping the sample in complete darkness before the exposure
to the laser excitation is crucial for the reported experiments.
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Figure 4. (a) Determination of the stability points in the exciton energy spectrum via the analytic model.
(b) Calculated stability zones in the QD population rate-exciton energy plan. (c–e) Numerically simulated QD
population rate- exciton energy maps of excitonic occurrence without and with CM at different energy. Solid
red lines in (c–e) trace the energies of the occurrence peak without the CM. (f) Purcell factor-energy map of
exciton occurrence and (g) normalized FWHM of exciton occurrence as a function of Purcell factor. Dashed red
lines in (d) and (e) show the transition (normalized) power above which the occurrence peak energy recovers
its values in (c). Dashed lines in (g) highlight Fp ≈ Fpth values at which exciton diffusion is only partially limited
to the CM side.
Charging/recombination numerical model. In the model, N traps, each either unfilled or filled with a single
→
electron or hole charge, form the charged environment of the QD. We choose an occupation vector B = {Bi }N
i=1
th
with Bi = 1 (Bi = 0) if i center is charged (not charged). Trap filling with a charge leads to either positive or nega→
tive spectral shift38–43 of the QD excitonic line (X). Vector S = {ΔEi}N
i = 1 describes spectral shifts provided by each
of the N centers while it is charged. Spectral shifts are generated randomly from the uniformly distributed
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numbers in the range [Emin, Emax]. The total excitonic energy shift from the unperturbed excitonic energy E0X is
→T →
defined as ΔE = B ∗ S .
∂B
The time evolution of trap occupation is driven by the system of differential equations i = fi(1 − Bi ) − l iBi,
∂t
th
i = 1, …, N where fi and li are charging and discharging rates of i trap. In our model, charging rate fi is proporf
=
Π
∗
F
tional to the probability Π of finding an electron-hole pair in the QD, that is, i
i where Fi is the charging
rate when the QD is occupied. The steady state solution of the rate equation dΠ = P(1 − Π) − γΠ provides the
dt
1
QD occupation probability Π = 1 + γ /P where γ and P are X recombination and pumping rates, respectively.
Charging and discharging rates {Fi}iN=1 and {l i}iN=1 are randomly generated from the uniform distribution in the
ranges [0, Fmax] and [0, lmax] tuned for better fitting of the experimental data.
The coupling of the X transition with the CM modifies the QD population depending on the X-CM detuning
δ = E − ECM, where ECM is the CM energy. The Purcell effect on the X recombination rate reads28–30
(
γ0FP 1 +
γ(δ) =
8
δ2
Δω CM 2
(
QCM
QX
+2 1+
)
QCM
QX
)
2
η2 + γleak
where γ0 and γleak are the X spontaneous emission rates in the bulk (open photonic environment) and in the PhC
ω
bandgap, FP is the Purcell factor, QCM = ω CM and Q X = ΔωX are the CM and the X quality factors defined
Δω CM
X
through the X and the CM angular frequencies ωCM and ωX and their FWHM ΔωCM and ΔωX, and η is a CM-X
44
spatial overlap parameter . For our structure, we chose the Purcell factor Fp = 100 and the exciton-CM overlap
η = 0.6 providing an emission rate γ enhancement of ~10 times at X-CM resonance (δ = 0) for our QD-PhC cavity
parameters: γ0 = 0.44 µeV, γleak = 0.22 µeV, ΔωCM = 600 µeV, QX = 7000 and QCM = 3000. Thus, we accounted for
around 90% linear emission polarization of the X transition observed in resonance with the CM for devices with
Δ = 120 nm and Δ = 180 nm.
As initial conditions, we set all traps filled at t = 0, that is, Bi = 1 for i = 1, …, N. At each computation interval
F ⁎ δt
δt = 0.01 s, charge centers lose or absorb single electrons with probabilities ploss
= l i ∗ δt and pabs
= 1 +i γ(δ)/P ,
i
i
shifting the X transition to a new excitonic energy E(t). This time evolution of the X energy is recorded during
t = 104 s providing modeled energy traces of X transition. The occurrence histograms are obtained counting the
generated energy values with 50 µeV bin width. The simulated effects did not depend on the particular set of
charging and discharging rates Fi and li. In the model we introduced both positive and negative shifts by choosing
Emin < 0 and Emax > 0 with in average bigger absolute values for positive shifts, that is, Emax > |Emin| > 0. This is in
accordance with the experimentally observed shifts to higher energies of the spectrally diffusing QD transitions
relative to the emission energy of the non-diffusing QDs.
Analytical model of excitonic occurrence depletion. To study analytically the observed occurrence depletion,
we assumed a linear dependence of the excitonic energy on the total charge Q accommodated in the QD environment: E − EX = const * Q where EX is the excitonic energy at zero charge in QD environment. Assuming continuous charging and discharging of environment with rates α and β, we get the following differential equation
driving the time evolution of the charge Q stored in the QD environment:
dQ
Pα
=
(Qmax − Q) − βQ
dt
γ(δ) + γleak + P
where Qmax is the maximum charge that can be stored. Then, the time evolution of excitonic energy reads
dE
=
dt
Pα(E max − E)
C1
(E − E CM)2 + C2
+ γleak + P
− β(E − E X)
where Emax is the excitonic energy corresponding to the maximum charge accommodated in the environment,
(
Q
)
Δω
2 2
η
(
Q
)
2
Δω
2
ω
CM
CM
and C2 = 1 + CM
where FP is the Purcell factor, QCM = CM and
C1 = γ0FP 1 + QCM
Δω CM
8
4
QX
X
ωX
Q X = Δω are the CM and the X quality factors defined through the X and the CM angular frequencies ωCM and
X
ωX and their FWHM values ΔωCM and ΔωX, and η is the CM-X spatial overlap. The right part of this equation can
be reduced to a single fraction with a numerator being a third degree polynomial of the excitonic energy E providing either one, two or three stationary points Estat of the time-energy curve. Among these points, stable points
d2E
are provided by the condition stab
< 0.
dt 2
The datasets generated and analyzed during the current study are available from the corresponding author on
reasonable request.
References
1. Bradac, C. et al. Observation and control of blinking nitrogen-vacancy centres in discrete nanodiamonds. Nat. Nanotechnol. 5,
345–9 (2010).
2. Ambrose, W. P. & Moerner, W. E. Fluorescence spectroscopy and spectral diffusion of single impurity molecules in a crystal. Nature
349, 225–227 (1991).
3. Htoon, H., O’Connell, M. J., Cox, P. J., Doorn, S. K. & Klimov, V. I. Low temperature emission spectra of individual single-walled
carbon nanotubes: multiplicity of subspecies within single-species nanotube ensembles. Phys. Rev. Lett. 93, 027401 (2004).
Scientific Reports |
(2019) 9:1195 | https://doi.org/10.1038/s41598-018-37677-2
7
www.nature.com/scientificreports/
4. Müller, J. et al. Monitoring surface charge movement in single elongated semiconductor nanocrystals. Phys. Rev. Lett. 93, 167402
(2004).
5. Nirmal, M. et al. Fluorescence intermittency in single cadmium selenide nanocrystals. Nature 383, 802–804 (1996).
6. Robinson, H. D. & Goldberg, B. B. Light-induced spectral diffusion in single self-assembled quantum dots. Phys. Rev. B 61,
R5086–R5089 (2000).
7. Cichos, F., von Borczyskowski, C. & Orrit, M. Power-law intermittency of single emitters. Curr. Opin. Colloid Interface Sci. 12,
272–284 (2007).
8. Pelton, M., Grier, D. G. & Guyot-Sionnest, P. Characterizing quantum-dot blinking using noise power spectra. Appl. Phys. Lett. 85,
819–821 (2004).
9. Shimizu, K. T. et al. Blinking statistics in single semiconductor nanocrystal quantum dots. Phys. Rev. B 63, 205316 (2001).
10. Schlegel, G., Bohnenberger, J., Potapova, I. & Mews, A. Fluorescence decay time of single semiconductor nanocrystals. Phys. Rev.
Lett. 88, 137401 (2002).
11. Tang, J. & Marcus, R. A. Mechanisms of fluorescence blinking in semiconductor nanocrystal quantum dots. J. Chem. Phys. 123,
054704 (2005).
12. Frantsuzov, P. A. & Marcus, R. A. Explanation of quantum dot blinking without the long-lived trap hypothesis. Phys. Rev. B Condens. Matter Mater. Phys. 72 (2005).
13. Kuno, M., Fromm, D. P., Hamann, H. F., Gallagher, A. & Nesbitt, D. J. “On”/“off ” fluorescence intermittency of single semiconductor
quantum dots. J. Chem. Phys. 115, 1028 (2001).
14. Verberk, R., van Oijen, A. M. & Orrit, M. Simple model for the power-law blinking of single semiconductor nanocrystals. Phys. Rev.
B 66, 233202 (2002).
15. Hohng, S. & Ha, T. Near-complete suppression of quantum dot blinking in ambient conditions. J. Am. Chem. Soc. 126, 1324–5
(2004).
16. Fomento, V. & Nesbitt, D. J. Solution control of radiative and nonradiative lifetimes: A novel contribution to quantum dot blinking
suppression. Nano Lett. 8, 287–293 (2008).
17. Chen, Y. et al. ‘Giant’ multishell CdSe nanocrystal quantum dots with suppressed blinking. J. Am. Chem. Soc. 130, 5026–7 (2008).
18. Mahler, B. et al. Towards non-blinking colloidal quantum dots. Nat. Mater. 7, 659–664 (2008).
19. Park, Y. S., Bae, W. K., Padilha, L. A., Pietryga, J. M. & Klimov, V. I. Effect of the core/shell interface on auger recombination
evaluated by single-quantum-dot spectroscopy. Nano Lett. 14, 396–402 (2014).
20. Ji, B. et al. Non-blinking quantum dot with a plasmonic nanoshell resonator. Nat. Nanotechnol. 10, 170–175 (2015).
21. Gurlek, B., Sandoghdar, V. & Martin Cano, D. Manipulation of quenching in nanoantenna-emitter systems enabled by external
detuned cavities: a path to enhance strong-coupling. ACS Photonics acsphotonics.7b00953, https://doi.org/10.1021/
acsphotonics.7b00953 (2017).
22. Peng, P. et al. Enhancing Coherent Light-Matter Interactions through Microcavity-Engineered Plasmonic Resonances. Phys. Rev.
Lett. 119, 233901 (2017).
23. Hu, F., Cao, Z., Zhang, C., Wang, X. & Xiao, M. Defect-induced photoluminescence blinking of single epitaxial InGaAs quantum
dots. Sci. Rep. 5, 8898 (2015).
24. Calic, M. et al. Phonon-mediated coupling of InGaAs/GaAs quantum-dot excitons to photonic crystal cavities. Phys. Rev. Lett. 106,
227402 (2011).
25. Jarlov, C. et al. Exciton dynamics in a site-controlled quantum dot coupled to a photonic crystal cavity. Appl. Phys. Lett. 107, 191101 (2015).
26. Türck, V. et al. Effect of random field fluctuations on excitonic transitions of individual CdSe quantum dots. Phys. Rev. B 61,
9944–9947 (2000).
27. Galland, C. et al. Two types of luminescence blinking revealed by spectroelectrochemistry of single quantum dots. Nature 479,
203–7 (2011).
28. Auffèves, A. et al. Controlling the dynamics of a coupled atom-cavity system by pure dephasing. Phys. Rev. B - Condens. Matter
Mater. Phys. 81, 1–10 (2010).
29. VanExter, M. P., Nienhuis, G. & Woerdman, J. P. Two simple expressions for the spontaneous emission factor beta. Phys. Rev. A 54,
3553–3558 (1996).
30. Xu, Y., Lee, R. K. & Yariv, A. Finite-difference time-domain analysis of spontaneous emission in a microdisk cavity. Phys. Rev. A 61,
33808 (2000).
31. Somaschi, N. et al. Near-optimal single-photon sources in the solid state. Nat. Photonics 10, 340–345 (2016).
32. Ding, X. et al. On-Demand Single Photons with High Extraction Efficiency and Near-Unity Indistinguishability from a Resonantly
Driven Quantum Dot in a Micropillar, https://doi.org/10.1103/PhysRevLett.116.020401.
33. Ma, X., Tan, H., Kipp, T. & Mews, A. Fluorescence enhancement, blinking suppression, and gray states of individual semiconductor
nanocrystals close to gold nanoparticles. Nano Lett. 10, 4166–74 (2010).
34. Lyasota, A. A. Interaction between site-controlled quantum dot systems and photonic cavity structures. PhD thesis, EPFL (2017).
35. Lyasota, A. et al. Integration of multiple site-controlled pyramidal quantum dot systems with photonic-crystal membrane cavities.
J. Cryst. Growth 414, 192–195 (2015).
36. Felici, M. et al. Site-controlled InGaAs quantum dots with tunable emission energy. Small 5, 938–943 (2009).
37. Mohan, A. et al. Record-low inhomogeneous broadening of site-controlled quantum dots for nanophotonics. Small 6, 1268–1272
(2010).
38. Finley, J. J. et al. Quantum-confined Stark shifts of charged exciton complexes in quantum dots. Phys. Rev. B - Condens. Matter Mater.
Phys. 70, 2–5 (2004).
39. Nakaoka, T. et al. Competing influence of an in-plane electric field on the Stark shifts in a semiconductor quantum dot. Appl. Phys.
Lett. 99, 81–84 (2011).
40. Nakaoka, T., Kako, S. & Arakawa, Y. Unconventional quantum-confined Stark effect in a single GaN quantum dot. Phys. Rev. B 73,
121305 (2006).
41. Abbarchi, M. et al. Spectral diffusion and line broadening in single self-assembled GaAsAlGaAs quantum dot photoluminescence.
Appl. Phys. Lett. 93 (2008).
42. Singh, R. & Bester, G. Effects of charged defects on the electronic and optical properties of self-assembled quantum dots. Phys. Rev.
B 85, 205405 (2012).
43. Saito, T., Nakaoka, T. & Arakawa, Y. Effect of lateral electric field on the transition energies of neutral and charged excitons in In 0.5
Ga 0.5 As/GaAs quantum dots. Phys. Rev. B 91, 115306 (2015).
44. Jarlov, C. et al. Effect of Pure Dephasing and Phonon Scattering on the Coupling of Semiconductor Quantum Dots to Optical
Cavities. Phys. Rev. Lett. 117, 076801 (2016).
Acknowledgements
This work was supported by the Swiss National Science Foundation. We thank Dr. Giorgio Biasiol of TASC,
Trieste, Italy, for the growth of the (111)B GaAs/AlGaAs membrane wafers employed. We thank Christophe
Galland for critical reading of the manuscript.
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Author Contributions
B.D. and E.K. designed the samples, A.L., B.D. and A.R. fabricated the samples, C.J. did modeling of PhC
structures and QD optical characterization, A.L. did optical measurements of fabricated devices and developed
the theoretical model, E.K. supervised the work, A.L. and E.K. interpreted the results and wrote the article.
Additional Information
Competing Interests: The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and
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© The Author(s) 2019
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https://openalex.org/W4391190318
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https://ojs.unud.ac.id/index.php/eum/article/download/80037/47163
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Indonesian
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KARAKTERISTIK KLINIKOPATOLOGI PENYAKIT HIRSCHSPRUNG DI RSUP SANGLAH DENPASAR TAHUN 2019-2020
|
E-Jurnal Medika Udayana
| 2,022
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cc-by
| 2,857
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Kata kunci : Penyakit Hirschsprung., megakolon kongenital., aganglionik., karakteristik klinikopatologi unci : Penyakit Hirschsprung., megakolon kongenital., aganglionik., karakteristik klinikopatologi. JMU
Jurnal medika udayana JMU
Jurnal medika udayana JMU
Jurnal medika udayana KARAKTERISTIK KLINIKOPATOLOGI
PENYAKIT HIRSCHSPRUNG DI RSUP SANGLAH DENPASAR TAHUN 2019-2020
Ida Bagus Ardya Kurnia Wilananda 1, I Gusti Ayu Sri Mahendra Dewi 2 ,Ni Wayan Winarti 2, Ni Putu Sriwidyani 2
Program Studi Sarjana Kedokteran dan Profesi Dokter, Fakultas Kedokteran, Universitas Udayana
Departemen Patologi Anatomi Fakultas Kedokteran Universitas Udayana/ RSUP Sanglah Denpasar
e-mail: ardyawilananda@gmail.com ABSTRAK Penyakit Hirschsprung merupakan keadaan abnormal kolon dengan ciri khas submukosus meissneri,
pleksus dan pleksus mienterikus aurbachi tanpa sel ganglion parasimpatis. Manifestasi penyakit hirschsprung
terlihat pada pengeluaran mekonium tidak keluar dalam 24 jam pada. Kemudian adanya tanda-tanda obstruksi
pada saluram gastrointestinal, muntah cairan empedu, konstipasi, dan defekasi tidak teratur. Pada tahun 2017
terdapat 2,5 juta kematian pada anak berumur kurang dari 1 bulan di seluruh dunia. 47% kematian anak di bawah
usia 5 tahun terjadi pada bayi baru lahir, yaitu sekitar 7.000 kematian. Kasus penyakit hirschsprung sendiri terjadi
dalam 1:5000-7000 kelahiran, dengan kasus pada bayi laki-laki 4 kali lebih banyak dari perempuan. Penelitian ini
bertujuan untuk mengetahui karakteristik klinikopatologi penyakit hirschsprung di RSUP Sanglah Denpasar pada
tahun 2019-2020 berdasarkan gejala klinis, usia, jenis kelamin, dan lokasi segmen usus aganglionik. Penelitian ini dilaksanakan pada tahun 2021 di Instalasi Patologi Anatomi (PA) RS Sanglah Denpasar,
dengan menggunakan data tahun 2019-2020. Penelitian dilakukan dengan metode potong lintang, dengan data
sekunder berupa data pasien yang ada di Laboratorium PA RSUP Sanglah Denpasar, dan total sampling. SPSS
versi 26 digunakan untuk menganalisis data. g
g
Hasil dari penelitian ini menunjukan bahwa pasien penyakit hirschsprung di RSUP Sanglah Denpasar
pada tahun 2019-2020 sejumlah 78 orang dan seluruhnya telah memenuhi kriteria inklusi. Kasus penyakit
hirschsprung terbanyak di RSUP Sanglah Denpasar paling banyak mengalami perut kembung yaitu sebanyak 38
kasus dengan persentase sebesar 36,5%. Kasus didominasi oleh pasien dengan rentang usia 0-12 bulan dengan
jumlah 64 orang dengan persentase 82,1%, serta didominasi oleh laki-laki sebanyak 50 orang dengan persentase
sebesar 64,1%,. Lokasi segmen usus aganglionik yang paling sering ditemukan adalah rektum sebanyak 49 orang
dengan persentase sebesar 62,8%. Dari hasil penelitian disimpulkan bahwa pasien penyakit hirschsprung di RSUP Sanglah pada tahun
2019-2020 paling banyak laki-laki, paling banyak pada rentang usia 0-12 bulan, dengan gejala klinis paling
banyak adalah perut kembung, dan lokasi segmen usus aganglionik paling banyak ditemukan pada rektum. Hasil
penelitian ini kemudian dapat dijadikan dasar dan juga bahan studi pada penelitian dan pembelajaran terkait
selanjutnya. 1.
PENDAHULUAN Risiko terjadinya penyakit ini lebih tinggi pada pasien
dengan riwayat keluarga Hirschsprung dan down. Lokasi paling
sering dalam terjadinya aganglionik adalah rektosigmoid yaitu
sekitar 75% kasus, dan 17% kasus pada kolon transversum atau
fleksura lienalis. Risiko terjadinya penyakit hirschsprung juga
meningkat pada anak kembar. Ibu yang aganglionosis lebih
cenderung menurunkan penyakit hirschsprung dibandingkan
ayah. Dibanding perempuan, laki-laki mempunyai kasus penyakit
hirschsprung 4 kali lebih banyak 3. Angka kematian anak di bawah 5 tahun di Indonesia masih
tinggi dan sebagian besar terjadi saat masa neonatus. Kematian
anak-anak 28 hari pertama berkaitan erat dengan kurangnya
penatalaksanaan pada anak tersebut sehingga menimbulkan
kondisi dan penyakit tertentu. Salah satu kondisi tersebut adalah
megacolon congenital atau penyakit hirschsprung. Penyakit Hirschsprung merupakan keadaan tidak adanya sel
ganglion parasimpatis pada kolon, tepatnya pada peksus
mienterikus aurbachi dan submukosus meissneri. Penyakit
ditemukan pada tahun 1886 oleh Herald Hirschsprung dengan
patofisiologinya yang masih belum jelas sampai Robertson dan
Kernohan menemukan pembesaran kolon ini disebabkan oleh
abnormalitas peristaltik usus pada bagian distal akibat tidak
adanya sel ganglion pada tahun 1938. Manifestasi penyakit
hirschsprung pada neonatus paling umum adalah mekonium yang
terlambat keluar. Kemudian adanya tanda-tanda obstruksi dan
gangguan defekasi1. Penyakit hirschsprung menyebabkan adanya obstruksi pada
saluran pencernaan, khususnya pada kolon sehingga pada bayi
masa neonatus, hal ini menyebabkan tidak keluarnya mekonium
atau feses pertama. Hal ini juga menyebabkan rasa tidak nyaman
di lambung, gangguan saluran pencernaan, sehingga dapat terjadi
distensi abdomen, dan bahkan bisa menyebabkan muntah cairan
empedu dan infeksi saluran pencernaan khususnya di kolon. Dan
hal ini bisa menyebabkan kematian, karena meyerang bayi pada
masa neonatusnya, jika tidak segera diberi tindakan medis 2. Hal
inilah yang menyebabkan pentingnya dilakukan penelitian
tentang karakteristik penyakit hirschsprung. Masa Neonatus merupakan masa pada umur bayi masih di
bawah 28 hari. Masa masa neonatus ini merupakan masa yang
paling rentan, di mana 47% kematian anak di bawah usia 5
tahun terjadi pada bayi baru lahir, yaitu sekitar 7.000
kematian, naik dari 40% pada tahun 1990, dan berjumlah hampir
sama jumlahnya dengan kematian bayi dalam masa kandungan
pada tahun 2015. Hal ini dibuktikan bahwa pada tahun 2017
terdapat 2,5 juta anak mengalami kematian pada usia satu
bulan atau kurang di seluruh dunia. Keywords : Hirschsprung's disease., congenital megacolon., aganglionic., clinicopathological characteristics. Keywords : Hirschsprung's disease., congenital megacolon., aganglionic., clinicopathological characteristics. KARAKTERISTIK KLINIKOPATOLOGI PENYAKIT HIRSCHSPRUNG DI RSUP., Ida Bagus Ardya Kurnia Wilananda 1, I
Gusti Ayu Sri Mahendra Dewi 2 ,Ni Wayan Winarti 2, Ni Putu Sriwidyani 2 KARAKTERISTIK KLINIKOPATOLOGI PENYAKIT HIRSCHSPRUNG DI RSUP., Ida Bagus Ardya Kurnia Wilananda 1, I
Gusti Ayu Sri Mahendra Dewi 2 ,Ni Wayan Winarti 2, Ni Putu Sriwidyani 2 KARAKTERISTIK KLINIKOPATOLOGI PENYAKIT HIRSCHSPRUNG DI RSUP., Ida Bagus Ardya Kurnia Wilananda 1, I
Gusti Ayu Sri Mahendra Dewi 2 ,Ni Wayan Winarti 2, Ni Putu Sriwidyani 2 This study was carried out in 2021 at the Sanglah Hospital Denpasar Anatomical Pathology (PA)
installation, using data from the years 2019-2020. It was conducted using a cross-sectional method, with
secondary data in the form of patient data available at the Sanglah Hospital Denpasar PA Laboratory, and total
sampling. SPSS version 26 was used to analyze the data. The results of this study showed that there were 78 patients with Hirschsprung's disease at Sanglah
Hospital Denpasar in 2019-2020 and all of them met the criteria of inclusion. Most cases of Hirschsprung's
disease in Sanglah Hospital Denpasar experienced the most flatulence, namely 38 cases with a percentage of
36.5%. Cases were dominated by patients within age of 0-12 months with total of 64 people with 82.1%
percentage, and dominated by men as many as 50 people with 64.1% percentage. The aganglionic most frequently
found in the rectum as many as 49 people with a percentage of 62.8%. It was concluded from the study result that Hirschsprung's disease patients at Sanglah Hospital in 2019-
2020 were mostly male, mostly in the age range 0-12 months, with the most clinical symptoms being flatulence,
and the most common aganglionic location found in the rectum. This result may then be used as a basis and also
study material for further related research and learning. ABSTRACT Hirschsprung's disease is an usual condition that affects the colon with characteristics of the Meissneri
submucosus, myenteric plexus and Aurbachi plexus without parasympathetic ganglion cells. Manifestations of
Hirschsprung's disease are seen in the discharge of meconium that does not come out within 24 hours. Then there
are signs of obstruction to the gastrointestinal tract, vomiting bile, constipation, and irregular bowel movements. In 2017, 2.5 million death of children under 1 month old. 47% of deaths of children under the age of 5 years occur
in newborns, which is about 7,000 deaths. Cases of Hirschsprung's disease itself occur in 1:5000-7000 births, with
cases in boys 4 times more than girls. This study aims to determine the clinicopathological characteristics of
Hirschsprung's disease at Sanglah Hospital Denpasar in 2019-2020 based on clinical symptoms, age, gender, and
location of the aganglionic bowel segment. 102 http://ojs.unud.ac.id/index.php/eum
doi:10.24843.MU.2022.V11.i11.P18 KARAKTERISTIK KLINIKOPATOLOGI PENYAKIT HIRSCHSPRUNG DI RSUP., Ida Bagus Ardya Kurnia Wilananda 1, I
Gusti Ayu Sri Mahendra Dewi 2 ,Ni Wayan Winarti 2, Ni Putu Sriwidyani 2 Tabel 3. Jenis Kelamin
Frekuensi (n=78) Persentase (%)
Laki-Laki
50
64,1
Perempuan
28
35,9
Total
78
100
Tabel
3
menunjukkan
bahwa
hasil
distribusi
berdasarkan jenis kelamin pasien terdiagnosis penyakit
hirschsprung di RSUP Sanglah paling banyak adalah laki-
laki dengan jumlah 50 kasus dengan persentase sebesar
64,1%, sedangkan pasien perempuan berjumlah sebanyak
28 orang dengan persentase sebesar 35,9%. Selain itu
peneliti juga mencari data berdasarkan lokasi terjadinya
aganglionik yang tercantum pada tabel 4. Jenis Kelamin
Frekuensi (n=78) Persentase (%) Jenis Kelamin
Frekuensi (n=78) Persentase (%)
Laki-Laki
50
64,1
Perempuan
28
35,9
Total
78
100
Tabel
3
menunjukkan
bahwa
hasil
dis 3.
HASIL Lokasi
Aganglionik
Frekuensi (n=78) Persentase (%)
Ileum
2
2,6
Kolon
Transversum
3
3,8
Kolon
Descenden
1
1,3
Kolon Sigmoid
6
7,7
Rektosigmoid
4
5,1
Rektum
49
62,8
Kolon lainnya
(tidak spesifik)
9
11,5
Anus
4
5,1
Total
78
100
Tabel
4
menjelaskan
bahwa
hasil
distribusi
karakteristik pasien penyakit hirschsprung di RSUP Sanglah
berdasarkan lokasi segmen usus terjadinya aganglionik
paling banyak pada rektum sebanyak 49 orang dengan
persentase sebesar 62,8% dan paling sedikit terjadi pada
colon descenden sebanyak 1 orang atau sebesar 1,3%. Tabel 1 menunjukkan bahwa terdapat 6 gejala klinis
yang ditunjukan pasien terdiagnosis penyakit hirschsprung
di RSUP Sanglah, yaitu tidak bisa BAB, sulit BAB, perut
kembung, mekonium keluar > 24 jam, muntah, dan BAB
dibantu dengan rectal wash. Dari keenam gejala ini, gejala
yang paling banyak ditemukan adalah perut kembung
dengan 38 kasus dan persentase sebesar 36,5%, dan gejala
klinis yang paling sedikit dialami adalah keterlambatan
meconium (lebih dari 24 jam), yaitu sebanyak 3 kasus
dengan persentase 2,9%. Selanjutnya penulis juga mencari
pasien berdasarkan usia yang dapat dilihat pada tabel 2. Tabel 2. Distribusi
Pasien
Penyakit
Hirschprung
Berdasarkan Usia di RSUP Sanglah Denpasar
Tahun 2019-2020 Distribusi
Pasien
Penyakit
Hirschprung
Berdasarkan Usia di RSUP Sanglah Denpasar
Tahun 2019-2020 Usia (Bulan)
Frekuensi (n=78) Persentase (%)
0-12
64
82,1
13-60
7
8,9
>60
7
8,9
Total
78
100 3.
HASIL Jumlah pasien yang terdiagnosa sebagai penyakit
hirschsprung sebanyak 78 orang. Data pasien terdiagnosa
penyakit hirschsprung dengan kriteria inklusi terpenuhi sebanyak
78 orang. Dalam penelitian ini data merupakan gejala klinis, usia,
jenis kelamin, dan lokasi terjadinya aganglionik yang terdapat
pada lembar pemeriksaan pasien terdiagnosis penyakit
hirschsprung di Laboratorium PA RSUP Sanglah Denpasar. Jumlah pasien yang terdiagnosa sebagai penyakit
hirschsprung sebanyak 78 orang. Data pasien terdiagnosa
penyakit hirschsprung dengan kriteria inklusi terpenuhi sebanyak Tabel
3
menunjukkan
bahwa
hasil
distribusi
berdasarkan jenis kelamin pasien terdiagnosis penyakit
hirschsprung di RSUP Sanglah paling banyak adalah laki-
laki dengan jumlah 50 kasus dengan persentase sebesar
64,1%, sedangkan pasien perempuan berjumlah sebanyak
28 orang dengan persentase sebesar 35,9%. Selain itu
peneliti juga mencari data berdasarkan lokasi terjadinya
aganglionik yang tercantum pada tabel 4. 78 orang. Dalam penelitian ini data merupakan gejala klinis, usia,
jenis kelamin, dan lokasi terjadinya aganglionik yang terdapat
pada lembar pemeriksaan pasien terdiagnosis penyakit
hirschsprung di Laboratorium PA RSUP Sanglah Denpasar. g
g
Frekuensi
Gejala
Klinis
Pasien
Penyakit
Hirschsprung di RSUP Sanglah Denpasar Tahun
2019-2020 Distribusi
Pasien
Penyakit
Hirschsprung
Berdasarkan Jenis Kelamin di RSUP Sanglah
Denpasar Tahun 2019-2020 Distribusi
Pasien
Penyakit
Hirschsprung
Berdasarkan Jenis Kelamin di RSUP Sanglah
Denpasar Tahun 2019-2020 Gejala Klinis
Frekuensi (n=78) Persentase (%)
Tidak BAB
20
19,2
Sulit BAB
32
30,8
Perut kembung
38
36,5
Mekonium > 24
jam
3
2,9
Muntah
4
3,8
BAB dengan
rectal wash
7
6,7
Tabel 1 menunjukkan bahwa terdapat 6 gejala k Gejala Klinis
Frekuensi (n=78) Persentase (%) Lokasi
Aganglionik
Frekuensi (n=78) Persentase (%)
Ileum
2
2,6
Kolon
Transversum
3
3,8
Kolon
Descenden
1
1,3
Kolon Sigmoid
6
7,7
Rektosigmoid
4
5,1
Rektum
49
62,8
Kolon lainnya
(tidak spesifik)
9
11,5
Anus
4
5,1
Total
78
100
Tabel
4
menjelaskan
bahwa
hasil
distribusi
karakteristik pasien penyakit hirschsprung di RSUP Sanglah
berdasarkan lokasi segmen usus terjadinya aganglionik
paling banyak pada rektum sebanyak 49 orang dengan
persentase sebesar 62,8% dan paling sedikit terjadi pada
colon descenden sebanyak 1 orang atau sebesar 1,3%. KARAKTERISTIK KLINIKOPATOLOGI PENYAKIT
HIRSCHSPRUNG DI RSUP., KARAKTERISTIK KLINIKOPATOLOGI PENYAKIT
HIRSCHSPRUNG DI RSUP., Tabel 3. Distribusi
Pasien
Penyakit
Hirschsprung
Berdasarkan Jenis Kelamin di RSUP Sanglah
Denpasar Tahun 2019-2020 diolah menggunakan program komputer Statistical Package
for the Social Science (SPSS), Microsoft Excel, dan
Microsoft Word. 2.
BAHAN DAN METODE Penelitian ini dilakukandi Laboratorium PA RSUP Sanglah
Denpasar dengan menggunakan data pada rentang tahun 2019-
2020. Penelitian ini telah mendapatkan persetujuan dari Komite
Etik Fakultas Kedokteran Universitas Udayana dengan nomor
referensi 87/UN14.2.2. VII.14/LT/2021. Penelitian ini merupakan
penelitian deskriptif observasional potong lintang dengan
menggunakan data sekunder dengan cara retrospektif yaitu
dengan menggunakan data rekam medis pasien di Laboratorium
Patologi Anatomi RSUP Sanglah Denpasar. Populasi target pada
penelitian ini yaitu pasien yang didiagnosis secara histopatologi
sebagai penyakit Hirschsprung di RSUP Sanglah Denpasar dari
tanggal 1 Januari 2019 – 31 Desember 2020. Sistem saraf enterik (ENS) adalah bagian dari sistem saraf
otonom yang secara langsung mengontrol saluran pencernaan
yang berasal dari neural crest, sistem saraf enterik lengkap
diperlukan untuk fungsi usus yang tepat. Gangguan yang timbul
sebagai akibat dari perkembangan sel neural crest yang rusak
disebut neurokristopati. Dan salah satu dari neurokristopati ini
adalah penyakit hirschsprung. Mutasi genetik dan kombinasi
mutasi genetik dan pengubah yang mungkin berkontribusi pada
etiologi dan patogenesis penyakit Hirschsprung 2. Data diambil dari pasien yang didiagnosis penyakit
hirschsprung di Laboratorium Patologi Anatomi RSUP
Sanglah Denpasar. Data yang terkumpul kemudian akan 103 http://ojs.unud.ac.id/index.php/eum
doi:10.24843.MU.2022.V11.i11.P18 http://ojs.unud.ac.id/index.php/eum
doi:10.24843.MU.2022.V11.i11.P18 http://ojs.unud.ac.id/index.php/eum
doi:10.24843.MU.2022.V11.i11.P18 5.
SIMPULAN Selanjutnya hasil pada tabel 2 mengindikasikan berdasarkan
kelompok usia, penyakit hirschsprung paling banyak terjadi kasus
pada pasien usia 0-12 bulan dengan 64 kasus (82,1%), dilanjutkan
dengan kasus 13-60 bulan (1-5 tahun) dengan 7 kasus (8,9%), dan
di atas 60 bulan (di atas 5 tahun) dengan 7 kasus (8,9%). Bukti ini
searah dengan penelitian yang dilakukan di RSUP H. Adam
Malik pada tahun 2016 dengan kasus 0-2 tahun sebanyak 30
kasus (63,8%) 6. Penytakit hirschsprung di RSUP Sanglah Denpasar tahun
2019-2020 ditemukan paling banyak mengalami gejala klinis
perut kembung yaitu 38 kasus dengan persentase 36,5% serta
paling banyak ditemukan pada rentang usia 0-12 bulan (64 orang)
selanjutnya didominasi oleh pasien dengan jenis kelamin laki-laki
(64,1%) dan jika dilihat berdasarkan lokasi aganglioniknya paling
banyak pada rektum yaitu 49 kasus dengan persentase 62,8%. Lalu pada tabel 3 menunjukkan bahwa karakteristik
penyakit hirschsprung berdasarkan jenis kelamin paling
banyak ditemukan pada laki-laki dengan 50 kasus (64,1%)
sementara pada perempuan ditemukan 28 kasus (35,9%). Penelitian ini sejalan dengan penelitian yang sebelumnya
dilakukan di RSUP Haji Adam Malik Medan oleh Nehemia
pada tahun 2016 dan Devi pada tahun 2017, dan penelitian
di RS Prof. Dr. Margono Soekarjo di Purwokerto oleh
Artathi. Dalam
ketiga
penelitian
tersebut,
didapati
karakteristik pasien penyakit hirschsprung berdasarkan
jenis kelamin, dengan kasus pasien laki-laki lebih dominan
daripada kasus pasien perempuan 4. 4.
PEMBAHASAN Hasil data tabel 1 menunjukkan bahwa karakterirtik pasien
penyakit hirschsprung di RSUP Sanglah Denpasar berdasarkan
gejala klinis paling banyak ditemukan pada gejala perut kembung
yaitu sebanyak 38 kasus dengan persentase 36,5% dan kasus
paling sedikit ditemukan pada gejala meconium yang keluar
setelah 24 jam sebanyak 3 orang sebesar 2,9%. Hal ini sejalan
dengan hasil penelitian pada 2017 di RSUP Haji Adam Malik
Medan dengan kasus gejala klinis terbanyak yaitu perut kembung,
tetapi berbeda dari segi kasus terendah, karena pada penelitian di
RSUP Haji Adam Malik Medan pada tahun 2017, kasus
terendahnya merupakan gejala klinis muntah kehijauan, dengan
gejala meconium keluar di atas 24 jam menjadi kasus terendah
ke-2 4. Hasil penelitian ini juga searah dengan penelitian pada
2020 di RSUD Al-Ihsan Bandung, yang menunjukkan bahwa
gejala perut kembung menempati kasus tertinggi dengan total 32
kasus dan persentase 61,5% 5. Pada tabel 2 ditunjukkan bahwa distribusi usia pasien
penyakit hirschsprung di RSUP Sanglah Denpasar sebagian
besar berusia 0-12 bulan dengan jumlah 64 orang dengan
persentase 82,1%, dan kasus paling sedikit terjadi pada
pasien dengan usia 13-60 bulan (1-5 tahun), dan pada pasien
berumur di atas 60 bulan (di atas 5 tahun) dengan jumlah
kasus masing-masing 7 kasus dengan persentase 8,9%. Selanjutnya peneloti juga mencari data distribusi pada
pasien berdasarkan jenis kelamin yang dapat dilihat pada
tabel 3. http://ojs.unud.ac.id/index.php/eum
doi:10.24843.MU.2022.V11.i11.P18 104 Ida Bagus Ardya Kurnia Wilananda 1, I Gusti Ayu Sri Mahendra
Dewi 2 ,Ni Wayan Winarti 2, Ni Putu Sriwidyani 2 DAFTAR PUSTAKA 1. Sergi C. Hirschsprung’s disease: Historical notes and
pathological diagnosis on the occasion of the 100th
anniversary of Dr. Harald Hirschsprung’s death. World
journal of clinical pediatrics. 2015 Nov 8;4(4):120. 2. Tjaden NE, Trainor PA. The developmental etiology
and pathogenesis of Hirschsprung disease. Translational
research. 2013 Jul 1;162(1):1-5. 3. Kartono, D., 2010. Penyakit Hirschsprung. Cetakan
Kedua. Sagung Seto. Jakarta p
p
p
p
Selanjutnya
pada
tabel
4
menunjukkan
bahwa
karakteristik penyakit hirschsprung berdasarkan lokasi
segmen usus aganglionik paling banyak ditemukan pada
rektum yaitu 49 kasus dengan persentase sebesar 62,8% dan
paling sedikit pada kolon descenden dengan jumlah 1 kasus
(1,3%). Penelitian ini sejalan dengan penelitian yang
sebelumnya telah dilakukan di RSUD Al-Ihsan Bandung
pada tahun 2020. Pada penelitian tersebut ditemukan kasus
terbanyak terjadi pada rektum dengan 53 kasus dengan
persentase 100%, dan dilanjutkan dengan kasus aganglionik
pada segmen rektosigmoid dan descenden dengan 0 kasus
dan persentase 0% 5. 4. Mangunsong
DS. Gambaran
Pasien
Penyakit
Hirschsprung pada Bayi Usia 0-12 Bulan di RSUP Haji
Adam Malik Medan Tahun 2012-2016. 5. Maidah
SA,
Nur
IM,
Santosa
D. Gambaran
Karakteristik Penyakit Hirschsprung di RSUD Al-Ihsan
Bandung Periode 1 Januari 2016-30 September 2019. 5. Maidah
SA,
Nur
IM,
Santosa
D. Gambaran
Karakteristik Penyakit Hirschsprung di RSUD Al-Ihsan
Bandung Periode 1 Januari 2016-30 September 2019. 6. Meliala NH. Gambaran Penderita Hirschsprung pada
Anak Usia 0-14 Tahun di RSUP H. Adam Malik Medan
Tahun 2013-2015. 6. Meliala NH. Gambaran Penderita Hirschsprung pada
Anak Usia 0-14 Tahun di RSUP H. Adam Malik Medan
Tahun 2013-2015. 105 http://ojs.unud.ac.id/index.php/eum
doi:10.24843.MU.2022.V11.i11.P18 http://ojs.unud.ac.id/index.php/eum
doi:10.24843.MU.2022.V11.i11.P18
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Impact of outdoor residual spraying on the biting rate of malaria vectors: A pilot study in four villages in Kayin state, Myanmar
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PloS one
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PLOS ONE
RESEARCH ARTICLE
Impact of outdoor residual spraying on the
biting rate of malaria vectors: A pilot study in
four villages in Kayin state, Myanmar
Victor Chaumeau ID1,2*, Ladda Kajeechiwa1, Thithiworada Kulabkeeree1, Ramesh
Kumar Vishwakarma2,3, Praphan Wasisakun1, Saw Nay Hsel1, Kyi Oo1, Tee Dah1,
Sunisa Sawasdichai1, Muesuwa Trakoolchengkaew1, Monthicha Phanaphadungtham1,
Aritsara Inta1, Yanada Akararungrot1, Naw Yu Lee1, Prasan Kankew1,
Jacher Wiladphaingern1, Mavuto Mukaka2,3, Gilles Delmas1,2, François Nosten ID1,2
a1111111111
a1111111111
a1111111111
a1111111111
a1111111111
1 Shoklo Malaria Research Unit, Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical
Medicine, Mahidol University, Mae Sot, Thailand, 2 Nuffield Department of Medicine, Centre for Tropical
Medicine and Global Health, University of Oxford, Oxford, United Kingdom, 3 Mahidol-Oxford Tropical
Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand
* victor@shoklo-unit.com
Abstract
OPEN ACCESS
Citation: Chaumeau V, Kajeechiwa L, Kulabkeeree
T, Vishwakarma RK, Wasisakun P, Hsel SN, et al.
(2020) Impact of outdoor residual spraying on the
biting rate of malaria vectors: A pilot study in four
villages in Kayin state, Myanmar. PLoS ONE
15(10): e0240598. https://doi.org/10.1371/journal.
pone.0240598
Editor: Clive Shiff, Johns Hopkins University,
UNITED STATES
Received: May 4, 2020
Accepted: September 29, 2020
Published: October 29, 2020
Copyright: © 2020 Chaumeau et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files.
Outdoor and early mosquito biters challenge the efficacy of bed-nets and indoor residual
spraying on the Thailand-Myanmar border. Outdoor residual spraying is proposed for the
control of exophilic mosquito species. The objective of this study was to assess the impact
of outdoor residual spraying on the biting rate of malaria vectors in Kayin state, Myanmar.
Outdoor residual spraying using lambda-cyhalothrin was carried out in two villages in
December 2016 (beginning of the dry season) and two villages were used as a control.
Malaria mosquitoes were captured at baseline and monthly for four months after the intervention using human-landing catch and cow-baited trap collection methods. The impact of
outdoor residual spraying on human-biting rate was estimated with propensity score
adjusted generalized linear mixed-effect regressions. At baseline, mean indoor and outdoor
human-biting rate estimates ranged between 2.12 and 29.16 bites /person /night, and
between 0.20 and 1.72 bites /person /night in the intervention and control villages respectively. Using model output, we estimated that human-biting rate was reduced by 91% (95%
CI = 88–96, P <0.0001) immediately after outdoor residual spraying. Human-biting rate
remained low in all sprayed villages for 3 months after the intervention. Malaria vector populations rose at month 4 in the intervention villages but not in the controls. This coincided with
the expected end of insecticide mist residual effects, thereby suggesting that residual effects
are important determinants of intervention outcome. We conclude that outdoor residual
spraying with a capsule suspension of lambda-cyhalothrin rapidly reduced the biting rate
malaria vectors in this area where pyrethroid resistance has been documented.
Funding: This work was supported by the
Wellcome Trust [101148]; and the Bill and Melinda
Gates Foundation [GH OPP 1177406]. The funders
had no role in study design, data collection and
analysis, decision to publish, or preparation of the
manuscript.
PLOS ONE | https://doi.org/10.1371/journal.pone.0240598 October 29, 2020
1 / 18
PLOS ONE
Competing interests: The authors have declared
that no competing interests exist.
Outdoor residual spraying for malaria vector-control
Introduction
The Thailand-Myanmar border is an area where malaria transmission is low, seasonal and
unstable [1]. Plasmodium falciparum was eliminated from most endemic villages with widespread deployment of community-wide access to early diagnostic and treatment with artemisinin-based combination therapies, and mass-drug administration campaigns in places where
submicroscopic malaria prevalence was high [2]. Although the endemicity of vivax malaria has
also declined in recent years [3], it is more difficult to tackle than falciparum malaria because
of some features in the biology of P. vivax [4–6].
In this area, the primary mosquito vectors are Anopheles minimus (s.s.) (Minimus Complex,
Funestus Group), An. maculatus (s.s.), An. sawadwongporni (Maculatus Group), An. dirus (s.
s.) and An. baimaii (Dirus Complex, Leucosphyrus Group). Anopheles pseudowillmori (Maculatus Group), An. aconitus (s.s.) (Aconitus Subgroup, Funestus Group) and some members in
the Annularis and Barbirostris Groups are secondary vectors [7–9]. Biting rate can be very
high, thereby playing a disproportionate role in driving transmission intensity in this setting
where Plasmodium-infection rates in mosquito populations are low [7, 10]. Bed-nets and
indoor residual spraying fail to prevent most of malaria infections [11–13] because of the ecology and biology of relevant Anopheles species, including exophily and exophagy, zoophagy
and opportunistic blood type selection, and activity peaks at dusk and dawn [7, 14, 15]. Larval
source management is difficult to implement because of the diverse and fragmented nature of
larval habitats [16], and because incredibly high densities of vector larvae can be found over
large areas covered with paddy fields [17]. Several vector species multiply in a variety of biotopes and at different times of the year, adding another layer of complexity to the dynamics of
entomological indices.
In order to avoid severe desiccation and heat stress during daytime, mosquitoes seek for
resting habitats that provide a fresh and humid microclimate [18]. Daytime resting habitats
have been identified both indoors (e.g. roof, wall, ceilings of houses and animal barns) and outdoors (e.g. tree holes, rodent holes, dense bushes, wells) [19]. We hypothesized that peridomestic dense bushes in and around the village are the main daytime resting habitat of Anopheles
mosquitoes in Kayin state, and therefore proposed outdoor residual spraying (ORS) for
malaria vector-control in this region. Several published studies have assessed the duration and
magnitude of the residual insecticidal effects of insecticide mists applied to outdoor vegetation,
and the subsequent impact on wild mosquito populations. Outcome measures included mosquito biting rate or abundance (assessed with human-landing catch and light traps respectively), oviposition [20, 21], recapture rate in mark-released experiments [22] and mortality of
laboratory-adapted or wild female imagoes exposed to extemporaneously collected insecticide-treated plant material [21, 23, 24]. Both failure and success were reported with effects lasting from days to months. Most studies were conducted in North America and only three were
for malaria vector-control in endemic areas [22, 25, 26]. After the studies on DDT in the 1940s
[22, 25, 27], synthetic pyrethroids such as permethrin, deltamethrin, lambda-cyhalothrin and
bifenthrin were preferred to other insecticide classes for their rapid knockdown effect and better toxicity profile [21, 23, 24, 28, 29]. ORS was carried out in a variety of settings ranging from
urban backyards to hectares of jungle bush using backpack mist blowers, truck-mounted
sprayers or aircrafts. Handling concentration and target dose of the active ingredient were usually that recommended on the product label. Gürtler et al. applied higher doses on peridomestic vegetation for the control of triatomine bugs that transmit Chagas disease in Argentina and
did not report any adverse event to the spraying operators, by-standers or livestock [30, 31].
Recently, we determined the longevity of the insecticidal effect of pyrethroid mists applied
to outdoor vegetation using a standard forced-contact assay and a pyrethroid-susceptible
PLOS ONE | https://doi.org/10.1371/journal.pone.0240598 October 29, 2020
2 / 18
PLOS ONE
Outdoor residual spraying for malaria vector-control
laboratory-adapted colony of Anopheles dirus [32]. Residual effects of a capsule suspension of
lambda-cyhalothrin sprayed at a target concentration of 500 g a.i. /ha lasted for 42 and 98 days
during the rainy and dry seasons respectively. The objective of the herein study was to assess
ORS impact on the biting rate of malaria vectors in Kayin state, Myanmar.
Methods
Risk assessment
A detailed risk assessment was performed before the study in order to evaluate the risk of causing toxicity to human health, disrupting the ecosystem and selecting insecticide resistances in
the mosquito vector populations, and to mitigate the negative effects of the spraying.
Use, toxicity and environmental fate of lambda-cyhalothrin. The insecticide chosen for
the intervention was the 2.5% capsule suspension of lambda-cyhalothrin Karate Zeon1 2.5 CS
(Syngenta, Basel, Switzerland). Lambda-cyhalothrin is a type II pyrethroid used for pest control in agriculture, forestry and public health. This insecticide was chosen because of the better
toxicity profile of pyrethroids when compared to other insecticide classes, it is registered for
outdoor use and it gives long-lasting insecticidal effects when applied to outdoor vegetation
[32]. Outdoor application rate ranges between 7 and 540 g a.i. /ha and crop pre-harvest interval ranges between 1 and 90 days [33, 34]. Lambda-cyhalothrin is acutely toxic to mammals
after ingestion, highly toxic after inhalation and it is of moderate toxicity upon contact with
the skin. It is classified as unlikely to be genotoxic, carcinogenic or to cause reproductive or
developmental effects. Lambda-cyhalothrin is highly toxic to aquatic organisms, terrestrial
arthropods (including honeybees) but slightly to non-toxic to birds [33, 35]. Toxicity to reptiles and amphibians is likely high and overlooked [36, 37]. Lambda-cyhalothrin has a low
vapour pressure, low solubility in water and high soil adsorption coefficient [35]. It is relatively
photostable under natural irradiation. In the environment, lambda-cyhalothrin is degraded
into inactive and non-toxic metabolites by photolysis, hydrolysis, microorganisms and plants
[35]. Its fate in an ecosystem therefore depends on the nature of system components including
soil, microbial community and plants, and of the climate. In surface water, the concentration
of lambda-cyhalothrin decreases rapidly if suspended solids or aquatic organisms are present
because lambda-cyhalothrin molecules are strongly adsorbed by particulates and plants. Some
aquatic plants were also shown to rapidly assimilate and metabolise lambda-cyhalothrin dissolved in surface water [35]. Lambda-cyhalothrin is immobile in soils and the risk of leaching
into streams and ground water is low [35]. A representative half-life in North American and
European soils is 30 days with values ranging between 9 and 163 days [38, 39]. Temperature,
humidity, aerobic microorganisms and plants are associated with shorter degradation time
and probably contribute to the shorter half-lives reported in some tropical settings [35, 40, 41].
Lambda-cyhalothrin is not considered to be absorbed by the roots of terrestrial plants because
it strongly binds to soil. When applied to vegetation, lambda-cyhalothrin can persist on foliage
with half-life estimates ranging between 1.6 and 9.1 days [42], but values up to 40 days were
reported [43]. The mechanisms of lambda-cyhalothrin clearance from plant surface are not
known precisely. It certainly involves photolysis, hydrolysis and microorganisms on the plant
surface, but uptake by leaves and plant metabolism may contribute.
Mitigation measures. In order to mitigate human health hazard and toxicity, the insecticide mist was not applied to human shelters and to plants used by local villagers for eating,
drinking and preparing medicines or cosmetics. Moreover, the community engagement team
of the Shoklo Malaria Research Unit did intensive preparatory work. The villagers and their
headman were invited to participate in individual interviews, group discussion and workshops
detailing no less than the exact nature of the study, and the implications, constraints, risks and
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benefits of the intervention. By-standers were asked to stay 100 meters or more from the operation and to wait 4 hours before entering into the treated area.
In order to mitigate toxicity to domestic animals and livestock, villagers shut it away under
their house or outside of the village for 15 days after the intervention, and insecticide was not
sprayed on plants used to feed livestock and on animal barns. In addition to domestic animals,
we identified that aquatic organisms, wild mammals, amphibians, reptiles and terrestrial
arthropods were the groups of non-target organisms most likely to be affected be the intervention because they are present in the sprayed area and lambda-cyhalothrin is toxic to those taxa.
We were less concerned by the toxicity to birds because lambda-cyhalothrin is classified as
slightly to non-toxic to birds, and most wild bird species nest in undisturbed habitats located
at distance of the villages [44]. In order to mitigate toxicity to non-target organism, insecticide
mist was not applied to flowering plants, beehives, and near streams. It is noteworthy that
lambda-cyhalothrin is immobile in in soils and unlikely to leach into ground water. Given the
timing of the intervention (beginning of the dry season) and the half-life of lambda-cyhalothrin in soils and foliage, most of the lambda-cyhalothrin was likely degraded before the next
rains. Moreover, the insecticide mist was applied only once in two villages. Application of the
insecticide mist was limited to some peridomestic vegetation in and around the village, it was
not sprayed to large areas of wild nature. Hence, the spayed area was small in comparison to
the size of the ecosystem.
Pyrethroid resistance was previously reported in malaria mosquito population collected in
Kayin state [45]. In order to mitigate insecticide resistance in mosquitoes, insecticide mist was
not applied around potential breeding sites thereby preventing insecticide leaching in water
bodies where mosquito multiplies and subsequent exposure of mosquito larvae to sub-lethal
concentration of lambda-cyhalothrin.
Study sites
Four malaria-endemic villages in Kayin state were involved in the study based on their accessibility and willingness to participate in the study. The study villages, namely Klay Mo Khee
(KM-500), Klay Poe Klo (KP-506), Mae Khel (MK-509) and Si Poe Khee (SP-513), were traditional Karen villages located on the foothills of the Dawna Range. The distance between the villages was 2.5 to 5 km. The land accessed is protected by the local Karen authorities. The
villagers grow vegetable, beans, yam, fruit trees and sometime tobacco in the vicinity of their
house whereas paddy fields cover large areas around the village. Villagers commonly own
chickens, pigs and dogs that are kept below or near the house. Some families also own small
goat flocks and rarely cows or buffaloes. The houses, built on silts, are made of wooden materials and roofed with leaves of antimony tree (Dipterocarpus tuberculatus Roxb), sometimes
with sheet metal. The number of households was 62 in KM-500, 40 in KP-506, 27 in MK-509
and 38 in SP-613 (approximate population size between 100 and 300 persons /village).
Intervention
ORS was carried out in two villages (MK-509 and SP-513) in December 2016 while the other
two villages (KM-500 and KP-506) were used as a control. Outdoor residual spraying was carried out on 3rd and 4th December 2016 in MK-509, and on 5th and 6th December 2016 in SP513. A capsule suspension of lambda-cyhalothrin (Karate zeon1 2.5% CS, Syngenta, Switzerland) was diluted at a concentration of 2 g a.i. /L with water, and sprayed on peridomestic
dense bushes with a mist blower model PM7650H1 (Makita, Anjo, Japan) at a target concentration of 500 g of a.i. /ha. This product was selected because it is widely used in agriculture
and its formulation is optimized for lasting long on outdoor vegetation. The dose was set-up
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based on a literature review and risk assessment. The treated area covered a 20-meter wide
band of dense bushes around the village and the peridomestic vegetation in between human
shelters. No sensitive animals or plants were sampled.
Entomological surveys
Malaria vectors were captured in all villages before and at one-month intervals for four months
after the intervention (cool months of the dry season). Baseline surveys were conducted at the
end of November (just before the intervention). Follow-up surveys started immediately after
the intervention (beginning of December, month 0) and were carried out at one-month intervals until April (month 4). The survey schedule is presented in the S1 Appendix. All surveys
were included in the analysis. Entomological surveys consisted of five consecutive nights of
collection from 06:00 pm to 06:00 am in five houses per village and on one cow, as described
previously [7]. In each village, five houses were randomly selected for mosquito sampling
using the human-landing catch collection method. In each house, one mosquito collector sat
indoors and one mosquito collector sat outdoors, yielding a total of 50 person-nights of collection per survey (25 person-nights indoors and 25 person-nights outdoors). Collectors were
asked to catch every mosquito landing on their uncovered legs for 50 min per hour and
allowed to rest for 10 min per hour. A cow-baited trap was also set-up yielding an additional
five cow-nights of collection. One cow was isolated from the herd and a 1m-wide mosquito
net was fenced around the animal, 30 cm above the ground level. One collector was asked to
capture mosquitoes resting on the net for 50 min per hour and allowed to rest for 10 min. Mosquitoes were collected individually into 5 mL plastic tubes and shipped at the Shoklo Malaria
Research Unit (Mae Sot, Thailand) at the end of each survey.
Laboratory processing of the mosquito samples
Laboratory processing of the mosquito samples was performed as described previously [7].
Mosquitoes were identified by morphology at the genus level and Anopheles specimens were
identified at the group level using the dichotomic key of Rattanarithikul et al. [46]. DNA was
extracted from mosquito specimens using the cetyl-trimethylammonium bromide method
[47]. Allele-specific polymerase chain reaction (AS-PCR) assays were used to discriminate
sensu stricto species in the Funestus and Maculatus Groups [48, 49]. In case AS-PCR gave a
negative result, and for the specimens in the Leucosphyrus Group, identification at the species
level was performed by sequencing the internal transcribed spacer 2 (ITS2) DNA marker [50].
Insecticide resistances in malaria mosquitoes were assessed using wild-caught female Anopheles imagoes still alive at the end of the survey and a standard susceptibility assay [51].
DNA extraction. Whole mosquitoes were crushed individually in 200 μl of cetyl-trimethylammonium bromide solution 2% (TrisHCl pH = 8, 20mM; EDTA 10mM; NaCl, 1.4
mM; N-cetyl-N,N,N-trimethyl ammonium bromide 2%) with a TissueLyser II™ (Qiagen) set
on 29 movements /second for 3 minutes. Samples were then warmed at 65˚C for 5 minutes
and 200 μl of chloroform were added. The aqueous phase was collected and DNA was precipitated with 200 μl of isopropanol. After centrifugation at 20,000 g for 15 minutes, the pellet was
washed twice with 200 μl of 70% ethanol and suspended in 50 μl of PCR grade water.
Allele-specific polymerase chain reaction assays. The PCR mix was composed of 1X
Goldstar™ DNA polymerase (Eurogentec, Seraing, Belgium) and 400 nM of each primer
(Funestus assay: ITS2A 5’-TGT GAA CTG CAG GAC ACA T-3’, MIA 5’-CCC GTG
CGA CTT GAC GA-3’, MIC 5’-GTT CAT TCA GCA ACA TCA GT-3’, ACO 5’ACA GCG TGT ACG TCC AGT-3’, PAM 5’-TGT ACA TCG GCC GGG GTA-3’,
VAR 5’-TTG ACC ACT TTC GAC GCA-3’; Maculatus assay: 5.8F 5’-TGT GAA
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CTG CAG GAC ACA T-3’, MAC 5’-CCC GTG CGA CTT GAC GA-3’, PSEU 5’GTT CAT TCA GCA ACA TCA GT-3’, SAW 5’-ACA GCG TGT ACG TCC AGT3’, K 5’-TGT ACA TCG GCC GGG GTA-3’, DRAV 5’-TTG ACC ACT TTC GAC
GCA-3’). The PCR was conducted in a total reaction volume of 25 μl (1 μl of DNA template
and 24 μl of PCR mix). The thermocycling protocol consisted in an initial activation step of 1
minute at 94˚C, followed by 40 amplification cycles of 20 seconds at 94˚C, 20 seconds at the
appropriate annealing temperature (45˚C and 55˚C for the Funestus and Maculatus assays
respectively), and 30 seconds at 72˚C. The length of the PCR product was determined by gel
electrophoresis in 2% agarose for 70 minutes at 120V.
Internal transcribed spacer 2 sequencing. Amplification of ITS2 was performed using
the primer pair ITS2A (5'-TGT GAA CTG CAG GAC ACA T-3') and ITS2B (5'-ATG
CTT AAA TTY AGG GGG T-3') described by Beebe and Saul [50]. The PCR mix was composed of 1X Goldstar™ DNA polymerase (Eurogentec, Seraing, Belgium) and 400 nM of each
primer. The PCR was conducted in a total reaction volume of 25 μl (4 μl of DNA template and
21 μl of PCR mix). The thermocycling protocol consisted in an initial activation step of 1 min
at 94˚C, followed by 40 amplification cycles of 20 s at 94˚C, 20 s at 51˚C and 30 s at 72˚C. The
PCR product was sequenced by Macrogen™ (Seoul, South Korea) using the ITS2A primer. The
sequence was analysed using the blastn algorithm of the online BLAST™ software in order to
determine the corresponding species.
Insecticide susceptibility assay. Insecticide susceptibility tests were performed with alive
mosquito specimens collected at the end of the follow-up (March and April surveys, i.e. three to
four months after ORS intervention). Female imagoes still alive at the end of the surveys were identified at the group level by morphology. Specimens which belonged to the same taxa were introduced into standard plastic cylinders and exposed for 60 minutes to filter papers impregnated the
1X discriminating concentration of deltamethrin (0.05% or 18 mg of a.i. /m2), permethrin (0.75%
or 275 mg of a.i. /m2) and lambda-cyhalothrin (0.05% or 18 mg of a.i. /m2) set by World Health
Organization. Mosquitoes collected in different villages and/or during different surveys were tested
independently. Mosquitoes collected in different sites (within the same village) and with different
methods (human-landing catch or cow-baited trap) were pooled in the same test cylinders. The
number of knocked down mosquitoes was recorded at the end of the exposure time. Mosquitoes
were then transferred into standard holding tubes and provided with a 10% sugar solution. Mortality was recorded 24 hours after exposure to the insecticide. If sufficient number of collected specimens was available, mosquitoes exposed for 1 hour to a paper impregnated with the carrier (Dow
556 mixed with acetone) were used as a control. Tests were performed at 25 ± 2˚C with a relative
humidity of 70–80%. All insecticide testing materials used in this study were provided by the Vector Control Research Unit (VCRU), Universiti Sains Malaysia.
Meteorological data
In this area, the rainy season usually starts in May and ends in November. Meteorological data
were obtained from the Thai Meteorological Department, including daily records of rainfall
and temperature in Mae Sot (Tak province) and Mae Sariang (Mae Hong Son province). The
cumulative rainfall and mean temperature were calculated over a 15-day period before each
collection date. Moon phase was taken into account in the analysis as a categorical variable
with four levels (first quarter, full moon last quarter and new moon).
Data analysis
Human-biting rate (HBR) and cow-biting rate (CBR) were defined as the number of collected
mosquitoes divided by the number of person-nights or cow-nights respectively. The exophagy
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index (EI) was defined as the outdoor HBR divided by the sum of the indoor and outdoor
HBRs. The cow-biting index (CBI) was defined as the CBR divided by the sum of the HBR and
the CBR. Knockdown (KD) rate was defined as the number of knocked down mosquitoes
divided by the number of exposed mosquitoes. Mortality rate was defined as the number of
mosquitoes dead at the end of the 24-hour observation time divided by the number of exposed
mosquitoes.
As expected, baseline characteristics of the two village pairs differed because of the small
number of clusters (4 villages) and the non-randomized nature of the study (intervention was
carried out in the villages with higher baseline HBR). In order to adjust for these differences, a
propensity score for ORS intervention was generated with the survey (baseline and month
0–4), collection method (indoor or outdoor human-landing), mean temperature in the 15
days before the collection date, cumulative rainfall in the 15 days before the collection date and
moon phase. ORS impact on the biting rate of malaria vectors was assessed using propensity
scores adjusted generalized linear mixed effect regressions with a negative binomial distribution for the number of collected malaria vectors /collector /night (i.e. the unit of the model was
one person-night of collection). Cow-baited trap collection method was not included in the
analysis because the cow-biting rate did not follow the same distribution than human-biting
rates and were not relevant to the understanding of ORS impact on human-vector contact.
The variance was estimated with a robust method. We included two-level random effects (village and collection site) to account for nested dependency within the data. Parameter estimates
may be non-robust to the failure of the assumed distribution of the random effects; therefore,
diagnostic checking of the residuals was performed. As the data deviated from the assumption
of equidispersion (average = variance) and from the dispersion parameter, it was evident that a
negative binomial model was more appropriate. The Vuong test was performed to decide
between zero inflated negative binomial and plain negative binomial model. The model was
selected as best model with unique covariance structure for G-side random effects that produces the lowest Bayesian Information Criterion value. The covariance structures considered
in the model were: AR(1) covariance structure, unstructured covariance structure, Toeplitz
covariance structure and variance component structure.
Ethics approval
The protocol for mosquito collection and analysis has been approved by the Oxford Tropical
Research Ethics Committee (1015–13, dated 29 Apr 2013), by the Ethics Review Committee
for Research Involving Human Research Subjects, Health Science Group, Chulalongkorn University (COA 154/2014), by the Karen Department of Health and Welfare, Karen National
Union and by the Tak Province Border Community Ethics Advisory Board [52]. All participants provided their written consent to participate in this study. This consent procedure was
approved by the ethics committees.
Results
Baseline assessment of Anopheles diversity, blood feeding behavior and
biting rates
Two thousand one hundred forty-seven Anopheles specimens were collected during baseline
surveys (200 person-nights and 20 cow-nights of collection) and identified by morphology.
Moreover, 97% (616/636), 98% (1350/1375) and 93% (13/14) of the specimens in the Funestus,
Maculatus and Leucosphyrus Groups respectively were identified with molecular assays. We
report the occurrence of five Anopheles species sensu lato and ten Anopheles species sensu
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Table 1. Village-collated mean estimates of mosquito human-biting rates determined during baseline surveys.
Genus
Group
Species
Mean human-biting rate estimate of the taxon in the indicated village expressed in bites /person /night [range]
KM-500 (Nov 30th to Dec
4th) a
KP-506 (Nov 29th to Dec
3rd) a
MK-509 (Dec 1st to Dec
5th) a
SP-513 (Nov 29th to Dec
3rd) a
Aedes
-
spp.
5.98 [0–37]
0.32 [0–3]
5.74 [0–36]
0.86 [0–8]
Culex
-
spp.
0.28 [0–2]
0.12 [0–2]
1.94 [0–12]
3.24 [0–24]
Anopheles
-
spp.
1.26 [0–12]
0.98 [0–7]
6.68 [0–44]
20.1 [0–128]
Annularis
spp. b
0 [0–0]
0 [0–0]
0.04 [0–1]
0.18 [0–3]
Barbirostris
spp. b
0 [0–0]
0.02 [0–1]
0 [0–0]
0.1 [0–2]
Funestus
spp.
0.64 [0–8]
0.14 [0–3]
2.6 [0–17]
5.16 [0–35]
An. minimus (s.s.) c
0.62 [0–7]
0.14 [0–3]
2.54 [0–17]
4.86 [0–34]
0.02 [0–1]
An. harrisoni
0 [0–0]
0 [0–0]
0 [0–0]
An. jeyporiensis
0 [0–0]
0 [0–0]
0.02 [0–1]
0 [0–0]
Hyrcanus
spp. d
0 [0–0]
0 [0–0]
0.08 [0–1]
0.1 [0–1]
Jamesii
spp. d
0 [0–0]
0 [0–0]
0.02 [0–1]
0.08 [0–2]
Kochi
spp.
0 [0–0]
0 [0–0]
0.12 [0–3]
0.02 [0–1]
Leucosphyrus
spp.
0.04 [0–1]
0 [0–0]
0.12 [0–1]
0.06 [0–1]
An. baimaii c
0.04 [0–1]
0 [0–0]
0.12 [0–1]
0.04 [0–1]
3.68 [0–28]
14.34 [0–90]
Maculatus
spp.
0.58 [0–4]
0.82 [0–7]
c
0.22 [0–2]
0.02 [0–1]
0.9 [0–14]
3.14 [0–21]
An. pseudowillmori b
0.26 [0–4]
0.76 [0–6]
2.56 [0–16]
10.42 [0–69]
An. sawadwongporni
0.1 [0–2]
0 [0–0]
0.18 [0–6]
0.6 [0–3]
Subpictus
spp. d
0 [0–0]
0 [0–0]
0 [0–0]
0 [0–0]
Tessellatus
spp. d
0 [0–0]
0 [0–0]
0 [0–0]
0 [0–0]
0 [0–0]
0 [0–0]
0.02 [0–1]
0 [0–0]
An. maculatus (s.s.)
c
Unclassified
a
An. karwari
d
each survey consisted of 25 person-nights of collection indoors and 25 person-nights of collection outdoors (total of 50 person-nights /village), the numbers of
mosquitoes collected indoors and outdoors were pooled to calculate the mean human-biting rate.
b
secondary malaria vectors.
c
primary malaria vectors.
d
some species in these Groups are efficient malaria vectors elsewhere, but were never found infected with human malaria parasites on the Thailand-Myanmar border (e.
g. An. karwari, An. sinensis, An. subpictus (s.s.), An. splendidus and An. tessellatus) [7].
https://doi.org/10.1371/journal.pone.0240598.t001
stricto including: An. annularis (s.l.) (Annularis Group), An. barbirostris (s.l.) (Barbirostris
Group), An. baimaii (s.s.) (Leucosphyrus Group, Dirus Complex), An. hyrcanus (s.l.) (Hyrcanus Group), An. jamesii (s.l.) (Jamesii Group), An. kochi (Kochi Group), An. maculatus (s.s.),
An. sawadwongporni, An. pseudowillmori (Maculatus Group), An. minimus (s.s.), An. harrisoni
(Funestus Group, Minimus Complex), An. jeyporiensis (Funestus Group), An. subpictus (s.l.)
(Subpictus Group), An. tessellatus (Tessellatus Group) and An. karwari (undefined Group).
The most abundant human-biting species were An. minimus (s.s.), An. maculatus (s.s.), An.
pseudowillmori and An. sawadwongporni (Table 1).
Malaria vectors were predominantly exophagic and zoophagic with village-collated EI and
CBI estimates ranging from 0.45 to 1.00 and from 0.33 to 0.95 respectively (Fig 1). The analysis
of hourly biting pattern showed activity peaks during the early evening and early morning (Fig
2). Only 24% (339/1409) of the specimens in the Funestus, Maculatus and Leucosphyrus
Groups were collected indoors between 9 PM and 5 AM. EI, CBI and hourly biting pattern of
a given species were significantly different from one village to another, hence suggesting the
importance of local ecological factors in shaping the blood seeking behaviour of malaria
mosquitoes.
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Fig 1. Exophagic and cow-biting index estimates of malaria mosquitoes at baseline. EI and CBI were not estimated if the total number
of collected mosquitoes was less than 15 for the corresponding index. Error bars indicate exact binomial 95% CIs. Abbreviations: CBI,
cow-biting index; CI, confidence interval; EI, exophagic index, pse: An. pseudowillmori, mac, An. maculatus (s.s.) min, An. minimus (s.s.),
saw: An. sawadwongporni.
https://doi.org/10.1371/journal.pone.0240598.g001
When taking into account only the specimens in the Funestus, Maculatus and Leucopshyrus Groups, the proportion of human-landing catches with a positive count of mosquito
vectors varied between 16% and 96% according to the village. Village-collated mean HBR estimates were consistently higher than the corresponding median and unaggregated HBR measurements ranged between 0 and 125 bites /person /night. Village-collated mean CBR
estimates varied between 12.2 and 50.4 bites /cow /night and unaggregated measurements ranged between 3 and 81 bites /cow /night (Table 2).
Impact of outdoor residual spraying on malaria vector human-biting rates
Only the specimens in the Funestus, Maculatus and Leucopshyrus Groups were taken
into account for assessing the impact of ORS on the biting rates of malaria mosquitoes.
Including other Anopheles species in the analysis did not change the results. The observed
biting rates of malaria mosquitoes are presented in the Fig 3 and S1 Appendix. Villagecollated mean biting rate estimates at baseline were higher in the intervention villages
than in the controls and drastically decreased in the intervention villages immediately
after ORS. The effect was observed indoors, outdoors and in the cow-baited trap. The
HBR remained low during 3 months after ORS in all villages and rose again at month 4 in
the intervention villages but not in the controls. The same trend was observed in the cowbaited traps except in SP-513 (intervention village) where the CBR increased gradually
after 2 months.
Using model outputs, we estimated that HBR in the sprayed villages was 9.6 times higher
than that in the controls (95%CI = 5.7–16.5, P <0.0001), and that outdoor HBR was 4.1 times
higher than indoor HBR (95%CI = 3.35–5.10, P = <0.0001). HBR naturally declined after baseline survey with incidence rate ratio (IRR) estimates varying between 0.26 (95%CI = 0.14–0.48,
P<0.0001) and 0.67 (95%CI = 0.34–1.30, P = 0.2364) during the follow-up. HBR was reduced
by 91% (95%CI = 88–96, P <0.0001) immediately after ORS. In the sprayed villages, the HBR
remained lower during the entire follow-up when compared to baseline with IRR ranging
between 0.02 (95%CI = 0.01–0.06, P <0.0001) and 0.19 (95%CI = 0.09–0.40, P <0.0001) at
month 3 and month 4 respectively (Table 3).
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Fig 2. Hourly biting pattern of malaria mosquitoes at baseline. (a) KM-500. (b) KP-506. (c) MK-509. (d) SP-513. Error bars indicate exact Poisson
95% CIs. Abbreviations: b/p/n: bites /person /night; CI, confidence interval; HLC, human-landing catch.
https://doi.org/10.1371/journal.pone.0240598.g002
Insecticide resistance
A subsample of 552 specimens collected during March and April surveys were used to perform
insecticide susceptibility tests, of which 434 were exposed to insecticides and 118 were used for
controls (Table 4 and S2 Appendix). Given the diversity of Anopheles mosquito species and the
relatively low biting rates, most of the tests were performed with small number of mosquitoes
and without control. The number of exposed An. maculatus (s.l.) and An. minimus (s.l.) was
large enough to estimate mortality with reasonable certainty. Mortality rate of permethrin, deltamethrin and lambda-cyhalothrin against An. maculatus (s.l.) was 97, 95 and 87% respectively. Mortality rate of lambda-cyhalothrin against An. minimus (s.l.) was 91%. The KD rate at
the end of the exposure time ranged between 85 and 91%. Only one out of eight An. barbirostris (s.l.) was dead at the end of the tests. Insecticide resistant mosquitoes were detected in all
villages.
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Table 2. Descriptive statistics of malaria vector biting rates at baseline.
Biting rate
Parameter
Parameter estimate in the indicated village
KM-500
KP-506
MK-509
SP-513
indoor HBR (in bites/person/night)
p (in %) a
52
16
68
80
minimum
0
0
0
0
10 percentile
0
0
0
0
median
1
0
2
3
mean
1.28
0.2
2.12
9.96
90th percentile
2.6
1
4.6
30.8
maximum
12
2
10
38
p (in %)
52
64
92
96
minimum
0
0
0
0
10th percentile
0
0
1
1.4
th
outdoor HBR in bites /person/night)
median
mean
CBR (in bites/cow/night)
1
1
5
17
1.24
1.72
10.68
29.16
90th percentile
2
4
25.8
83
maximum
9
7
42
125
100
p (in %)
100
100
100
minimum
3
14
26
13
10th percentile
5
16.4
26.4
20.2
median
13
28
38
40
mean
12.2
26
50.4
34.6
90th percentile
19.4
34.8
80.6
45
23
38
81
47
maximum
Abbreviations: CBR, cow-biting rate; HBR, human-biting rate.
a
p, proportion of catches with positive counts of malaria vectors expressed as a percentage. Only the specimens in the Funestus, Maculatus and Leucopshyrus Groups
were taken into account for parameter estimations.
https://doi.org/10.1371/journal.pone.0240598.t002
Discussion
To our knowledge, this is the first assessment of ORS for malaria vector-control in a Southeast
Asian transmission setting since Nair studies on DDT in the 1940s [25]. We estimated that the
HBR of primary malaria vector species was divided by 91% (95%CI = 88–96, P <0.0001)
immediately after applying a lambda-cyhalothrin mist to outdoor vegetation, thereby strongly
suggesting that peridomestic dense bushes in and around the village are an important resting
site of malaria mosquitoes in this area. Interestingly, the HBR decreased both indoors and outdoors. This result confirms that mosquito vectors seeking a blood meal indoors spend most of
their life cycle outside. As expected, there was a rich diversity of malaria mosquito species in
the area of the study [7, 53]. This diversity translates into complex transmission dynamics and
challenges the efficacy malaria vector-control with mosquito bed nets, indoor residual spraying
or larval source management [8, 16, 54, 55]. Therefore, the impact of ORS on multiple malaria
mosquito vector species is an important feature of this intervention.
The impact of ORS on mosquito biting rate may involve several mechanisms. The sharp
decrease in biting rates immediately after the intervention implies that an important proportion of the adult population was affected by the intervention. At the concentration used in this
study, lethal effect is to be expected if insecticide mist reaches resting mosquitoes, thus ORS
may result in mass killing of the vector population. In addition, pyrethroids have irritant and
excito-repellent properties that deter mosquitoes and compete with the lethal effect during
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Outdoor residual spraying for malaria vector-control
Fig 3. Impact of ORS on malaria vector biting rates. ORS intervention was carried out on 3rd and 4th December 2016 in MK-509 and on 5th
and 6th December 2016 in SP-513. Gradient-filled panels show previous estimate of the longevity of the residual insecticidal effects of lambdacyhalothrin mist applied to outdoor vegetation (98 days) [32]. Error bars indicate the negative binomial regression 95% confidence intervals.
Abbreviations: b/h/n: bites /host /night; CI, confidence interval; HBR, human-biting rate; ORS, outdoor residual spraying.
https://doi.org/10.1371/journal.pone.0240598.g003
operational deployment of vector-control interventions [56]. This mechanism may also lower
mosquito density inside the village after the spraying, especially when using lambda-cyhalothrin which is irritant to susceptible and resistant mosquitoes [57]. The rise in malaria vector
abundance observed 4 months after ORS in the intervention villages but not in the controls
coincided with the expected end of the residual effects of this insecticide formulation applied
to outdoor vegetation [32]. This result suggests the importance of the residual effects in determining intervention efficacy. Noteworthy, both male and female Anopheles mosquitoes rest on
outdoor vegetation [18] and intervention impact on male mosquitoes is likely to be an important factor.
There were several limitations to this study. We did not collect data on the resting sites,
residual effect and biting rate outside of the intervention area because of the logistic constrains
it would have implied and the pilot nature of the study, therefore it is not possible to draw
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Table 3. Generalized linear mixed-effect model output for the multivariable analysis of the number of collected malaria vectors /person /night including location,
visits and outdoor residual spraying as predictors.
Category
Study arm
Control (reference)
0
1
-
ORS
2.3 (0.27)
9.66 (5.66–16.50)
<0.0001
Collection method
Survey
Estimate (SE)
IRR (95%CI)
p-value a
Variable
indoor HLC (reference)
0
1
-
outdoor HLC
1.4 (0.11)
4.13 (3.35–5.10)
<0.0001
baseline (reference)
0
1
month 0
-0.8 (0.32)
0.45 (0.24–0.84)
month 1
-0.4 (0.34)
0.67 (0.34–1.30)
0.2364
month 2
-1.4 (0.32)
0.26 (0.14–0.48)
<0.0001
Survey� Arm
0.0124
month 3
-1.2 (0.32)
0.31 (0.17–0.58)
0.0003
month 4
-0.4 (0.29)
0.64 (0.36–1.14)
0.1269
baseline:ORS
0
1
-
month 0:ORS
-2.4 (0.46)
0.09 (0.04–0.22)
<0.0001
month 1:ORS
-2.7 (0.44)
0.06 (0.03–0.15)
<0.0001
month 2:ORS
-2.5 (0.49)
0.08 (0.03–0.21)
<0.0001
month 3:ORS
-3.8 (0.48)
0.02 (0.01–0.06)
<0.0001
month 4:ORS
-1.7 (0.39)
0.19 (0.09–0.40)
<0.0001
Abbreviations: CI, confidence interval; HLC, human-landing catch; IRR, incidence rate ratio; ORS, outdoor residual spraying.
The P-value was calculated from mixed effect negative binomial regression model after adjusting for propensity scores.
a
https://doi.org/10.1371/journal.pone.0240598.t003
conclusion on the mechanism of action of ORS. Moreover, the intervention was not randomized (villages with higher HBR estimates at baseline were selected for ORS) and only two pairs
of villages were included in this pilot study. Given the small number of clusters and the timing
of ORS intervention (transition period during the rainy and the dry seasons, when vector populations naturally declines), it was not possible to determine precisely the duration and
Table 4. Results of the standard suceptibility tests performed with wild-caught female imagoes using deltamethrin, lambda-cyhalothrin (“0.05%” or 18 mg of a.i.
/m2) or permethrin (“0.75%” or 275 mg of a.i. /m2).
Species
Insecticide
No. exposed
mosquitoes
No. knocked-down mosquitoes
after 60 min
No. dead mosquitoes after
24 hours
KD60 in % (95% Mortality in % (95%
CI) a
CI) a
An. barbirostris
(s.l.)
lambdacyhalothrin
8
0
1
0 (0–37)
12 (0–53)
An. dirus (s.l.)
lambdacyhalothrin
1
0
1
0 (0–98)
100 (3–100)
An. jamesii (s.l.)
lambdacyhalothrin
20
19
18
95 (75–100)
90 (68–99)
An. kochi (s.s.)
lambdacyhalothrin
1
1
1
100 (3–100)
100 (3–100)
102
89
97
87 (79–93)
95 (89–98)
lambdacyhalothrin
163
144
142
88 (82–93)
87 (81–92)
permethrin
58
53
56
91 (81–97)
97 (88–100)
lambdacyhalothrin
88
75
80
85 (76–92)
91 (83–96)
An. maculatus (s. deltamethrin
l.)
An. minimus (s.
l.)
Abbreviations: CI, confidence interval; KD60, knockdown rate at the end of the 60 min period of exposure to insecticide.
95% binomial confidence intervals were calculated for KD60 and mortality rates.
a
https://doi.org/10.1371/journal.pone.0240598.t004
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magnitude of ORS impact on malaria vector biting rate. We did not assess intervention effects
on transmission intensity and malaria incidence. Falciparum malaria has been eliminated
from these villages before the study [2] and the incidence of symptomatic vivax malaria is
mostly driven by relapses [58], thereby confounding the relationship between malaria incidence and the P. vivax entomological inoculation rate. Importantly, vector longevity is a key
parameter in the equation of vectorial capacity [59, 60] and should also be used as an outcome
measure in future studies. The toxicity of the insecticide mist to non-target organisms is likely
to be high given the nature of sprayed sites. Although we did not collect data, the villages were
closely monitored during and after the intervention by the study team and villagers. We did
not observe any dramatic impact of the intervention on the ecosystem (especially on the taxa
at high risk including aquatic organisms, wild mammals, amphibians, reptiles and terrestrial
arthropods). Noteworthy, the risk that insecticide residues were washed into streams when the
rains came was deemed very low given the high soil adsorption coefficient of lambda-cyhalothrin, its half-life in soils, the timing of the intervention (beginning of the dry season) and that
insecticide mist was not applied near streams.
Deltamethrin and permethrin resistances were reported in Kayin state and may pose an
additional challenge to effective vector-control intervention in this region [45]. We did not
perform insecticide susceptibility tests during baseline surveys precluding the assessment of
ORS effect on resistance dynamics. Mortality rates were lower with lambda-cyhalothrin (type
II pyrethroid) than with deltamethrin and permethrin (type I pyrethroids). Type II pyrethroids
are intensively used for agriculture in this region [61]. This observation suggests that agriculture may play an important role in the emergence and/or selection of resistance to insecticides
used in public health. The KD rate after 60 min of exposure to insecticide was high in An.
minimus (s.l.) and An. maculatus (s.l.) suggesting the involvement of metabolic pathways
rather than kdr mutations in these species.
Conclusions
Outdoor residual spraying with a capsule suspension of lambda-cyhalothrin rapidly decreases
exophilic malaria vector biting rates in this area where pyrethroid resistance has been
documented.
Supporting information
S1 Appendix. Raw data of mosquito catches.
(XLSX)
S2 Appendix. Raw data of the insecticide susceptibility tests.
(XLSX)
Acknowledgments
We thank the team of the entomology department of the Shoklo Malaria Research Unit for
their work, the local communities for their support to the study and the Thai Meteorological
Department for sharing their data. The Shoklo Malaria Research Unit is part of the Mahidol
Oxford University Research Unit, supported by the Wellcome Trust of Great Britain.
Author Contributions
Conceptualization: Victor Chaumeau, Gilles Delmas, François Nosten.
Data curation: Victor Chaumeau, Sunisa Sawasdichai, Jacher Wiladphaingern.
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Formal analysis: Victor Chaumeau, Ramesh Kumar Vishwakarma.
Funding acquisition: Victor Chaumeau, Gilles Delmas, François Nosten.
Investigation: Victor Chaumeau, Ladda Kajeechiwa, Thithiworada Kulabkeeree, Praphan
Wasisakun, Saw Nay Hsel, Kyi Oo, Tee Dah, Sunisa Sawasdichai, Muesuwa Trakoolchengkaew, Monthicha Phanaphadungtham, Aritsara Inta, Yanada Akararungrot, Naw Yu Lee,
Prasan Kankew, Gilles Delmas.
Methodology: Victor Chaumeau, Praphan Wasisakun.
Project administration: Victor Chaumeau, Gilles Delmas, François Nosten.
Resources: Ladda Kajeechiwa, François Nosten.
Supervision: Victor Chaumeau, Ladda Kajeechiwa, Praphan Wasisakun, Kyi Oo, Tee Dah,
Sunisa Sawasdichai, Prasan Kankew, Mavuto Mukaka, Gilles Delmas, François Nosten.
Validation: Victor Chaumeau, François Nosten.
Visualization: Victor Chaumeau.
Writing – original draft: Victor Chaumeau.
Writing – review & editing: Victor Chaumeau, François Nosten.
References
1.
Luxemburger C, Thwai KL, White NJ, Webster HK, Kyle DE, Maelankirri L, et al. The epidemiology of
malaria in a Karen population on the western border of Thailand. Trans R Soc Trop Med Hyg. 1996; 90
(2):105–11. https://doi.org/10.1016/s0035-9203(96)90102-9 PMID: 8761562
2.
Landier J, Parker DM, Thu AM, Lwin KM, Delmas G, Nosten FH. Effect of generalised access to early
diagnosis and treatment and targeted mass drug administration on Plasmodium falciparum malaria in
Eastern Myanmar: an observational study of a regional elimination programme. Lancet (London,
England). 2018; 391(10133):1916–26. https://doi.org/10.1016/S0140-6736(18)30792-X PMID:
29703425
3.
Ashley EA, Phyo AP, Carrara VI, Tun KM, Nosten F, Smithuis F, et al. Plasmodium vivax relapse rates
following falciparum malaria reflect previous transmission intensity. J Infect Dis. 2019; 220(1): 100–104.
https://doi.org/10.1093/infdis/jiz052 PMID: 30698794
4.
Jeffery GM. The infection of mosquitoes by Plasmodium vivax (Chesson strain) during the early primary
parasitemias. Am J Trop Med Hyg. 1952; 1(4):612–7. https://doi.org/10.4269/ajtmh.1952.1.612 PMID:
14943909
5.
Boyd M. Malariology. Philadelphia: Saunders; 1949.
6.
Chu CS, White NJ. Management of relapsing Plasmodium vivax malaria. Expert Rev Anti Infect Ther.
2016; 14(10):885–900. https://doi.org/10.1080/14787210.2016.1220304 PMID: 27530139
7.
Chaumeau V, Fustec B, Nay Hsel S, Montazeau C, Naw Nyo S, Metaane S, et al. Entomological determinants of malaria transmission in Kayin state, Eastern Myanmar: A 24-month longitudinal study in four
villages. Wellcome Open Research. 2018; 3(109). https://doi.org/10.12688/wellcomeopenres.14761.4
PMID: 31206035
8.
Somboon P, Aramrattana A, Lines J, Webber R. Entomological and epidemiological investigations of
malaria transmission in relation to population movements in forest areas of north-west Thailand. Southeast Asian J Trop Med Public Health. 1998; 29(1):3–9. PMID: 9740259
9.
Sriwichai P, Samung Y, Sumruayphol S, Kiattibutr K, Kumpitak C, Payakkapol A, et al. Natural human
Plasmodium infections in major Anopheles mosquitoes in western Thailand. Parasit Vectors. 2016; 9
(1):17. https://doi.org/10.1186/s13071-016-1295-x PMID: 26762512
10.
Chaumeau V, Kajeechiwa L, Fustec B, Landier J, Nyo SN, Hsel SN, et al. The contribution of asymptomatic Plasmodium infections to the transmission of malaria in Kayin state, Myanmar. J Infect Dis.
2018.
11.
Dolan G, ter Kuile FO, Jacoutot V, White NJ, Luxemburger C, Malankirii L, et al. Bed nets for the prevention of malaria and anaemia in pregnancy. Trans R Soc Trop Med Hyg. 1993; 87(6):620–6. https://doi.
org/10.1016/0035-9203(93)90262-o PMID: 8296357
PLOS ONE | https://doi.org/10.1371/journal.pone.0240598 October 29, 2020
15 / 18
PLOS ONE
Outdoor residual spraying for malaria vector-control
12.
Smithuis FM, Kyaw MK, Phe UO, van der Broek I, Katterman N, Rogers C, et al. Entomological determinants of insecticide-treated bed net effectiveness in Western Myanmar. Malar J. 2013; 12:364. https://
doi.org/10.1186/1475-2875-12-364 PMID: 24119994
13.
Smithuis FM, Kyaw MK, Phe UO, van der Broek I, Katterman N, Rogers C, et al. The effect of insecticide-treated bed nets on the incidence and prevalence of malaria in children in an area of unstable seasonal transmission in western Myanmar. Malar J. 2013; 12:363. https://doi.org/10.1186/1475-2875-12363 PMID: 24119916
14.
Killeen GF. Characterizing, controlling and eliminating residual malaria transmission. Malar J. 2014;
13:330. https://doi.org/10.1186/1475-2875-13-330 PMID: 25149656
15.
Somboon P, Lines J, Aramrattana A, Chitprarop U, Prajakwong S, Khamboonruang C. Entomological
evaluation of community-wide use of lambdacyhalothrin-impregnated bed nets against malaria in a border area of north-west Thailand. Trans R Soc Trop Med Hyg. 1995; 89(3):248–54. https://doi.org/10.
1016/0035-9203(95)90525-1 PMID: 7660424
16.
Rattanarithikul R, Green CA, Panyim S, Noigamol C, Chanaimongkol S, Mahapibul P. Larval habitats of
malaria vectors and other Anopheles mosquitoes around a transmission focus in northwestern Thailand. J Am Mosq Control Assoc. 1995; 11(4):428–33. PMID: 8825502
17.
James SP. Extermination of Mosquitos. Ind Med Gaz. 1899; 34(11):428. PMID: 29002452
18.
Dewald JR, Fuller DO, Muller GC, Beier JC. A novel method for mapping village-scale outdoor resting
microhabitats of the primary African malaria vector, Anopheles gambiae. Malar J. 2016; 15(1):489.
https://doi.org/10.1186/s12936-016-1534-9 PMID: 27659918
19.
Silver JB. Mosquito Ecology—Field Sampling Methods: Springer Netherlands; 2008.
20.
Richards SL, Volkan JK, Balanay JAG, Vandock K. Evaluation of bifenthrin and deltamethrin barrier
sprays for mosquito control in Eastern North Carolina. J Med Entomol. 2017; 54(6):1659–65. https://doi.
org/10.1093/jme/tjx152 PMID: 28968745
21.
Trout RT, Brown GC, Potter MF, Hubbard JL. Efficacy of two pyrethroid insecticides applied as barrier
treatments for managing mosquito (Diptera: Culicidae) populations in suburban residential properties. J
Med Entomol. 2007; 44(3):470–7. https://doi.org/10.1603/0022-2585(2007)44[470:eotpia]2.0.co;2
PMID: 17547233
22.
Ludvik GF. Barrier strip and pre-flood treatments with DDT to control Anopheles quadrimaculatus. Journal of Economical Entomology. 1950; 43:516–9.
23.
Hurst TP, Ryan PA, Kay BH. Efficacy of residual insecticide Biflex AquaMax applied as barrier treatments for managing mosquito populations in suburban residential properties in southeast Queensland.
J Med Entomol. 2012; 49(5):1021–6. https://doi.org/10.1603/me11278 PMID: 23025182
24.
Unlu I, Williams GM, Rochlin I, Suman D, Wang Y, Chandel K, et al. Evaluation of lambda-cyhalothrin
and pyriproxyfen barrier treatments for Aedes albopictus (Diptera: Culicidae) management in urbanized
areas of New Jersey. J Med Entomol. 2018; 55(2):472–6. https://doi.org/10.1093/jme/tjx216 PMID:
29244157
25.
Nair CP. Studies on DDT barrier spray with reference to local rainfall and seasonal incidence of mosquitoes. Trans R Soc Trop Med Hyg. 1951; 44(6):741–6. https://doi.org/10.1016/0035-9203(51)90010-7
PMID: 14855622
26.
Perich MJ, Tidwell MA, Dobson SE, Sardelis MR, Zaglul A, Williams DC. Barrier spraying to control the
malaria vector Anopheles albimanus: laboratory and field evaluation in the Dominican Republic. Med
Vet Entomol. 1993; 7(4):363–8. https://doi.org/10.1111/j.1365-2915.1993.tb00706.x PMID: 8268492
27.
Madden AH, Shoeder HO, Lindquist AW. Residual sprays applications to salt-marsh and jungle vegetation for control of mosquitoes. Journal of economic entomology. 1947; 40(1):119–23. https://doi.org/10.
1093/jee/40.1.119 PMID: 20240407
28.
Cilek JE, Hallmon CF. Residual effectiveness of pyrethroid-treated foliage against adult Aedes albopictus and Culex quinquefasciatus in screened field cages. J Am Mosq Control Assoc. 2006; 22(4):725–
31. https://doi.org/10.2987/8756-971X(2006)22[725:REOPFA]2.0.CO;2 PMID: 17304943
29.
Cilek JE, Hallmon CF. Residual effectiveness of three pyrethroids on vegetation against adult Aedes
albopictus and Culex quinquefasciatus in screened field cages. J Am Mosq Control Assoc. 2008; 24
(2):263–9. https://doi.org/10.2987/5653.1 PMID: 18666535
30.
Gurtler RE, Canale DM, Spillmann C, Stariolo R, Salomon OD, Blanco S, et al. Effectiveness of residual
spraying of peridomestic ecotopes with deltamethrin and permethrin on Triatoma infestans in rural western Argentina: a district-wide randomized trial. Bull World Health Organ. 2004; 82(3):196–205. PMID:
15112008
31.
Cecere MC, Vazquez-Prokopec GM, Ceballos LA, Gurevitz JM, Zarate JE, Zaidenberg M, et al. Comparative trial of effectiveness of pyrethroid insecticides against peridomestic populations of Triatoma
PLOS ONE | https://doi.org/10.1371/journal.pone.0240598 October 29, 2020
16 / 18
PLOS ONE
Outdoor residual spraying for malaria vector-control
infestans in northwestern Argentina. J Med Entomol. 2006; 43(5):902–9. https://doi.org/10.1603/00222585(2006)43[902:ctoeop]2.0.co;2 PMID: 17017227
32.
Chaumeau V, Wisisakun P, Sawasdichai S, Kankew P, Htoo GN, Saithanmettajit S, et al. Longevity of
the insecticidal effect of three pyrethroid formulations applied to outdoor vegetation on a laboratoryadapted colony of the Southeast Asian malaria vector Anopheles dirus. PLoS One. 2020; 15(4):
e0231251. https://doi.org/10.1371/journal.pone.0231251 PMID: 32287300
33.
European Food Safety Agency. Peer review of the pesticide risk assessment of the active substance
lambda-cyhalothrin. European Food Safety Agency; 2014.
34.
Sieke C. Joint FAO/WHO Meeting on Pesticide Residues—Lambda-cyhalothrin. 2008.
35.
He LM, Troiano J, Wang A, Goh K. Environmental chemistry, ecotoxicity, and fate of lambda-cyhalothrin. Rev Environ Contam Toxicol. 2008; 195:71–91. https://doi.org/10.1007/978-0-387-77030-7_3
PMID: 18418954
36.
Kenko Nkontcheu DB, Tchamadeu NN, Ngealekeleoh F, Nchase S. Ecotoxicological effects of imidacloprid and lambda-cyhalothrin (insecticide) on tadpoles of the African common toad, Amietophrynus
Regularis (Reuss, 1833) (Amphibia: Bufonidae). Italian Journal of Science & Engineering. 2017; 1
(2):49–53.
37.
Khan Z, Zaheer M, Fatima F. Effect of lambda-cyhalothrin (pyrethroid) and monocrotophos (organophosphate) on cholinesterase activity in liver, kidney and brain of Rana cyanophlyctis. Korean Journal
of Biological Sciences. 2003; 7(2):165–8.
38.
Hornsby AG, Herner AE, Don Wauchope R. Pesticide properties in the environment. New-York:
Springer; 1996.
39.
Hill BD, Inaba DJ. Dissipation of lambda-cyhalothrin on fallow vs cropped Soil. Journal of Agricultural
and Food Chemistry. 1991;39(12).
40.
Laabs V, Wehrhan A, Pinto A, Dores E, Amelung W. Pesticide fate in tropical wetlands of Brazil: an
aquatic microcosm study under semi-field conditions. Chemosphere. 2007; 67(5):975–89. https://doi.
org/10.1016/j.chemosphere.2006.10.067 PMID: 17166548
41.
Daam MA, Chelinho S, Niemeyer JC, Owojori OJ, De Silva PMCS, Sousa JP, et al. Environmental risk
assessment of pesticides in tropical terrestrial ecosystems: test procedures, current status and future
perspectives. Ecotoxicol Environ Saf. 2019; 181:534–47. https://doi.org/10.1016/j.ecoenv.2019.06.038
PMID: 31234068
42.
Fantke P, Juraske R. Variability of pesticide dissipation half-lives in plants. Environ Sci Technol. 2013;
47(8):3548–62. https://doi.org/10.1021/es303525x PMID: 23521068
43.
International Programme on Chemical Safety and World Health Organization. Cyhalothrin and lambdacyhalothrin: health and safety guide. World Health Organization; 1990.
44.
Robson C. Field guide to birds of South-East Asia: Bloomsbury Publishing; 2020.
45.
Chaumeau V, Cerqueira D, Zadrozny J, Kittiphanakun P, Andolina C, Chareonviriyaphap T, et al. Insecticide resistance in malaria vectors along the Thailand-Myanmar border. Parasit Vectors. 2017; 10
(1):165. https://doi.org/10.1186/s13071-017-2102-z PMID: 28359289
46.
Rattanarithikul R, Harrison BA, Harbach RE, Panthusiri P, Coleman RE. Illustrated keys to the mosquitoes of Thailand. IV. Anopheles. Southeast Asian J Trop Med Public Health. 2006; 37 Suppl 2:1–128.
47.
Chaumeau V, Andolina C, Fustec B, Tuikue Ndam N, Brengues C, Herder S, et al. Comparison of the
performances of five primer sets for the detection and quantification of Plasmodium in anopheline vectors by real-time PCR. PLoS One. 2016; 11(7):e0159160. https://doi.org/10.1371/journal.pone.
0159160 PMID: 27441839
48.
Walton C, Somboon P, O’Loughlin SM, Zhang S, Harbach RE, Linton YM, et al. Genetic diversity and
molecular identification of mosquito species in the Anopheles maculatus group using the ITS2 region of
rDNA. Infect Genet Evol. 2007; 7(1):93–102. https://doi.org/10.1016/j.meegid.2006.05.001 PMID:
16782411
49.
Garros C, Koekemoer LL, Coetzee M, Coosemans M, Manguin S. A single multiplex assay to identify
major malaria vectors within the African Anopheles funestus and the Oriental An. minimus groups. Am J
Trop Med Hyg. 2004; 70(6):583–90. PMID: 15210996
50.
Beebe NW, Saul A. Discrimination of all members of the Anopheles punctulatus complex by polymerase
chain reaction—restriction fragment length polymorphism analysis. Am J Trop Med Hyg. 1995; 53
(5):478–81. https://doi.org/10.4269/ajtmh.1995.53.478 PMID: 7485705
51.
World Health Organization. Test procedures for insecticide resistance monitoring in malaria vector mosquitoes Geneva: World Health Organization; 2016.
52.
Cheah PY, Lwin KM, Phaiphun L, Maelankiri L, Parker M, Day NP, et al. Community engagement on
the Thai-Burmese border: rationale, experience and lessons learnt. Int Health. 2010; 2(2):123–9.
https://doi.org/10.1016/j.inhe.2010.02.001 PMID: 22984375
PLOS ONE | https://doi.org/10.1371/journal.pone.0240598 October 29, 2020
17 / 18
PLOS ONE
Outdoor residual spraying for malaria vector-control
53.
Morgan K, Somboon P, Walton C. Understanding Anopheles Diversity in Southeast Asia and Its Applications for Malaria Control. In: Manguin S, editor. Anopheles mosquitoes—New insights into malaria
vectors. Rijeka, Croatia: InTech; 2013. p. 327–56.
54.
Meek SR. Vector control in some countries of Southeast Asia: comparing the vectors and the strategies.
Ann Trop Med Parasitol. 1995; 89(2):135–47. https://doi.org/10.1080/00034983.1995.11812944 PMID:
7605123
55.
Ismail IA, Notananda V, Schepens J. Studies on malaria and responses of Anopheles balabacensis
balabacensis and Anopheles minimus to DDT residual spraying in Thailand. Acta Trop. 1975; 32
(3):206–31. PMID: 1984
56.
Killeen GF, Chitnis N, Moore SJ, Okumu FO. Target product profile choices for intra-domiciliary malaria
vector control pesticide products: repel or kill? Malar J. 2011; 10:207. https://doi.org/10.1186/14752875-10-207 PMID: 21798023
57.
Hougard JM, Duchon S, Darriet F, Zaim M, Rogier C, Guillet P. Comparative performances, under laboratory conditions, of seven pyrethroid insecticides used for impregnation of mosquito nets. Bull World
Health Organ. 2003; 81(5):324–33. PMID: 12856050
58.
Adekunle AI, Pinkevych M, McGready R, Luxemburger C, White LJ, Nosten F, et al. Modeling the
dynamics of Plasmodium vivax infection and hypnozoite reactivation in vivo. PLoS Negl Trop Dis. 2015;
9(3):e0003595. https://doi.org/10.1371/journal.pntd.0003595 PMID: 25780913
59.
Cohuet A, Harris C, Robert V, Fontenille D. Evolutionary forces on Anopheles: what makes a malaria
vector? Trends Parasitol. 2010; 26(3):130–6. https://doi.org/10.1016/j.pt.2009.12.001 PMID: 20056485
60.
Pongtavornpinyo W, Yeung S, Hastings IM, Dondorp AM, Day NP, White NJ. Spread of anti-malarial
drug resistance: mathematical model with implications for ACT drug policies. Malar J. 2008; 7:229.
https://doi.org/10.1186/1475-2875-7-229 PMID: 18976503
61.
Pesticides in Southeast Asia: environmental, biomedical and economic uses and effects. Bangkok;
2007.
PLOS ONE | https://doi.org/10.1371/journal.pone.0240598 October 29, 2020
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English
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Identification of lptA, lpxE, and lpxO, Three Genes Involved in the Remodeling of Brucella Cell Envelope
|
Frontiers in microbiology
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Edited by:
Axel Cloeckaert,
Institut National de la Recherche
Agronomique (INRA), France
Reviewed by:
Diego J. Comerci,
Instituto de Investigaciones
Biotecnológicas (IIB-INTECH),
Argentina
Roy Martin Roop II,
East Carolina University, United States
*Correspondence:
Ignacio Moriyón
imoriyon@unav.es
Maite Iriarte
miriart@unav.es Edited by:
Axel Cloeckaert,
Institut National de la Recherche
Agronomique (INRA), France 1 Universidad de Navarra, Facultad de Medicina, Departamento de Microbiología y Parasitología, Instituto de Salud Tropical
(ISTUN) e Instituto de Investigación Sanitaria de Navarra (IdISNA), Pamplona, Spain, 2 Instituto de Agrobiotecnología,
Consejo Superior de Investigaciones Científicas – Universidad Pública de Navarra – Gobierno de Navarra, Pamplona, Spain,
3 Unidad de Producción y Sanidad Animal, Instituto Agroalimentario de Aragón, Centro de Investigación y Tecnología
Agroalimentaria de Aragón – Universidad de Zaragoza, Zaragoza, Spain, 4 Wellcome-Wolfson Institute for Experimental
Medicine, Queen’s University Belfast, Belfast, United Kingdom, 5 Institut National de la Santé et de la Recherche Médicale,
U1104, Centre National de la Recherche Scientifique UMR7280, Centre d’Immunologie de Marseille-Luminy, Aix-Marseille
University UM2, Marseille, France, 6 Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México,
Cuernavaca, Mexico Reviewed by:
Diego J. Comerci,
Instituto de Investigaciones
Biotecnológicas (IIB-INTECH),
Argentina Roy Martin Roop II,
East Carolina University, United States The brucellae are facultative intracellular bacteria that cause a worldwide extended
zoonosis. One of the pathogenicity mechanisms of these bacteria is their ability to
avoid rapid recognition by innate immunity because of a reduction of the pathogen-
associated molecular pattern (PAMP) of the lipopolysaccharide (LPS), free-lipids, and
other envelope molecules. We investigated the Brucella homologs of lptA, lpxE, and
lpxO, three genes that in some pathogens encode enzymes that mask the LPS
PAMP by upsetting the core-lipid A charge/hydrophobic balance. Brucella lptA, which
encodes a putative ethanolamine transferase, carries a frame-shift in B. abortus but
not in other Brucella spp. and phylogenetic neighbors like the opportunistic pathogen
Ochrobactrum anthropi. Consistent with the genomic evidence, a B. melitensis lptA
mutant lacked lipid A-linked ethanolamine and displayed increased sensitivity to
polymyxin B (a surrogate of innate immunity bactericidal peptides), while B. abortus
carrying B. melitensis lptA displayed increased resistance. Brucella lpxE encodes a
putative phosphatase acting on lipid A or on a free-lipid that is highly conserved in all
brucellae and O. anthropi. Although we found no evidence of lipid A dephosphorylation,
a B. abortus lpxE mutant showed increased polymyxin B sensitivity, suggesting the
existence of a hitherto unidentified free-lipid involved in bactericidal peptide resistance. ORIGINAL RESEARCH
published: 10 January 2018
doi: 10.3389/fmicb.2017.02657 Identification of lptA, lpxE, and lpxO,
Three Genes Involved in the
Remodeling of Brucella Cell
Envelope Raquel Conde-Álvarez1, Leyre Palacios-Chaves2, Yolanda Gil-Ramírez1,
Miriam Salvador-Bescós1, Marina Bárcena-Varela1, Beatriz Aragón-Aranda1,
Estrella Martínez-Gómez1, Amaia Zúñiga-Ripa1, María J. de Miguel3,
Toby Leigh Bartholomew4, Sean Hanniffy5, María-Jesús Grilló2,
Miguel Ángel Vences-Guzmán6, José A. Bengoechea4, Vilma Arce-Gorvel5,
Jean-Pierre Gorvel5, Ignacio Moriyón1* and Maite Iriarte1* Gene lpxO putatively encoding an acyl hydroxylase carries a frame-shift in all brucellae
except B. microti and is intact in O. anthropi. Free-lipid analysis revealed that lpxO
corresponded to olsC, the gene coding for the ornithine lipid (OL) acyl hydroxylase
active in O. anthropi and B. microti, while B. abortus carrying the olsC of O. anthropi
and B. microti synthesized hydroxylated OLs. Interestingly, mutants in lptA, lpxE, or olsC *Correspondence:
Ignacio Moriyón
imoriyon@unav.es
Maite Iriarte
miriart@unav.es *Correspondence:
Ignacio Moriyón
imoriyon@unav.es
Maite Iriarte
miriart@unav.es Specialty section:
This article was submitted to
Infectious Diseases,
a section of the journal
Frontiers in Microbiology
Received: 26 October 2017
Accepted: 20 December 2017
Published: 10 January 2018
Citation:
Conde-Álvarez R, Palacios-Chaves L,
Gil-Ramírez Y, Salvador-Bescos M,
Bárcena-Varela M, Aragón-Aranda B,
Martínez-Gómez E, Zúñiga-Ripa A,
de Miguel MJ, Bartholomew TL,
Hanniffy S, Grilló M-J,
Vences-Guzmán MÁ,
Bengoechea JA, Arce-Gorvel V,
Gorvel J-P, Moriyón I and Iriarte M
(2018) Identification of lptA, lpxE,
and lpxO, Three Genes Involved
in the Remodeling of Brucella Cell
Envelope. Front. Microbiol. 8:2657. doi: 10.3389/fmicb.2017.02657 Specialty section:
This article was submitted to
Infectious Diseases,
a section of the journal
Frontiers in Microbiology Specialty section:
This article was submitted to
Infectious Diseases,
a section of the journal
Frontiers in Microbiology
Received: 26 October 2017
Accepted: 20 December 2017
Published: 10 January 2018 Received: 26 October 2017
Accepted: 20 December 2017
Published: 10 January 2018 INTRODUCTION is a eukaryotic-type phospholipid required for Brucella full
virulence (Comerci et al., 2006; Conde-Alvarez et al., 2006). Among the amino lipids, only the ornithine lipids (OL) have
been investigated which unlike their counterparts in Bordetella,
do not trigger the release of IL-6 or TNF-α by macrophages,
possibly on account of their longer acyl chains that reduce the OL
PAMP (Palacios-Chaves et al., 2011). Concerning the LPS, most
bacteria carry C1 and C4′ glucosamine disaccharides with C12
and C14 acyl and acyl-oxyacyl chains. This highly amphipathic
structure, named lipid A, is adjacent to additional negatively
charged groups of the core oligosaccharide, namely the heptose
phosphates and 2-keto-3-deoxyoctulosonate carboxyl groups
(Kastowsky et al., 1992; Moriyón, 2003). This lipid A-core PAMP
is so efficiently detected by the innate immunity system that some
pathogens partially conceal it by removing phosphate groups or
substituting them with arabinosamine and/or ethanolamine, or
by hydroxylating the acyl chains (Takahashi et al., 2008; Lewis
et al., 2013; Moreira et al., 2013; Needham and Trent, 2013; Llobet
et al., 2015; Trombley et al., 2015). In contrast, Brucella lipid
A is a diaminoglucose disaccharide amide-linked to long (C16,
C18) and very long (C28–C30) acyl chains (Velasco et al., 2000;
Iriarte et al., 2004; Fontana et al., 2016). Furthermore, negative
charges in lipid A phosphates and 2-keto-3-deoxyoctulosonate
are counterbalanced by four glucosamine units present in the
core (Kubler-Kielb and Vinogradov, 2013; Fontana et al., 2016). As illustrated by the unusually reduced endotoxicity of the
Brucella LPS this structure is defectively detected by the innate
immune response (Lapaque et al., 2005; Martirosyan et al., 2011;
Conde-Álvarez et al., 2012). It remains unknown, however,
whether Brucella LPS undergoes post-synthetic modifications
that have been described for other bacteria that could alter
its PAMP potential and contribution to virulence. In this
work, we investigated in Brucella the role of gene homologs to
phosphatases, phospho-ethanolamine (pEtN) transferases, and
acyl hydroxylases (Figure 1) that have been shown in other
Gram-negative pathogens to act on LPS and to contribute to
overcoming innate immunity defenses. Brucellosis is the collective name of a group of zoonotic
diseases afflicting a wide range of domestic and wild mammals
(Whatmore, 2009; Zheludkov and Tsirelson, 2010). INTRODUCTION In domestic
livestock brucellosis is manifested mostly as abortions and
infertility, and contact with infected animals and consumption
of unpasteurized dairy products are the sources of human
brucellosis, an incapacitating condition that requires prolonged
antibiotic treatment (Zinsstag et al., 2011). Eradicated in a
handful of countries, brucellosis is endemic or even increasing in
many areas of the world (Jones et al., 2013; Ducrotoy et al., 2017;
Lai et al., 2017). This disease is caused by facultative intracellular parasites
of the genus Brucella. Taxonomically placed in the α-2
Proteobacteria (Moreno et al., 1990), the brucellae are close
to plant pathogens and endosymbionts such as Agrobacterium,
Sinorhizobium, and Rhizobium and to soil bacteria such
as Ochrobactrum, the latter including some opportunistic
pathogens, and comparative analyses suggest that soil bacteria
of this group are endowed with properties that represent a first
scaffold on which an intracellular life style develops (Velasco
et al., 2000; Moreno and Moriyón, 2007; Barquero-Calvo et al.,
2009). The brucellae owe their pathogenicity mainly to their
ability to multiply within dendritic cells, macrophages, and a
variety of other cells. Due to their ability to control intracellular
trafficking and be barely detected by innate immunity, these
bacteria are able to reach a safe intracellular niche before
an effective immune response is mounted, and to multiply
extensively (Gorvel and Moreno, 2002; Barquero-Calvo et al.,
2007). A mechanism used by Brucella to scape from the
host immune response is the interference with the toll-like
receptor (TLR) signaling pathway by the injection of active
effectors such as BtpA and BtpB through the Type IV secretion
system T4SS. Both effector proteins contain a TIR domain
that interferes with TLR signaling by directly interacting with
MyD88 (Cirl et al., 2008; Salcedo et al., 2008, 2013; Chaudhary
et al., 2012) and contribute to the control of dendritic cell (DC)
activation during infection. Moreover, Brucella has modified
outer membrane (OM) components in order to reduce the
pathogen-associated molecular patterns (PAMP) of the cell
envelope. In Gram-negative bacteria, these PAMP are created by
the conserved composition of the OM lipopolysaccharide (LPS)
and the free lipids on which the topology of the OM also depends. However, in addition to free-lipid species present in most Gram-
negative
bacteria
(i.e.,
cardiolipin,
phosphatidylglycerol,
and
phosphatidylethanolamine),
Brucella
also
possesses
phosphatidylcholine and amino lipids. Phosphatidylcholine Citation: Conde-Álvarez R, Palacios-Chaves L,
Gil-Ramírez Y, Salvador-Bescos M,
Bárcena-Varela M, Aragón-Aranda B,
Martínez-Gómez E, Zúñiga-Ripa A,
de Miguel MJ, Bartholomew TL,
Hanniffy S, Grilló M-J,
Vences-Guzmán MÁ,
Bengoechea JA, Arce-Gorvel V,
Gorvel J-P, Moriyón I and Iriarte M
(2018) Identification of lptA, lpxE,
and lpxO, Three Genes Involved
in the Remodeling of Brucella Cell
Envelope. Front. Microbiol. 8:2657. doi: 10.3389/fmicb.2017.02657 January 2018 | Volume 8 | Article 2657 1 Frontiers in Microbiology | www.frontiersin.org Conde-Álvarez et al. Genes Remodeling Brucella Cell Envelope were not attenuated in dendritic cells or mice. This lack of an obvious effect on virulence
together with the presence of the intact homolog genes in O. anthropi and B. microti but
not in other brucellae suggests that LptA, LpxE, or OL β-hydroxylase do not significantly
alter the PAMP properties of Brucella LPS and free-lipids and are therefore not positively
selected during the adaptation to intracellular life. Keywords: lipopolysaccharide, Brucella, lipids, cell envelope, PAMP January 2018 | Volume 8 | Article 2657 Keywords: lipopolysaccharide, Brucella, lipids, cell envelope, PAMP MATERIALS AND METHODS Bacterial Strains and Growth Conditions
The bacterial strains and plasmids used in this study are listed
in Supplementary Table S1. Bacteria were routinely grown in
standard tryptic soy broth or agar either plain or supplemented
with kanamycin at 50 µg/ml, or/and nalidixic at 5 or 25 µg/ml
or/and 5% sucrose. All strains were stored in skim milk
at −80◦C. or/and 5% sucrose. All strains were stored in skim milk
at −80◦C. January 2018 | Volume 8 | Article 2657 Frontiers in Microbiology | www.frontiersin.org 2 Genes Remodeling Brucella Cell Envelope Conde-Álvarez et al. FIGURE 1 | Brucella lipid A and hypothetical sites of action of putative LpxE, LptA, and LpxO. The structure proposed is based on acyl-chain and mass
spectrophotometry analyses and genomic predictions. The predicted sites of action of LpxE (phosphatase), LptA (pEtN transferase), and LpxO (acyl chain
hydroxylase) are indicated, and the corresponding ORF of B. microti (green), B. melitensis (blue), and B. abortus (red) presented (NA, not annotated). The B. abortus
lptA homolog and the B. melitensis and B. abortus lpxO homologs carry a frame-shift mutation. FIGURE 1 | Brucella lipid A and hypothetical sites of action of putative LpxE, LptA, and LpxO. The structure proposed is based on acyl-chain and mass
spectrophotometry analyses and genomic predictions. The predicted sites of action of LpxE (phosphatase), LptA (pEtN transferase), and LpxO (acyl chain
hydroxylase) are indicated, and the corresponding ORF of B. microti (green), B. melitensis (blue), and B. abortus (red) presented (NA, not annotated). The B. abort
lptA homolog and the B. melitensis and B. abortus lpxO homologs carry a frame-shift mutation. 1http://www.genome.jp/kegg/
2http://www.ncbi.nlm.nih.gov/
3http://www.ebi.ac.uk/ DNA Manipulations Genomic sequences were obtained from the Kyoto Encyclopedia
of Genes and Genomes (KEGG) database1. Searches for DNA
and protein homologies were carried out using the National
Center
for
Biotechnology
Information
(NCBI2)
and
the
European Molecular Biology Laboratory (EMBL) – European
Bioinformatics Institute server3. Primers were synthesized
by
Sigma-Genosys
(Haverhill,
United
Kingdom). DNA
sequencing was performed by the “Servicio de Secuenciación
del Centro de Investigación Médica Aplicada” (Pamplona,
Spain). Restriction–modification enzymes were used under the
conditions recommended by the manufacturer. Plasmid and
chromosomal DNA were extracted with Qiaprep Spin Miniprep
(Qiagen) and Ultraclean Microbial DNA Isolation Kits (Mo Bio
Laboratories), respectively. When needed, DNA was purified
from agarose gels using the Qiack Gel Extraction Kit (Qiagen). To obtain Bme1lptA, Ba1lpxE, and Bmi1olsC in-frame deletion
mutants, directed mutagenesis by overlapping PCR were
performed using genomic DNA as template and pJQK (Scupham
and Triplett, 1997) as the suicide vector. The corresponding gene
was deleted using allelic exchange by double recombination as
previously described (Conde-Alvarez et al., 2006). p
y
For the construction of the Bme1lptA mutant, we first
generated two PCR fragments: oligonucleotides lptA-F1 (5′-GAA
CGCGAGACTATGGAAAC-3′)
and
lptA-R2
(5′-TGGT
GAACGCCAGAAGATAGA-3′)
were
used
to
amplify
a
400-bp fragment including codons 1–26 of BmelptA ORF,
as well as 324 bp upstream of the BmelptA start codon, and
oligonucleotides
lptA-F3
(5′-TCTATCTTCTGGCGTTCACC
GCACGACAATCTCTTC-3′) and lptA-R4 (5′-AATATTCCAT
GGCGCATTTC-3′) were used to amplify a 472-bp fragment
including codons 506–544 of the lptA ORF and 353-bp
downstream of the lptA stop codon. Both fragments were ligated
by overlapping PCR using oligonucleotides lptA-F1 and lptA-R4
for amplification, and the complementary regions between 1http://www.genome.jp/kegg/
2http://www.ncbi.nlm.nih.gov/
3http://www.ebi.ac.uk/ 1http://www.genome.jp/kegg/
2http://www.ncbi.nlm.nih.gov/
3http://www.ebi.ac.uk/ January 2018 | Volume 8 | Article 2657 Frontiers in Microbiology | www.frontiersin.org 3 Genes Remodeling Brucella Cell Envelope Conde-Álvarez et al. lptA-R2 and lptA-F3 for overlapping. The resulting fragment,
containing the lptA deleted allele, was cloned into pCR2.1
(Invitrogen, Barcelona, Spain), sequenced to ensure maintenance
of the reading frame, and subcloned into the BamHI and the
XbaI sites of the suicide plasmid pJQK. The resulting mutator
plasmid (pRCI-32) was introduced in B. melitensis 16M by
conjugation using the Escherchia coli S.17 strain (Simon et al.,
1983). the corresponding ORF was subcloned in plasmid pRH001
(Hallez et al., 2007) to produce pBMElpxE and pBMElptA. For pBMIolsC, olsC was amplified using genomic DNA of
Bmi-parental as DNA template. The primers used were olsC-
F6 (5′-GCTTTCCGAACAAGCACTGA-3′) and olsC-R7 (5′-
GCCTCCCTTCACCGGTTATT-3′). LPS Preparation Lipopolysaccharide was obtained by methanol precipitation of
the phenol phase of a phenol–water extract (Leong et al., 1970). This fraction [10 mg/ml in 175 mM NaCl, 0.05% NaN3, 0.1 M
Tris–HCl (pH 7.0)] was then purified by digestion with nucleases
[50 µg/ml each of DNase-II type V and RNase-A (Sigma, St. Louis, MO, United States), 30 min at 37◦C] and three times
with proteinase K (50 µg/ml, 3 h at 55◦C), and ultracentrifuged
(6 h, 100,000 × g) (Aragón et al., 1996). Free lipids (OLs and
phospholipids) were then removed by a fourfold extraction with
chloroform–methanol [2:1 (vol/vol)] (Velasco et al., 2000). For the construction of the Bmi1olsC mutant, we first
generated two PCR fragments: oligonucleotides olsC-F1 (5′-
TGCTGGATCGTATTCGTCTG-3′) and olsC-R2 (5′-GCCATAA
GCCGATGGAACTA-3′) were used to amplify a 334-bp fragment
including codons 1–15 of olsC ORF, as well as 289 bp upstream
of the olsC start codon, and oligonucleotides olsC-F3 (5′-TA
GTTCCATCGGCTTATGGCAGGAGGGGCTAGACAACCAC-
3′) and olsC-R4 (5′-AACCAGCGACAGGGTAAGC-3′) were
used to amplify a 320-bp fragment including codons 286–313
of the olsC ORF and 237-bp downstream of the olsC stop
codon. Both fragments were ligated by overlapping PCR using
oligonucleotides olsC-F1 and olsC-R4 for amplification, and
the complementary regions between olsC-R2 and olsC-F3 for
overlapping. The resulting fragment, containing the lptA deleted
allele, was cloned into pCR2.1 (Invitrogen, Barcelona, Spain),
sequenced to ensure maintenance of the reading frame, and
subcloned into the BamHI and the XbaI sites of the suicide
plasmid pJQK (Scupham and Triplett, 1997). The resulting
mutator plasmid (pRCI-65) was introduced in B. microti CM445
by conjugation using the E. coli S.17 strain (Simon et al., 1983). Sensitivity to Cationic Peptides Exponentially growing bacteria were adjusted to an optical
density equivalent to one of the McFarland scale and the
minimal inhibitory concentrations (MICs) of polymyxin B were
determined by the e-test method on Müller–Hinton agar (Izasa)
or by the serial dilution method in a similar broth. Infections in Mice Seven-week-old female BALB/c mice (Charles River, Elbeuf,
France) were kept in cages with water and food ad libitum and
accommodated under biosafety containment conditions 2 weeks
before the start of the experiments. To prepare inocula, tryptic
soy agar (TSA) grown bacteria were harvested and suspended in
10 mM phosphate buffered saline (pH 6.85), and 0.1 ml/mouse
containing approximately 5 × 104 colony forming units (CFU)
for B. melitensis or B. abortus and 1 × 104 CFU for B. microti
was administered intraperitoneally. The exact doses assessed
retrospectively by plating dilutions of the inocula. Number of
CFU in spleens was determined at diferent time after inoculation. For this, the spleens were aseptically removed and individually
weighed and homogenized in 9 volumes of PBS. Serial 10-fold
dilutions of each homogenate were performed and each dilution
was plated by triplicate. Plates were incubated at 37◦C for 5 days. At several points during the infection process, the identity of
the spleen isolates was confirmed by PCR. The individual data
were normalized by logarithmic transformation, and the mean
log CFU/spleen values and the standard deviations (n = 5) were
calculated. Seven-week-old female BALB/c mice (Charles River, Elbeuf,
France) were kept in cages with water and food ad libitum and
accommodated under biosafety containment conditions 2 weeks
before the start of the experiments. To prepare inocula, tryptic
soy agar (TSA) grown bacteria were harvested and suspended in
10 mM phosphate buffered saline (pH 6.85), and 0.1 ml/mouse
containing approximately 5 × 104 colony forming units (CFU)
for B. melitensis or B. abortus and 1 × 104 CFU for B. microti
was administered intraperitoneally. The exact doses assessed
retrospectively by plating dilutions of the inocula. Number of
CFU in spleens was determined at diferent time after inoculation. Deletion of each gene was checked with oligonucleotides
gene-F1 and gene-R4 and internal primers hybridizing in the
non-deleted regions. DNA Manipulations The resulting PCR product,
containing the ORF from 342 bp upstream to 84 bp downstream,
was then cloned into pCR2.1 TOPO (Invitrogen) plasmid by
“TA cloning” (Life Technologies). The resulting plasmid was
sequenced to ensure that the gene was correctly cloned. Then,
the gene was subcloned into the BamHI and the XbaI sites
of the replicative plasmid pBBR1 MCS (Kovach et al., 1994)
pBMElpxE, pBMElptA, and pBMIolsC were introduced into
Brucella by conjugation using E. coli S.17-1 strain and the
conjugants harboring corresponding plasmid were selected by
plating onto TSA-Nal-Cm plates. For the construction of the Ba1lpxE mutant, we first
generated two PCR fragments: oligonucleotides lpxE-F1 (5′-
CGCGTGTGCCATAGGTATATT-3′) and lpxE-R2 (5′-TATAGG
CAGGGCGCAGAA-3′) were used to amplify a 482-bp fragment
including codons 1–29 of lpxE ORF, as well as 394 bp upstream of
the lpxE-1 start codon, and oligonucleotides lpxE-F3 (5′-TTCTG
CGCCCTGCCTATAGATTCGTTTCCGCATGGT-3′) and lpxE-
R4 (5′-CCAATACAC CCGTCATGAGA-3′) were used to amplify
a 577-bp fragment including codons 226–255 of the lpxE ORF
and 488-bp downstream of the lpxE stop codon. Both fragments
were ligated by overlapping PCR using oligonucleotides lpxE-F1
and lpxE-R4 for amplification, and the complementary regions
between lpxE-R2 and lpxE-F3 for overlapping. The resulting
fragment, containing the lpxE deleted allele, was cloned into
pCR2.1 (Invitrogen, Barcelona, Spain), sequenced to ensure
maintenance of the reading frame, and subcloned into the BamHI
and the XbaI sites of the suicide plasmid pJQK (Scupham and
Triplett, 1997). The resulting mutator plasmid (pRCI-36) was
introduced in B. abortus 2308 by conjugation using the E. coli
S.17 strain (Simon et al., 1983). Complementation of Deleted Genes For pBMElpxE and pBMElptA construction we took advantage
of the Brucella ORFeome constructed with the Gateway cloning
Technology (Invitrogen) (Dricot et al., 2004). The clones carrying
BmelpxE or BmelptA were extracted and the DNA containing January 2018 | Volume 8 | Article 2657 Frontiers in Microbiology | www.frontiersin.org 4 Genes Remodeling Brucella Cell Envelope Conde-Álvarez et al. Flow Cytometry To assess activation and maturation, BMDC were analyzed
for surface expression of classical maturation markers at 24 h
post-treatment with the different Brucella strains and derived
mutants. Cells were labeled with fluorochrome-conjugated
antibodies specific for mouse CD11c:APC-Cy7 (clone N418),
IA-IE:PE (MHC class II clone M5/114.15.2) (PE), CD86:FITC
(Clone GL-1), CD40:APC (clone 3/23), and CD80:PE-Cy5 (clone
16-10A1), all from BioLegend. Labeled cells were then subjected
to multi-color cytometry using a LSR II UV (Becton Dickinson)
and the data analyzed using FlowJo Software by first gating on
the CD11c+ population (100,000 events) prior to quantifying
expression of receptors. Cells were stimulated with E. coli LPS
(055:B5) as a positive control. A genomic search in the KEGG database revealed that all Brucella
spp. carry an ORF (BMEI0118 in B. melitensis) homologous
to Neisseria meningitidis lptA, a pEtN transferase that modifies
lipid A (Cox et al., 2003). Strikingly, in B. abortus but not
in other Brucella spp., all genomic sequences available at
KEGG show a deletion of a thymine in position 774 that
should result in a truncated protein lacking the amino acids
related to the enzymatic activity (Naessan et al., 2008; Figure 1
and Supplementary Figure S1 and Supplementary Table S2). In addition to LptA, two other pEtN transferases have been
identified in N. meningitidis: Lpt-3 and Lpt-6, which, respectively,
modify the LPS core at the third and sixth position of heptose II
(Mackinnon et al., 2002; Wright et al., 2004). By multiple
sequence alignment, the B. melitensis putative pEtN transferase
showed highest homology with Neisseria LptA and also displayed
the LptA membrane-associated domains not present in Lpt-3 and
Lpt-6 (ORFs NMB1638, NMB2010, and NMA0408, respectively). Accordingly, it can be predicted that ORF BMEI0118 (henceforth
BMElptA) encodes a pEtN transferase that acts on lipid A, a
hypothesis fully consistent with the absence of heptose in the
Brucella LPS core (Iriarte et al., 2004; Fontana et al., 2016). Lipid A Extraction Five milligrams of LPS was hydrolyzed in 5 ml 1% acetic acid by
sonication, heating to 100◦C for 30 min, and cooling to room
temperature. Concentrated HCl was added to the mixture until
the pH was 1–2. The solution was converted to a two-phase
acidic Bligh–Dyer mixture by adding 5.6 ml of chloroform and
5.6 ml of methanol. Phases were mixed by inverting the tubes
and separated by centrifugation at 4000 × g for 20 min. The
lower phases containing lipid A were collected, washed two times
with water, and dried under a stream of nitrogen. Extraction was
repeated, and the lower phases (11.2 ml) were combined and
neutralized with a drop of pyridine. Samples were evaporated to
dryness under a stream of nitrogen. To test this hypothesis, we constructed a B. melitensis
non-polar mutant (Bme1lptA) lacking the LptA enzymatic
domain (amino acids 26–506), which as expected maintained a
smooth (S) phenotype (negative crystal violet test and positive
coagglutination with anti-S-LPS antibodies). As a consequence
of the increased positive charge of the amino group, pEtN has
been shown to decrease binding of the polycationic lipopeptide
polymyxin B to LPS, and to increase resistance to this antibiotic
in a variety of bacteria (Needham and Trent, 2013; Trombley
et al., 2015; Herrera et al., 2017). In keeping with this possibility,
the Bme1lptA mutant was more sensitive to polymyxin B
than the parental strain B. melitensis 16M (Bme-parental)
(Figure 2A). In contrast, and consistent with the frame-shift
in its lptA homolog, B. abortus 2308 (Ba-parental) displayed
polymyxin B sensitivity similar to that of Bme1lptA. Moreover,
complementation of Bme1lptA with the multi-copy plasmid Intracellular Multiplication Assays ntracellular Multiplication Assays Intracellular Multiplication Assays
Bone
marrow
cells
were
isolated
from
femurs
of
7–8-
week-old C57Bl/6 female and differentiated into dendritic
cells
[bone-marrow
derived
dendritic
cells
(BMDCs)]
as
described by Inaba et al. (1992). Infections were performed
by centrifuging the bacteria onto the differentiated cells
(400 x g for 10 min at 4◦C; bacteria:cells ratio of 30:1
followed by incubation at 37◦C for 30 min under a 5%
CO2 atmosphere). BMDCs were gently washed with medium
to remove extracellular bacteria before incubating in medium
supplemented with 50 µg/ml gentamicin for 1 h to kill
extracellular bacteria. Thereafter, the antibiotic concentration
was decreased to 10 µg/ml. To monitor Brucella intracellular
survival at different time-points post-infection, BMDC were
lysed with 0.1% (vol/vol) Triton X-100 in H2O and serial
dilutions of lysates were plated onto TSA plates to enumerate
the CFU. The free-lipid fraction was extracted as described by Bligh
and Dyer (1959), and analyzed on a silica gel 60 high-
performance thin layer chromatography (HPLC) plates (Merck,
Darmstadt, Germany). Chromatography was performed either
monodimensionally with chloroform–methanol–water [14:6:1
(volume)] or bidimensionally with chloroform–methanol–water
[14:6:1 (volume)] first and chloroform–methanol–acetic acid
[13:5:2 (volume)] in the second dimension (Weissenmayer et al.,
2002). Plates were developed with 0.2% ninhydrin in acetone at
180◦C or 15% sulfuric acid in ethanol at 180◦C. Mass Spectrometry Mass spectrometra were acquired on a Bruker Autoflex
R⃝Speed
TOF/TOF Mass Spectrometer (Bruker Daltonics Inc.) in negative
reflective mode with delayed extraction. The ion-accelerating
voltage was set at 20 kV. Each spectrum was an average of 300
shots. A peptide calibration standard (Bruker Daltonics Inc.) was
used to calibrate the Matrix Assisted Laser Desorption/Ionization
Time-of-Flight (MALDI-TOF), and lipid A extracted from E. coli
strain MG1655 grown in LB medium at 37◦C. January 2018 | Volume 8 | Article 2657 Frontiers in Microbiology | www.frontiersin.org 5 Genes Remodeling Brucella Cell Envelope Conde-Álvarez et al. FIGURE 2 | The Brucella lptA orthologs are involved in polymyxin B resistance
and code for a phosphate-ethanolamine transferase acting on lipid A. (A) Polymyxin B sensitivity of B. melitensis wild-type (Bme-parental),
B. melitensis non-polar lptA mutant (BmelptA), the cognate complemented
mutant (Bme1lptApBMElptA), B. abortus wild-type (Ba-parental), and
B. abortus wild-type carrying a plasmid with the B. melitensis lptA gene
(Ba-parentalpBMElptA) (the results are representative of three independent
experiments). (B) MALDI-TOF analysis of the lipid A of Bme-parental,
Bme1lptA, and Ba-parental. pBMElptA or its introduction into B. abortus 2308 leads to both constructs kept the S type features (negative crystal violet
test and positive coagglutination with anti-S-LPS antibodies) of
the parental strains. N. gonorrhoeae shows increased resistance
to the action of complement in non-immune serum that is
dependent on lipid A-linked pEtN (Lewis et al., 2013). Testing for
a similar contribution here, we found that Bme1lptA was more
sensitive than either the parental strain or the complemented
mutant (25% vs. no decrease in viability after 3 h of incubation
in normal sheep serum) relevant given that B. melitensis is
characteristically resistant to killing by normal serum. y
g y
By MALDI-TOF analysis, the lipid A of Bme-parental was
found to contain four main clusters of ions (A, B, C, and D
in Figure 2B). Bme1lptA lipid A was qualitatively identical to
Bme-parental with respect to groups A, B, and C but clearly
differed in group D (Figure 2B and Supplementary Table S3). In group D, the 2191 m/z′′ species of Ba-parental was consistent
with the isotopic mass of a molecule (C120H232N4O25P2)
formed by a hexaacylated and bisphosphorylated diaminoglucose
disaccharide carrying the hydroxylated long and very long chain
acyl groups characteristic of Brucella (Velasco et al., 2000;
Ferguson et al., 2004). Mass Spectrometry According to this interpretation, the
signal(s) at 2112 m/z (mass of - H2PO3 o - HPO3 -, 80.9 - 79.9)
could correspond to a monophosphorylated (C120H232N4O25P)
2191 m/z′′ equivalent. Substitution of this monophosphorylated
form with pEtN (+H3NCH2-CH2- HPO3 mass 125) should
account for signal m/z 2237, in keeping with the fact that m/z
2237 did not appear in the spectrum of the lipids A from either
Bme1lptA or Ba-parental (Figure 2B). Although a clear cut
demonstration requires direct analyses of the enzymatic analyses
of LptA, these results and the homologies with LptA of other
bacteria are consistent with the hypothesis that LptA acts as
a pEtN transferase in B. melitensis and lacks functionality in
B. abortus. It is remarkable that pEtN activity was detected for
only a fraction (D) of lipid A species. This could be explained
by a preferential activity of the enzyme for higher MW lipid A
molecules. The Brucella lpxE Orthologs Encode a
Phosphatase Involved in the Remodeling
of the OM As
described
above,
MALDI-TOF
analyses
showed
the
presence
of
molecular
species
with
a
mass
compatible
with monophosphorylated lipid A. Since lipid A synthesis
produces C1 and C4′ bisphosphorylated disaccharide backbones
(Qureshi et al., 1994), a possible explanation could be its
dephosphorylation by a phosphatase such as LpxE, an inner
membrane enzyme that in the phylogenetic neighbor Rhizobium
leguminosarum removes the lipid A phosphate at C1 (Raetz
et al., 2009). A search in KEGG showed that all Brucella
spp. carry an ORF homologous to R. leguminosarum lpxE
(Supplementary Table S2). However, the start codon in the
B. melitensis 16M homolog (BMEI1212) is annotated to a
position different from that determined for other brucellae
(Supplementary Table S2), including other B. melitensis strains. Thus, whereas the B. abortus homolog (BAB1_0671) is predicted
to encode a protein of 255 amino acids, the B. melitensis FIGURE 2 | The Brucella lptA orthologs are involved in polymyxin B resistance
and code for a phosphate-ethanolamine transferase acting on lipid A. (A) Polymyxin B sensitivity of B. melitensis wild-type (Bme-parental),
B. melitensis non-polar lptA mutant (BmelptA), the cognate complemented
mutant (Bme1lptApBMElptA), B. abortus wild-type (Ba-parental), and
B. abortus wild-type carrying a plasmid with the B. melitensis lptA gene
(Ba-parentalpBMElptA) (the results are representative of three independent
experiments). (B) MALDI-TOF analysis of the lipid A of Bme-parental,
Bme1lptA, and Ba-parental. pBMElptA or its introduction into B. abortus 2308 leads to
restoration of polymyxin B resistance in Bme1lptA or an increase
up to B. melitensis level in B. abortus (Figure 2A). As expected January 2018 | Volume 8 | Article 2657 Frontiers in Microbiology | www.frontiersin.org 6 Genes Remodeling Brucella Cell Envelope Conde-Álvarez et al. this ORF presents a frame-shift leading to a truncated protein that
lacks the consensus of the aspartyl/asparaginyl β-hydroxylases
family to which LpxO belongs (Figure 1 and Supplementary
Table S2). These characteristics are consistent with chemical
studies that previously failed to observe S2 hydroxylated fatty
acids in B. abortus lipid A (Velasco et al., 2000). Moreover, a
lpxO homolog is present in Ochrobactrum anthropi where S2
hydroxylated fatty acids were also not observed in the lipid A
(Velasco et al., 2000), indicating that a role similar to that of
Salmonella LpxO is unlikely. Thus, the lpxO homologs present
in these B. microti and O. anthropi could be acting on a free
lipid and, in fact, it has been reported that the corresponding
R. The Brucella lpxE Orthologs Encode a
Phosphatase Involved in the Remodeling
of the OM tropici homolog is a β-hydroxylase acting on OLs (Vences-
Guzmán et al., 2011). If this were the case in O. anthropi
and the brucellae, the end product [a hydroxylated OL (OH–
OL)] of the pathway described previously in members of the
Rhizobiaceae (Figure 3A) should be observed in O. anthropi
and B. microti (and B. vulpis) but not in other Brucella
spp. one could encode a protein of either 235 or 255 amino acids
(Figure 1). Both proteins conserve the consensus sequence of
the lipid phosphatase superfamily [KX6RP-(X12–54)-PSGH-
(X31–54)-SRX5HX3D] (Stukey and Carman, 2008) which is also
present in LpxE from R. leguminosarum, Sinorhizobium meliloti,
and Agrobacterium tumefaciens (Karbarz et al., 2003). Although
BAB1_0671 and BMEI1212 code for proteins that contain the
three motifs conserved in the LpxF phosphatase from Francisella,
they lack two amino acids of the central motif, NCSFX2G, which
seems LpxF specific (Wang et al., 2006, 2007). Thus, the Brucella
proteins were named BALpxE and BMELpxE. To study whether BALpxE actually acts as a lipid A
phosphatase, we constructed a non-polar mutant (Ba1lpxE)
and tested it against polymyxin B, since the permanence of a
phosphate group in an OM molecule should increase sensitivity
to this antibiotic. Mutant Ba1lpxE was eight times more sensitive
than the parental strain (MIC 0.2 and 1.6 µg/ml, respectively). Moreover, when we introduced a plasmid containing the
BMElpxE ortholog into Ba1lpxE, the resistance to polymyxin B
was restored (MIC 1.6 µg/ml). Although final confirmation of
this interpretation would require to assay the enzymatic activity
of the protein, these results are consistent with the predicted role
of lpxE as a phosphatase and its functionality in both B. abortus
and B. melitensis 16M, a strain where the annotation of the start
codon was a source of ambiguity. To investigate these hypotheses, we compared the free lipids
of B. abortus, B. melitensis, B. suis, B. ovis, B. microti, and
O. anthropi. As can be seen in Figure 3B, B. microti but
not B. abortus produced an amino lipid with the migration
pattern predicted for OH-OL (Vences-Guzmán et al., 2011),
and results similar to those of B. microti were obtained for
O. anthropi but not for the other Brucella spp. tested (not
shown). These observations support the interpretation that
O. anthropi and B. microti LpxO are OL hydroxylases and are
fully consistent with the aforementioned genomic and chemical
evidence. Accordingly, Brucella lpxO should be named olsC. The Brucella lpxE Orthologs Encode a
Phosphatase Involved in the Remodeling
of the OM To
confirm this, we examined the amino lipids of a non-polar olsC
mutant in B. microti (Bmi1olsC). As predicted, this mutant did
not synthesize OH–OL and complementation with a plasmid
containing B. microti olsC restored the wild-type phenotype
(Figure 3C). Furthermore, introducing this plasmid or a plasmid
carrying O. anthropi olsC into B. abortus resulted in the
synthesis of OH–OL (Figure 3C and Supplementary Figure S2). No difference in polymyxin sensitivity was observed in these
constructs or the mutant Bmi1olsC when compared to the
corresponding parental strains. g
y
By MALDI-TOF analysis, the Ba-parental lipid A spectrum
showed three of the four predominant clusters of ions (A, B,
and C) found in B. melitensis (Figure 2B and Supplementary
Table S3). Cluster A (m/z 2173) was consistent with an
hexaacylated
bisphoshorylated
diaminoglucose
disaccharide
(C120H232N4O24P2) and the signal at 2093 m/z, which differed
in the mass of one phosphate group (i.e., 80), was consistent with
the cognate monophosphorylated lipid A (C120H232N4O25P)
(A-Pi, Figure 2). Other signals differing in a mass of 14 or 28
units should result from the heterogeneity in acyl chain length
that is typical of lipid A. The B and C clusters also contained
signals differing in 80 mass units that could correspond to
bis- and mono-phosphorylated species. The mass spectrum of
Ba1lpxE lipid A (not shown) did not differ significantly from
that of Ba-parental, and again showed acyl chain heterogeneity
in the A, B, C clusters, as well as the −80 m/z signals indicative
of mono- and bisphoshorylated lipid A species. As mutation of
lpxE is concomitant with an increase in polymyxin B sensitivity,
it is tempting to speculate that LpxE directly or indirectly
modulates Brucella cell envelope by removing an accessible
phosphate group from a substrate different from lipid A. Further studies need to be performed to clarify the role of
LpxE. LptA, LpxE, and OlsC Are Not Required
for Brucella Virulence in Laboratory
Models Brucella abortus, B. melitensis, and B. suis have been shown
to multiply in murine and human monocyte-derived dendritic
cells while interfering with their activation and maturation and
reducing both antigen presentation and an effective adaptive
response (Billard et al., 2007; Martirosyan et al., 2011; Conde-
Álvarez et al., 2012; Gorvel et al., 2014; Papadopoulos et al.,
2016). To assess whether LptA, LpxE, and OL β-hydroxylase
(OlsC) were involved, we compared parental and mutant strains
of B. melitensis, B. abortus, and B. microti in mouse BMDCs. As shown in Figure 4, the kinetics of multiplication of the
mutants and wild-type strains were similar. We also performed
a phenotypic characterization of MHC II and co-stimulatory The Brucella lpxO Orthologs Encode an
Acyl Hydroxylase Acting on Ornithine
Lipids carry
a frame-shift in the olsC homolog. (B) Lipid profile of B. abortus wild-type
(Ba-parental) and B. microti wild-type (Bmi-parental) showing the absence or
presence, respectively, of OH–OL. (C) Amino lipid profile of B. abortus
wild-type (Ba-parental), B. microti wild-type (Bmi-parental), B. microti deleted
in olsC (Bmi1olsC), the cognate reconstituted mutant (Bmi1olsCpOlsC), and
B. abortus wild-type carrying a plasmid with the B. microti olsC gene
(Ba-parentalpOlsC). on ornithine lipids. (A) Pathway of synthesis of ornithine lipids in α 2
Proteobacteria (adapted from Geiger et al., 2010); the ORFs of B. abortus and
B. microti are indicated, whereas B. microti, B. vulpis, and O. anthropi contain
an intact olsC acyl hydroxylase gene, B. abortus and other Brucella spp. carry
a frame-shift in the olsC homolog. (B) Lipid profile of B. abortus wild-type
(Ba-parental) and B. microti wild-type (Bmi-parental) showing the absence or
presence, respectively, of OH–OL. (C) Amino lipid profile of B. abortus
wild-type (Ba-parental), B. microti wild-type (Bmi-parental), B. microti deleted
in olsC (Bmi1olsC), the cognate reconstituted mutant (Bmi1olsCpOlsC), and
B. abortus wild-type carrying a plasmid with the B. microti olsC gene
(Ba-parentalpOlsC). FIGURE 4 | LptA, LpxE, and OlsC deletions do not alter the Brucella
interaction with dendritic cells. Intracellular replication in BMDCs (each point
represents the mean ± standard error of the logarithm of CFU in dendritic
cells). receptors CD86 and CD80 (Figure 5). In agreement with
previous studies, these analyses showed that activation and
maturation was only partially induced in BMDC infected with
B. melitensis and B. abortus (Martirosyan et al., 2011). In
addition, a similar partial-activation profile was evident both for
B. microti, for which no previous studies exist in infected BMDC,
and all of the tested mutants obtained for each of the three
Brucella spp. alter the CFU/spleen profile produced by this species which is
characterized by a lower lethal dose in mice as well as a faster
clearance from mouse spleens (Jiménez de Bagüés et al., 2010;
Figure 6, lower left panel). Moreover, when we tested whether
the expression of B. microti olsC in B. abortus could affect
virulence, we found no differences between the B. microti olsC-
carrying and the wild-type B. abortus strains (Figure 6, lower
right panel). The mouse model has been widely used for testing Brucella
virulence (Grilló et al., 2012). In this model, the LptA and
LpxE mutants and the parental strains behaved identically
(Figure 6 upper panels). Frontiers in Microbiology | www.frontiersin.org The Brucella lpxO Orthologs Encode an
Acyl Hydroxylase Acting on Ornithine
Lipids The genomes of all Brucella species available at KEGG contain
an ORF homologous to Salmonella lpxO (Gibbons et al., 2000),
which encodes an enzyme hydroxylating the 3′-secondary acyl
chain of lipid A. In all Brucella spp. except B. microti and B. vulpis January 2018 | Volume 8 | Article 2657 Frontiers in Microbiology | www.frontiersin.org 7 Genes Remodeling Brucella Cell Envelope Conde-Álvarez et al. FIGURE 3 | The Brucella lpxO orthologs encode an acyl hydroxylase acting
on ornithine lipids. (A) Pathway of synthesis of ornithine lipids in α-2
Proteobacteria (adapted from Geiger et al., 2010); the ORFs of B. abortus and
B. microti are indicated, whereas B. microti, B. vulpis, and O. anthropi contain
an intact olsC acyl hydroxylase gene, B. abortus and other Brucella spp. carry
a frame-shift in the olsC homolog. (B) Lipid profile of B. abortus wild-type
(Ba-parental) and B. microti wild-type (Bmi-parental) showing the absence or
presence, respectively, of OH–OL. (C) Amino lipid profile of B. abortus
wild-type (Ba-parental), B. microti wild-type (Bmi-parental), B. microti deleted
in olsC (Bmi1olsC), the cognate reconstituted mutant (Bmi1olsCpOlsC), and
B. abortus wild-type carrying a plasmid with the B. microti olsC gene
(Ba-parentalpOlsC). receptors CD86 and CD80 (Figure 5)
In agreement with
FIGURE 4 | LptA, LpxE, and OlsC deletions do not alter the Brucella
interaction with dendritic cells. Intracellular replication in BMDCs (each point
represents the mean ± standard error of the logarithm of CFU in dendritic FIGURE 4 | LptA, LpxE, and OlsC deletions do not alter the Brucella
interaction with dendritic cells. Intracellular replication in BMDCs (each point
represents the mean ± standard error of the logarithm of CFU in dendritic
cells). FIGURE 4 | LptA, LpxE, and OlsC deletions do not alter the Brucella
interaction with dendritic cells. Intracellular replication in BMDCs (each point
represents the mean ± standard error of the logarithm of CFU in dendritic
cells). FIGURE 3 | The Brucella lpxO orthologs encode an acyl hydroxylase acting
on ornithine lipids. (A) Pathway of synthesis of ornithine lipids in α-2 FIGURE 3 | The Brucella lpxO orthologs encode an acyl hydroxylase acting
on ornithine lipids. (A) Pathway of synthesis of ornithine lipids in α-2
Proteobacteria (adapted from Geiger et al., 2010); the ORFs of B. abortus and
B. microti are indicated, whereas B. microti, B. vulpis, and O. anthropi contain
an intact olsC acyl hydroxylase gene, B. abortus and other Brucella spp. The Brucella lpxO Orthologs Encode an
Acyl Hydroxylase Acting on Ornithine
Lipids Deletion of olsC in B. microti did not January 2018 | Volume 8 | Article 2657 Frontiers in Microbiology | www.frontiersin.org 8 Genes Remodeling Brucella Cell Envelope Conde-Álvarez et al. FIGURE 5 | LptA, LpxE, and OlsC deletions do not significantly impact the intrinsic immunogenicity of Brucella. Each point represents the mean ± standard error of
the median intensity of surface receptor expression in dendritic cells treated with Brucella strains or derived mutants. E. coli LPS was used as a positive control for
dendritic cell activation. FIGURE 5 | LptA, LpxE, and OlsC deletions do not significantly impact the intrinsic immunogenicity of Brucella. Each point represents the mean ± standard error of
the median intensity of surface receptor expression in dendritic cells treated with Brucella strains or derived mutants. E. coli LPS was used as a positive control for
dendritic cell activation. FIGURE 5 | LptA, LpxE, and OlsC deletions do not significantly impact the intrinsic immunogenicity of Brucella. Each point represents the mean ± standard error of
the median intensity of surface receptor expression in dendritic cells treated with Brucella strains or derived mutants. E. coli LPS was used as a positive control for
dendritic cell activation. sC deletions do not significantly impact the intrinsic immunogenicity of Brucella. Each point represents the mean ± standard error o
eceptor expression in dendritic cells treated with Brucella strains or derived mutants. E. coli LPS was used as a positive control for Frontiers in Microbiology | www.frontiersin.org DISCUSSION BALB/c mice were inoculated
intraperitoneally with 5 × 104 (Bme-parental, Bme1lptA, Ba-parental, and Ba1lpxE) or 1 × 104 (Bmi-parental and Bmi1olsC) CFU/mouse and CFU/spleen
determined at the indicated times. Each point represents the mean ± standard error of the logarithm of CFU in the spleens of five animals. FIGURE 6 | The OM properties that depend on LptA, LpxE, and OlsC are not required for Brucella virulence in the mouse model. BALB/c mice were inoculated
intraperitoneally with 5 × 104 (Bme-parental, Bme1lptA, Ba-parental, and Ba1lpxE) or 1 × 104 (Bmi-parental and Bmi1olsC) CFU/mouse and CFU/spleen
determined at the indicated times. Each point represents the mean ± standard error of the logarithm of CFU in the spleens of five animals. FIGURE 6 | The OM properties that depend on LptA, LpxE, and OlsC are not required for Brucella virulence in the mouse model. BALB/c mice were inoculated
intraperitoneally with 5 × 104 (Bme-parental, Bme1lptA, Ba-parental, and Ba1lpxE) or 1 × 104 (Bmi-parental and Bmi1olsC) CFU/mouse and CFU/spleen
determined at the indicated times. Each point represents the mean ± standard error of the logarithm of CFU in the spleens of five animals. very long chain fatty acids (VCLFA). Although further chemical
and enzymatic analyses are necessary for a definite conclusion,
our results strongly suggest that, if present, pEtN is in much
less amounts in B. abortus than in B. melitensis lipid A. It is
also worth noting that such genetic and phenotypic differences
in the lipid A of B. abortus and B. melitensis could relate to
differences in biological properties. The LPS of B. abortus and
B. melitensis is a poor activator of the complement cascade, and
this property has been traced to the core and lipid A structure
(Moreno et al., 1981; Conde-Álvarez et al., 2012; Fontana et al.,
2016). Since B. abortus is less resistant than B. melitensis to
normal serum (González et al., 2008), it is tempting to suggest
that, like in N. gonorrhoeae, B. melitensis pEtN could sequester
regulatory elements enhancing complement resistance in this
species. displayed by the LPS of B. abortus and B. melitensis (Martínez
de Tejada et al., 1995; Lapaque et al., 2006; Conde-Álvarez
et al., 2012), our results do not support a role for BALpxE as
a lipid A phosphatase. DISCUSSION pEtN promotes binding to N. gonorrhoeae lipid A of factors that
downregulate the complement cascade and thwart building of
the membrane-attack complex and opsonophagocytosis (Lewis
et al., 2013). N. meningitidis pEtN also promotes adhesion of
non-encapsulated bacteria to endothelial cells (Takahashi et al.,
2008). Indeed, properties that parallel some of those observed
for the above-listed pathogens can also be attributed to the pEtN
transferase counterpart in Brucella. An intact lptA was related to
polymyxin B resistance in B. melitensis and the introduction of
B. melitensis lptA into B. abortus increases polymyxin B resistance
to the level of B. melitensis, suggesting that LptA function is
severally impaired in B. abortus. This is in agreement with
the presence of a frame-shift in B. abortus lptA encompassing
the consensus sequence, which makes likely that it codes for a
protein with no or residual enzymatic activity. Previous analyses
are contradictory with regard to the presence (Casabuono et al.,
2017) or absence (Moreno et al., 1990) of ethanolamine in
B. abortus lipid A but the materials analyzed differ in methods
of extraction and presence of B. abortus lipid A markers, such as In this work we investigated three Brucella ORFs that according
to homologies with genes of known function in other pathogens
could modify the lipid A and contribute to further altering the
LPS PAMP of representative Brucella species. The results show
that, whereas Brucella LptA modifies the lipid A, this is not the
case for lpxE and lpxO (redesignated olsC), the former encoding a
putative phosphatase acting on an unidentified OM molecule and
the latter for an enzyme with OlsC activity. Our data strongly suggest that B. melitensis LptA is involved
in the addition of pEtN to lipid A, homologous proteins
carrying out this function are not uncommon in Gram-negative
pathogens and modulate the properties of lipid A. In Salmonella
Typhimurium,
Shigella
flexneri,
E. coli,
Vibrio
cholerae,
Helicobacter
pylori,
Haemophilus
ducreyi,
N. gonorrhoeae,
and N. meningitidis pEtN reduces the binding of cationic
bactericidal peptides by balancing the negative charge of lipid A
(Needham and Trent, 2013; Trombley et al., 2015). Conversely, January 2018 | Volume 8 | Article 2657 Frontiers in Microbiology | www.frontiersin.org 9 Conde-Álvarez et al. Genes Remodeling Brucella Cell Envelope FIGURE 6 | The OM properties that depend on LptA, LpxE, and OlsC are not required for Brucella virulence in the mouse model. Frontiers in Microbiology | www.frontiersin.org DISCUSSION This is consistent with genomic analysis
showing that, whereas in bacteria where LpxE acts on lipid
A the gene is located together with lptA in an operon (Tran
et al., 2006; Renzi et al., 2015), Brucella lpxE is instead located
upstream of three sequences annotated as pseudogenes and
downstream, but in the opposite direction, of a cystathionine
beta-lyase. On the basis of the data shown here, the origin of
monophosphoryl lipid A in Brucella remains to be explained. Further, we believe it unlikely to be an artifact resulting from
the hydrolytic steps used to obtain lipid A and instead favor
the hypothesis of the existence of an as yet unidentified lipid A
phosphatase. LpxE belongs to the type 2 family of phosphatases that can
act on lipid A but also on phosphatidylglycerol phosphate,
phosphatidic acid, sphingosine phosphate, and lysophosphatidic
acid (Brindley and Waggoner, 1998; Sciorra and Morris,
2002). Significantly,
LpxE
from
Agrobacterium,
although
predicted to be a lipid A phosphatase, dephosphorylates Concerning LpxE, phosphatases acting on lipid A have at least
been shown in Francisella tularensis, H. pylori, Porphyromonas
gingivalis, and Capnocytophaga canimorsus, bacteria where lipid
A dephosphorylation is involved both in resistance to bactericidal
peptides and the reduction of TLR-4-dependent recognition
(Needham and Trent, 2013). Although these properties are January 2018 | Volume 8 | Article 2657 Frontiers in Microbiology | www.frontiersin.org 10 Genes Remodeling Brucella Cell Envelope Conde-Álvarez et al. phosphatidyl glycerophosphate (Karbarz et al., 2009) to generate
phosphatidylglycerol, a cell envelope phospholipid. Indeed,
a
hypothetical
phosphatidyl
glycerophosphate
phosphatase
activity of Brucella LpxE could account for both the polymyxin
B sensitivity of the mutated bacteria and the unaltered mass
spectra of the lipid A of the mutant. Such a modification
of a phospholipid could be meaningful by itself on account
of the LpxE-dependent bactericidal peptide resistance but
there are other possibilities. In some bacteria (i.e., Rhizobium)
phosphatidylglycerol is a precursor for the synthesis of amino
lipids such as lysyl-phosphatidylglycerol. This synthesis is
induced by acid pH and brings about resistance to daptomycin
and polymyxin B (Sohlenkamp et al., 2007; Ernst and Peschel,
2011; Arendt et al., 2012). Interestingly, whereas the Ba1lpxE
mutant is impaired for growth at pH 6, the parental B. abortus
becomes more resistant to cationic peptides (L. Palacios-Chaves
and
R. Conde-Álvarez,
Unpublished
observations). These
observations suggest the existence in Brucella of pH-dependent
envelope modifications that require a functional LpxE. DISCUSSION Research
is in progress to elucidate the mechanisms behind the increased
resistance at acid pH and the implication regarding a role for
LpxE. lpxE, or olsC do not play a role in the ability of Brucella to replicate
in BMDC and do not modulate the activation and maturation
profile in these cells. Similarly, the mouse model did not reveal
any effect on its ability to colonize and multiply in the spleen. However, further experimental work in the natural hosts and
alternative routes of infection might provide evidence on the
role in virulence of these genes. The fact that lptA and olsC are
not functional in all Brucella spp. must therefore be considered
in the context of the models used. While the absence of a
functional lptA in B. abortus suggests that the gene is not essential
for the virulence of this species we cannot conclude it to be
totally irrelevant. Differences between B. melitensis and B. abortus
related to lptA could explain the higher invasiveness of the former
species noted by early researchers in studies carried out in guinea
pigs, animals that are highly susceptible to brucellosis (Braude,
1951). This possibility together with the presence of intact lptA
and olsC in Ochrobactrum and B. microti is also compatible with
the hypothesis that they represent ancestral characters that are
liable to be lost in the absence of a selective pressure during
the intracellular life cycle or, in the case of lptA, that is no
longer present in the ruminant host species (i.e., cattle) to which
B. abortus is characteristically associated. p
In S. Typhimurium, Pseudomonas aeruginosa, Bordetella
bronchispetica,
Legionella
pneumophila,
and
Klebsiella
pneumonia,
LpxO
is
a
Fe2+/α-ketoglutarate-dependent
dioxygenase that catalyzes the hydroxylation of the 3′-secondary
acyl chain of lipid A. LpxO has been implicated indirectly in
stress responses at the envelope level (Needham and Trent,
2013) and, in K. pneumoniae, it has been shown to be relevant
in vivo by increasing bactericidal peptide resistance and reducing
the inflammatory responses (Llobet et al., 2015). However, as
discussed above, previous chemical analysis (Velasco et al.,
2000) of lipid A and the evidence presented here indicate that
the Brucella lpxO homolog is not a lipid A hydroxylase but
rather an OlsC whose mutation, in contrast with LpxO, does
not result in increased sensitivity to polymyxin B. ETHICS STATEMENT Female BALB/c mice (Charles River, France) were kept in
cages with water and food ad libitum under P3 biosafety
conditions in the facilities of “Centro de Investigación Médica
Aplicada” (registration code ES31 2010000132) 2 weeks before
and during the experiments. The procedures were in accordance
with the current European (directive 86/609/EEC) and Spanish
(RD 53/2013) legislations, supervised by the Animal Welfare
Committee of the University of Navarra, and authorized by the
“Gobierno de Navarra” [CEEA045/12 and E36-14 (045-12E1)]. DISCUSSION This absence
of an effect on polycation resistance is in keeping with both
the lack of activity on lipid A and the fact that OL do no play
a major role in resistance to polycationic bactericidal peptides
in B. abortus (Palacios-Chaves et al., 2011). At the same time, it
would also appear to rule out, the involvement of this protein in
the metabolism of succinate in B. microti as has been previously
suggested (Audic et al., 2009). AUTHOR CONTRIBUTIONS IM, MI, J-PG, JB, and RC-Á conceived the study. RC-Á, LP-C,
YG-R, MB-V, MS-B, BA-A, EM-G, AZ-R, MdM, TLB, SH, M-JG, IM, MI, J-PG, JB, and RC-Á conceived the study. RC-Á, LP-C,
YG-R, MB-V, MS-B, BA-A, EM-G, AZ-R, MdM, TLB, SH, M-JG, MV-G, and VA-G carried out the experimental work. IM, MI,
and RC-Á wrote the paper. All authors participated in the
presentation and discussion of results. gg
Previous data showing lptA, lpxE, and lpxO to be involved
in modulating the properties of the OM in a way that in some
cases confers in vitro resistance to innate immunity bactericidal
peptides, complement, and cytokine responses (Needham and
Trent, 2013) have been drawn upon as evidence for a role
in virulence. However, to the best of our knowledge, a
role in vivo has thus far been shown only for lpxO from
K. pneumoniae (Llobet et al., 2015). Moreover, contrasting results
have been obtained with mutants both showing bactericidal
peptide sensitivity in vitro and no phenotype in vivo have been
reported for at least H. ducreyi (Trombley et al., 2015) and may
reflect the complexities of the infection processes and/or the
inadequacies of the currently available in vivo models. Despite
their effect on the envelope, our results show that Brucella lptA, Frontiers in Microbiology | www.frontiersin.org REFERENCES Brucella suis prevents human
dendritic cell maturation and antigen presentation through regulation of tumor
necrosis factor alpha secretion. Infect. Immun. 75, 4980–4989. doi: 10.1128/IAI. 00637-07 Geiger, O., González-Silva, N., López-Lara, I. M., and Sohlenkamp, C. (2010). Amino acid-containing membrane lipids in bacteria. Prog. Lipid Res. 49, 46–60. doi: 10.1016/j.plipres.2009.08.002 Bligh, E. M., and Dyer, W. J. (1959). A rapid method of total lipid extraction and
purification. Can. J. Biochem. Physiol. 37, 911–917. doi: 10.1139/y59-099 Gibbons, H. S., Lin, S., Cotter, R. J., and Raetz, C. R. H. (2000). Oxygen
requirement for the biosynthesis of the S -2-hydroxymyristate moiety in
Salmonella typhimurium lipid A. J. Biol. Chem. 275, 32940–32949. doi: 10.1074/
jbc.M005779200 Braude, A. I. (1951). Studies in the pathology and pathogenesis of experimental
brucellosis: I. A comparison of the pathogenicity of Brucella abortus, Brucella
melitensis, and Brucella suis for Guinea pigs. J. Infect. Dis. 89, 76–86. doi: 10.1093/infdis/89.1.76 González, D., Grilló, M.-J., De Miguel, M.-J., Ali, T., Arce-Gorvel, V., Delrue,
R.-M., et al. (2008). Brucellosis vaccines: assessment of Brucella melitensis
lipopolysaccharide rough mutants defective in core and O-polysaccharide
synthesis and export. PLOS ONE 3:e2760. doi: 10.1371/journal.pone.000
2760 Brindley, D. N., and Waggoner, D. W. (1998). Mammalian lipid phosphate
phosphohydrolases. J. Biol. Chem. 273, 24281–24284. doi: 10.1074/JBC.273.38. 24281 Casabuono, A. C., Czibener, C., Del Giudice, M. G., Valguarnera, E., Ugalde, J. E.,
and Couto, A. S. (2017). New features in the lipid A structure of Brucella
suis and Brucella abortus lipopolysaccharide. J. Am. Soc. Mass Spectrom. 28,
2716–2723. doi: 10.1007/s13361-017-1805-x Gorvel, J.-P., and Moreno, E. (2002). Brucella intracellular life: from invasion
to intracellular replication. Vet. Microbiol. 90, 281–297. doi: 10.1016/S0378-
1135(02)00214-6 Gorvel, L., Textoris, J., Banchereau, R., Ben Amara, A., Tantibhedhyangkul, W.,
von Bargen, K., et al. (2014). Intracellular bacteria interfere with dendritic
cell functions: role of the type I interferon pathway. PLOS ONE 9:e99420. doi: 10.1371/journal.pone.0099420 Chaudhary, A., Ganguly, K., Cabantous, S., Waldo, G. S., Micheva-Viteva, S. N.,
Nag, K., et al. (2012). The Brucella TIR-like protein TcpB interacts with the
death domain of MyD88. Biochem. Biophys. Res. Commun. 417, 299–304. doi: 10.1016/j.bbrc.2011.11.104 Grilló, M.-J., Blasco, J. M., Gorvel, J. P., Moriyón, I., and Moreno, E. (2012). What have we learned from brucellosis in the mouse model? Vet. Res. 43:29. doi: 10.1186/1297-9716-43-29 Cirl, C., Wieser, A., Yadav, M., Duerr, S., Schubert, S., Fischer, H., et al. (2008). REFERENCES Cox, A. D., Wright, J. C., Li, J., Hood, D. W., Moxon, E. R., and Richards,
J. C. (2003). Phosphorylation of the lipid A region of meningococcal
lipopolysaccharide: identification of a family of transferases that add
phosphoethanolamine to lipopolysaccharide. J. Bacteriol. 185, 3270–3277. doi: 10.1128/jb.185.11.3270-3277.2003 Aragón, V., Díaz, R., Moreno, E., and Moriyón, I. (1996). Characterization of
Brucella abortus and Brucella melitensis native haptens as outer membrane
O-type polysaccharides independent from the smooth lipopolysaccharide. J. Bacteriol. 178, 1070–1079. doi: 10.1128/jb.178.4.1070-1079.1996 Dricot, A., Rual, J. F., Lamesch, P., Bertin, N., Dupuy, D., Hao, T., et al. (2004). Generation of the Brucella melitensis ORFeome version 1.1. Genome Res. 14,
2201–2206. doi: 10.1101/gr.2456204 Arendt, W., Hebecker, S., Jäger, S., Nimtz, M., and Moser, J. (2012). Resistance phenotypes mediated by aminoacyl-phosphatidylglycerol synthases. J. Bacteriol. 194, 1401–1416. doi: 10.1128/JB.06576-11 Ducrotoy, M., Bertu, W. J., Matope, G., Cadmus, S., Conde-Álvarez, R., Gusi,
A. M., et al. (2017). Brucellosis in sub-saharan Africa: current challenges for
management, diagnosis and control. Acta Trop. 165, 179–193. doi: 10.1016/j. actatropica.2015.10.023 Audic, S., Lescot, M., Claverie, J.-M., and Scholz, H. C. (2009). Brucella microti:
the genome sequence of an emerging pathogen. BMC Genomics 10:352. doi: 10.1186/1471-2164-10-352 Barquero-Calvo, E., Chaves-Olarte, E., Weiss, D. S., Guzmán-Verri, C., Chacón-
Díaz, C., Rucavado, A., et al. (2007). Brucella abortus uses a stealthy strategy
to avoid activation of the innate immune system during the onset of infection. PLOS ONE 2:e631. doi: 10.1371/journal.pone.0000631 Ernst, C. M., and Peschel, A. (2011). Broad-spectrum antimicrobial peptide
resistance by MprF-mediated aminoacylation and flipping of phospholipids. Mol. Microbiol. 80, 290–299. doi: 10.1111/j.1365-2958.2011.07576.x Ferguson, G. P., Datta, A., Baumgartner, J., Roop, R. M., Carlson, R. W., Walker,
G. C., et al. (2004). Similarity to peroxisomal-membrane protein family reveals
that Sinorhizobium and Brucella BacA affect lipid-A fatty acids. Proc. Natl. Acad. Sci. U.S.A. 101, 5012–5017. doi: 10.1073/pnas.0307137101 Barquero-Calvo, E., Conde-Alvarez, R., Chacón-Díaz, C., Quesada-Lobo, L.,
Martirosyan, A., Guzmán-Verri, C., et al. (2009). The differential interaction
of Brucella and Ochrobactrum with innate immunity reveals traits related to the
evolution of stealthy pathogens. PLOS ONE 4:e5893. doi: 10.1371/journal.pone. 0005893 Fontana, C., Conde-Álvarez, R., Ståhle, J., Holst, O., Iriarte, M., Zhao, Y.,
et al. (2016). Structural studies of lipopolysaccharide-defective mutants from
Brucella melitensis identify a core oligosaccharide critical in virulence. J. Biol. Chem. 291, 7727–7741. doi: 10.1074/jbc.M115.701540 Billard, E., Dornand, J., and Gross, A. (2007). ACKNOWLEDGMENT The
authors
thank
A. Delgado-López
for
excellent
technical
assistance
in
the
extraction
and
purification
of LPS. The Supplementary Material for this article can be found
online
at:
https://www.frontiersin.org/articles/10.3389/fmicb. 2017.02657/full#supplementary-material The
authors
thank
A. Delgado-López
for
excellent
technical
assistance
in
the
extraction
and
purification
of LPS. FUNDING This research was supported by the Institute for Tropical Health
funders (Obra Social la CAIXA, Fundaciones Caja Navarra
and Roviralta, PROFAND, Ubesol, ACUNSA, and Artai) and
grants MINECO (AGL2014-58795-C4-1-R, Bru-Epidia 291815-
FP7/ERANET/ANIHWA), Aragón Government (Consolidated
Group A14), and Marie Curie Career Integration Grant U-KARE
(PCIG13-GA-2013-618162). TLB is the recipient of a Ph.D. Fellowship funded by the Department for Employment and
Learning (Northern Ireland, United Kingdom). January 2018 | Volume 8 | Article 2657 11 Genes Remodeling Brucella Cell Envelope Conde-Álvarez et al. REFERENCES The study of the core part and
non-repeating elements of the O-antigen of Brucella lipopolysaccharide. Carbohydr. Res. 366, 33–37. doi: 10.1016/j.carres.2012.11.004 Qureshi, N., Takayama, K., Seydel, U., Wang, R., Cotter, R. J., Agrawal,
P. K., et al. (1994). Structural analysis of the lipid A derived from
the lipopolysaccharide of Brucella abortus. J. Endotoxin Res. 1, 137–148. doi: 10.1177/096805199400100303 Lai, S., Zhou, H., Xiong, W., Gilbert, M., Huang, Z., Yu, J., et al. (2017). Changing
epidemiology of human brucellosis, China, 1955-2014. Emerg. Infect. Dis. 23,
184–194. doi: 10.3201/eid2302.151710 Raetz, C. R. H., Guan, Z., Ingram, B. O., Six, D. A., Song, F., Wang, X., et al. (2009). Discovery of new biosynthetic pathways: the lipid A story. J. Lipid Res. 50(Suppl.), S103–S108. doi: 10.1194/jlr.R800060-JLR200 Lapaque, N., Moriyon, I., Moreno, E., and Gorvel, J.-P. (2005). Brucella
lipopolysaccharide acts as a virulence factor. Curr. Opin. Microbiol. 8, 60–66. doi: 10.1016/j.mib.2004.12.003 Renzi, F., Zähringer, U., Chandler, C. E., Ernst, R. K., Cornelis, G. R., and Ittig,
S. J. (2015). Modification of the 1-phosphate group during biosynthesis of
Capnocytophaga canimorsus lipid A. Infect. Immun. 84, 550–561. doi: 10.1128/
IAI.01006-15 Lapaque, N., Takeuchi, O., Corrales, F., Akira, S., Moriyon, I., Howard, J. C., et al. (2006). Differential inductions of TNF-alpha and IGTP, IIGP by structurally
diverse classic and non-classic lipopolysaccharides. Cell. Microbiol. 8, 401–413. doi: 10.1111/j.1462-5822.2005.00629.x Salcedo, S. P., Marchesini, M. I., Degos, C., Terwagne, M., Von Bargen, K.,
Lepidi, H., et al. (2013). BtpB, a novel Brucella TIR-containing effector
protein with immune modulatory functions. Front. Cell. Infect. Microbiol. 3:28. doi: 10.3389/fcimb.2013.00028 Leong, D., Díaz, R., Milner, K., Rudbach, J., and Wilson, J. B. (1970). Some
structural and biological properties of Brucella endotoxin. Infect. Immun 1,
174–182. Lewis, L. A., Shafer, W. M., Dutta Ray, T., Ram, S., and Rice, P. A. (2013). Phosphoethanolamine residues on the lipid A moiety of Neisseria gonorrhoeae
lipooligosaccharide modulate binding of complement inhibitors and resistance
to complement killing. Infect. Immun. 81, 33–42. doi: 10.1128/IAI.00751-12 Salcedo, S. P., Marchesini, M. I., Lelouard, H., Fugier, E., Jolly, G., Balor, S.,
et al. (2008). Brucella control of dendritic cell maturation is dependent on the
TIR-containing protein Btp1. PLOS Pathog. 4:e21. doi: 10.1371/journal.ppat. 0040021 g
g
to complement killing. Infect. Immun. 81, 33–42. doi: 10.1128/IAI.00751-12 Sciorra, V. A., and Morris, A. J. (2002). Roles for lipid phosphate phosphatases
in regulation of cellular signaling. Biochim. Biophys. Acta 1582, 45–51. REFERENCES Subversion of Toll-like receptor signaling by a unique family of bacterial
Toll/interleukin-1 receptor domain–containing proteins. Nat. Med. 14,
399–406. doi: 10.1038/nm1734 Hallez, R., Letesson, J.-J., Vandenhaute, J., and De Bolle, X. (2007). Gateway-based
destination vectors for functional analyses of bacterial ORFeomes: application
to the min system in Brucella abortus. Appl. Environ. Microbiol. 73, 1375–1379. doi: 10.1128/AEM.01873-06 Comerci, D. J., Altabe, S., de Mendoza, D., and Ugalde, R. A. (2006). Brucella abortus synthesizes phosphatidylcholine from choline provided
by the host. J. Bacteriol. 188, 1929–1934. doi: 10.1128/JB.188.5.1929-1934. 2006 Herrera, C. M., Henderson, J. C., Crofts, A. A., and Trent, M. S. (2017). Novel
coordination of lipopolysaccharide modifications in Vibrio cholerae promotes
CAMP resistance. Mol. Microbiol. 106, 582–596. doi: 10.1111/mmi.13835 Conde-Álvarez, R., Arce-Gorvel, V., Iriarte, M., Man˘cek-Keber, M., Barquero-
Calvo, E., Palacios-Chaves, L., et al. (2012). The lipopolysaccharide core of
Brucella abortus acts as a shield against innate immunity recognition. PLOS
Pathog. 8:e1002675. doi: 10.1371/journal.ppat.1002675 Inaba, K., Inaba, M., Romani, N., Aya, H., Deguchi, M., Ikehara, S.,
et al. (1992). Generation of large numbers of dendritic cells from mouse
bone marrow cultures supplemented with granulocyte/macrophage colony-
stimulating factor. J. Exp. Med. 176, 1693–1702. doi: 10.1084/jem.176. 6.1693 Conde-Alvarez, R., Grilló, M. J., Salcedo, S. P., de Miguel, M. J., Fugier, E.,
Gorvel, J. P., et al. (2006). Synthesis of phosphatidylcholine, a typical eukaryotic
phospholipid, is necessary for full virulence of the intracellular bacterial parasite
Brucella abortus. Cell. Microbiol. 8, 1322–1335. doi: 10.1111/j.1462-5822.2006. 00712.x Iriarte, M., González, D., Delrue, R. M., Monreal, D., Conde-Álvarez, R., López-
Goñi, I., et al. (2004). “Brucella LPS: structure, biosynthesis and genetics,” in January 2018 | Volume 8 | Article 2657 Frontiers in Microbiology | www.frontiersin.org 12 Genes Remodeling Brucella Cell Envelope Conde-Álvarez et al. Moreno, E., and Moriyón, I. (2007). “The genus Brucella,” in The Prokaryotes:
Proteobacteria, Vol. 5, eds S. Falkow, E. Rosenberg, K. H. Schleifer, E. Stackebrandt, and M. Dworkin (Berlin: Springer-Verlag), 315–455. Brucella: Molecular and Cellular Biology, eds I. López-Goñi and I. Moriyón
(Gurgaon: Horizon Bioscience), 159–192. (Gurgaon: Horizon Bioscience), 159–192. Jiménez de Bagüés, M. P., Ouahrani-Bettache, S., Quintana, J. F., Mitjana, O., (
g
),
Jiménez de Bagüés, M. P., Ouahrani-Bettache, S., Quintana, J. F., Mitjana, O., Moreno, E., Stackebrandt, E., Dorsch, M., Wolters, J., Busch, M., and
Mayer, H. (1990). Brucella abortus 16S rRNA and lipid A reveal a
phylogenetic relationship with members of the alpha-2 subdivision of the class
Proteobacteria. J. Bacteriol. REFERENCES 172, 3569–3576. doi: 10.1128/jb.172.7.3569-3576. 1990 Hanna, N., Bessoles, S., et al. (2010). The new species Brucella microti replicates
in macrophages and causes death in murine models of infection. J. Infect. Dis. 202, 3–10. doi: 10.1086/653084 Jones, B. A., Grace, D., Kock, R., Alonso, S., Rushton, J., Said, M. Y., et al. (2013). Zoonosis emergence linked to agricultural intensification and environmental
change. Proc. Natl. Acad. Sci. U.S.A. 110, 8399–8404. doi: 10.1073/pnas. 1208059110 Moriyón, I. (2003). “Against gram-negative bacteria: the lipopolysaccharide case,”
in Intracellular Pathogens in Membrane Interactions and Vacuole Biogenesis, ed. change. Proc. Natl. Acad. Sci. U.S.A. 110, 8399–8404. doi: 10.1073/pnas. 1208059110 Moriyón, I. (2003). “Against gram-negative bacteria: the lipopolysaccharide cas
in Intracellular Pathogens in Membrane Interactions and Vacuole Biogenesis,
J. P. Gorvel (Dordrecht: Kluwer Academic Publishers), 204–230. Karbarz, M. J., Kalb, S. R., Cotter, R. J., and Raetz, C. R. H. (2003). Expression
cloning and biochemical characterization of a Rhizobium leguminosarum
lipid A 1-phosphatase. J. Biol. Chem. 278, 39269–39279. doi: 10.1074/jbc. M305830200 J. P. Gorvel (Dordrecht: Kluwer Academic Publishers), 204–230. Naessan, C., Egge-Jacobsen, W., Heiniger, R., Wolfgang, M., Aas, F., Røhr, A., et al. (2008). Genetic and functional analyses of PptA, a phospho-form transferase
targeting type IV pili in Neisseria gonorrhoeae. J. Bacteriol. 190, 387–400. doi: 10.1128/JB.00765-07 Karbarz, M. J., Six, D. A., and Raetz, C. R. H. (2009). Purification and
characterization
of
the
lipid
A
1-phosphatase
LpxE
of
Rhizobium
leguminosarum. J. Biol. Chem. 284,
414–425. doi:
10.1074/jbc.M8083
90200 Needham, B. D., and Trent, M. S. (2013). Fortifying the barrier: the impact of
lipid A remodelling on bacterial pathogenesis. Nat. Rev. Microbiol. 11, 467–481. doi: 10.1038/nrmicro3047 Kastowsky, M., Gutberlet, T., and Bradaczek, H. (1992). Molecular modelling
of the three-dimensional structure and conformational flexibility of bacterial
lipopolysaccharide. J. Bacteriol. 174, 4798–4806. doi: 10.1128/jb.174.14.4798-
4806.1992 Palacios-Chaves, L., Conde-Álvarez, R., Gil-Ramírez, Y., Zuñiga-Ripa, A.,
Barquero-Calvo, E., Chacón-Díaz, C., et al. (2011). Brucella abortus ornithine
lipids are dispensable outer membrane components devoid of a marked
pathogen-associated molecular pattern. PLOS ONE 6:e16030. doi: 10.1371/
journal.pone.0016030 Kovach, M. E., Phillips, R. W., Elzer, P. H., Roop, R. M., and Peterson, K. M. (1994). pBBR1MCS: a broad-host-range cloning vector. Biotechniques 16,
800–802. Papadopoulos, A., Gagnaire, A., Degos, C., de Chastellier, C., and Gorvel, J.-
P. (2016). Brucella discriminates between mouse dendritic cell subsets upon
in vitro infection. Virulence 7, 33–44. doi: 10.1080/21505594.2015.1108516 Kubler-Kielb, J., and Vinogradov, E. (2013). REFERENCES doi: 10.1016/S1388-1981(02)00136-1 Llobet, E., Martínez-Moliner, V., Moranta, D., Dahlström, K. M., Regueiro, V.,
Tomás, A., et al. (2015). Deciphering tissue-induced Klebsiella pneumoniae lipid
A structure. Proc. Natl. Acad. Sci. U.S.A. 112, E6369–E6378. doi: 10.1073/pnas. 1508820112 Scupham, A. J., and Triplett, E. W. (1997). Isolation and characterization of the
UDP-glucose 4‘-epimerase-encoding gene, galE, from Brucella abortus 2308. Gene 202, 53–59. doi: 10.1016/S0378-1119(97)00453-8 Mackinnon, F. G., Cox, A. D., Plested, J. S., Tang, C. M., Makepeace, K., Coull,
P. A., et al. (2002). Identification of a gene (lpt-3) required for the addition
of phosphoethanolamine to the lipopolysaccharide inner core of Neisseria
meningitidis and its role in mediating susceptibility to bactericidal killing and
opsonophagocytosis. Mol. Microbiol. 43, 931–943. doi: 10.1046/j.1365-2958. 2002.02754.x Simon, L. D., Randolph, B., Irwin, N., and Binkowski, G. (1983). Stabilization of
proteins by a bacteriophage T4 gene cloned in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 80, 2059–2062. doi: 10.1073/pnas.80.7.2059 Sohlenkamp, C., Galindo-Lagunas, K. A., Guan, Z., Vinuesa, P., Robinson, S.,
Thomas-Oates, J., et al. (2007). The lipid Lysyl-phosphatidylglycerol is present
in membranes of Rhizobium tropici CIAT899 and confers increased resistance
to polymyxin B under acidic growth conditions. Mol. Plant Microbe Interact. 20, 1421–1430. doi: 10.1094/MPMI-20-11-1421 Martínez de Tejada, G., Pizarro-Cerdá, J., Moreno, E., and Moriyón, I. (1995). The
outer membranes of Brucella spp. are resistant to bactericidal cationic peptides. Infect. Immun. 63, 3054–3061. Martirosyan, A., Moreno, E., and Gorvel, J.-P. (2011). An evolutionary strategy
for a stealthy intracellular Brucella pathogen. Immunol. Rev. 240, 211–234. doi: 10.1111/j.1600-065X.2010.00982.x Stukey, J., and Carman, G. M. (2008). Identification of a novel phosphatase
sequence
motif. Protein
Sci. 6,
469–472. doi:
10.1002/pro.55600
60226 Moreira, C. G., Herrera, C. M., Needham, B. D., Parker, C. T., Libby, S. J., Fang,
F. C., et al. (2013). Virulence and stress-related periplasmic protein (VisP)
in bacterial/host associations. Proc. Natl. Acad. Sci. U.S.A. 110, 1470–1475. doi: 10.1073/pnas.1215416110 Takahashi, H., Carlson, R. W., Muszynski, A., Choudhury, B., Kim, K. S.,
Stephens, D. S., et al. (2008). Modification of lipooligosaccharide with
phosphoethanolamine
by
LptA
in
Neisseria
meningitidis
enhances
meningococcal adhesion to human endothelial and epithelial cells. Infect. Immun. 76, 5777–5789. doi: 10.1128/IAI.00676-08 Moreno, E., Berman, D. T., and Boettcher, L. A. (1981). Biological activities of
Brucella abortus lipopolysaccharides. Infect. Immun. 31, 362–370. January 2018 | Volume 8 | Article 2657 Frontiers in Microbiology | www.frontiersin.org 13 Genes Remodeling Brucella Cell Envelope Conde-Álvarez et al. Tran, A. X., Whittimore, J. D., Wyrick, P. REFERENCES B., Mcgrath, S. C., Cotter, R. J., and Trent,
M. S. (2006). The lipid A 1-phosphatase of Helicobacter pylori is required for
resistance to the antimicrobial peptide polymyxin. J. Bacteriol. 188, 4531–4541. doi: 10.1128/JB.00146-06 Whatmore, A. M. (2009). Current understanding of the genetic diversity of
Brucella, an expanding genus of zoonotic pathogens. Infect. Genet. Evol. 9,
1168–1184. doi: 10.1016/j.meegid.2009.07.001 Wright, J. C., Hood, D. W., Randle, G. A., Makepeace, K., Cox, A. D., Li, J., et al. (2004). lpt6, a gene required for addition of phosphoethanolamine to inner-
core lipopolysaccharide of Neisseria meningitidis and Haemophilus influenzae. J. Bacteriol. 186, 6970–6982. doi: 10.1128/JB.186.20.6970-6982.2004 Trombley, M. P., Post, D. M. B., Rinker, S. D., Reinders, L. M., Fortney,
K. R., Zwickl, B. W., et al. (2015). Phosphoethanolamine transferase LptA
in Haemophilus ducreyi modifies lipid A and contributes to human defensin
resistance in vitro. PLOS ONE 10:e0124373. doi: 10.1371/journal.pone.0124373 Zheludkov, M. M., and Tsirelson, L. E. (2010). Reservoirs of Brucella infection in
nature. Biol. Bull. 37, 709–715. doi: 10.1134/S106235901007006X Velasco, J., Bengoechea, J. A., Brandenburg, K., Lindner, B., Seydel, U.,
González, D., et al. (2000). Brucella abortus and its closest phylogenetic relative,
Ochrobactrum spp., differ in outer membrane permeability and cationic peptide
resistance. Infect. Immun. 68, 3210–3218. doi: 10.1128/IAI.68.6.3210-3218. 2000 Zinsstag, J., Schelling, E., Solera, J., Blasco, J. M., and Moriyón, I. (2011). “Brucellosis,” in Handbook of Zoonoses, eds S. R. Palmer, L. Soulsby, P. R. Torgeson, and D. G. Brown (Oxford: Oxford University Press), 54–62. Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest. Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest. Vences-Guzmán, M. Á., Guan, Z., Ormeño-Orrillo, E., González-Silva, N., López-
Lara, I. M., Martínez-Romero, E., et al. (2011). Hydroxylated ornithine lipids
increase stress tolerance in Rhizobium tropici CIAT899. Mol. Microbiol. 79,
1496–1514. doi: 10.1111/j.1365-2958.2011.07535.x Copyright © 2018 Conde-Álvarez, Palacios-Chaves, Gil-Ramírez, Salvador-Bescós,
Bárcena-Varela, Aragón-Aranda, Martínez-Gómez, Zúñiga-Ripa, de Miguel,
Bartholomew, Hanniffy, Grilló, Vences-Guzmán, Bengoechea, Arce-Gorvel, Gorvel,
Moriyón and Iriarte. This is an open-access article distributed under the terms
of the Creative Commons Attribution License (CC BY). January 2018 | Volume 8 | Article 2657 Frontiers in Microbiology | www.frontiersin.org REFERENCES The use, distribution or
reproduction in other forums is permitted, provided the original author(s) or licensor
are credited and that the original publication in this journal is cited, in accordance
with accepted academic practice. No use, distribution or reproduction is permitted
which does not comply with these terms. Wang, X., McGrath, S. C., Cotter, R. J., and Raetz, C. R. H. (2006). Expression
cloning and periplasmic orientation of the Francisella novicida lipid A 4’-
phosphatase LpxF. J. Biol. Chem. 281, 9321–9330. doi: 10.1074/jbc.M600435200 Wang, X., Ribeiro, A. A., Guan, Z., Abraham, S. N., and Raetz, C. R. H. (2007). Attenuated virulence of a Francisella mutant lacking the lipid A 4’-phosphatase. Proc. Natl. Acad. Sci. U.S.A. 104, 4136–4141. doi: 10.1073/pnas.0611606104 Weissenmayer, B., Gao, J. L., Lopez-Lara, I. M., and Geiger, O. (2002). Identification of a gene required for the biosynthesis of ornithine-derived lipids. Mol. Microbiol. 45, 721–733. doi: 10.1046/j.1365-2958.2002.03043.x January 2018 | Volume 8 | Article 2657 Frontiers in Microbiology | www.frontiersin.org 14
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Correspondence: Paul Laris (paul.laris@csulb.edu) Correspondence: Paul Laris (paul.laris@csulb.edu) Received: 17 December 2020 – Discussion started: 22 March 2021
Revised: 15 October 2021 – Accepted: 19 October 2021 – Published: 1 December 2021 Received: 17 December 2020 – Discussion started: 22 March 2021
Revised: 15 October 2021 – Accepted: 19 October 2021 – Published: 1 December 2021 ties than backfires – 1.203 vs. 0.708 g m−2 – respectively, a
significant difference. We suggest the reason for the higher
methane emissions from head fires, which have higher in-
tensity, is the longer flame lengths that burn green leaves on
trees, releasing methane. We conclude that policies aimed at
shifting the burning regime earlier to reduce methane emis-
sions will not have the desired effects, especially if fire type
is not considered. Future research should consider the state
and amount of leafy biomass combusted in savanna fires. ties than backfires – 1.203 vs. 0.708 g m−2 – respectively, a
significant difference. We suggest the reason for the higher
methane emissions from head fires, which have higher in-
tensity, is the longer flame lengths that burn green leaves on
trees, releasing methane. We conclude that policies aimed at
shifting the burning regime earlier to reduce methane emis-
sions will not have the desired effects, especially if fire type
is not considered. Future research should consider the state
and amount of leafy biomass combusted in savanna fires. Abstract. Savanna fires contribute significantly to green-
house gas emissions. While it is recognized that these fires
play a critical role in the global methane cycle, there are
too few accurate estimates of emissions from West Africa,
the continent’s most active fire region. Most estimates of
methane emissions contain high levels of uncertainty as they
are based on generalizations of diverse landscapes that are
burned by complex fire regimes. To improve estimates we
used an approach grounded in the burning practices of peo-
ple who set fires to working landscapes. We collected and
analyzed smoke samples for 36 experimental fires using a
canister method for the early dry season (EDS) and mid-dry
season (MDS). We also collected data for savanna type, grass
type, biomass composition and amount consumed, scorch
height, speed of fire front, fire type, and ambient air condi-
tions for two sites in Mali. We report values for fire intensity,
combustion completeness, patchiness, modified combustion
efficiency (MCE), emission factor (EF) and methane emis-
sion density. 1
Introduction The African savannas are the Earth’s most extensively and
frequently burned regions (Giglio et al., 2010) and account
for some 64 % of the global extent of area burnt annually
(Grégoire et al., 2013). Indeed, African savanna fires regu-
larly burn such large areas that they are visible from space,
so much so that NASA scientists refer to Africa as the “burn
center of the planet” (National Aeronautics and Space Ad-
ministration, 2005). Savanna fires are a major source of
greenhouse gases (GHGs) including carbon dioxide, carbon
monoxide, methane and nitrous oxide (Koppmann et al.,
2005; Russell-Smith et al., 2021). y
Our study found that mean methane EFs ranged from
3.83 g kg−1 in the EDS to 3.18 g kg−1 in the MDS, but the
small sample did not provide enough power for this effect
to be significant. We found head fires had nearly double
the CH4 EF of backfires (5.12 g kg−1 to 2.74), a significant
difference. Byram’s fire intensity was a significant driver
of CH4 EF but with weak effect. Methane emission den-
sity increased marginally from 0.839 g m−2 in the EDS to
0.875 g m−2 in the MDS, a difference that was not signifi-
cant. Head fires, however, had much higher emission densi- Methane, a critical GHG, is responsible for about 20 %
of the warming induced by long-lived gases. Although most
sources and sinks of methane are known, their relative con-
tributions to atmospheric methane levels remain highly un-
certain (Kirschke et al., 2013; Saunois et al., 2016, 2020). Biogeosciences, 18, 6229–6244, 2021
https://doi.org/10.5194/bg-18-6229-2021
© Author(s) 2021. This work is distributed under
the Creative Commons Attribution 4.0 License. Biogeosciences, 18, 6229–6244, 2021
https://doi.org/10.5194/bg-18-6229-2021
© Author(s) 2021. This work is distributed under
the Creative Commons Attribution 4.0 License. Paul Laris1, Moussa Koné2, Fadiala Dembélé3, Christine M. Rodrigue1, Lilian Yang1, Rebecca Jacobs1, and
Quincy Laris4 1Geography Department, California State University Long Beach, 1250 Bellflower Blvd., Long Beach, CA 90840, USA
2Geography, Université Félix Houphouët-Boigny, BP V34, Abidjan, République de Côte d’Ivoire
3Institut Polytechnique Rural de Formation et de Recherché Appliquée de Katibougou (IPR/IFRA), BP 06 Bamako, Mali
4Department of Civil and Environmental Engineering, University of California, 760 Davis Hall, Berkeley, CA 94720, USA P. Laris et al.: Methane gas emissions from savanna fires There is thus a need to im-
prove land-based estimates of emissions from savanna fires. Nowhere is this truer than for West Africa, the continent’s
most active fire region. To date, measurements of emissions
from African savannas are limited to a few broad-scale stud-
ies, largely based in the continent’s southeast and that rarely
adequately account for changes in fuel classes, seasonality
or a host of other key factors including fire type and intensity
(Bonsang et al., 1995; Lacaux et al., 1995; Hoffa et al., 1999;
Korontzi, 2005). Indeed, the most recent catalogs of EFs and
fuel consumption (FC) for savannas includes a single data
point from West Africa (van Leeuwen et al., 2014; Andreae,
2019). Studies from other regions find there is great variation
in study results (Russel-Smith, 2009; van Leeuwen and van
der Werf, 2011), and, as Murphy et al. (2012) note, the vari-
ability between samples collected within fires can be greater
than the differences between fires of different seasons. These
authors were unable to draw general conclusions about sea-
sonal variation in methane emissions and EFs. Among the
key issues cited were the variations in the fraction of tree-
leaf litter in the fuels of different savanna environments. In general, the crucial parameters for determining GHG
emissions from fires include burned area (BA), fuel con-
sumption (FC) and the species-specific emission factor (EF),
usually defined as the amount of gas or particle mass emitted
per kilogram of dry fuel burned, expressed in units of grams
per kilogram (g kg−1) dry matter (van Leeuwen and van der
Werf, 2011). The precise emissions from savanna fires de-
pend on a variety of factors including those associated with
fuel, specifically vegetation type (the mix of grassy, leafy and
woody fuels) and fuel moisture (a function of climate, soil
and fire season), as well as factors directly related to a fire’s
properties, most of which change during a long fire season. In fact, there are not a lot of data in the literature on fine
fuel mixtures (the primary fuel for savanna fires) used to es-
timate EFs in Africa, although the amount of woody vegeta-
tion clearly affects emissions (Korontzi, 2005; van Leeuwen
and van der Werf, 2011). In the Brazilian Cerrado, for exam-
ple, Vernooij et al. P. Laris et al.: Methane gas emissions from savanna fires 6230 Yet, although scientists and policy makers increasingly
recognize the important role these fires play in the global
carbon cycle, there are few accurate estimates of their emis-
sions especially in terms of the key factors that determine
the type and quantity of GHG emissions. Critically, most
studies of emissions are global scale and use average biome
level EFs. EFs show large variability, however, between and
within biomes due to differences in fuel type and composi-
tion, burning conditions, and tree density (Andreae and Mer-
let, 2001; Korontzi, 2005; van Leeuwen and van der Werf,
2011; Russell-Smith et al., 2021). There are few regionally
specific emission estimates because accurate quantification
of such emissions is difficult, being dependent upon reliable
estimation of the various parameters, many of which require
intense fieldwork (Russell-Smith et al., 2009). Our lack of understanding of the global methane cycle con-
tributed to the recent “methane enigma”, a dramatic observed
decline in the rate of increase in atmospheric methane, which
triggered a search for “missing methane” (Heimann, 2011). Although the decrease was originally and mistakenly at-
tributed to a decline in fossil fuel burning and a shift in farm-
ing practices (Kirschke et al., 2013), it was eventually deter-
mined that the missing methane was due to a decline in area
burned by savanna fires. As NASA researchers determined,
the missing methane from a drop in savanna burned area
caused a decrease of 3.7 Tg CH4 per year – a value nearly
twice the decrease expected (Worden et al., 2018). The missing methane event demonstrates two important
aspects of emissions from savanna fires. First, these emis-
sions are significant, so much so that they can offset increases
from the key sources (fossil fuels and agriculture). Second,
our knowledge of the processes and factors that regulate the
amount of methane emissions from savannas is limited to
the point that a large decrease went virtually undetected. Although eventually scientists discovered the source, there
remains a high level of uncertainty for many key variables
that determine the amounts of methane emitted from savanna
fires (Worden et al., 2018). In addition, there remains con-
cern about the gap between “top-down” (atmospheric mea-
surements) and “bottom-up” (land-based models) estimates
of global methane emissions, which differ by 30 %; Saunois
et al. (2020) suggest the reason is an overestimation of emis-
sions from bottom-up models. Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. P. Laris et al.: Methane gas emissions from savanna fires P. Laris et al.: Methane gas emissions from savanna fires It is clear that any effort to predict future changes in emis-
sions or to implement policy to reduce emissions requires
more detailed information on how emissions vary according
to the key factors noted above, many of which are a func-
tion of human land management practices (see Appendix). Specifically, given the spatiotemporal complexity of savanna
environments, whether a shift to an earlier fire regime will
result in a decrease in methane emissions for a given savanna
must be determined empirically, and proposed policies to ap-
ply generalized findings from one continent to another may
not achieve desired emission reductions. Savannas are patch mosaic landscapes in which vegeta-
tion and soil types vary dramatically – on the order of tens
of meters – across landscapes (Duvall, 2011). This variation
creates a seasonal-mosaic landscape in which the fuel condi-
tions (fuel moisture, fuel load and mix, and fuel stature) vary
over space and time (Fig. 1). As the fuel conditions change,
people commonly set fire to different patches in accordance
with grass species drying rates, as well as other land man-
agement concerns (e.g., crop harvests and grazing patterns). In many parts of West Africa, people control the time (time
of day and season), location and type of savanna, and type
of fires they set. By controlling these variables, human acts
determine the conditions of the fuels, as well as the ambi-
ent air conditions and the specific fire properties. To take one
simple example, a fire in wooded savanna with tall perennial
grasses (Fig. 1d) will have very different fuel moisture levels
as the fire season progresses (shifting high to low), different
percentages of leaf litter and total fine fuel biomass (shifting
low to high) (Fig. 1f), and variable wind conditions (peak-
ing in mid-dry season, MDS). Critically, the seasonal timing
of a fire not only affects the fuel moisture of grasses but also
the leafy biomass because green leaves on small trees burn in
EDS, while fallen leaf litter, which creates a bed of compact
and less-aerated fuel, burns in LDS. As such, the incomplete
combustion of leafy biomass is a function of high fuel mois-
ture in one season and low oxygen conditions in another, with
unknown implications for methane gas emissions. P. Laris et al.: Methane gas emissions from savanna fires (Fig. 1d) until later, often igniting them in a flurry of fires at
the end of the harvest in late December (below). This creates
a seasonal-mosaic fire regime in which some patches burn
early, others later and some not at all. Purposeful fires are
most often set as backfires in the late afternoon as temper-
atures and wind speeds are falling and as humidity is ris-
ing, which limits fire intensity (Laris et al., 2020). Lower-
intensity fires tend to self-extinguish at the edge of moister
vegetation patches and in the evening; they have lower flame
heights reducing the reach of fires into leafy tree canopies
(Laris et al., 2021). Later in the fire season, fires are less
likely to be purposefully set and are more likely to burn as
intense, uncontrolled head fires. in the CH4 EF. Elsewhere in southern Africa, Russell-Smith
et al. (2021) found that emission factors varied significantly
by season for some vegetation types but not others, although
notably the latter study involved only “cured” grasses. We
would argue that “early” fires burn uncured fuels by defini-
tion. in the CH4 EF. Elsewhere in southern Africa, Russell-Smith
et al. (2021) found that emission factors varied significantly
by season for some vegetation types but not others, although
notably the latter study involved only “cured” grasses. We
would argue that “early” fires burn uncured fuels by defini-
tion. In sum, while savannas undoubtedly harbor great theoreti-
cal potential to sequester more carbon and emit less through
a change in fire regime, there exists a great deal of uncer-
tainty as to what the actual carbon shifts might be should
regimes change. Fire regimes are themselves complex; we
define them as the characteristic fire activity prevailing in a
region, typically determined by frequency, intensity, season-
ality, size distribution, type of fire and fuels consumed (Kee-
ley and Pausas, 2019). Changes in one or more of these fac-
tors can alter fire emissions. We suggest the key sources of
uncertainty in terms of carbon emissions arise largely from
the spatiotemporal complexity of savanna vegetation patterns
and fire regimes combined with many unknowns or biases
associated with a lack of consideration of human fire setting
and land management practices in these complex landscapes
(Laris, 2021). P. Laris et al.: Methane gas emissions from savanna fires (2021) found that the seasonal effect on
methane EF was stronger in more woody savanna vegetation,
with late dry season (LDS) fires having 20 % lower EF than
early dry season (EDS) ones in shrub-dominated areas. By
one
estimate
savanna
fires
contribute
62 %
(4.92 Pg CO2 per year) of gross global mean fire emis-
sions (Lipsett-Moore et al., 2018). Due to their high rates
of burning and vast extents, savannas are thought to hold
potential as major carbon sinks if the fire regime could be
modified to reduce emissions. The most commonly proposed
change in the regime to reduce the impacts of fires is to
shift burning to an earlier period in the dry season because
early fires generally burn less completely and more patchily. Indeed, Lipsett-Moore et al. (2018, p. 1) recently argued
that there are “global opportunities for significant emission
reductions by simply shifting the fire period in African
savannas to early dry season”. Fuel moisture is also an issue; Russel-Smith et al. (2009)
noted there are currently no comprehensive measurements of
the seasonality of emission gas composition, yet fuel mois-
ture is a key determinant. This is a critical problem be-
cause, although evidence suggests that EDS fires consume
less biomass and burn more patchily, they also tend to have
a lower combustion efficiency than later fires due to their
higher fuel moisture levels. A lower combustion efficiency
theoretically causes a higher emission factor for CH4. In-
deed, one study in Africa found that the bulk of CH4 emis-
sions come from EDS fires (Hoffa et al., 1999) because the
decrease in area burned is more than offset by the increase Biogeosciences, 18, 6229–6244, 2021 https://doi.org/10.5194/bg-18-6229-2021 6231 P. Laris et al.: Methane gas emissions from savanna fires P. Laris et al.: Methane gas emissions from savanna fires https://doi.org/10.5194/bg-18-6229-2021 P. Laris et al.: Methane gas emissions from savanna fires This study aims to fill a knowledge gap by incorporat-
ing data on human burning practices, the characteristics of
the fire regimes they produce, the vegetation conditions on
the landscapes they burn and the resulting emissions of key
GHGs. Through a novel geographic approach, we designed
our experiments to gather data in ways that reflect actual
on-the-ground burning practices of people living in working
landscapes at two mesic savanna sites in Mali, West Africa. By “working landscapes”, we mean savanna lands that are
occupied and worked by people as opposed to areas managed
as reserves (e.g., Charnley et al., 2014); the latter are most
often used in fire research. The biomass (fuels) in working
landscapes is a function of land use practices including rota-
tional agriculture, annual burning and animal grazing and can
differ significantly from those found on non-working lands
(Fig. 1d and e), which can affect fire intensity, combustion
completeness and combustion efficiency with implications
for gas emissions. The burning regimes studied, which are
determined by such factors as seasonality, time of day, (am-
bient weather), fire type (with or counter to the wind), grass
type and woody vegetation cover, were selected to reflect lo-
cal practices and are based on over a decade of field and
remote sensing research. To determine the factors that influence fire emissions of
methane gas from anthropogenic fires we conducted experi-
mental fires using a field-based method to measure key fac-
tors. We collected canister samples of smoke emissions for
36 fires during the early and middle seasons, which we report
on here. We also collected data for savanna type, grass type,
biomass composition and amount consumed, scorch height, Human fire uses determine the pyrogeography of fire – the
specific location, timing, type of fire and vegetation burned –
creating complex spatiotemporal patterns of fires and emis-
sions. People tend to set fires to fine annual grasses (Fig. 1a)
as soon as they are dry, while they wait to burn perenni-
als (which are too moist to burn in the early dry season) https://doi.org/10.5194/bg-18-6229-2021 https://doi.org/10.5194/bg-18-6229-2021 Biogeosciences, 18, 6229–6244, 2021 Biogeosciences, 18, 6229–6244, 2021 6232 P. Laris et al.: Methane gas emissions from savanna fires P. Laris et al.: Methane gas emissions from savanna fires Figure 1. Different savanna vegetation types used in fire experiments for the study areas of Tabou and Faradiélé, Mali. Note that grass
species, height and density, woody cover, and leaf litter amounts vary dramatically over space and time. Figure 1. Different savanna vegetation types used in fire experiments for the study areas of Tabou and Faradiélé, Mali. Note that grass
species, height and density, woody cover, and leaf litter amounts vary dramatically over space and time. speed of fire front, fire type, and ambient air conditions for
two mesic savanna sites in Mali. Figure 2. Study areas in southern Mali (figure by Scott Winslow). 2.1
Data collection We studied 36 experimental fires. Data on the following vari-
ables were collected in the field for each fire: average plot
biomass, grass proportion of biomass, grass species, biomass
consumed, fuel moisture, wind speed, scorch height, ambient
humidity, temperature, fire type, time of day, fire duration,
burn patchiness and fire season. Vegetation characteristics in-
cluding grass type (annual or perennial), grass species and
leaf height were also recorded for each site. Fuel load (plot
biomass) was measured in each of the experimental plots by
delineating three representative pre-fire quadrats of 1 × 1 m. Grasses were cut at the base using a scythe and weighed
with an electronic balance to determine the average. When
present, we weighed leaf litter separately. A total of 16 of the
36 fires were set in the EDS and 20 in the MDS. As the pur-
pose of the study was to replicate local burning practices, the
majority of these fires were set as backfires (25) with head
fires (11) set for the purpose of comparison. We noted ignition time, and each fire was timed until the
flaming front reached the end of the 10 m plot. We set the ma-
jority of fires in late afternoon, which is in accordance with
local practice, although we set some fires earlier for compar-
ative purposes. Post-fire ash and any unburned material were
weighed for areas of similar composition to the 1 × 1 m pre-
fire quadrats to determine the amount of biomass consumed. Scorch height was averaged for each plot by measuring the
height of scorch marks on several small trees. Burn patchi-
ness – the percentage of the plot affected by fire – was esti-
mated by two observers. Most grasses burned were fully cured; however, for those
that were not, we cut a sample and weighed it wet and then
dried and reweighed it to determine the cure rate, which was
taken as the average for the plot. Fuel moisture content for
the cured fuels was calculated using the method developed
by Viney (1991) for savanna fuels: There are several key limitations of this study. First, the
number of gas samples is relatively small due to the high
price of shipping gas samples (we collected only 36 emission
samples for a total of 97 experimental burns, and no samples
were from the LDS). P. Laris et al.: Methane gas emissions from savanna fires unique patterns of land cover (Duvall, 2011; Laris, 2011). Ferricrete outcrops on hard pan cover considerable areas. Soil in these areas generally has high gravel content and is
very shallow, creating xeric conditions. Vegetation is domi-
nated by short, annual grasses (principally Loudetia togoen-
sis but also Andropogon pseudapricus), and there are usually
a few widely scattered trees. They form up to 25 % of the sa-
vanna in southern Mali. Except for the intensively cultivated
areas, a near-continuous layer of tall (over 1 m in height)
perennial grasses (principally Andropogon gayanus, Hypar-
rhenia dissolute, Cymbopogon giganteus and Schizachyrium
pulchellum) covers the more fertile soils, although there are
pockets where the tree canopy is closed and there is little
grass cover. The land cover in settled areas has been signif-
icantly modified. Perennial grasses are less common (except
on long-fallow plots), and large portions of the landscape are
covered by annual grasses, particularly Andropogon pseu-
dapricus and Pennisetum pedicellatum with scattered trees. We used a Kestrel 5500 Weather Meter station (Kestrel-
Meters.com, Boothwyn, Pennsylvania, USA) to collect wind
speed, ambient humidity and temperature during the burn-
ing of each plot. We recorded values every 5 s and averaged
them for each burn. The weather station was placed upwind
and near each experimental plot 2 m off the ground in an open
area. We recorded wind speed relative to the direction of each
fire. 2.2
Plot design We selected plots to represent an array of savanna vegetation
types dominated by different amounts of woody cover and
grass species. To aid in the selection of the burn plots, we
used a long-term fire database to select sites with known fire
seasonality – fires known to burn during the early, middle or
late fire season on an annual basis (Laris, 2011). We divided
the sites into plots of 10 × 10 m and applied fire treatments
of head and back burns. Fire timing was set according to the
historical pattern of burning, with early fires set in November
through December, middle fires in January, and late fires in
late February and March (Laris et al., 2016). We conducted
multiple burns per site to account for plot-level heterogene-
ity. Plots at each site were located in close proximity with
attention paid to maintaining consistency in grass type and
woody cover. Head-fire and backfire plots were located di-
rectly adjacent. 2
Study area and methods We based our research on two working landscapes located in
the southern Sudanian savanna of southern Mali (Fig. 2). We
chose areas with annual precipitation over 900 mm because
they burn frequently and are typical of this broad mesic sa-
vanna belt in West Africa. The climate is divided into two
seasons: a wet period from approximately June through Oc-
tober and a dry season from November through May. We also
recognize a cool dry period from approximately November
through February and a hot dry period from March through
May. This distinction is important because the Harmattan
wind, which is dry and desiccates vegetation while creat-
ing unique fire weather, dominates in the cool season. The
mean annual rainfall is 991 mm for Tabou and 1177 mm for
Faradiélé (based on data from the nearby urban centers of
Bamako (latitude 12.64◦, longitude −8.00◦) and Bougouni
(latitude 11.42◦, longitude −7.47◦) for each study area, re-
spectively) (Henry, 2011). The fire season follows the rains
and typically runs from November through April. The regime
follows a regular annual spatiotemporal pattern with the ma-
jority of fires occurring in late December and early January
(Laris et al., 2016). The vegetation is southern Sudanian savanna and is pre-
dominantly composed of a mixture of grasses, trees and
shrubs in a complex mosaic. The landscape heterogeneity is
a function of topography, underlying soil and hydrology, as
well agricultural uses, the combinations of which produces Figure 2. Study areas in southern Mali (figure by Scott Winslow). https://doi.org/10.5194/bg-18-6229-2021 https://doi.org/10.5194/bg-18-6229-2021 Biogeosciences, 18, 6229–6244, 2021 6233 P. Laris et al.: Methane gas emissions from savanna fires https://doi.org/10.5194/bg-18-6229-2021 × Emission Factor (gkg−1) × 10−3
(5 Here, “emission” is the gas or aerosol flux in tons (t),
“area” is the total area burnt in hectares (ha), “fuel load”
is the amount of burnt biomass in tons per hectare (t ha−1),
“combustion completeness” is the fraction of fuel affected by
fire that was pyrolyzed in percent (%), and “emission factor”
of a gas is the amount of this gas generated when 1 kg of
fuel is burnt. We have revised the formula to include season-
ally specific values for area, fuel load, combustion complete-
ness and emission factor and add the variable burn patchiness
(BP), which is the fraction of the surface area affected by the
fire. We suggest the following revision for determining emis-
sions by fire season in savannas (Es). Emissions (t) = BAs (ha) × FLs (tha−1)
× CCs (%) × EFxs × BPs (%)
(6) (6) We calculated EF as follows: Here, BA is burned area, FL is fuel load, CC is combustion
completeness, and EFx is emission factor of species x. We
propose using seasonal values for these key factors because
these variables vary significantly by season as a function of
ambient weather, as well as fuel moisture and fuel type (frac-
tion of leaf litter or shrubs) and fuel conditions. We have
added BPs because patchiness varies by season (as well as
fuel) and because most estimates of burned area are based on
satellite image analysis, which is too coarse to determine the
actual surface area burned due to burn patchiness (the actual
area burned is thus BP × BA) (Russell-Smith et al., 2009). Note that even medium-resolution burned area estimates us-
ing Landsat contain errors in the percent of area burned due EFx = Fc1000
MMx
MMcarbon
Cx
CT
. (3) (3) EFx is the emission factor for species x (g kg−1). Fc is the
mass fraction of carbon in the fuel for which we use the value
of 0.5 (the majority of studies find the carbon fraction to vary 1We used the value of 20 000 kJ kg−1 following Williams et
al. (1998, p. 2.3
Field data analysis between 0.425 and 0.50; the latter is used most often for pur-
poses of comparison (Ward et al., 1996), although Lacaux et
al. (1995) found a value of 0.425 for West Africa). MM is the
molecular mass of species x (g), and 1000 g kg−1 is a conver-
sion factor. MMcarbon is the molecular mass of carbon (12 g),
and Cx / CT is the ratio of the number of moles of species x
in the emission sample divided by the total number of moles
of carbon, calculated as follows: To quantify intensity we used the fire-line intensity of
Byram (1959), which is defined as follows: (2) I = Hwr, I = Hwr, where I is Byram’s fire-line intensity (kW m−1), H is the net
low heat of combustion (kJ kg−1), w is the fuel consumed in
the active flaming front (kg m−2), and r is the linear rate of
fire spread (m s−1). The net low heat of combustion (H) was
selected following Williams et al. (1998) with 20 000 kJ kg−1
as an appropriate value for savanna fires.1 The amount of fuel
consumed was calculated by subtracting the average ash and
unburned material remaining in three quadrats per plot from
the pre-fire measurement of dry biomass. Variable r was de-
rived from the time it took for the base of the first flaming
front to reach the end of the 10 m plot. We calculated fire-line
intensity for all samples possessing all the variables for anal-
ysis. Finally, combustion completeness was calculated by di-
viding the biomass consumed by the pre-fire biomass. Cx
CT
=
ERx/CO2
nP
j=1
(NCjERj/CO2)
,
(4) (4) (4) where ERx/CO2 is the emission ratio of species x to CO2,
NCj is the number of carbon atoms in compound j, and the
sum is over all carbonaceous species (approximated as CO2,
CO and CH4 for this study). The general equation used to quantify the gas species
emitted from vegetation fires is the basic biomass burning
emission model of the Intergovernmental Panel on Climate
Change (IPCC) (IPCC, 2003: 49; IPCC, 2006: A2.13). Emission (t) = Burned Area (ha) × Fuel (tha−1)
× Completeness (%) 2.1
Data collection The majority of the samples were for
backfires to replicate local practices, with head-fire samples
taken for comparative purposes. We only sampled each fire
once and thus caution against assuming a single sample rep-
resents the typical emissions for the entire fire (see Murphy et
al., 2012, above), but we do think the mean values for the data
we collected provide a useful sample of typical West African
fire emissions. In addition, we burned different savanna veg-
etation types (with different grasses and woody vegetation
amounts) at different times of the fire season in accordance
with local practices; as such, we do not have systematic re-
sults for burning all grass types for all fire seasons (e.g., few
perennial grasses burn in the EDS). m = 5.658 + (0.04651H)
+
0.0003151H 3
T
−
0.1854T 0.77
,
(1) (1) where H is relative humidity, and T is ambient temperature
at the time of the burn. We calculated dry biomass weight by
subtracting the fuel moisture content from the wet biomass
weight, and the amount of fuel consumed was calculated by
subtracting the average ash and unburned fuels remaining in
three quadrats per plot from the pre-fire dry biomass weight. https://doi.org/10.5194/bg-18-6229-2021 Biogeosciences, 18, 6229–6244, 2021 6234 P. Laris et al.: Methane gas emissions from savanna fires 2.4
Gas emission sampling and analysis We collected samples of smoke from a sample of 36 of our
experimental fires for the early and middle seasons along
with background air samples for each different site prior to
burning. (The high cost of shipping canisters prohibited col-
lecting data for the late season using this method.) Samples
were collected in stainless steel vacuum canisters by mount-
ing the canister on a pole and holding the canister with open
flow-restricting valve about 40 cm above the flame. We sam-
pled all fires a single time once the flaming front had devel-
oped (approximately two-thirds of the way through the burn
plot). Once filled, the canisters were shipped directly back
to California for analysis at the laboratory of the Department
of Chemistry at the University of California at Irvine. Mass
spectrometer (MS), thermal conductivity and flame ioniza-
tion after separation by gas chromatography were used to
detect, inventory and measure the quantities of the different
species of gas contained in the samples (Katzenstein et al.,
2003; Koné et al., 2020). (5) × Emission Factor (gkg−1) × 10−3
(5) P. Laris et al.: Methane gas emissions from savanna fires Table 1. Mean plot characteristics by study period (n and standard
deviations in parentheses) (2014–2016, Mali). to the fragmented patterns fires create across a heterogeneous
savanna landscape. We did not determine total BA for this
work but have done so previously using Landsat data cov-
ering the study area (Laris, 2011). As such, we present our
results in terms of emissions per square meter according to
season of the burn. to the fragmented patterns fires create across a heterogeneous
savanna landscape. We did not determine total BA for this
work but have done so previously using Landsat data cov-
ering the study area (Laris, 2011). As such, we present our
results in terms of emissions per square meter according to
season of the burn. Mean plot
EDS
MDS
All fires
characteristics (n = 36)
(16)
(20)
(36)
Dry biomass (g m−2)
340
349
345
(137)
(71.3)
(110)
Grass biomass (%)
83.1
78.9
80.8
(21.1)
(17.2)
(19.1)
Total fuel moisture (%)
10.8
15.1
13.2
(10.7)
(12.0)
(11.7)
Temperature (◦C)
33.0
29.2
30.9
(3.03)
(3.62)
(3.86)
Relative humidity (%)
28.7
30.6
29.8
(4.02)
(12.2)
(9.51)
Wind speed (m s−1)
0.99
1.63
1.35
(0.61)
(0.58)
(0.68) Complete combustion of vegetation results in the release
of carbon in the form of CO2, while incomplete combustion
leads to the emission of CO, CH4 and a large variety of or-
ganic compounds (Koppmann et al., 2005). Because many
of the factors that control EFs also regulate combustion ef-
ficiency, determining the latter is a useful proxy for predict-
ing how individual emission factors will vary under different
fire conditions. Modified combustion efficiency (MCE) – the
ratio of CO2 to CO + CO2 – is frequently used to estimate
combustion completeness as values for MCE are related to
different phases in the combustion process. In general, when
the MCE exceeds 90 %, a fire is flaming and combustion tem-
peratures are high. When MCE is less than 85 %, combustion
is smoldering. A savanna fire is typically characterized by a
flaming front moving across the landscape leaving smolder-
ing material. As such, the smoke emitted from savanna fires
is typically a product of both flaming and smoldering on dif-
ferent fragments of fuel. son progresses, but the mid-season humidity is considerably
more variable than in the early season. 3.2
Fire characteristics The characteristics of the fires also vary by season (Table 2). The mean BP increased as the dry season progressed from
77.4 % in the EDS to 92.3 % in the MDS, as expected due to
the gradual desiccation of the biomass and slight rise in wind
speed. CC also increased from the early to middle season
(81.3 % to 86.2 %). These variables showed great variability
in both seasons. Spread rate and intensity increased from the
early to the middle season, with the high variation in inten-
sity values reflecting the wide variety of fuel, weather and
fire conditions. The data also show that fire type has a large
influence on fire intensity, as expected; head-fire mean inten-
sity was much greater than that for backfires (242.4 W m−1 P. Laris et al.: Methane gas emissions from savanna fires Calculated fuel mois-
ture content based on Viney declines over the course of the
dry season, but when combined with the measured mean cure
rates for moist grasses, the total fuel moisture means rose
from 10.8 % in the EDS to 15.1 % in the MDS with high vari-
ability. Mean wind speed peaks mid-season during the Har-
mattan, although the wind speeds are relatively low – clas-
sified as a light breeze on the Beaufort scale. The percent of
grass of the total plot biomass is greatest in the early season,
while the total biomass (total fine fuels – grasses and leaves)
is higher in mid-season, reflecting an increase in leaf litter
as the dry season progresses. The increase in dry biomass
also reflects the changes in species types burned – the taller
perennials often burn later in the dry season. Some perennials
are too moist to burn during the early months of the dry sea-
son and burn less completely in the mid-season due to higher
moisture content. Bivariate statistical analyses were performed to test the
significance of the difference of means (t tests) in CH4 EF by
season (EDS and MDS) and by fire direction (head fires and
backfires) and in MCE by season and fire direction. F tests
established the similarity of variances, and all t tests were
done with pooled estimates of variance. These were done in
the OpenOffice Calc spreadsheet (Apache Software Founda-
tion, version 4.1, available at: https://www.openoffice.org/,
last access: 29 November 2021) and PAleontological STatis-
tics (Hammer et al., 2001), with effect sizes (Cohen’s d) and
post hoc power calculated in G*Power (Faul et al., 2009). We
used bivariate regression analysis to look for correlations be-
tween the two dependent variables – methane EF and density
– and independent variables – Byram’s fire intensity, propor-
tion of grass biomass (to woody biomass), total fuel moisture
and Viney fuel moisture (a function of ambient temperature
and humidity). These were done in Calc, and power was es-
timated in G*Power. × Emission Factor (gkg−1) × 10−3
(5 230) who note: “Given the range and lack of consis-
tency between studies in the value of H, and, in the view of the
authors, the misleading precision implied by values rounded to the
nearest 100 kJ kg−1, 20 000 kJ kg−1 is within the range of reported
vales and is easy and convenient to apply.”. https://doi.org/10.5194/bg-18-6229-2021 Biogeosciences, 18, 6229–6244, 2021 6235 3.3
Methane emissions and MCE The mean EF for methane was 3.47 g kg−1, and the mean
MCE was 0.90, which is considered to be on the cusp of flam-
ing and smoldering (Table 2). Our study found that methane
EFs ranged from 3.83 g kg−1 in the EDS to 3.18 g kg−1 in the
MDS. These differences yield a weak effect size of 0.25 (Co-
hen’s d), but the small sample did not provide enough power
(1−β = 0.11) for this effect to be significant (p = 0.45). The
results indicate that fire type has a larger impact on methane
EF than fire season. Head fires had nearly double the CH4 EF
of backfires (5.12 to 2.74 g kg−1), and this held regardless of
fire season. This difference is both significant (p = 0.02) and
dramatic in effect (Cohen’s d = 0.92) despite the relatively
small sample (1 −β = 0.69). MCE was also slightly lower
for head fires than for backfires and lower for the EDS (0.87
compared to 0.92 for MDS). Figure 5. Carbon monoxide EF as a function of Byram’s fire inten-
sity for all fires. Arrows indicate fire type (2014–2016, Mali). between CH4 EF and either total moisture, calculated Viney
moisture, or percent of grass in the biomass. We did find a
negative and significant relationship between MCE and CH4
EF, as expected (P = 0.000001; R2
adj = 0.436); however, the
effects of fire type can be seen here as well. When head fires
and backfires are examined separately, the relationship be-
tween CH4 EF and MCE for backfires is much stronger than
head fires (Fig. 6). Similarly, for MCE and methane density
we found a stronger relationship for backfires than head fires. between CH4 EF and either total moisture, calculated Viney
moisture, or percent of grass in the biomass. We did find a
negative and significant relationship between MCE and CH4
EF, as expected (P = 0.000001; R2
adj = 0.436); however, the
effects of fire type can be seen here as well. When head fires
and backfires are examined separately, the relationship be-
tween CH4 EF and MCE for backfires is much stronger than
head fires (Fig. 6). Similarly, for MCE and methane density
we found a stronger relationship for backfires than head fires. P. Laris et al.: Methane gas emissions from savanna fires 6236 Figure 3. Methane EF as a function of Byram’s fire intensity for all
fires. Arrows indicate fire type (2014–2016, Mali). Figure 3. Methane EF as a function of Byram’s fire intensity for all
fires. Arrows indicate fire type (2014–2016, Mali). Figure 3. Methane EF as a function of Byram’s fire intensity for all
fires. Arrows indicate fire type (2014–2016, Mali). Figure 4. Methane density as a function of Byram’s fire intensity
for all fires. Arrows indicate fire type (2014–2016, Mali). Figure 5. Carbon monoxide EF as a function of Byram’s fire inten-
sity for all fires. Arrows indicate fire type (2014–2016, Mali). to 100.0 W m−1). In addition, we found a large variation in
the fire-line intensity values especially for head fires. 3.3
Methane emissions and MCE Despite the small sample (1 −β = 0.64), fire intensity
(Byram’s) was a significant driver of CH4 EF (p = 0.03), but
the correlation was modest (R = 0.38), and the effect size
was weak (R2
adj = 0.09) (Fig. 3). There was a similar rela-
tionship for fire intensity and methane density (P = 0.006;
R2
adj = 0.165) (Fig. 4). Methane emission density increased
marginally from 0.839 g m−2 in the EDS to 0.875 g m−2 in
the MDS. This was not significant (p = 0.88), the effect
size was trivial (Cohen’s d = 0.05), and the sample size
was underpowered (1 −β = 0.05). Head fires, however, had
much higher emission densities than backfires (1.203 vs. 0.708 g m−2, respectively). This difference yields a strong ef-
fect (Cohen’s d = 0.81), which is significant (p = 0.04) even
though the study was underpowered (1 −β = 0.58). 3.1
Plot characteristics The mean plot characteristics for biomass and weather con-
ditions demonstrate the importance of the inclusion of the
mid-season in this study (Table 1) (see Laris et al., 2021, for
late dry season values). Average temperature generally in-
creases over the course of the dry season but dips by 0.4 ◦C
in mid-season, which is an established phenomenon in West
African climates. Average humidity decreases as the dry sea- https://doi.org/10.5194/bg-18-6229-2021 Biogeosciences, 18, 6229–6244, 2021 P. Laris et al.: Methane gas emissions from savanna fires
Figure 4. Methane density as a function of Byram’s fire intensity
for all fires. Arrows indicate fire type (2014–2016, Mali). 6236
P. Laris et al.: Methane gas emissions from savanna fires
Figure 3. Methane EF as a function of Byram’s fire intensity for all
fires. Arrows indicate fire type (2014–2016, Mali). Figure 4. Methane density as a function of Byram’s fire intensity
for all fires. Arrows indicate fire type (2014–2016, Mali). P. Laris et al.: Methane gas emissions from savanna fires P. Laris et al.: Methane gas emissions from savanna fires P. Laris et al.: Methane gas emissions from savanna fires
Table 2. Mean fire characteristics and emissions by study period and fire type (2014–2016, Mali). Mean fire
EDS
MDS
Head
Back
All fires
characteristics and
(16)
(20)
(11)
(25)
(36)
emissions (n = 36)
Spread rate (m s−1)
0.024
0.027
0.046
0.017
0.026
(0.019)
(0.035)
(0.043)
(0.012)
(0.029)
Scorch height (m)
1.20
1.14
1.37
1.07
1.16
(0.45)
(0.53)
(0.42)
(0.50)
(0.50)
Burn patchiness (%)
77.4
92.4
82.1
87.3
85.7
(15.5)
(8.13)
(16.1)
(12.9)
(14.1)
Combustion completeness (%)
81.3
86.2
82.0
84.9
84.0
(12.3)
(13.7)
(12.4)
(13.6)
(13.3)
Byram’s fire intensity (kW m−1)
118.3
163.7
242.4
100.0
143.5
(84.5)
(191.9)
(230.9)
(71.5)
(155.3)
Modified combustion efficiency (MCE)
0.87
0.92
0.88
0.90
0.90
(0.18)
(0.19)
(0.19)
(0.20)
(0.18)
Methane emission factor (CH4 EF g kg−1)
3.83
3.18
5.12
2.74
3.47
(2.67)
(2.31)
(1.74)
(2.43)
(2.50)
Methane emission density (g m−2)
0.839
0.875
1.203
0.708
0.859
(0.651)
(0.667)
(0.658)
(0.602)
(0.660) Table 2. Mean fire characteristics and emissions by study period and fire type (2014–2016, Mali). intensity increased, and head fires, which have higher fire
intensity, had higher methane EFs regardless of season. In-
creased fire intensity results in taller flame heights, which
reach into the tree canopies of the numerous small trees and
burn greater amounts of fresh green leaves (Fig. 7). Indeed,
our field observations recorded the highest methane emis-
sions (over 5000 ppm) during the combustion of green leaves
on small trees. We were not able to determine the amount of
leaves on trees that were combusted in this study, although it
is reasonable to estimate that more green leaves would burn
on trees in the EDS than other seasons. Interestingly, we did
not find a correlation between Byram’s fire intensity and CO
EF, although CH4 and CO EFs did correlate with each other,
as expected. We suggest the latter finding supports our argu-
ment that higher flame heights result in increased CH4 emis-
sions, and this suggests that CH4 and CO EFs may not be as
coupled as some research suggests. vannas” as opposed to the “grassland” values used in most
savanna biome estimates. Wooded savannas contain small
trees, shrubs and leaf litter, which tend to reduce MCE and
increase methane EF values (e.g., Vernooij et al., 2021) due
to the fraction of ligneous fuel in the mix. 4
Discussion Our study finds that methane EF means were highest for
EDS, as expected, and dropped by about 20 % by the MDS. We found, however, that fire type had a greater (and more
significant) impact on methane EF than season; head-fire
methane EFs were nearly double those for backfires (5.12
to 2.74 g kg−1). In general, methane EFs increased as fire We found no significant relationship between Byram’s in-
tensity and CO EF (Fig. 5) and no significant relationship Biogeosciences, 18, 6229–6244, 2021 https://doi.org/10.5194/bg-18-6229-2021 6237 P. Laris et al.: Methane gas emissions from savanna fires P. Laris et al.: Methane gas emissions from savanna fires 6238 Table 3. A comparison of methane EF values for seasonal savanna and woodland fire studies. Methane EF
EDS
MDS∗
LDS
Ratio (E/L)
Mean
This study
3.82
3.18
NA
1.20
3.47 (± 2.5)
Wooded savanna
Korontzi (2005)
3.82
NA
2.61
1.47
3.22
Miombo woodlands
Korontzi (2005)
3.14
NA
0.80
3.93
1.97
Dambo grasslands
Russell-Smith et al. (2021)
NA
6.12
1.45
4.22
3.72
Dambo grass savanna
Russell-Smith et al. (2021)
NA
1.34
1.31
1.02
1.33
Dry savanna
Russell-Smith et al. (2021)
NA
1.51
2.22
0.68
1.87
Wooded savanna
Andrea (2019)
NA
NA
NA
2.71 (± 2.2)
Savanna biome
∗Although Russell-Smith et al. (2021) refer to their fires as EDS for comparative purposes, they are more in line
with MDS burning for reasons noted below. NA – not available. omparison of methane EF values for seasonal savanna and woodland fire studies. ∗Although Russell-Smith et al. (2021) refer to their fires as EDS for comparative purposes, they are more in line
with MDS burning for reasons noted below. NA – not available. Figure 6. MCE as a function of CH4 EF for head fires (a) and back-
fires (b). Green fires are EDS, and orange are MDS. Arrows indicate
fire type (2014–2016, Mali). Figure 7. A head fire extending from a bed of dry grasses into
the green leaves in the tree canopy, Tabou Village, Mali (Photo by
Paul Laris). from 0.24 g m−2 in the EDS to 0.55 g m−2 in the LDS. By
comparison, we found smaller increases by season – less than
5 %, which was insignificant. The larger range in emission
density values estimated by Korontzi is derived largely from
the higher range of CC used in their model. Korontzi (2005)
also notes that the lower emission densities for CH4 in the
EDS were mainly a result of the larger effect of the increased
fuel moisture content on lowering CC compared with its ef-
fect on MCE. They note this was the opposite of what they
found for their grassland fires, indicating that the amount of
woody vegetation is a critical determinant of CH4 EF. It is important to note that Korontzi’s values for early sea- Figure 6. MCE as a function of CH4 EF for head fires (a) and back-
fires (b). Green fires are EDS, and orange are MDS. Arrows indicate
fire type (2014–2016, Mali). P. Laris et al.: Methane gas emissions from savanna fires Our results compare favorably for seasonal changes in
methane EFs found by Korontzi (2005) for East African
woodland savanna. Importantly, our data are more in line
with Korontzi’s values for “woodlands” (Miombo) than for
“grassland” (Dambo) savannas (Table 3). We attribute this to
the fact that both areas have mesic rainfall regimes and high
tree and shrub cover. As noted, we found the percent of grass
of the total plot biomass is greatest in the EDS, while the to-
tal biomass is higher in the MDS, reflecting a tripling of leaf
litter biomass. Korontzi (2005) argued that because EDS fires have a
much lower CC – near 0 % in the early EDS to 80 % by LDS
– total emissions from EDS fires would be less than those
from LDS ones. This is in spite of the fact that they found
EDS fires have higher methane EFs by a factor of 1.47 for
woodlands and 3.93 for grasslands (compared with our ratio
of 1.20). They also found that fuel load increased by about
20 % from EDS to LDS with a big increase in MDS (we
found a smaller increase). Finally, they found that methane
EFs were at their peak in MDS as opposed to LDS. As a re-
sult, Korontzi concludes that for southeastern Africa, early
fires produce lower methane emissions than either mid- or
late-season fires in contrast to our results. Korontzi found the
regional average CH4 emission densities more than doubled Our results compare favorably with the biome averages
from Andreae (2019). Andreae’s savanna biome mean MCE
was 0.94 (± 0.02), and mean CH4 EF was 2.7 (± 2.2) g kg−1,
which can be compared with our values for our MCE (f )
of 0.90 and methane EF (f ) of 3.47 g kg−1 (Table 3). If
we use the lower-percent carbon value for West African sa-
vannas (42.5 % based on Lacaux et al. (1995) as opposed
to 50 % used for comparative purposes), then our methane
EF (adjusted) values (2.95 g kg−1) are quite close to the
biome means. It is not surprising that our values had a lower
MCE and higher CH4 EF than the biome means because
we based our values on emissions from “wooded mesic sa- https://doi.org/10.5194/bg-18-6229-2021 Biogeosciences, 18, 6229–6244, 2021 P. Laris et al.: Methane gas emissions from savanna fires (2021) note, trees in wooded
savanna had already begun dropping leaves, and grasses were
fully cured at the time of their “EDS” fires – characteristics
we would not associate with early burning in West Africa. As
such, we would argue that Russell-Smith et al. (2021) pro-
vide good evidence that MDS (not EDS) fires produce lower
methane emissions than LDS fires in Africa. When comparing our results for EDS and MDS fires, we
find evidence for an emission trade-off. That is, while EDS
fires have a lower BP than MDS fires, as well as lower CC,
the lower amount of biomass burned by EDS fires is offset
by the higher methane EF resulting in statistically insignifi-
cant differences in methane emission densities by season2. We must reiterate, however, that an unknown quantity of
standing leaves are combusted during fires (especially the
EDS when leaves remain on trees), meaning the fuel loads
we measured for the EDS are an underestimation of the leafy
fuels consumed. Small trees often dominate mesic savanna
woodlands, such as those in West Africa. These so-called
“Gulliver” trees are often less than 2 m tall because they
are repeatedly burned back to the rootstock by annual fires
(Laris and Dembele, 2012). We argue that the burning of
small trees contributes significantly to methane release. As
such, we cannot support the policy suggestion put forth by
Lipsett-Moore (2018) who promote increased early burning
in African savannas to reduce methane emissions. While it
is theoretically possible that very early fires would burn a Unfortunately, there is no recognized standard for what
distinguishes the early from the middle or late dry season
in the savanna literature – a problem hampering fire science. Elsewhere we have argued that the dichotomous (EDS/LDS)
view of savanna burning is problematic because the point at
which the fire season shifts from early to late has not been
adequately defined and varies by context (Laris et al., 2017;
Laris, 2021). We note that although adding a third, middle
season is potentially useful for research on gas emissions, the
fundamental problem of typology remains. While the EDS
clearly begins when the rains end, there are no recognized
standards for determining when the MDS or LDS begins. P. Laris et al.: Methane gas emissions from savanna fires Indeed, we have rarely seen
burned landscapes with more than 50 % of the biomass stand-
ing post fire. )
In our previous research involving a large sample of 97
experimental fires, we found that fires set in the MDS (the
peak moment in local burning) differed from those set in the
EDS or LDS (the seasons used in most fire studies) in key
ways (Laris et al., 2020). In our larger study we found that
when fires are set in accordance with local practices, MDS
fires had the lowest fire intensity and scorch heights, while
CC increased only slightly from the early to the middle sea-
son with a larger jump in the late season. Mean values for
BP gradually increased from the early to the middle to late
season due to the drying of the biomass. Fuel moisture was
also slightly higher in the MDS than EDS (due to the burn-
ing of more perennials) before dropping dramatically in the
LDS. Fuel loads increased in the MDS largely due to a rise
in the percentage of leaf litter in the total biomass. It should
be noted that an increase in leaf litter means a decrease in the
amount of green leaves burned on trees. In sum, we conclude
that seasonal distinctions can be useful if clearly defined, but
they should be limited to specific savannas and not used for
comparative purposes between regions for the purposes of
determining the effects of fire on emissions. By comparison, Russell-Smith et al. (2021) found that
emission factors varied by season for Dambo grasslands but
not for savanna woodlands (Table 3). They found that Dambo
grasses burned in the EDS had a methane EF of over 4 times
that of those burned in LDS (nearly identical with results
from Korontzi). Contrastingly, they found little difference
by season in methane EF for dry wooded savannas and a
surprisingly higher EF in late season for more wooded sa-
vannas, which contrasts with our findings and those of Ko-
rontzi (2005) for wooded savannas. We must note, how-
ever, that the date chosen for the EDS of Russell-Smith et
al. (2021) is more comparable with the MDS date used in our
study – both dates represent the “middle” of the dry season. Indeed, as Russell-Smith et al. 2We note that results from our larger study of 97 fires found a
less dramatic rise in BP and CC from EDS to MDS to LDS than for
the sample of 36 fires used here. In the larger study, BP increased
marginally as the dry season progressed to a near-complete burn
by the late season (85.3 % to 92.3 % to 99.2 %). CC increased very
slightly from the early to middle season before increasing substan-
tially in the late season (85.1 % to 86.4 % to 92.8 %) (Laris et al.,
2020). These findings suggest a stronger emission trade-off than re-
ported here. P. Laris et al.: Methane gas emissions from savanna fires able marker for a division between EDS and MDS. Weather
conditions also play a critical role, and these differ by region. For example, in West Africa winds peak in MDS, but they are
stronger in the LDS in southern Africa (Russell-Smith et al.,
2021). tically speaking, people do not set fires when grasses are too
moist to carry fire in West Africa. As such, we argue that us-
ing CC values less than 50 %, while theoretically useful, are
not at all practical because people are unlikely to set such
fires as they would not achieve the desired goals of burning
(Laris, 2005, 2011). Indeed, it is probable that a fire which
burns less than 50 % of biomass will burn a second time late
in the season because a fire consuming such a small frac-
tion will not break fuel connectivity. It is also important to
note that breaking fuel connectivity is a key reason for set-
ting early fires and a critical reason why a mosaic fire regime
burns less total area (Laris et al., 2018). As such, although
theoretically possible, we do not agree that using such low
CC values is reasonable for determining emissions from fires
in actual African landscapes. Indeed, we have rarely seen
burned landscapes with more than 50 % of the biomass stand-
ing post fire. tically speaking, people do not set fires when grasses are too
moist to carry fire in West Africa. As such, we argue that us-
ing CC values less than 50 %, while theoretically useful, are
not at all practical because people are unlikely to set such
fires as they would not achieve the desired goals of burning
(Laris, 2005, 2011). Indeed, it is probable that a fire which
burns less than 50 % of biomass will burn a second time late
in the season because a fire consuming such a small frac-
tion will not break fuel connectivity. It is also important to
note that breaking fuel connectivity is a key reason for set-
ting early fires and a critical reason why a mosaic fire regime
burns less total area (Laris et al., 2018). As such, although
theoretically possible, we do not agree that using such low
CC values is reasonable for determining emissions from fires
in actual African landscapes. P. Laris et al.: Methane gas emissions from savanna fires Figu
the g
Paul
from
com
5 %
dens
the h
also
EDS
fuel
fect
foun
woo
It Figure 7. A head fire extending from a bed of dry grasses into
the green leaves in the tree canopy, Tabou Village, Mali (Photo by
Paul Laris). Figure 7. A head fire extending from a bed of dry grasses into
the green leaves in the tree canopy, Tabou Village, Mali (Photo by
Paul Laris). Figure 7. A head fire extending from a bed of dry grasses into
the green leaves in the tree canopy, Tabou Village, Mali (Photo by
Paul Laris). from 0.24 g m−2 in the EDS to 0.55 g m−2 in the LDS. By
comparison, we found smaller increases by season – less than
5 %, which was insignificant. The larger range in emission
density values estimated by Korontzi is derived largely from
the higher range of CC used in their model. Korontzi (2005)
also notes that the lower emission densities for CH4 in the
EDS were mainly a result of the larger effect of the increased
fuel moisture content on lowering CC compared with its ef-
fect on MCE. They note this was the opposite of what they
found for their grassland fires, indicating that the amount of
woody vegetation is a critical determinant of CH4 EF. Figure 6. MCE as a function of CH4 EF for head fires (a) and back-
fires (b). Green fires are EDS, and orange are MDS. Arrows indicate
fire type (2014–2016, Mali). It is important to note that Korontzi’s values for early sea-
son CC were derived using a model and based on fuel mois-
ture levels alone, not experimental data. We argue that, prac- Biogeosciences, 18, 6229–6244, 2021 https://doi.org/10.5194/bg-18-6229-2021 6239 P. Laris et al.: Methane gas emissions from savanna fires P. Laris et al.: Methane gas emissions from savanna fires Surprisingly, there are but a few studies of African sa-
vanna emission factors for which head-fire and backfire data
are available. Wooster et al. (2011) found mean EF methane
for head fires to be higher than the backfire mean by 3.35
to 1.88 g kg−1 (a similar ratio to what we found). Several
laboratory results also support our finding that MCE and
EF methane are functions of fire type. Keene et al. (2006)
used laboratory fires of fuels from southeastern Africa and
found that the type of fire (backing, heading or mixed), as
well as fuel moisture, influenced MCE. The lowest MCE val-
ues they recorded were all for heading fires with relatively
low moisture content, while MCE fell and EF methane rose
as fuel moisture increased. Similarly, Surawski et al. (2015)
found that heading fires exhibited the lowest MCE and higher
methane EF. In conclusion, our study finds that several factors influ-
ence the emissions from savanna fires including the fire sea-
son, fuel load and type, and, most importantly, fire type. Each
of these factors are a function of human land and fire man-
agement practices. We also conclude there is an emission
trade-off in setting fires earlier, and, as such, a policy to in-
crease the amount of early burning in West African would
be very difficult to implement because much burning is al-
ready “early” and because earlier burning of uncured grasses
and green leaves would likely result in higher methane EFs. Moreover, any policy aimed at increasing the amount of early
burning would likely require setting head fires, which would
decrease burn patchiness and increase combustion complete-
ness, further negating the effects of any reduction in burned
area while also causing an undesired increase in uncontrolled
fires. While the primary purpose of this study was to deter-
mine realistic values for methane emissions for a represen-
tative working savanna in West Africa, the findings can in-
form broader-scale modeling efforts for savanna fire burning
(Hanston et al., 2016) in three ways. First, we do not rec-
ommend making crude assumptions about the effects of fire
season on methane emissions as these will vary by savanna. Second, there is a need to map fires according to type be-
cause head fires cause higher methane emissions for the two
reasons noted above. P. Laris et al.: Methane gas emissions from savanna fires lower fraction of the landscape than we have observed, we
argue that such a policy is just as likely to cause an increase
in methane emissions due to higher methane EF of earlier
burning, which may be a function of green leaf combustion
(see Korontzi, 2005). It is also important to note that higher-
intensity head fires would be required to increase the burned
area of moist perennial grasses in the EDS3, and because
head fires have a methane EF nearly double that of backfires,
burning with head fires would likely counter any advantage
of burning early to reduce emissions. In addition, local inhab-
itants would be very reluctant to set such fires due to the in-
creased risk that setting head fires could damage field crops,
which remain unharvested in the EDS. dry season to the mid-dry season (although the results were
not significant). We also found that methane emission den-
sity increased only marginally from EDS to the MDS, a dif-
ference that was not significant. We found that fire type had
a much greater effect on methane emissions than fire season,
with head fires having significantly higher methane EF com-
pared to backfires and significantly higher methane densities
due to higher fire intensity. We note that we are unaware of
any estimates for area burned according to fire type for any
of the world’s savannas. It is important to reiterate that several key findings of
this study arise from documented burning practices of peo-
ple living in working landscapes. People set fires in West
Africa later in the day resulting in fires with lower inten-
sity due to lower wind and air temperature and higher hu-
midity, and people predominantly set backfires, all of which
contribute to lower-intensity burning, which results in lower
methane emissions. In addition, we note that the fuel loads
we recorded are nearly 50 % lower on working savanna lands
compared to reserve lands used in some other studies (Laris
et al., 2020). Finally, the number of fires peaks in the West
African region in the MDS, and although the methane emis-
sion density values for the EDS were slightly lower than for
the MDS, a significant reason for this was the increased fuel
load from leaf litter in the MDS. We should note that EDS
fires tend to burn more green leaves on trees, which are not
accounted for in this study. P. Laris et al.: Methane gas emissions from savanna fires Third, modelers need to make distinc-
tions between savanna types because there are large differ-
ences between emissions from grass-dominated and wooded
savanna landscapes as both theory and empirical results sug-
gest. In sum, modelers should focus on developing methods
to determine the direction (type) of fire remotely in addition
to other key factors such as fire intensity, fuel moisture, sa-
vanna woody cover (especially small trees) and burn severity. 3We made several attempts to burn perennial grasses in Decem-
ber and could not get them to ignite. Only under windy, head-fire
conditions will perennial grasses burn in the EDS. P. Laris et al.: Methane gas emissions from savanna fires While fuel moisture level holds some promise for develop-
ing a typology, the patchy heterogeneous nature of savan-
nas means that some patches could burn “early” (higher fuel
moisture) and some “late” (lower fuel moisture) in the same
fire. Other factors must be considered as well, the clearest of
which is leaf fall. We suggest the onset of leaf fall is a reason- https://doi.org/10.5194/bg-18-6229-2021 Biogeosciences, 18, 6229–6244, 2021 6240 P. Laris et al.: Methane gas emissions from savanna fires 5
Conclusions This study finds that when fires are set in working landscapes
in accordance with well-documented burning practices of
West African people, methane EFs decreased from the early 3We made several attempts to burn perennial grasses in Decem-
ber and could not get them to ignite. Only under windy, head-fire
conditions will perennial grasses burn in the EDS. Biogeosciences, 18, 6229–6244, 2021 People play a critical role deter-
mining the point at which fires
are set often according to fuel
moisture level of grasses at fine
spatial resolution. Medium. Total emissions are a
function of fuel load, including
the type of fuel and amounts
(below). Appendix A A1. Factors affecting methane emissions from West African savanna fires, human influence and uncertainty. Level of uncertainty High. There is no agreed upon
definition for distinguishing fire
seasons. Approaches vary from
the use of crude typologies
based on month of low rain-
fall to more sophisticated ones
based on vegetation or soil
moisture level. Physiological
measures could also include
leaf fall or leaf flush. People often begin burning the
moment rains end. They de-
termine the seasonal timing of
fires by selecting to burn when
grasses are just dry enough to
carry a fire with consideration
given to the agricultural calen-
dar. In occupied areas, human land
uses determine fuel load. Graz-
ing and rotational agricultural
practices have significant ef-
fects. In other areas, wild ani-
mals graze and browse. Medium. Much research has
been conducted on reserves or
protected areas where grassy
fuel loads are higher. Realistic
values for fuel loads on work-
ing lands should be used based
on ground or remotely sensed
methods. Medium. Higher fuel moisture
(in grasses or leaves) can reduce
combustion efficiency and raise
CH4 EF. Higher fuel moisture
can also increase patchiness of
burning. Fuel moisture declines
over the fire season. High. Fuel moisture is often
considered to be a function of
seasonality; however, there is
high spatial heterogeneity in sa-
vannas. A single fire can burn
one type of grass with high fuel
moisture and another with low
moisture with implications for
CH4 EF. High. Fires tend to burn in a
patchy manner especially when
vegetation is not uniformly dry
and when burning as a backfire. Patchiness created by earlier
fires, and prevents the spread of
later ones. Low. Advances in remote sens-
ing and image processing al-
gorithms have improved esti-
mates of patchy burning, al-
though the smaller, often ear-
lier fires are still most often un-
derestimated. Higher-resolution
data eliminate this problem. People create a patch mosaic
by systematically burning the
driest patches on the landscape
first, fragmenting the landscape
and creating a patch mosaic
with new, old and unburned
patches. High. Amounts of leaf litter
vary by savanna type and sea-
son. While amounts of dry leaf
litter have been estimated in
some cases, green leaf com-
bustion on standing trees and
shrubs is relatively understud-
ied. People determine the timing of
fires which has implications for
whether leaves are burned green
(early dry season) or dry (later
dry season). People determine the timing of
fires which has implications for
whether leaves are burned green
(early dry season) or dry (later
dry season). People play a critical role deter-
mining the point at which fires
are set often according to fuel
moisture level of grasses at fine
spatial resolution. P. Laris et al.: Methane gas emissions from savanna fires P. Laris et al.: Methane gas emissions from savanna fires https://doi.org/10.5194/bg-18-6229-2021 https://doi.org/10.5194/bg-18-6229-2021 https://doi.org/10.5194/bg-18-6229-2021 6241 Appendix A The authors wish to thank Fakuru Camara for
his never-ending help in the field, Umu Kante for keeping us all
happy and fed, and the people of Tabou and Faradiélé for supporting
this research. Financial support. This research has been supported by the Na-
tional Science Foundation (grant nos. 1313820 and G181115100). Review statement. This paper was edited by Fang Li and reviewed
by Sally Archibald and three anonymous referees. Author contributions. PL was principal investigator on the project,
supervised all aspects of the research and wrote the manuscript. MK
was involved in the fieldwork and the gathering, cleaning and orga-
nizing of all data, as well as commenting on the manuscript. FD
was head of the field research team and advisor in the field. RJ and
LY were involved in data organization and analysis, as well as dis-
cussing and commenting on the manuscript. CMR was responsible
for the statistical analysis with assistance from QL. Appendix A People determine the timing of
fires which has implications for
whether leaves are burned green
(early dry season) or dry (later
dry season). High. There is a potentially
large and unknown impact on
emissions of methane. There
are few studies of fire type for
savannas, but remotely sensed
methods offer potential. People purposefully set back-
fires, although fires can change
direction, and accidental fires
may more often burn as head
fires. https://doi.org/10.5194/bg-18-6229-2021 Biogeosciences, 18, 6229–6244, 2021 6242
P. Laris et al.: Methane gas emissions from savanna fires
Table A1. Continued. Impact on emissions
Human influence
Level of uncertainty
Fire time of day
Medium. Ambient
weather
conditions can affect fire inten-
sity and combustion, and these
are a function of time of day. People determine the time of
day to set fires, most often late
afternoon. Low. Although rarely consid-
ered in the literature, satellite
data can provide an estimate of
fire timing. Grass type
Low. Perennial grasses hold
moisture longer and are often
taller than annuals. Grass types
vary dramatically on savanna
landscapes. Human actions modify grass
species over the short and long
term. Perennials are highly val-
ued but are being replaced by
annuals. High. Few
studies
consider
variations in grassy vegetation
cover at fine resolution. Re-
motely
sensed
methods
can
potentially distinguish between
annuals and perennials. Woody vegetation type
Medium. Savannas are highly
heterogeneous
with
varying
levels of tree cover, which
affects
CH4
EF
especially
when small trees burn. Woody vegetation type is par-
tially a function of long-term
human land use patterns of agri-
culture and grazing. Medium. Improved
remote
sensing techniques can increase
accuracy of vegetation mapping
including canopy cover. P. Laris et al.: Methane gas emissions from savanna fires 6242 Table A1. Continued. Woody vegetation type is par-
tially a function of long-term
human land use patterns of agri-
culture and grazing. Medium. Improved
remote
sensing techniques can increase
accuracy of vegetation mapping
including canopy cover. Data availability. Notes
are
available
at
https://cla.csulb.edu/
departments/geography/savannalabo/wp-content/uploads/2021/01/
Notes-for-Mali-fire-data-2016-2018.docx (Laris, 2016–2018a). Data
are
available
at
https://cla.csulb.edu/departments/
geography/savannalabo/wp-content/uploads/2021/01/Cannister_
data-with-EF-and-MCE-good.xlsx (Laris, 2016–2018b) and https:
//cla.csulb.edu/departments/geography/savannalabo/wp-content/
uploads/2021/01/Mali-2015-2018-final-merge-all-gases-by-plot. xlsx (Laris, 2016–2018c). Data availability. Notes
are
available
at
https://cla.csulb.edu/
departments/geography/savannalabo/wp-content/uploads/2021/01/
Notes-for-Mali-fire-data-2016-2018.docx (Laris, 2016–2018a). Data
are
available
at
https://cla.csulb.edu/departments/
geography/savannalabo/wp-content/uploads/2021/01/Cannister_
data-with-EF-and-MCE-good.xlsx (Laris, 2016–2018b) and https:
//cla.csulb.edu/departments/geography/savannalabo/wp-content/
uploads/2021/01/Mali-2015-2018-final-merge-all-gases-by-plot. xlsx (Laris, 2016–2018c). Acknowledgements. P. Laris et al.: Methane gas emissions from savanna fires Laris, P.: Spatiotemporal problems with detecting and mapping mo-
saic fire regimes with coarse-resolution satellite data in savanna
environments, Rem. Sens. Environ., 99, 412–424, 2005. Heimann, M.: Enigma of the recent methane budget, Nature, 476,
157–158, 2011. Laris, P.: Humanizing savanna biogeography: Linking human prac-
tices with ecological patterns in a frequently burned savanna of
southern Mali, Ann. Assoc. Am. Geogr., 101, 1067–1088, 2011. Henry, C.: An integrated approach to estimating groundwater stor-
age, variability and recharge in Southern Mali, Africa, M.Sc
Thesis, Department of Earth Sciences, Simon Fraser University,
Canada, 2011. Laris,
P.:
Notes
for
Mali
Fire
Data
(2016–2018),
Mali
Fire
Data
[data
set],
available
at:
https://cla.csulb.edu/
departments/geography/savannalabo/wp-content/uploads/
2021/01/Notes-for-Mali-fire-data-2016-2018.docx (last access:
26 November 2021), 2016–2018a. Hoffa, E. A., Wakimoto, R. H., Ward, D. E., Hao, W. M., and Susott,
R. A.: Seasonality of carbon emissions from biomass burning in
a Zambian savanna, J. Geophys. Res., 104, 13841–13853, 1999. Laris, P.: Canister Data with EF, Mali Fire Data [data set],
available
at:
https://cla.csulb.edu/departments/geography/
savannalabo/wp-content/uploads/2021/01/Cannister_
data-with-EF-and-MCE-good.xlsx (last access: 26 Novem-
ber 2021), 2016–2018b. IPCC:
Good
Practice
Guidance
for
Land
Use,
Land-Use
Change and Forestry, available at: http://www.ipcc-nggip. iges.or.jp/public/gpglulucf/gpglulucf_contents.html (last access:
25 November 2021), 2003. IPCC: Guidelines for National Greenhouse Gas Inventories,
Agriculture, Forestry and other Land Use (AFOLU), Vol. 4, available at: http://www.ipcc-nggip.iges.or.jp/public/2006gl/
vol4.html (last access: 25 November 2021), 2006. Laris, P.: Final Merge – All Gases by Plot, Mali Fire Data
[data
set],
available
at:
https://cla.csulb.edu/departments/
geography/savannalabo/wp-content/uploads/2021/01/
Mali-2015-2018-final-merge-all-gases-by-plot.xlsx
(last
access: 26 November 2021), 2016–2018c. Katzenstein, A. S., Doezema, L. A., Simpson, I. J., Blake, D. R.,
and Rowland, F. S.: Extensive regional atmospheric hydrocarbon
pollution in the southwestern United States, P. Natl. Acad. Sci. USA, 100, 11975–11979, 2003. Laris,
P.:
On
the
problems
and
promises
of
sa-
vanna
fire
regime
change,
Nat. Commun.,
12,
4891,
https://doi.org/10.1038/s41467-021-25141-1, 2021. Keeley, J. E. and Pausas, J. G.: Distinguishing disturbance from
perturbations in fire-prone ecosystems, Int. J. Wildland Fire, 28,
282–287, 2019. Laris, P. and Dembele, F.: Humanizing savanna models: integrating
natural factors and anthropogenic disturbance regimes to deter-
mine tree-grass dynamics in savannas, J. Land Use Sci., 7, 459–
482, 2012. Keene, W. C., Lobert, J. M., Crutzen, P. J., Maben, J. R., Scharffe,
D. H., Landmann, T., Hély, C., and Brain, C.: Emissions of ma-
jor gaseous and particulate species during experimental burns
of southern African biomass, J. Geophys. Res., 111, D04301,
https://doi.org/10.1029/2005JD006319, 2006. P. Laris et al.: Methane gas emissions from savanna fires 6243 I. J., Spahni, R., Steele, L. P., Strode, S. A., Sudo, K., Szopa, S.,
van der Werf, G. R., Voulgarakis, A., van Weele, M., Weiss, R. F.,
Williams, J. E., and Zeng, G.: Three decades of global methane
sources and sinks, Nat. Geosci., 6, 813–823, 2013. Faul, F., Erdfelder, E., Buchner, A., and Lang, A.-G.: Statistical
power analyses using G*Power 3.1: Tests for correlation and re-
gression analyses, Behav. Res. Methods, 41, 1149–1160, 2009. Giglio, L., Randerson, J. T., van der Werf, G. R., Kasibhatla, P. S., Collatz, G. J., Morton, D. C., and DeFries, R. S.: Assess-
ing variability and long-term trends in burned area by merging
multiple satellite fire products, Biogeosciences, 7, 1171–1186,
https://doi.org/10.5194/bg-7-1171-2010, 2010. Koné, M., Dembele, F., and Laris, P.: Inventaire, typologie et esti-
mation quantitative des gaz émis par les feux de brousse en sa-
vane soudanaise dans le sud Mali, Revue de Géographie Tropi-
cale et d’Environnement, 2, 26–39, 2020. Koppmann, R., von Czapiewski, K., and Reid, J. S.: A review of
biomass burning emissions, part I: gaseous emissions of carbon
monoxide, methane, volatile organic compounds, and nitrogen
containing compounds, Atmos. Chem. Phys. Discuss., 5, 10455–
10516, https://doi.org/10.5194/acpd-5-10455-2005, 2005. Grégoire, J.-M., Eva, H. D., Belward, A. S., Palumbo, I., Simonetti,
D., and Brink, A.: Effect of land-cover change on Africa’s burnt
area, Int. J. Wildland Fire, 22, 107–120, 2013. Hammer, O., Harper, D. A. T., and Ryan, P. D.: PAST: Paleontologi-
cal Statistics Software Package for Education and Data Analysis,
Palaeontol. Electron., 4, 1–9, 2001. Korontzi, S.: Seasonal patterns in biomass burning emissions from
southern African vegetation fires for the year 2000, Glob. Change
Biol., 11, 1680–1700, 2005. Hantson, S., Arneth, A., Harrison, S. P., Kelley, D. I., Prentice, I. C.,
Rabin, S. S., Archibald, S., Mouillot, F., Arnold, S. R., Artaxo,
P., Bachelet, D., Ciais, P., Forrest, M., Friedlingstein, P., Hickler,
T., Kaplan, J. O., Kloster, S., Knorr, W., Lasslop, G., Li, F., Man-
geon, S., Melton, J. R., Meyn, A., Sitch, S., Spessa, A., van der
Werf, G. R., Voulgarakis, A., and Yue, C.: The status and chal-
lenge of global fire modelling, Biogeosciences, 13, 3359–3375,
https://doi.org/10.5194/bg-13-3359-2016, 2016. Lacaux, J. P., Brustet, M., and Delmas, R.: Biomass burning in the
tropical savannas of Ivory Coast: An overview of the field experi-
ment Fire of Savannas (FOS/DECAFE 91), J. Atmos. Chem., 22,
195–216, 1995. References Andreae, M. O.: Emission of trace gases and aerosols from biomass
burning – an updated assessment, Atmos. Chem. Phys., 19,
8523–8546, https://doi.org/10.5194/acp-19-8523-2019, 2019. Andreae, M. O. and Merlet, P.: Emission of trace gases and aerosols
from biomass burning, Global Biogeochem. Cy., 15, 955–966,
2001. Competing interests. The contact author has declared that neither
they nor their co-authors have any competing interests. Bonsang, B., Boissard, C., Le Cloarec, M. F., Rudolph, J., and La-
caux, J. P.: Methane, carbon monoxide and light non-methane
hydrocarbon emissions from African savanna burnings during
the FOS/DECAFE experiment, J. Atmos. Chem., 22, 149–162,
1995. Disclaimer. Publisher’s note: Copernicus Publications remains
neutral with regard to jurisdictional claims in published maps and
institutional affiliations. Byram, G. M.: Combustion of forest fuels, edited by: Davis, K. P.,
Forest fire: Control and use, McGraw-Hill, New York, 61–89,
1959. Special issue statement. This
article
is
part
of
the
special
issue
“The
role
of
fire
in
the
Earth
system:
understand-
ing
interactions
with
the
land,
atmosphere,
and
society
(ESD/ACP/BG/GMD/NHESS inter-journal SI)”. It is a result
of the EGU General Assembly 2020, 3–8 May 2020. Charnley, S., Sheridan, T. E., and Nabhan, G. P. (Eds.): Stitching
the West Back Together: Conservation of Working Landscapes,
The University of Chicago Press, Chicago, 2014. Duvall, C. S.: Biocomplexity from the ground up: Vegetation pat-
terns in a west African savanna landscape, Ann. Assoc. Am. Ge-
ogr., 101, 497–522, 2011. https://doi.org/10.5194/bg-18-6229-2021 Biogeosciences, 18, 6229–6244, 2021 Biogeosciences, 18, 6229–6244, 2021 P. Laris et al.: Methane gas emissions from savanna fires 6244 ing landscapes of an African savanna, Fire Ecol., 16, 27,
https://doi.org/10.1186/s42408-020-00085-x, 2020. Surawski, N. C., Sullivan, A. L., Meyer, C. P., Roxburgh, S. H.,
and Polglase, P. J.: Greenhouse gas emissions from laboratory-
scale fires in wildland fuels depend on fire spread mode and
phase of combustion, Atmos. Chem. Phys., 15, 5259–5273,
https://doi.org/10.5194/acp-15-5259-2015, 2015. Laris, P., Yang, L., Dembele, F., and Rodrigue, C. M.: Fire and wa-
ter: the role of grass competition on juvenile tree growth and
survival rates in a mesic savanna, Plant Ecol., 222, 861–875,
https://doi.org/10.1007/s11258-021-01149-x, 2021. van Leeuwen, T. T. and van der Werf, G. R.: Spatial and
temporal variability in the ratio of trace gases emitted
from biomass burning, Atmos. Chem. Phys., 11, 3611–3629,
https://doi.org/10.5194/acp-11-3611-2011, 2011. Lipsett-Moore, G. J., Wolff, N. H., and Game, E. T.: Emis-
sions mitigation opportunities for savanna countries from
early dry season fire management, Nat. Commun., 9, 2247,
https://doi.org/10.1038/s41467-021-25042-3, 2018. van Leeuwen, T. T., van der Werf, G. R., Hoffmann, A. A., Det-
mers, R. G., Rücker, G., French, N. H. F., Archibald, S., Car-
valho Jr., J. A., Cook, G. D., de Groot, W. J., Hély, C., Kasischke,
E. S., Kloster, S., McCarty, J. L., Pettinari, M. L., Savadogo,
P., Alvarado, E. C., Boschetti, L., Manuri, S., Meyer, C. P.,
Siegert, F., Trollope, L. A., and Trollope, W. S. W.: Biomass
burning fuel consumption rates: a field measurement database,
Biogeosciences, 11, 7305–7329, https://doi.org/10.5194/bg-11-
7305-2014, 2014. Murphy, B., Russell-Smith, J., Edwards, A., Meyer, M., and Meyer,
C. P. (Eds.): Carbon Accounting and Savanna Fire Management,
CSIRO Press, Australia, 2012. National
Aeronautics
and
Space
Administration
(NASA),
Biomass
burning,
https://earthobservatory.nasa.gov/features/
BiomassBurning/biomass_burning3.php (last access: 20 Novem-
ber 2021), 2005. Russell-Smith, J., Murphy, B. P., Meyer, C. P., Cook, G. D., Maier,
S., Edwards, A. C., Schatz, J., and Brocklehurst, P.: Improving
estimates of savanna burning emissions for greenhouse account-
ing in northern Australia: Limitations, challenges, applications,
Int. J. Wildland Fire, 18, 1–18, 2009. Vernooij, R., Giongo, M., Borges, M. A., Costa, M. M., Bar-
radas, A. C. S., and van der Werf, G. R.: Intraseasonal vari-
ability of greenhouse gas emission factors from biomass burn-
ing in the Brazilian Cerrado, Biogeosciences, 18, 1375–1393,
https://doi.org/10.5194/bg-18-1375-2021, 2021. P. Laris et al.: Methane gas emissions from savanna fires Russell-Smith, J., Yates, C., Vernooij, R., Eames, T., van der
Werf, G., Ribeiro, N., Edwards, A., Beatty, R., Lekoko,
O., Mafoko, J., Monagle, C., and Johnston, S.: Opportu-
nities and challenges for savanna burning emissions abate-
ment in southern Africa, J. Environ. Manage., 288, 112414,
https://doi.org/10.1016/j.jenvman.2021.112414, 2021. Viney, N. R.: A review of fine fuel moisture modelling, Int. J. Wildland Fire, 1, 215–234, https://doi.org/10.1071/WF9910215,
1991. Williams, R. J., Gill, A. M., and Moore, P. H.: Seasonal changes in
fire behavior in a tropical savanna in northern Australia, Int. J. Wildland Fire, 8, 227–239, 1998. Saunois, M., Jackson, R. B., Bousquet, P., Poulter, B., and
Canadell, J. G.: The growing role of methane in anthro-
pogenic climate change, Environ. Res. Lett., 11, 120207,
https://doi.org/10.1088/1748-9326/11/12/120207, 2016. Wooster, M. J., Freeborn, P. H., Archibald, S., Oppenheimer, C.,
Roberts, G. J., Smith, T. E. L., Govender, N., Burton, M.,
and Palumbo, I.: Field determination of biomass burning emis-
sion ratios and factors via open-path FTIR spectroscopy and
fire radiative power assessment: headfire, backfire and residual
smouldering combustion in African savannahs, Atmos. Chem. Phys., 11, 11591–11615, https://doi.org/10.5194/acp-11-11591-
2011, 2011. Saunois, M., Stavert, A. R., Poulter, B., Bousquet, P., Canadell, J. G., Jackson, R. B., Raymond, P. A., Dlugokencky, E. J., Houwel-
ing, S., Patra, P. K., Ciais, P., Arora, V. K., Bastviken, D., Berga-
maschi, P., Blake, D. R., Brailsford, G., Bruhwiler, L., Carl-
son, K. M., Carrol, M., Castaldi, S., Chandra, N., Crevoisier, C.,
Crill, P. M., Covey, K., Curry, C. L., Etiope, G., Frankenberg,
C., Gedney, N., Hegglin, M. I., Höglund-Isaksson, L., Hugelius,
G., Ishizawa, M., Ito, A., Janssens-Maenhout, G., Jensen, K. M., Joos, F., Kleinen, T., Krummel, P. B., Langenfelds, R. L.,
Laruelle, G. G., Liu, L., Machida, T., Maksyutov, S., McDon-
ald, K. C., McNorton, J., Miller, P. A., Melton, J. R., Morino,
I., Müller, J., Murguia-Flores, F., Naik, V., Niwa, Y., Noce, S.,
O’Doherty, S., Parker, R. J., Peng, C., Peng, S., Peters, G. P.,
Prigent, C., Prinn, R., Ramonet, M., Regnier, P., Riley, W. J.,
Rosentreter, J. A., Segers, A., Simpson, I. J., Shi, H., Smith, S. J., Steele, L. P., Thornton, B. F., Tian, H., Tohjima, Y., Tubiello,
F. N., Tsuruta, A., Viovy, N., Voulgarakis, A., Weber, T. S.,
van Weele, M., van der Werf, G. R., Weiss, R. P. Laris et al.: Methane gas emissions from savanna fires Laris, P., Dadashi, S., Jo, A., and Wechsler S.: Buffering the sa-
vanna: Fire regimes and disequilibrium ecology in West Africa,
Plant Ecol., 217, 583–596, 2016. Kirschke, S., Bousquet, P., Ciais, P., Saunois, M., Canadell, J. G.,
Dlugokencky, E. J., Bergamaschi, P., Bergmann, D., Blake, D. R., Bruhwiler, L., Cameron-Smith, P., Castaldi, S., Chevallier,
F., Feng, L., Fraser, A., Heimann, A., Hodson, E. L., Houweling,
S., Josse, B., Fraser, P. J., Krummel, P. B., Lamarque, J.-F., Lan-
genfelds, R. L., Le Quéré, C., Naik, V., O’Doherty, S., Palmer,
P. I., Pison, I., Plummer, D., Poulter, B., Prinn, R. G., Rigby, M.,
Ringeval, B., Santini, M., Schmidt, M., Shindell, D. T., Simpson, Laris, P., Koné, M., Dadashi, S., and Dembele, F.: The early/late fire
dichotomy: Time for a reassessment of Aubreville’s savanna fire
experiments, Prog. Phys. Geogr., 41, 68–94, 2017. Laris, P., Jo, A., Wechsler, S., and Dadashi, S.: The Effects of Land-
scape Pattern and Vegetation Type on the Fire Regime of a Mesic
Savanna in Mali, J. Environ. Manag., 227, 134–145, 2018. Laris,
P.,
Jacobs,
R.,
Koné,
M.,
Dembele,
F.,
and
Ro-
drigue,
C. M.:
Determinants
of
fire
intensity
in
work- https://doi.org/10.5194/bg-18-6229-2021 Biogeosciences, 18, 6229–6244, 2021 Biogeosciences, 18, 6229–6244, 2021 P. Laris et al.: Methane gas emissions from savanna fires F., Worthy, D.,
Wunch, D., Yin, Y., Yoshida, Y., Zhang, W., Zhang, Z., Zhao,
Y., Zheng, B., Zhu, Q., Zhu, Q., and Zhuang, Q.: The Global
Methane Budget 2000–2017, Earth Syst. Sci. Data, 12, 1561–
1623, https://doi.org/10.5194/essd-12-1561-2020, 2020. Saunois, M., Stavert, A. R., Poulter, B., Bousquet, P., Canadell, J. G., Jackson, R. B., Raymond, P. A., Dlugokencky, E. J., Houwel-
ing, S., Patra, P. K., Ciais, P., Arora, V. K., Bastviken, D., Berga-
maschi, P., Blake, D. R., Brailsford, G., Bruhwiler, L., Carl-
son, K. M., Carrol, M., Castaldi, S., Chandra, N., Crevoisier, C.,
Crill, P. M., Covey, K., Curry, C. L., Etiope, G., Frankenberg,
C., Gedney, N., Hegglin, M. I., Höglund-Isaksson, L., Hugelius,
G., Ishizawa, M., Ito, A., Janssens-Maenhout, G., Jensen, K. M., Joos, F., Kleinen, T., Krummel, P. B., Langenfelds, R. L.,
Laruelle, G. G., Liu, L., Machida, T., Maksyutov, S., McDon-
ald, K. C., McNorton, J., Miller, P. A., Melton, J. R., Morino,
I., Müller, J., Murguia-Flores, F., Naik, V., Niwa, Y., Noce, S.,
O’Doherty, S., Parker, R. J., Peng, C., Peng, S., Peters, G. P.,
Prigent, C., Prinn, R., Ramonet, M., Regnier, P., Riley, W. J.,
Rosentreter, J. A., Segers, A., Simpson, I. J., Shi, H., Smith, S. J., Steele, L. P., Thornton, B. F., Tian, H., Tohjima, Y., Tubiello,
F. N., Tsuruta, A., Viovy, N., Voulgarakis, A., Weber, T. S.,
van Weele, M., van der Werf, G. R., Weiss, R. F., Worthy, D.,
Wunch, D., Yin, Y., Yoshida, Y., Zhang, W., Zhang, Z., Zhao,
Y., Zheng, B., Zhu, Q., Zhu, Q., and Zhuang, Q.: The Global
Methane Budget 2000–2017, Earth Syst. Sci. Data, 12, 1561–
1623, https://doi.org/10.5194/essd-12-1561-2020, 2020. Worden, J. R., Bloom, A. A., Pandey, S., Jiang, Z., Worden, H. M., Walker, T. W., Houweling, S., and Röckmann, T.: Reduced
biomass burning emissions reconcile conflicting estimates of the
post-2006 atmospheric methane budget, Nat. Commun., 8, 2227,
https://doi.org/10.1038/s41467-017-02246-0, 2017. Ward, D. E., Hao, W. M., Susott, R. A., Babbitt, R. E., Shea, R. W.,
Kauffman, J. B., and Justice, C. O.: Effect of fuel composition on
combustion efficiency and emission factors for African savanna
ecosystems, J. Geophys. Res., 101, 23569–23576, 1996. Biogeosciences, 18, 6229–6244, 2021 https://doi.org/10.5194/bg-18-6229-2021
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Using concept mapping to explore why patients become lost to follow up from an antiretroviral therapy program in the Zomba District of Malawi
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RESEARCH ARTICLE Open Access © 2013 Rachlis et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited. Rachlis et al. BMC Health Services Research 2013, 13:210
http://www.biomedcentral.com/1472-6963/13/210 Rachlis et al. BMC Health Services Research 2013, 13:210
http://www.biomedcentral.com/1472-6963/13/210 Using concept mapping to explore why patients
become lost to follow up from an antiretroviral
therapy program in the Zomba District of Malawi Beth Rachlis1, Farah Ahmad1,2, Monique van Lettow1,3*, Adamson S Muula4, Medson Semba5 and Donald C Cole1 Abstract Background: Retention in antiretroviral therapy (ART) programmes remains a challenge in many settings including
Malawi, in part due to high numbers of losses to follow-up. Concept Mapping (CM), a mix-method participatory
approach, was used to explore why patients on ART are lost to follow-up (LTFU) by identifying: 1) factors that
influence patient losses to follow-up and 2) barriers to effective and efficient tracing in Zomba, Malawi. Methods: CM sessions (brainstorming, sorting and rating, interpretation) were conducted in urban and rural
settings in Zomba, Malawi. Participants included ART patients, ART providers, Health Surveillance Assistants, and
health managers from the Zomba District Health Office. In brainstorming, participants generated statements in
response to “A specific reason why an individual on ART becomes lost to follow-up is…” Participants then sorted
and rated the consolidated list of brainstormed items. Analysis included inductive qualitative methods for grouping
of data and quantitative cluster identification to produce visual maps which were then interpreted by participants. Results: In total, 90 individuals brainstormed 371 statements, 64 consolidated statements were sorted (participant
n = 46), and rated on importance and feasibility (participant n = 69). A nine-cluster concept map was generated and
included both patient- and healthcare-related clusters such as: Stigma and Fears, Beliefs, Acceptance and Knowledge
of ART, Access to ART, Poor Documentation, Social and Financial Support Issues, Health Worker Attitudes, Resources
Needed for Effective Tracing, and Health Worker Issues Related to Tracing. Strategies to respond to the clusters were
generated in Interpretation. Conclusions: Multiple patient- and healthcare focused factors influence why patients become LTFU. Findings have
implications particularly for programs with limited resources struggling with the retention of ART patients. Keywords: Concept mapping, HIV/AIDS, Antiretroviral therapy (ART), Losses to follow-up, Malawi * Correspondence: m.vanlettow@dignitasinternational.org
1Dalla Lana School of Public Health, University of Toronto, Toronto, Canada
3Dignitas International, Zomba, Malawi
Full list of author information is available at the end of the article Background Losses to follow-up (LTFU) from antiretroviral therapy
is a major cause of patient attrition [7]. LTFU can be
considered a catch-all category for patients who miss
scheduled appointments or medication pick-ups (over a
period of time) although operational definitions vary [8]. In Malawi, the term ‘LTFU from ART’ refers to a patient
who is overdue for their appointment and is not known
to have stopped ART, died, or transferred to another
facility [9]. Importantly, patients who discontinue treat-
ment are vulnerable to drug resistance, AIDS-related ill-
nesses, and death [7,8,10-12]. Globally, 34 million people were living with HIV/AIDS
by December 2010 and the majority resided in sub-
Saharan Africa [1]. In Malawi, the prevalence of HIV is
estimated as 11% [2,3] and over 650,000 children have
been orphaned by AIDS [4]. By June 2010, approxi-
mately 58% of those in need were receiving antiretroviral
therapy (ART) [5] although disparities continue to exist
in access to treatment options, particularly for individ-
uals living in rural areas [6]. While various reasons are known, demonstrating asso-
ciations between various variables and risk of becoming
LTFU remains challenging. Reported reasons for LTFU * Correspondence: m.vanlettow@dignitasinternational.org
1Dalla Lana School of Public Health, University of Toronto, Toronto, Canada
3Dignitas International, Zomba, Malawi
Full list of author information is available at the end of the article Rachlis et al. BMC Health Services Research 2013, 13:210
http://www.biomedcentral.com/1472-6963/13/210 Page 2 of 11 pants were selected based on their previous experience
with ART and included patient and provider groups. Pa-
tient participants included patients who had become
LTFU while on ART as well as expert patients (patients
on ART trained to assist with clinical tasks and counsel-
ling). Provider participants consisted of ART providers
(clinical officers, medical assistants, and nurses), Health
Surveillance Assistants (HSAs) involved with patient
tracing, and members of the Zomba District Health
Office (DHO) health management team involved in ART
provision. We sought a balanced mix of participants
working or receiving care either at Zomba Central
Hospital (centralized) or in one of three decentralized
rural clinics that varied in terms of distance to Zomba
town and patient volume (small versus large). Separate
sessions were held for patient and provider groups to
avoid responder bias (e.g., patients may not speak freely
or may provide responses they think providers want to
hear). Background The brainstorming and rating sessions were ar-
ranged with a larger number of diverse participants to
capture
heterogeneity
whereas
sorting
and
rating
sessions were conducted with smaller and more alike
participants for homogeneity [32]. Sessions were con-
ducted in English (provider groups) or Chichewa (pa-
tient groups). Inclusion criteria included being at least
18 years of age and having experience with ART as a
patient, provider, manager, or
through
tracing. An
additional criterion of having at least a secondary school
education was applied for sorting which required read-
ing and comprehension skills. All CM activities took
place in May-July 2011. All CM activities were facilitated
by an experienced Malawian research assistant who was
briefly trained in CM methodology and was fluent in
both English and Chichewa. in Malawi and elsewhere have included: food insecurity
[13-15], financial constraints [14,16,17], religious and
family influences [14,16,18,19], wanting to access trad-
itional medicine [20], stigma and fear of disclosure of
HIV status [10,19-22], living far from clinics and trans-
port-related costs [10,14,18,20,23], and poor patient-
provider
relationships
[10,14,20]. Treatment
literacy
(e.g., understanding the natural course of treatment and
the need for adherence to ART) [24,25], experiencing an
improvement in health and believing that treatment is
no longer necessary [10] may also matter. Some of these
are consistent with the findings of our 2011 systematic
review exploring livelihood factors and ART adherence
[25] although we were limited by the available literature
and could not identify new factors that may be relevant
or specific to Malawi. Furthermore, our understanding
of how these various factors potentially interact to influ-
ence whether a patient becomes LTFU over time, re-
mains rudimentary [21]. Zomba is one of Malawi’s most populated districts
(population: 670,500) and is predominantly rural [26]. Within Zomba, HIV prevalence is 14.5% although esti-
mates have varied by location and population group,
ranging from 12% in an urban centre to >30% at a rural
hospital [27,28]. In collaboration with the Malawi Minis-
try of Health, Dignitas International (DI), a humanitarian
non-governmental organization, has delivered compre-
hensive HIV/AIDS care in Zomba since 2004, enrolling
over 20,000 patients on ART. The decentralization of
services to rural areas has led to a rapid increase in the
number of people accessing ART in the district. Recruitment
ll b In collaboration with local partners at DI, the University
of Malawi, and the DHO, the research team and a bilin-
gual research assistant distributed a study information
sheet (SIS) to potential participants. To reach provider
participants, the SIS was distributed to staff at participat-
ing clinics and the DHO. To reach the patient popula-
tion, collaborating providers disseminated the study
information to their patients. Interested individuals
contacted the research team for further details. Patients
who were currently LTFU (i.e. had not been seen in
care) were identified through a master list of currently-
missing patients, generated by DI (and shared with the
DHO). Those patients who were identified as LFTU
were then contacted (via phone or in person during
tracing with HSAs) and if successfully found, were
invited to participate in the study. Willing partici-
pants provided informed written consent. Participants
recruited in brainstorming were invited to participate in Background In order to improve patient retention in DI’s ART pro-
gram, we sought to comprehensively identify factors that
influence why patients become LTFU as well as factors
that impede successful tracing efforts. Methods
C Concept
Mapping
(CM)
is
a
participatory
mixed-
methods approach that enables diverse groups of stake-
holders to share their ideas, representing them in various
quantitatively derived visual concept maps [29,30]. CM
has three phases: brainstorming of statements in re-
sponse to a focus prompt; sorting and rating of the
consolidated list of brainstormed statements; and inter-
pretation where participants discuss and interpret the
maps identifying priority areas for action. The use of
CM in this context was advantageous as our aim was to
identify the most important and feasible interventions/
strategies that address patient retention [31]. Brainstorming
O
f Upon completion of the sorting and rating activities,
data were exported into Concept Systems Software
(Concept System Program, Concept Mapping Incorpo-
rated, 2005) to create a final cluster solution prior to the
interpretation session. Using the statistical technique of
a similarity index, the program first determined the
number of participants who sorted each pair of state-
ments together. Each statement was then located in
relation to all others on a two-dimensional point map
(statements sorted together frequently were located close
together). Next, a cluster concept map was generated
from the point map to show how the statements could
be organized into clusters with common themes using
hierarchical cluster analysis [29]. In this step, the re-
searchers examined several cluster maps by forcing an
upper and lower limit to the number of clusters in a
map. The team began with the upper limit of 15 clusters
and merged one cluster at a time until the lower limit of
5 clusters was reached. They examined changes in the
cluster solution at each merging step. The final cluster
map
was
identified
through
consensus
among
re-
searchers on the number of clusters that retained the
most meaningful detail between clusters. Clusters that
are smaller in size indicate that statements that make up
the cluster were more similarly grouped by participants
during sorting relative to larger sized clusters. A stress
value statistic reflecting a goodness-of-fit of the point
map to the similarity matrix was calculated. Generally,
indices range from 0.15 to 0.35 [29,33]. In the present
study, the stress value was 0.28 indicating an acceptable
value. Bridge maps were generated to explore the Our focus prompt was developed in consultation with
DI and pilot-tested with patients at Zomba Central
Hospital. Participants were prompted to generate state-
ments in response to “Why do individuals on ART be-
come lost to follow-up over time?” To help participants
understand ‘lost to follow-up’, several descriptions were
provided including when patients: stop taking their ART,
stop coming for appointments, or can’t be found by
health providers. Each statement generated in brain-
storming was captured by the facilitator. Overall, 90 par-
ticipants (ten group sessions; five individual sessions)
generated 371 statements. Once all sessions were completed, statement consoli-
dation was initiated. Statements in Chichewa were first
translated into English. Brainstorming
O
f Each statement generated was
read aloud in sequence with the focus prompt (i.e., A
specific reason why an individual on ART becomes lost
to follow-up is… statement generated in brainstorming). Statements that did not answer the prompt were consid-
ered irrelevant and were removed. The remaining state-
ments were organized into broad themes (e.g., tracing
issues), duplicates eliminated, and similar statements
merged to create a final consolidated list. The refined list
of 64 statements was then back-translated into Chichewa
for sorting and rating. Translations were verified by a
bilingual DI staff member. Sample To ensure a variety of viewpoints, purposive sampling
was used to include a heterogeneous participant popula-
tion with respect to patient losses to follow-up. Partici- Rachlis et al. BMC Health Services Research 2013, 13:210
http://www.biomedcentral.com/1472-6963/13/210 Page 3 of 11 sorting (if met additional criterion), rating, and inter-
pretation. To allow flexibility to participate, both group
sessions and one-on-one sessions were conducted for all
phases although generally brainstorming and interpret-
ation was done in group sessions to generate discussion. Sessions were conducted in private rooms at participat-
ing clinics and at the DHO. At the time of recruitment,
all patients were informed that by participating in group
sessions, they were disclosing their HIV positive status
to the other members attending the session. Further-
more, participants were informed that any concerns or
issues they had before, during (any phase), or after the
study would be addressed confidentially by the study
team as outlined in the SIS and consent form. Snacks
were provided and all patient participants received a
1 kg bag of sugar or a chitenje (local fabric) and 200
Malawian Kwacha (approximately $1.20 Canadian at the
time of the study) to help cover travel costs. Ethical ap-
proval was obtained from the University of Toronto HIV
Research Ethics Board and the National Health Sciences
Research Committee in Malawi. statement per card. Participants were instructed to make
piles of similar statements in ways that made sense to
them and to give labels to each pile they created to
reflect the statements within. Prior to each session, the
facilitator provided an example based on food (e.g. crunchy, soft, sweet, and sour) to explain how different
people may sort the same item in different ways. Rating
was completed by 69 participants (six group sessions;
twenty-three individual sessions). Sample Participants rated each
statement using a Likert 5-point
scale, along two
dimensions: Importance: (i.e., how important the statement is with
respect to why patients become lost to follow-up)
(1- not at all important to 5- extremely important,
compared to all other statements); Importance: (i.e., how important the statement is with
respect to why patients become lost to follow-up)
(1- not at all important to 5- extremely important,
compared to all other statements);
Feasibility: (i.e., how feasible it would be to address this
statement in order to prevent patients on ART from
becoming lost to follow-up) (1- not at all feasible to 5-
extremely feasible, compared to all other statements). Feasibility: (i.e., how feasible it would be to address this
statement in order to prevent patients on ART from
becoming lost to follow-up) (1- not at all feasible to 5-
extremely feasible, compared to all other statements). Participant interpretation
Clusters in the healthcare-related region g
In relation to Poor Documentation, provider participants
noted that the date and outcomes (e.g., health status,
ART supply given) of patient visits are often not
recorded accurately, if at all. This was attributed to a
high volume of patients, a shortage of health staff, and a
lack of proper filing space. ART providers further real-
ized that while patients may be given their ART, if the
visit was not recorded, the patient may be incorrectly la-
belled as LTFU. Participants noted that some patients
provide false contact information to hide from follow-
up. Without an accurate address, patients can’t be found
and remain LTFU. While improving documentation was
rated a feasible issue to address, participants struggled to
come up with potential strategies that could be used to
address documentation issues. Rating and bridge map relative agreement on rating variables between clusters
and across provider and patient groups. relative agreement on rating variables between clusters
and across provider and patient groups. g
g
p
Table 2 presents the mean cluster ratings with associated
standard deviations. Table 3 presents all statements. For
the rating of “importance” of items, the clusters with the
highest rating values were Poor Documentation, Resources
Needed for Effective Tracing, and Social and Financial
Support Issues. For the rating of “feasibility” of items to ad-
dress, the clusters with the highest rating values were Re-
sources Needed for Effective Tracing, Poor Documentation,
and Health Worker Issues Related to Tracing. Generally,
ratings were higher for “feasibility” although agreement
between the rating variables at the cluster level was rela-
tively high. Patient and providers both gave higher rating
values to clusters in the healthcare-related region versus
the patient-related region with the exception of Social and
Financial Support Issues. Provider groups rated clusters in
the healthcare-related region as more feasible to address
although this pattern was less clear for patient groups
(data not shown). Interpretation of maps Overall, 35 participants interpreted the maps (five group
sessions; 3 individual sessions) documenting key findings
with the facilitator. Participants discussed the content of
the main clusters and reached agreement through consen-
sus on the most appropriate cluster labels out of the sev-
eral labels generated individually by participants during
the sorting activity. Through the examination and com-
parison of the “Importance” and “Rating” variables for
each cluster, the most actionable clusters were identified
(e.g. where efforts to address LTFU should be directed). Clusters with a rating value of ≥3 are discussed in detail. Sorting and rating Sorting activity was completed individually by 46 partici-
pants (six group sessions; eight individual sessions). Each participant received a deck of 64 cards with one Page 4 of 11 Rachlis et al. BMC Health Services Research 2013, 13:210
http://www.biomedcentral.com/1472-6963/13/210 Participants Participant characteristics are presented in Table 1. Fifty-four percent were women and 49% were under the
age of 35 (median: 32, IQR: 27–38). An overview of the
CM methods and the number of participants in each
stage is presented in Figure 1. Cluster solution A
lack of housing options or the need to move with family
may help to partially explain this finding. Timing of tra-
cing was also discussed and according to one ART pro-
vider: “by waiting more than 2 months to start tracing, a
patient is already lost.” Provider participants suggested
that tracing should be initiated earlier and patients
should be prioritized for tracing. A master list of pa-
tients expecting to be seen each week could be gener-
ated at each clinic. When a patient doesn’t show up,
participants felt that tracing should begin immediately. ART providers suggested that lessons could be drawn
from successes with Tuberculosis (TB) tracing. bicycle, they often face rocky terrains and washed out
roads. Some HSAs noted that they do not even live in
their catchment areas and so have to travel far to get to
areas where they then can start tracing. Interestingly,
this was contested by DHO participants who mentioned
that HSAs should be living in their catchment areas. A
lack of housing options or the need to move with family
may help to partially explain this finding. Timing of tra-
cing was also discussed and according to one ART pro-
vider: “by waiting more than 2 months to start tracing, a
patient is already lost.” Provider participants suggested
that tracing should be initiated earlier and patients
should be prioritized for tracing. A master list of pa-
tients expecting to be seen each week could be gener-
ated at each clinic. When a patient doesn’t show up,
participants felt that tracing should begin immediately. ART providers suggested that lessons could be drawn
from successes with Tuberculosis (TB) tracing. patients in this line’). Participants generally agreed that
enhanced one-on-one counselling could help improve a
patient’s overall knowledge of the importance of adher-
ence as well as help to manage fears and expectations. Cluster solution A nine cluster concept map was generated (Figure 2). Clusters included: Stigma and Fears (8 statements);
Beliefs (5 statements); Lack of Knowledge and Acceptance
(11 statements); Access to ART (5 statements); Poor
Documentation (5 statements); Social and Financial
Support Issues (8 statements); Health Worker Attitudes
(8 statements); Resources Needed for Effective Tracing
(6 statements); and Health Worker Issues Related to Tra-
cing (8 statements). The map was further divided into
two cognitive regions: upper region clusters generally
focused on patient-related factors whereas lower region
clusters were more healthcare-focused. With respect to the clusters on Tracing, transportation
challenges emerged. While HSAs mostly walk or use a Table 1 Number of participants in brainstorming, sorting, rating, and interpretation activities Table 1 Number of participants in brainstorming, sorting, rating, and interpretation activities
Groups
# Participants
Task
Participant type
Urban area
Rural area (Pirimiti, Mayaka, Chingale)
Total
Brainstorming
Patients
18
23 (including n=9 LTFU)
41
Providers
29
20
49
Sorting
Patients
11
9 (including n = 1 LTFU)
20
Providers
14
12
26
Rating
Patients
14
20 (including n = 2 LTFU)
34
Providers
17
18
35
Interpretation
Patients
20 patients (including n = 1 LTFU)
20
Providers
15
15
Note: Provider participants included ART providers, Zomba District Health Office Management Team, and Health Surveillance Assistants; LTFU = patient lost
to follow-up. participants in brainstorming, sorting, rating, and interpretation activities Note: Provider participants included ART providers, Zomba District Health Office Management Team, and Health Surveillance Assistants; LTFU = patient lost
to follow up Note: Provider participants included ART providers, Zomba District Health Office Management Team, and Health Surveillance Assistants; LTFU = patient lost
to follow-up. Rachlis et al. BMC Health Services Research 2013, 13:210
http://www.biomedcentral.com/1472-6963/13/210 Page 5 of 11 Consolidation of 371 statements
Brainstorming
(n=90)
Sorting (n=46) and
Rating (n=69) of 64
statements
Interpretation
(n=35)
Multidimensional scaling &
Hierarchical cluster analysis
Figure 1 Overview of the concept mapping process. Sorting (n=46) and
Rating (n=69) of 64
statements Multidimensional scaling &
Hierarchical cluster analysis Consolidation of 371 statements Figure 1 Overview of the concept mapping process. bicycle, they often face rocky terrains and washed out
roads. Some HSAs noted that they do not even live in
their catchment areas and so have to travel far to get to
areas where they then can start tracing. Interestingly,
this was contested by DHO participants who mentioned
that HSAs should be living in their catchment areas. Clusters in the patient-related region Social and Financial Support Issues were also discussed. Many patients referred to strict hospital policies includ-
ing the need to come to care with a guardian (e.g., family
member) as a reason why patients may miss scheduled
visits. This would be particularly problematic for pa-
tients who have not disclosed their HIV status or who
lack a guardian. Interestingly, providers in our study
noted that this specific policy was no longer in effect. Being too sick to come to the clinic was the most im-
portant and feasible statement to address (data not
shown). HSAs spoke to experiences where they traced
patients that had been too sick to make it to their ap-
pointments. To minimize the likelihood that patients
(particularly those without guardians) stop taking ART, In relation to Health Worker Attitudes patients noted
that providers often use exposing language (e.g., ‘ART Stigma and Fears
Beliefs
Lack of Acceptance
and Knowledge of ART
Access to ART
Poor Documentation
Social and Financial
Support Issues
Health Worker
Attitudes
Resources Needed
for Effective Tracing
Health Worker Issues
Related to Tracing
Figure 2 Final nine cluster concept map. Lack of Acceptance
and Knowledge of ART Health Worker
Attitudes Health Worker Issues
Related to Tracing Access to ART Poor Documentation Resources Needed
for Effective Tracing Figure 2 Final nine cluster concept map. Figure 2 Final nine cluster concept map. Page 6 of 11 Rachlis et al. BMC Health Services Research 2013, 13:210
http://www.biomedcentral.com/1472-6963/13/210 Table 2 Cluster characteristics and ratings
Cluster
Importance rating
Feasibility rating
Mean (SD)
Mean (SD)
Poor documentation
3.3 (0.18)
3.5 (0.31)
Resources needed for
effective tracing
3.3 (0.23)
3.6 (0.14)
Social and financial
support issues
3.3 (0.35)
3.4 (0.34)
Health worker attitudes
3.1 (0.25)
3.5 (0.27)
Health worker issues
related to tracing
3.1 (0.40)
3.5 (0.20)
Stigma and fears
2.9 (0.44)
3.4 (0.29)
Access to ART
2.7 (0.23)
3.1 (0.22)
Beliefs
2.6 (0.35)
3.0 (0.17)
Lack of knowledge
and acceptance
2.6 (0.52)
2.9 (0.33) Table 2 Cluster characteristics and ratings gement can be particularly challenging in resource-poor
settings [34,35,37-39]. This has been attributed to poor
infrastructure, a lack of trained personnel, and clinic
characteristics including patient volume [35,38]. Often
front line health workers involved in data collection lack
a clear understanding as to how the data they collect will
be used and how particularly, it is relevant for their day-
to-day activities [34,40]. Clusters in the patient-related region However, other
patients suggested that even if food supplement were
provided at the clinics, individuals may choose to not
come given that others would know where they got their
food from and identify them as HIV positive. Finally, participants discussed the need for education
for church leaders as often it is church leaders who dis-
courage patients from taking ART. Having a chaplain at
the hospital to provide social and religious support was
also suggested as a way to encourage ART adherence
and prevent losses to follow-up. Tracing clusters were identified as important and feas-
ible to address in the present study. Participants noted
that, based on current definitions and guidelines, tracing
may be initiated too late in Malawi. Some studies have
suggested that patient retention can be improved when
tracing is initiated as soon as possible (dependent on
available resources) as this can minimize the likelihood
that patients remain LTFU [17,42]. Prioritizing who gets
traced and when could mean that fewer patients need to
be traced as many patients may ultimately return on
their own. Prioritization can be determined through the
identification of particular clinical markers that are asso-
ciated with an increased likelihood of becoming lost or
an increased risk of death. In Malawi, for example, risk
of death is highest in patients who have recently initiated
ART. This suggests that time on ART may be an import-
ant way to prioritize patients for tracing [41]. Clusters in the patient-related region Improving quality data collection,
however, may require incentives and ongoing training
and supervision for personnel [35,38,40], specifically
because data collection can be burdensome [35,37]. Programs with electronic monitoring systems have
demonstrated better quality data [35,36] and it is worth
noting that DI has improved their electronic system
since 2007 although challenges remain. Indeed, ART pro-
viders in our study spoke to limited storage space as one
reason why files become misplaced and/or lost. Import-
antly, our findings suggest that while there are patients
who truly become LTFU, poor documentation may lead
some patients to be incorrectly labelled as LTFU (e.g., the
files were lost, visit was not recorded). For example, miss-
ing patients may have also transferred to another clinic
and therefore may only be LTFU from the perspective of
their original clinic [35]. Therefore, identifying where (e.g.,
which clinics) and when (e.g., data entry, filing) errors
occur is a necessary first step in order to determine how
it can be addressed although enhanced documentation,
improved coordination between clinics (when there are
transfers), and effective tracing of missing patients are all
necessary components [7,41]. the HSAs suggested that they could bring ART directly
to the patients in their homes. Patients suggested that
there could be a phone number that they can call if they
are too sick to come for care. As elaborated by one pa-
tient, the hospital can then make alternate arrangements
for them (e.g., providing transport). Some patients re-
ferred to a lack of food needed to take with ART as a
reason why patients may become LTFU. However, other
patients suggested that even if food supplement were
provided at the clinics, individuals may choose to not
come given that others would know where they got their
food from and identify them as HIV positive. the HSAs suggested that they could bring ART directly
to the patients in their homes. Patients suggested that
there could be a phone number that they can call if they
are too sick to come for care. As elaborated by one pa-
tient, the hospital can then make alternate arrangements
for them (e.g., providing transport). Some patients re-
ferred to a lack of food needed to take with ART as a
reason why patients may become LTFU. Discussion
h In the present study, we used CM to incorporate both pa-
tient and provider perspectives to comprehensively and
systematically identify important and feasible issues to ad-
dress with respect to why patients on ART become LTFU
over time. The final 9 cluster concept map consisted of
both patient- and healthcare-related clusters. The findings
of the present study can inform the development of strat-
egies that work to prevent patients from becoming LTFU
including improved tracing efforts. Drawing from the experiences of TB programs, tracers
specific to ART could be trained [43] although important
differences in the management and perceptions of HIV
are important considerations. For example, the largely
sexual transmission and labelling of HIV as a ‘moral dis-
ease’ makes anonymity and confidentiality of those living
with HIV extremely important [22]. Home visits by Poor Documentation and Resources Needed for Effect-
ive Tracing were rated important and feasible clusters to
address. Importantly, the quality and accuracy of data
(including data on patients who become LTFU) can vary
across large longitudinal cohort studies [34,35]. While
reliable ART monitoring is critical for measuring patient
outcomes and program performance [35,36], data mana- Page 7 of 11 Page 7 of 11 Rachlis et al. BMC Health Services Research 2013, 13:210
http://www.biomedcentral.com/1472-6963/13/210 Rachlis et al. Stigma and fears Health worker attitudes Health worker issues related to tracing Social and financial support issues Table 3 The consolidated list of 64 statements (Continued) Patients on ART face mental health issues
Patients stop coming to the clinic because they get their ARV drugs from somewhere else
Fear dismissal at the work place because their employer may discover that they are on ART
Patients have difficulty finding ARVs when they travel both within and outside the country
ARV medicines are not always accessible because of inconsistent drug availability
Not being able to meet strict hospital policies (e.g., coming with a guardian)
The patient just picked up a guardian at the market and the guardian cannot be used to trace the patient
Patients are mobile and they move around a lot
Religion and the belief that prayer, not ARVs, will heal them
Church fellowships discourage them from taking their ARVs and tell them they are healed from HIV
Patients on ART believe that they are HIV negative because they gave birth
to a negative baby or re-tested negative for HIV
Beliefs in traditional medicines
Patients on ART believe that they are healed from HIV if they sleep with a virgin or a younger person
Patients feel tired of taking their drugs
Patients on ART don’t see the need to be on drugs anymore when
they feel better and their health has improved
When there is no improvement in their health, patients on ART are
frustrated because they were expecting a quick recovery
The patient on ART has not accepted their HIV status
Patients on ART want to have a normal life and feel the medicine is a burden
Patients on ART are just not serious about their lives
Patients see no value in taking their ARVs anymore because they have given up on life
Patients find new marriage partners and do not want them to find out that they are on ART
Pregnancy and wanting to protect the unborn child from ART
Patients prefer alcohol over taking ART
Patients have too many lovers and not enough time to take their ARVs Patients have too many lovers and not enough time to take their ARVs ARV antiretroviral, HSA Health Surveillance Assistant. been previously associated with stopping ART and/or an
increased risk of becoming LTFU [10,48,49]. During
interpretation, participants determined that HSAs and
guardians can help support patients who are unwell by
bringing ART to patients in their homes when they are
too sick to travel [50]. Lack of knowledge
and acceptance Table 3 The consolidated list of 64 statements (Continued) Table 3 The consolidated list of 64 statements (Continued) Table 3 The consolidated list of 64 statements (Continued)
Patients on ART face mental health issues
Patients stop coming to the clinic because they get their ARV drugs from somewhere else
Fear dismissal at the work place because their employer may discover that they are on ART
Access to ART
Patients have difficulty finding ARVs when they travel both within and outside the country
ARV medicines are not always accessible because of inconsistent drug availability
Not being able to meet strict hospital policies (e.g., coming with a guardian)
The patient just picked up a guardian at the market and the guardian cannot be used to trace the patient
Patients are mobile and they move around a lot
Beliefs
Religion and the belief that prayer, not ARVs, will heal them
Church fellowships discourage them from taking their ARVs and tell them they are healed from HIV
Patients on ART believe that they are HIV negative because they gave birth
to a negative baby or re-tested negative for HIV
Beliefs in traditional medicines
Patients on ART believe that they are healed from HIV if they sleep with a virgin or a younger person
Lack of knowledge
and acceptance
Patients feel tired of taking their drugs
Patients on ART don’t see the need to be on drugs anymore when
they feel better and their health has improved
When there is no improvement in their health, patients on ART are
frustrated because they were expecting a quick recovery
The patient on ART has not accepted their HIV status
Patients on ART want to have a normal life and feel the medicine is a burden
Patients on ART are just not serious about their lives
Patients see no value in taking their ARVs anymore because they have given up on life
Patients find new marriage partners and do not want them to find out that they are on ART
Pregnancy and wanting to protect the unborn child from ART
Patients prefer alcohol over taking ART
Patients have too many lovers and not enough time to take their ARVs
ARV antiretroviral, HSA Health Surveillance Assistant. Discussion
h BMC Health Services Research 2013, 13:210
http://www.biomedcentral.com/1472-6963/13/210 e 3 The consolidated list of 64 statements
er
Statements
documentation
Poor filing means that patient files can get lost when patients on ART are being transferred
The patient has died but their death is not reported
Patients have little social support in the villages due to lack of counsellors,
support groups and community-based organizations
The patient has moved away to another clinic without being properly transferred
Patient visits are not being recorded accurately
urces needed for
ive tracing
Tracing starts too late because of the way a ‘defaulter’ is defined
(e.g., missing an appointment by 2 months or more)
The HSAs have difficulty locating patients because they do not live in
their catchment areas and therefore do not know the villages where people come from
Patients on ART can’t be found if the HSAs do not have their proper address and/or their locator forms
They live in areas that are difficult to reach so that the HSAs have difficulty tracing them
Health workers have difficulty locating patients without working phones or phone numbers
There is no fuel or dedicated transport for tracing
and financial support issues
Being too sick to come to the clinic
The distance to the clinic is too far from some patients on ART
The guardians of the patient refuse to go the hospital to collect their ARVs from them
Patients on ART can’t afford transport means to come to the clinic because they face poverty
A lack of support for ART patients especially when they are orphans
Patients on ARV medicines may also have to deal with other diseases such as chronic illness
Patients on ARV medicines feel hungry but they can’t afford to buy extra food
They are attending to another sick relative (e.g., child)
h worker attitudes
Patients are on ART feel that there is a lack of confidentiality on behalf of
the health workers (e.g., drugs given without privacy)
Patients on ART get disappointed when they are not put on a different
ART regimen to help manage their side effects
Patients are not properly educated on ART because of little one-on-one counselling with the health workers
Patients on ART do not like the way they are treated by health workers
Patients feel shy to come to the health centre because there is limited space and no privacy
Patients on ART get frustrated because it takes too long before they are seen at the hospital
Anxiety about going back to the clinic after missing many appointments
To hide from follow-up, patients change their names and identities
h worker issues related to tracing
There are too many patients needing to be traced and not enough health workers to trace them
Poor communication and coordination between the HSA and ART providers
There is no coordinator for ART tracing and no specific follow-up health workers
There is no training on how to do the follow-up of ART patients
The health workers just wait for the patients to come back on their own
because their are no consequences for them if they don’t trace them
There is no deliberate effort to trace ART patients because there are no incentives for the HSAs
The HSAs do not actually trace patients and instead write fake information about patient visits
The HSAs don’t value tracing because they have too much other work
a and fears
The patient on ART fears stigmatization because they are transferred to a
health centre that is near to where they live
Fear of divorce if their spouse or loved one discover that they are on ART
The patient doesn’t want to be associated with ARV drugs because of stigma and fear of isolation
Patients on ART experience side effects from the medicine Table 3 The consolidated list of 64 statements
Cluster
Statements Resources needed for
effective tracing Rachlis et al. Discussion
h BMC Health Services Research 2013, 13:210
http://www.biomedcentral.com/1472-6963/13/210 Rachlis et al. BMC Health Services Research 2013, 13:210
http://www.biomedcentral.com/1472-6963/13/210 Page 8 of 11 Table 3 The consolidated list of 64 statements (Continued) The identification of the potentially
important role of guardians was, to our knowledge, a find-
ing unique to our study. Disclosure of one’s HIV status has
been recognized as a double-edged sword [51] - having
the potential to yield needed social support but that
may also result in stigmatization and abandonment
[51-53]. Through participation in support groups, pa-
tients have a safe place to share experiences and support
one another which can also strongly motivate patients
to adhere to ART [22]. HSAs may lead to involuntary disclosure of their HIV
status [21]. These factors have important implications
for how ART tracing efforts should be implemented
although as participants in the present study, as well as
elsewhere [35,41] have noted, accurate contact informa-
tion is needed to ensure that patients can actually be
found. While the number of patients with cell phones is
growing (by 2008, approximately 15% of Malawians were
reportedly using cell phones), many patients, particularly
those in remote settings, continue to be without working
phones [44]. Importantly, while a working phone num-
ber is one of the strongest predictors of successfully
finding patients [41,45] many still do not return to care
[40,42,46,47]. With enhanced training on ART counsel-
ling, HSAs may be better equipped to encourage the
patients they trace to return to care. Currently, many
struggle with questions from patients related to the need
for adherence and/or management of side effects. Findings around transport-related costs are consistent
with previous studies [10,14,17,18,20]. Patients often
have to choose between using their limited income on
transport or food for themselves/families [54]. Indeed
food insecurity and the perception that ART needs to be Participants rated Social and Financial Support Issues
high on importance. Poor health and feeling sick have Page 9 of 11 Rachlis et al. BMC Health Services Research 2013, 13:210
http://www.biomedcentral.com/1472-6963/13/210 participated in sorting and rating which may further
bias our findings more towards a provider perspec-
tive. Participant fatigue/sorting burden may have been
particularly
important
during
sorting
although
we
attempted to limit the number of statements to be
sorted. Rating may have also been impacted by par-
ticipant fatigue which may help to explain the small
range of rating values. Table 3 The consolidated list of 64 statements (Continued) Social desirability bias may be
present in interpretation activities where participants
were asked to interpret results in which many applied
to shortfalls in healthcare although participants were
informed at the start of the study that all perspectives
were valuable. Individuals who chose to volunteer and
participate in this study may have been fundamentally
different from those who declined to participate, par-
ticularly if they have had poor experiences in the past
with researchers or their service providers in general. Related to this is the recruitment of patients who
were already LTFU. Those that were reachable by
phone or in person and agreed to participate may be
different from those we could not find. Regardless,
differences
in
participation
across
individuals
we
attempted to
recruit
may
impact
on
the
genera-
lizability of our findings to the broader population of
interest (e.g., ART patients in Zomba). Finally, while
our findings may not be generalizable to individuals
outside of Malawi, the concept map presented here
identified
linkages
between
various
factors
that
influence
losses
to
follow-up
and
therefore,
may
be
of
interest
to
ART
managers
and
providers
internationally. taken with food have been shown to influence whether
patients can make it to their visits and keep taking their
meds [13-15,55,56]. While providing food supplements
at the clinics may encourage patients to remain in care
[15], this may also deter others, particularly when indi-
viduals do not want to be associated with ART. Future
research is needed to identify strategies that improve pa-
tient retention while supporting patients with logistical
challenges that may place them at increased risk for be-
coming LTFU. A brief discussion on Health Worker Attitudes and
Stigma and Fears is warranted given rating values. It is
worth noting that these clusters included both patient-
and healthcare- related statements. Within Health Worker
Attitudes for example, statements largely speak to pa-
tient frustrations with the healthcare they receive
particularly around the use of exposing language [21]. Stigma has been previously associated with patients
stopping treatment in Malawi [10]. While taking ART
can provide patients with a renewed sense of life,
allowing them to get back to work and take care of their
families, stigma continues to play a powerful role [22]. Experiences of HIV-related stigma can not only affect
adherence to ART but may also impact on social aspira-
tions and trajectories linked to a positive identity [21]. Table 3 The consolidated list of 64 statements (Continued) While we were surprised that Stigma and Fears was not
rated higher, participants in the present study may view
stigma as an underlying issue rather than a more imme-
diate cause of why patients become LTFU. For example,
a patient may seek care further from his/her home for
fear of stigmatization, resulting in having a farther dis-
tance to travel, thereby increasing transport costs. The
latter issue may be seen to be the more immediate issue
and as a result, given a higher rating value. In fact, in a
recent study, transport costs and other logistical bar-
riers were more commonly reported reasons for why
patients become lost when compared to stigma [49]. Healthcare delivery models that acknowledge patient
fears related to stigmatization and isolation are critical
as these can undermine relationships that are essential
for survival [57,58]. References Chalker J, Andualem T, Minzi O, Ntaganira J, Ojoo A, Waako P, Ross-Degnan
D: Monitoring adherence and defaulting for antiretroviral therapy in 5
East African countries: an urgent need for standards. J Int Assoc Physic
AIDS Care 2009, 7:193–199. 9. Malawi Ministry of Health: Clinical management of HIV in children and adults. Lilongwe, Malawi: Government of Malawi; 2011. 8. Chalker J, Andualem T, Minzi O, Ntaganira J, Ojoo A, Waako P, Ross-Degnan
D: Monitoring adherence and defaulting for antiretroviral therapy in 5
East African countries: an urgent need for standards. J Int Assoc Physic
AIDS Care 2009, 7:193–199. 9. Malawi Ministry of Health: Clinical management of HIV in children and adults. Lilongwe, Malawi: Government of Malawi; 2011. 8. Chalker J, Andualem T, Minzi O, Ntaganira J, Ojoo A, Waako P, Ross-Degnan
D: Monitoring adherence and defaulting for antiretroviral therapy in 5
East African countries: an urgent need for standards. J Int Assoc Physic
AIDS Care 2009, 7:193–199. y
29. Kane M, Trochim WM: Concept Mapping for Planning and Evaluation. Thousand Oaks: Sage Publications; 2007. 29. Kane M, Trochim WM: Concept Mapping
Thousand Oaks: Sage Publications; 2007 ,
9. Malawi Ministry of Health: Clinical management of HIV in children and adults. Lilongwe, Malawi: Government of Malawi; 2011. 30. National Institute for Health: The Contributions of Behavioural and Social
Sciences Research to Improving the Health of the Nation: a Prospectus for the
Future. 2009. http://obssr.od.nih.gov/pdf/OBSSR_Prospectus.pdf. 10. McGuire M, Munyenembe T, Szumilin E, Heinzelmann A, Le Paih M, Bouithy
N, Pujades- Rodriquez M: Vital status of pre-ART and ART patients
defaulting from care in rural Malawi. Trop Med Int Health 2009,
15(Suppl 1):55–62. 31. Jackson KM, Trochim WMK: Concept mapping as an alternative approach
for the analysis of open ended survey responses. Organ Res Methods
2002, 5:307–336. 11. Bisson GP, Stringer JSA: Lost but not forgotten-the economics of
improving patient retention treatment programs. PLoS Med 2009,
6:e1000174. 32. Burke JG, O’Campo P, Peak GL, Gielen AC, McDonnell KA, Trochim WM: An
introduction to concept mapping as a participatory public health
research method. Qual Health Res 2005, 15:1392–1410. 12. Bangsberg DR, Charlebois ED, Grant RM, Holodniy M, Deeks SG, Perry S,
Conroy KN, Clark R, Guzman D, Zolopa A, Moss A: High levels of adherence
do not prevent accumulation of HIV drug resistance mutations. AIDS 2003, 17:1925–1932. 33. Trochim WM: Reliability of Concept Mapping. Competing interests 15. Hardon AP, Akurut D, Comoro C, Ekezie C, Irunde HF, Gerrits T, Kglatwane J,
Kinsman J, Kwasa R, Maridadi J, Moroka TM, Moyo S, Nakiyemba A, Nsimba
S, Ogenyi R, Oyabba T, Temu F, Laing R: Hunger, waiting time and
transport costs: time to confront challenges to ART adherence in Africa. AIDS Care 2007, 19:658–665. The authors have declared that they have no competing interests The authors have declared that they have no competing interests Authors’ contributions BR conceived the study, developed and conducted concept mapping
activities and generated a first draft. FA conceived the study, assisted with
study development, supported CM activities and contributed to the drafting
of the manuscript. MvL assisted with the development of the study and
supported CM activities in Malawi and assisted with manuscript drafting. AM
assisted with the conceptual design of the study. MS provided support
during field activities. DCC assisted with conceptualization, study design and
development. All authors read and approved the final manuscript. 16. Dalal RP, Macphail C, Mghayi M, Wing J, Feldman C, Chersich MF, Venter
WD: Characteristics and outcomes of adult patients lost to follow-up at
an antiretroviral treatment clinic in Johannesburg, South Africa. J Acquir
Immune Defic Syndr 2008, 47:101–107. 17. Yu JKL, Chen SCC, Wang KY, Chang CS, Makombe SD, Schouten EJ, Harries
AD: True outcomes for patients on antiretroviral therapy Who are ‘lost to
follow-up’ in Malawi. Bull WHO 2007, 85:550–554. 18. Brinkhof MW, Dabis F, Myer L, Bangsberg DR, Boulle A, Nash D, Schechter
M, Laurent C, Keiser O, May M, Sprinz E, Egger M, Anglaret X, ART-LINC
IeDEA: Early loss of HIV-infected patients on potent antiretroviral therapy
programmes in lower-income countries. Bull WHO 2008, 86:559–567. Acknowledgements The authors wish to acknowledge Praise Mhango for his assistance with the
preparation, facilitation, and interpretation of Concept Mapping activities in
Zomba, Malawi. Funding support was received through a Centre for 19. Murray LK, Semrau K, McCurley E, Thea DM, Scott N, Mwiya M, Kankasa C,
Bass J, Bolton P: Barriers to acceptance and adherence of antiretroviral
therapy in urban Zambian women: a qualitative study. AIDS Care 2009,
21:78–86. International Governance and Innovation Africa Initiative Graduate Research
Grant. 20. Palombi L, Marazzi MC, Guidotti G, Germano P, Buonomo E, Scarcella P,
Doro Atlan A, Zimba Ida V, San Lio MM, De Luca A, DREAM Program:
Incidence and predictors of death, retention, and switch to second-line
regimens in antiretroviral-treated patients in sub-Saharan Africa. Clin Infect Dis 2009, 48:115–122. Author details
1 1Dalla Lana School of Public Health, University of Toronto, Toronto, Canada. 2School of Health Policy and Management, York University, Toronto, Canada. 3Dignitas International, Zomba, Malawi. 4College of Medicine, University of
Malawi, Blantyre, Malawi. 5District Health Office, Zomba, Malawi. 21. Merten S, Kenter E, McKenzie O, Musheke M, Ntalasha H, Martin A: Patient-
reported barriers and drivers of adherence to antiretrovirals in sub-Saharan
Africa: a meta- ethnography. Trop Med Int Health 2010, 15(Suppl 1):16–33. Received: 2 October 2012 Accepted: 1 June 2013
Published: 11 June 2013 22. Campbell C, Skovdal M, Madanhire C, Mugurungi O, Gregson S, Nyamukapa
C: “We, the AIDS people…”: How antiretroviral therapy enables
Zimbabweans living with AIDS to cope with stigma. Am J Public Health
2011, 101:1004–1010. References 1. Joint United Nations Programme on HIV/AIDS: Report on the Global AIDS
Pandemic. http://www.unaids.org/globalreport/global_report.htm. 1. Joint United Nations Programme on HIV/AIDS: Report on the Global AIDS
Pandemic. http://www.unaids.org/globalreport/global_report.htm. 23. Geng EH, Emenyonu N, Bwana MB, Glidden DV, Martin JN: Sampling-based
approach to determining outcomes of patients lost to follow-up in
antiretroviral therapy scale-up programs in Africa. JAMA 2008,
300:506–507. 2. Joint United Nations Programme on HIV/AIDS: Report on the the Global AIDS
Pandemic. http://www.unaids.org/en/media/unaids/contentassets/
dataimport/pub/report/2009/jc1700_epi_update_2009_en.pdf. 2. Joint United Nations Programme on HIV/AIDS: Report on the the Global AIDS
Pandemic. http://www.unaids.org/en/media/unaids/contentassets/
dataimport/pub/report/2009/jc1700_epi_update_2009_en.pdf. 24. Mills EJ, Nachega JB, Bangsberg DR, Singh S, Rachlis B, Wu P, Wilson K,
Buchan I, Gill CJ, Cooper C: Adherence to HAART: a systematic review of
developed and developing nation patient-reported barriers and
facilitators. PLoS Med 2006, 3:e438. 3. Office MNS: Malawi Demographic and Health Survey. Zomba, Malawi:
Government of Malawi; 2010. 3. Office MNS: Malawi Demographic and Health Survey. Zomba, Malawi:
Government of Malawi; 2010. 4. UNICEF/UNAIDS: Children and AIDS. Fifth Stocktaking Report. 2010. http://www.childinfo.org/files/ChildrenAndAIDS_Fifth_Stocktaking_Report_
2010_EN.pdf. 4. UNICEF/UNAIDS: Children and AIDS. Fifth Stocktaking Report. 2010. http://www.childinfo.org/files/ChildrenAndAIDS_Fifth_Stocktaking_Report_
2010_EN.pdf. 25. Rachlis B, Mills EJ, Cole DCC: Livelihood security and adherence to
antiretroviral therapy in Low and middle income settings: a systematic
review. PLoS One 2011, 6:e18948. 5. Malawi Ministry of Health, Department of HIV/AIDS: Quarterly ART Cohort
Report, Quarter. Lilongwe: Government of Malawi; 2010. 26. Office MNS: Population and Housing Census Report. Zomba, Malawi:
Government of Malawi; 2008. 6. Office of the President and Cabinet, Malawi: Malawi HIV and AIDS
Monitoring and Evaluation Report: Follow up to the UN Declaration of
Committment on HIV/AIDS. Lilongwe, Malawi: Government of Malawi; 2007. 27. Geubbels E, Bowie E: Chapter 2: Epidemiology of HIV/AIDS in Adults in
Malawi. In The Epidemiology of Malawi. 2nd edition. Edited by Guebbels E,
Bowie C. Blantyre, Malawi: College of Medicine, University of Malawi; 2009. 7. Rosen S, Fox MP, Gill CJ: Patient retention in an antiretroviral therapy
programs in sub-Saharan Africa: a systematic review. PLoS Med 2007, 4:e298. 28. Chan AK, Mateyu G, Jahn A, Schouten E, Arora P, Mlotha W, Kambanji M,
van Lettow M: Outcome assessment of decentralization of antiretroviral
therapy provision in a rural district of Malawi using an integrated
primary care model. Trop Med Int Health 2010, 15(Suppl 1):90–97. 8. Conclusions Through the incorporation of patient (including those
who were LTFU), provider, and decision-maker per-
spectives, we determined that individuals can become
LTFU for a multitude of patient- and healthcare-
related reasons. In general, nine major themes related
to why individuals become LTFU emerged and were
identified. Poor documentation and issues related to
tracing were identified as being particularly important
in our context. CM offered a unique approach that
combined the strengths of qualitative methodologies
of data collection with quantitative methods of ana-
lysis to further our understanding of LTFU in ART
programs in the Malawian setting. Both patient and
provider perspectives were brought to the forefront,
and offered insight into potential priority areas for
action. Findings of the present study have implica-
tions for strategies that maximize patient retention in
ART programs including improved documentation of
patient visits, enhanced coordination between clinics
involved in transfers, and more comprehensive coun-
selling for patients. There were several limitations in our study. Firstly,
while we attempted to include the perspectives of several
stakeholder groups, numerous potentially relevant stake-
holders with important perspectives may not have been
adequately represented including family members of
ART patients and religious leaders. Excluding partici-
pants without a secondary school education from
sorting activities may have biased our final cluster
map towards a more highly educated population al-
though it is worth noting that a secondary school
education
was
not
required
for
the
other
study
phases. While sampling was purposive, due in part
to
feasibility
issues,
more
providers
than
patients Page 10 of 11 Page 10 of 11 Rachlis et al. BMC Health Services Research 2013, 13:210
http://www.biomedcentral.com/1472-6963/13/210 Rachlis et al. BMC Health Services Research 2013, 13:210
http://www.biomedcentral.com/1472-6963/13/210 Ochieng D, Ochieng V, Braitstein P, Holdsworth M, Kimaiyo S, Wools-
Kaloustian K, Sang E, Musick B, Mwangi A, Sidle J: Patient Tracking and
Retention in a Resource- constrained Setting: The AMPATH Experience in
Western Kenya. Sydney, Australia: Proceedings of the 4th Annual
International AIDS Society Conference on HIV Pathogenesis, Treatment and
Prevention; 2007. 48. Geng EH, Bangsberg DR, Musinguzi N, Emenyonu N, Bwana MB,
Yiannoutsos CT, Glidden DV, Deeks SG, Martin JN: Understanding reasons
for and outcomes of patients lost to follow-up in antiretroviral therapy
programs in Africa through a sampling based approach. J Acquir Immune
Defic Syndromes 2010, 53:405–411. 49. Miller CM, Ketlhapile M, Rybasack-Smith H, Rosen S: Why Are antiretroviral
treatment patients lost to follow-up? a qualitative study from South
Africa. Trop Med Int Health 2010, 15(Suppl 1):48–54. 50. Kagee A, Remien RH, Berkman A, Hoffman S, Campos L, Swartz L: Structural
barriers to ART adherence in Southern Africa. Glob Public Health 2012,
6:83–97. 50. Kagee A, Remien RH, Berkman A, Hoffman S, Campos L, Swartz L: Structural
barriers to ART adherence in Southern Africa. Glob Public Health 2012,
6:83–97. Submit your next manuscript to BioMed Central
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Submit your manuscript at
www.biomedcentral.com/submit Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit Submit your next manuscript to BioMed Central
and take full advantage of: Submit your next manuscript to BioMed Central
and take full advantage of: 51. Rachlis et al. BMC Health Services Research 2013, 13:210
http://www.biomedcentral.com/1472-6963/13/210 Rachlis et al. BMC Health Services Research 2013, 13:210
http://www.biomedcentral.com/1472-6963/13/210 Rachlis et al. BMC Health Services Research 2013, 13:210
http://www.biomedcentral.com/1472-6963/13/210 55. Anema A, Vogenthaler N, Frongillo EA, Kadiyala S, Weiser SD: Food
insecurity and HIV/AIDS: current knowledge, gaps, and research
priorities. Curr HIV/AIDS Rep 2009, 6:224–231. 56. Sanjobo N, Frich JC, Freitheim A: Barriers and facilitators to Patients’
adherence to antiretroviral adherence to antiretroviral treatment in
Zambia. Sahara J 2008, 5:136–143. 57. Ncama BP, McInerney PA, Bhengu BR, Corless IB, Wantland DJ, Nicholas PK,
McGibbon CA, Davis SM: Social support and medication adherence in HIV
disease in KwaZulu-Natal, South Africa. Int J Nurs Studies 2008, 45:1757–1763. 58. Ware NC, Idoko J, Kaaya S, Biraro IA, Wyatt MA, Agbaji O, Chalamilla G,
Bangsberg DR: Explaining adherence successes in sub-Saharan Africa: an
ethnographic study. PLoS Med 2009, 6:e1000011. doi:10.1186/1472-6963-13-210
Cite this article as: Rachlis et al.: Using concept mapping to explore why
patients become lost to follow up from an antiretroviral therapy
program in the Zomba District of Malawi. BMC Health Services Research
2013 13:210. 55. Anema A, Vogenthaler N, Frongillo EA, Kadiyala S, Weiser SD: Food
insecurity and HIV/AIDS: current knowledge, gaps, and research
priorities. Curr HIV/AIDS Rep 2009, 6:224–231. to prevent mother-to-child HIV transmission in South Africa. PLoS One
2009, 4:e583. to prevent mother-to-child HIV transmission in South Africa. PLoS One
2009, 4:e583. 36. Shaw V: Health information system reform in South Africa: developing an
essential data Set. Bull WHO 2005, 83:632–636. 56. Sanjobo N, Frich JC, Freitheim A: Barriers and facilitators to Patients’
adherence to antiretroviral adherence to antiretroviral treatment in
Zambia. Sahara J 2008, 5:136–143. 37. Garrib A, Stoops N, McKenzie A, Dlamini L, Govender T, Rohde J, Herbst K:
An evaluation of the district health information system in rural South
Africa. South Afr Med 2008, 98:549–552. 57. Ncama BP, McInerney PA, Bhengu BR, Corless IB, Wantland DJ, Nicholas PK,
McGibbon CA, Davis SM: Social support and medication adherence in HIV
disease in KwaZulu-Natal, South Africa. Int J Nurs Studies 2008, 45:1757–1763. 38. Makombe SD, Hochgesang M, Jahn A, Tweya H, Hedt B, Chuka S, Yu JK,
Aberle-Grasse J, Pasulani O, Bailey C, Kamoto K, Schouten EJ, Harries AD:
Assessing the quality of data aggregated by antiretroviral treatment
clinics in Malawi. Bull WHO 2008, 86:310–314. 58. Rachlis et al. BMC Health Services Research 2013, 13:210
http://www.biomedcentral.com/1472-6963/13/210 Ware NC, Idoko J, Kaaya S, Biraro IA, Wyatt MA, Agbaji O, Chalamilla G,
Bangsberg DR: Explaining adherence successes in sub-Saharan Africa: an
ethnographic study. PLoS Med 2009, 6:e1000011. 39. Forster M, Bailey C, Brinkhof MW: Electronic medical record systems, data
quality, and lost to follow-up: survey of antiretroviral therapy
programmes in resource-limited settings. Bull WHO 2008, 86:939–947. doi:10.1186/1472-6963-13-210
Cite this article as: Rachlis et al.: Using concept mapping to explore why
patients become lost to follow up from an antiretroviral therapy
program in the Zomba District of Malawi. BMC Health Services Research
2013 13:210. 40. Clements CJ, Nshimirimanda D, Gasaira A: Using immunization delivery
strategies to accelerate Africa towards achieving the millenium
development goals. Vaccine 2008, 26:1926–1933. 41. Weigel R, Hochgesang M, Brinkhof MW, Hosseinipour MC, Boxshall M,
Mhango E, Nkwazi B, Tweya H, Kamlaka M, Chagwera F, Phiri S: Outcomes
and associated risk factors of patients traced after being lost to
follow-up from antiretroviral treatment in Lilongwe, Malawi. BMC Infect
Dis 2011, 11:31–39. 42. Tweya H, Gareta D, Chagwera F, Ben-Smith A, Mwenyemasi J, Chiputula F,
Boxshall M, Weigel R, Jahn A, Hosseinipour M, Phiri S: Early active follow-up
of patients on antiretroviral therapy (ART) Who Are lost to follow-up: the
‘back-to-Care’ project in Lilongwe, Malawi. Trop Med Int Health 2010,
15(Suppl 1):82–89. 43. Zachariah R, Teck R, Buhendwa L, Labana S, Chinji C, Humblet P, Harries AD:
How can the community contribute in the fight against HIV/AIDS and
tuberculosis? an example from a rural district in Malawi. Trans Royal Soc
Trop Med Hygiene 2006, 100:167–175. 44. Star Programme. http://www.starprogramme.org/index.php? option=com_content&view=article&id=79malawi-case-study&catid=33:mid-
term-review&Itemid=136. 44. Star Programme. http://www.starprogramme.org/index.php? option=com_content&view=article&id=79malawi-case-study&catid=33:mid-
term-review&Itemid=136. 45. Maskew M, Macphail C, Menezes C, Rubel D: Lost to follow-up:
contributing factors and challenges in South African patients on
antiretroviral therapy. South Afr J 2007, 97:853–857. 46. Krebs DW, Chi BH, Mulenga Y, Morris M, Cantrell RA, Mulenga L, Levy J,
Sinkala M, Stringer JS: Community-based follow-up for late patients
enrolled in a district-wide programme for antiretroviral therapy in
Lusaka, Zambia. AIDS Care 2008, 20:311–317. 47. Ochieng D, Ochieng V, Braitstein P, Holdsworth M, Kimaiyo S, Wools-
Kaloustian K, Sang E, Musick B, Mwangi A, Sidle J: Patient Tracking and
Retention in a Resource- constrained Setting: The AMPATH Experience in
Western Kenya. Sydney, Australia: Proceedings of the 4th Annual
International AIDS Society Conference on HIV Pathogenesis, Treatment and
Prevention; 2007. 47. References Dallas, Texas: Paper presented
at the Annual Conference of the American Evaluation Association; 1993. 13. Weiser SD, Tuller DM, Frongillo EA, Senkungu J, Mukiibi N, Bangsberg DR:
Food insecurity as a barrier to sustained antiretroviral therapy
adherence in Uganda. PLoS One 2010, 5:e10340. 34. Egger M, Ekouevi DK, Williams C, Lyamuya RE, Mukumbi H, Braitstein P,
Hartwell T, Graber C, Chi BH, Boulle A, Dabis F, Wools-Kaloustian K: Cohort
profile: the international epidemiological databases to evaluate AIDS
(IeDEA) in sub-Saharan Africa. Int Epidemiology 2011, 40:1–9. 14. Deribe K, Hailekiros F, Biadgilign S, Amberbir A, Beyene BK: Defaulters from
antiretroviral treatment in jimma university specialized hospital,
Southwest Ethiopia. Trop Med Int Health 2009, 13:328–333. 35. Mate KS, Bennett B, Mphatswe W, Barker P, Rollins N: Challenges for
routine health system data management in a large public programme Page 11 of 11 Rachlis et al. BMC Health Services Research 2013, 13:210
http://www.biomedcentral.com/1472-6963/13/210 Birbeck GL, Chomba E, Kvalsund M, Bradbury R, Mang’ombe C, Malama K,
Kaile T, Byers PA, Organek N, RAAZ Study Team: Antiretroviral adherence
in rural Zambia: the first year of treatment availability. Am J Tropl Med
Hygiene 2009, 80:669–674. • Convenient online submission 52. Kalichman SC, Ramachandran B, Catz S: Adherence to combination
antiretroviral therapies in HIV patients of Low health literacy. J Gen Intern
Med 1999, 14:267–273. 53. Norman A, Chopra M, Kadiyala S: Factors related to HIV disclosure in 2
South African communities. Am J Public Health 2007, 97:1775–1781. 54. Tuller DM, Bangsberg DR, Senkungu J, Ware NC, Emenyonu N, Weiser SD:
Transportation costs impede sustained adherence and access to HAART
in a clinic population in Southwestern Uganada: a qualitative study. AIDS
Behav 2010, 14:778–784.
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https://openalex.org/W4294307916
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https://www.frontiersin.org/articles/10.3389/fphys.2022.934163/pdf
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Cold exposure and musculoskeletal conditions; A scoping review
|
Frontiers in physiology
| 2,022
|
cc-by
| 9,702
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TYPE Review
PUBLISHED 01 September 2022
DOI 10.3389/fphys.2022.934163 TYPE Review
PUBLISHED 01 September 2022
DOI 10.3389/fphys.2022.934163 Background: Musculoskeletal conditions are major contributors to years lived
with disability. Cold exposure can be a risk factor, but any conclusion is obscure. Background: Musculoskeletal conditions are major contributors to years lived
with disability. Cold exposure can be a risk factor, but any conclusion is obscure. Background: Musculoskeletal conditions are major contributors to years lived
with disability. Cold exposure can be a risk factor, but any conclusion is obscure. Aim: The aim of the present scoping review was to identify the existing evidence
of an association between cold exposure and musculoskeletal conditions. The
aim also included to consider pain in different regions and their assessment, as
well as different measures of cold exposure, effect sizes, and to assess the
feasibility of future systematic reviews and meta-analyses. Eligibility criteria: The studies must have: an epidemiological design, defined
cold exposure to come prior to the health outcome, defined exposure and
outcome(s), existence of effect estimate(s) or data that made it possible to
calculate such an estimate. Further, studies were required to be in English
language and published in peer-reviewed journals. Studies that had a specific
goal of studying cold exposure as an aggravator of already existing health
problems were excluded. Front. Physiol. 13:934163. doi: 10.3389/fphys.2022.934163 COPYRIGHT
© 2022 Farbu, Höper, Reierth, Nilsson
and Skandfer. This is an open-access
article distributed under the terms of the Sources: We searched Ovid MEDLINE(R) and Epub Ahead of Print, In-Process
and Other Non-Indexed Citations, Daily and Versions(R), and Embase Classic +
Embase for original studies. Creative Commons Attribution License
(CC BY). The use, distribution or
reproduction in other forums is
permitted, provided the original
author(s) and the copyright owner(s) are
credited and that the original
publication in this journal is cited, in
accordance with accepted academic
practice. No use, distribution or
reproduction is permitted which does
not comply with these terms. Charting method: The included studies were reviewed for study population,
measurement of exposure and outcome, and effect size. Each publication was
assessed for risk of bias. Results: The included studies were heterogeneous in populations, measures of
cold exposure and musculoskeletal conditions. Most studies used self-reported
data. They were mostly cross-sectional studies, only two were prospective and
one was a case-control study. Associations were found for different cold
exposures and regional musculoskeletal conditions, but the heterogeneity
and lack of studies impeded valid synthesis of risk magnitude, or meta-analyses. Conclusion: The studies identified in this review indicate that cold exposure
increases the risk of musculoskeletal conditions. Cold exposure and
musculoskeletal conditions;
A scoping review OPEN ACCESS
EDITED BY
Sanjoy Deb,
University of Westminster,
United Kingdom
REVIEWED BY
Wilton Remigio,
Clarkson University, United States
Chayakrit Krittanawong,
Faculty of Medicine and Surgery, New
York University, United States
*CORRESPONDENCE
Erlend Hoftun Farbu,
erlend.h.farbu@uit.no
SPECIALTY SECTION
This article was submitted to
Environmental, Aviation and Space
Physiology,
a section of the journal
Frontiers in Physiology
RECEIVED 09 May 2022
ACCEPTED 30 June 2022
PUBLISHED 01 September 2022
CITATION
Farbu EH, Höper AC, Reierth E, Nilsson T
and Skandfer M (2022), Cold exposure
and musculoskeletal conditions;
A scoping review. Front. Physiol. 13:934163. doi: 10.3389/fphys.2022.934163 EDITED BY
Sanjoy Deb,
University of Westminster,
United Kingdom
REVIEWED BY
Wilton Remigio,
Clarkson University, United States
Chayakrit Krittanawong,
Faculty of Medicine and Surgery, New
York University, United States
*CORRESPONDENCE
Erlend Hoftun Farbu,
erlend.h.farbu@uit.no
SPECIALTY SECTION
This article was submitted to
Environmental, Aviation and Space
Physiology,
a section of the journal
Frontiers in Physiology
RECEIVED 09 May 2022
ACCEPTED 30 June 2022
PUBLISHED 01 September 2022
CITATION
Farbu EH, Höper AC, Reierth E, Nilsson T
and Skandfer M (2022), Cold exposure
and musculoskeletal conditions;
A scoping review. Front. Physiol. 13:934163. doi: 10.3389/fphys.2022.934163 Erlend Hoftun Farbu 1*, Anje Christina Höper1,2, Eirik Reierth3,
Tohr Nilsson4 and Morten Skandfer1 1Department of Community Medicine, UiT The Arctic University of Norway, Tromsø, Norway,
2Department of Occupational and Environmental Medicine, University Hospital of North Norway,
Tromsø, Norway, 3Department of Library Services, UiT The Arctic University of Norway, Tromsø,
Norway, 4Department of Public Health and Clinical Medicine, Occupational and Environmental
Medicine, Umeå University, Umeå, Sweden Background: Musculoskeletal conditions are major contributors to years lived
with disability. Cold exposure can be a risk factor, but any conclusion is obscure. However, there is a need for
studies that better assess temporality between exposure and outcome. Future
studies should also include better exposure assessment, including both
objective measurements and measures of subjective experience of cold Frontiers in Physiology 01 frontiersin.org Farbu et al. 10.3389/fphys.2022.934163 exposure. The heterogeneity in measurement of exposure and outcome
impeded any meta-analysis. cold exposure, cold environment, cold temperature, musculoskeletal conditions, pain,
regional pain, scoping review cold exposure, cold environment, cold temperature, musculoskeletal conditions, pain,
regional pain, scoping review Introduction contact cooling. Various studies use proxies for cold exposure
either as measured temperature, estimated cooling effect,
isotherms, climate, latitude, and contrasts from season, or
outdoor/ indoor exposures. Musculoskeletal conditions are among the most common
causes of severe pain (Woolf and Pfleger, 2003) and are some of
the leading contributors to the global burden of years lived with
disability (Blyth et al., 2019). As a group, musculoskeletal
conditions are estimated to cause 21.3% of the total years
lived with disability in the world (Hoy et al., 2015). Most of
the musculoskeletal conditions increase with age, and the present
population age structure and the predicted increase in longevity
imply augmented forthcoming occurrence. In addition, the risk
of pain increases with age-related comorbidity, thus the global
burden related to pain can be expected to continue to increase
(Blyth et al., 2019). The aim of the present scoping review was to identify the
existing evidence of an association between cold exposure and
musculoskeletal conditions. The aim also included to consider
pain in different regions and their assessment, as well as different
measures of cold exposure, effect sizes, and to assess the
feasibility of future systematic reviews and meta-analyses. p
(Blyth et al., 2019). This review was planned as a systematic review and
registered in PROSPERO (ID: CRD42018108223). However,
due to large heterogeneity in the measurement of exposure
and outcome it became evident that it was not feasible to
perform a systematic review. This will be described and
discussed in Results and Discussion. It was therefore carried
out as a scoping review. The following eligibility criteria were
defined: epidemiological design, defined cold exposure to come
prior to the health outcome, defined exposure and outcome(s),
existence of effect estimate(s) or data that made it possible to
calculate such an estimate. Further, studies were required to be in
English language and published in peer-reviewed journals. Studies that had a specific goal of studying cold exposure as
an aggravator of already existing health problems were excluded. Musculoskeletal conditions are highly diverse regarding
aetiology, pathophysiology, anatomy, and impact on physical
function. The collective concept entails all complaints related to
muscles, joints, tendons, ligaments, and bone structures. The
conditions may be systemic or regional. The latter include
neuropathic disorders such as radiculopathies with pain or
regional
entrapment
pain
[e.g.,
Carpal
tunnel
syndrome
(CTS)]. Many musculoskeletal conditions are defined by
symptoms rather than clinical findings (e.g., low back pain),
and pain is the major symptom. However, the aetiology of the
pain can
be difficult to identify
(Treede
et al., 2015). Consequently,
the
differentiation
between
musculoskeletal
conditions
and
other
pain
conditions
can
be
difficult. Therefore, pain or regional pain are often used terms. The majority of studies on risk factors for musculoskeletal
pain in the occupational context have explored the impact of job
task, physical load, repetitiveness, static strain (NIOSH, 1997), or
person related modifiers such as stress, anthropometry, BMI,
vitamin status, or genetics (Mills et al., 2019). Concurrent
exposures are the rule in epidemiological studies, obscuring
any effects of the multitude of risk factors and modifiers not
included in the study. The hypothesis claiming to account for the
findings include metabolic mechanisms, pathologic muscle
physiology, deteriorated blood supply or failing muscular
control (Visser and van Dieën, 2006). Self-reported working in
cold environments <25%
of the time Search Cold exposure was defined by search terms addressing
temperature, climate, region and cold effects, while health
outcome
was
defined
by
concepts
on
musculoskeletal
conditions subclassified by body regions. The search string
developed in Medline and EMBASE is presented in Figure 1. Databases searched were Ovid MEDLINE(R) and Epub Ahead of
Print, In-Process and Other Non-Indexed Citations, Daily and
Versions(R), and Embase Classic + Embase. We used controlled
vocabulary search terms (MeSH- and Emtree-index), whenever
applicable. In addition, we used search fields, with title, abstract
and keyword heading, as the preferred fields to search. Our
search was performed with a time limit set for publications
between the years 1980 and 2022. The search was last
updated in February 2022. The identified publications were Original studies on cold environment as a risk factor for
musculoskeletal conditions or pain are rare, and they are
divergent in designs, assessment methods and case definitions. The human reaction to cold exposure is relative and relates to
differences in temperature and is as such influenced by adaption,
acclimatisation, coping and physical status. The impact relates to
cold ambient temperature modified by wind chill, humidity, and Frontiers in Physiology frontiersin.org 02 Farbu et al. 10.3389/fphys.2022.934163 FIGURE 1
Systematic literature search of February 2022 in the following databases: Ovid MEDLINE(R) and Epub Ahead of Print, In-Process and Other Non-
Indexed Citations, Daily and Versions(R), and Embase Classic + Embase. 1980 to Present. Medline subject headings; ti, titles; ab, abstracts; kw,
keywords. FIGURE 1
Systematic literature search of February 2022 in the following databases: Ovid MEDLINE(R) and Epub Ahead of Print, In-Process and Other Non-
Indexed Citations, Daily and Versions(R), and Embase Classic + Embase. 1980 to Present. Medline subject headings; ti, titles; ab, abstracts; kw,
keywords. FIGURE 2
Prisma flow-chart displaying the literature search history. Of totally 3,580 references, we included 17 studies. FIGURE 1
Systematic literature search of February 2022 in the following databases: Ovid MEDLINE(R) and Epub Ahead of Print, In-Process and Other Non-
Indexed Citations, Daily and Versions(R), and Embase Classic + Embase. 1980 to Present. Medline subject headings; ti, titles; ab, abstracts; kw,
keywords. FIGURE 2
Prisma flow-chart displaying the literature search history. Of totally 3,580 references, we included 17 studies. FIGURE 2
Prisma flow-chart displaying the literature search history. Of totally 3,580 references, we included 17 studies. 03 Frontiers in Physiology frontiersin.org 10.3389/fphys.2022.934163 Farbu et al. TABLE 1 The included studies in alphabetical order. Search Study
Population
Country
Design
Exposure
Comparison
Outcome
Altuntas and
Cankaya
(2020) a
Poultry workers
Turkey
Cross-
sectional
Number of years working in an
environment <9°C
Less than 2 years
Self-reported, nordic
questionnaire, neck, shoulder,
elbow, wrist/hand, upper back,
low back, hip/thighs, knees,
ankles/feet
Bodin et al. (2012)
General working
population
France
Cross-
sectional
Self-experienced cold
exposure >4 h/day
Self-experienced cold
exposure ≤4 h/day
Rotator cuff syndrome
diagnosed with clinical
examination
Shoulder pain from Nordic Questionnaire
Burstrom et al. (2013)
Male construction
workers
Sweden
Cross-
sectional
Geographical regions with
different mean temperature
Warmest region
Self-reported back/neck pain
last 12 months that have
reduced work capacity
North
Central
Chiang et al. (1990)
Office workers,
non-frozen food
packers, frozen
food packers
China
Cross-
sectional
Handling frozen food
(−12 to −15°C), or handling
non-frozen food
Office workers
Carpal tunnel syndrome
diagnosed with clinical and
electrophysiological
examination
Dovrat and
Katz-Leurer
(2007)
Store workers
Israel
Cross-
sectional
Cold store work, −20°C
Store workers working in
20°–25°C
Self-reported, Nordic
Questionnaire, low back pain
Farbu et al. (2019)
General working
population
Norway
Cross-
sectional
Self-reported working in cold
environments ≥25% of the time
Self-reported working in
cold environments <25%
of the time
Self-reported chronic pain. Categorized into 1–2 pain
sites, ≥3 pain sites, and at
specific site neck, back,
shoulder, arm, hand, hip, leg,
foot
Farbu et al. (2021a)
General working
population
Norway
Prospective
Self-reported working in cold
environments ≥25% of the time
Self-reported working in
cold environments <25%
of the time
Musculoskeletal complaints
(MSC) lasting 3 months or
more
Ghani et al. (2020)
Store workers
Pakistan
Cross-
sectional
Working in −20°C to −30°C
Working outdoors or in
office
Musculoskeletal pain, adopted
from the Nordic questionnaire
Milgrom et al. (2003)
Army recruits
Israel
Prospective
Winter
Summer
Clinically verified Achilles
tendinosis or paratendinopathy
Piedrahı´;ta
et al. (2004)
Meat processing
workers
Colombia
Cross-
sectional
Ambient air temperature 2°C,
measured at workplace
Ambient air temperature
11°C, measured at
workplace
Self-reported, Nordic
questionnaire
Pinar et al. (2013)
Ammunition
factory workers
Turkey
Cross-
sectional
Self-reported cold environment
at workplace
No cold environment at
workplace
Self-reported categorized as
Yes/No
Pope et al. (1997)
General population
United Kingdom
Cross-
sectional
Self-reported sometimes or
always exposed to cold at work
Never exposed to cold at
work
Self-reported, shoulder pain
lasting 24 h or more last month
Raatikka et al. Store workers working in
20°–25°C Self-reported neck pain, back
pain, and lumbar radiculopathy (Continued on following page) Self-reported working in cold
environments ≥25% of the time Perceiving none cooling
experience of body parts
First tertile Self-reported pain affecting
activities of daily life
Self-reported neck pain, back
pain, and lumbar radiculopathy Office workers Search (2007)
General population
Finland
Cross-
sectional
Self-reported cold exposure by
hours/week exposed in past
winter
Self-reported, repeated pain
believed to be caused by cold
Skandfer et al. (2014)
Mine workers
Russia
Cross-
sectional
Self-reported
Not working in <10°C ≥
20 h a week
Self-reported low back pain,
Nordic questionnaire
Temperature at
workplace <10°C ≥20 h a week
Not wet clothes >5 h a
week
Wet clothes >5 h a week
Sormunen
et al. (2009)
Food processing
workers
Finland
Cross-
sectional
Perceiving slight/some/
extensive cooling experience of
body parts
Perceiving none cooling
experience of body parts
Self-reported pain affecting
activities of daily life
Stjernbrandt
and Farbu
(2022)
General working
population
Sweden
Cross-
sectional
Exposure to outdoor or cold
environment at work reported
on NRS from 0 (Do not agree) to
10 (Fully agree). Categorized
into tertiles
First tertile
Self-reported neck pain, back
pain, and lumbar radiculopathy
General population
Sweden
Cross-
sectional
Exposure to outdoor or cold
environment in leisure time
reported on NRS from 0 (Do not
First tertile
Self-reported neck pain, back
pain, and lumbar radiculopathy TABLE 1 The included studies in alphabetical order. Populations Six of the studies recruited from the general population (Pope
et al., 1997; Raatikka et al., 2007; Bodin et al., 2012; Farbu et al.,
2019; Farbu et al., 2021a; Stjernbrandt and Farbu, 2022), one
study recruited cases and controls from a clinic (Yagev et al.,
2007), and the remaining 10 studies comprised of specific
working populations, such as construction workers (Burstrom
et al., 2013), miners (Skandfer et al., 2014), food processing
workers (Piedrahı´;ta et al., 2004; Sormunen et al., 2009; Altuntas
and Cankaya, 2020), store workers (Chiang et al., 1990; Dovrat
and Katz-Leurer, 2007; Ghani et al., 2020), army recruits
(Milgrom et al., 2003) or factory workers (Pinar et al., 2013). The number of participants ranged from 162 to 118 258
(Tables 2, 3). Results Out of 2,770 unique hits, 46 studies were assessed in full text
(Figure 2). A total of 17 studies were identified as eligible for
inclusion (Table 1). One of the studies provided data that
contained what appears to be a logical flaw, there were more
participants not working due to musculoskeletal pain than
participants
having
musculoskeletal
pain
(Altuntas
and
Cankaya, 2020). We therefore chose to not calculate and
present any effect estimates from this study. Data extraction The 17 studies were examined by four authors (ACH, MS,
TN, and EHF) in full text for obtaining type of epidemiological
design, assessment of cold exposure and musculoskeletal
condition. Further, the number of included subjects, incidence
or prevalence, as well as association measures as odds ratio (OR),
relative risk (RR) or incidence rate ratio (IRR) and confidence
intervals (CI) were extracted by one author (EHF). Any missing
effect estimate were calculated using the inbuilt risk-ratio/odds-
ratio calculator in STATA 16. Exposure measures were classified
either as using measured temperature or proxy for it, or self-
reported cold exposure. The method of collecting outcomes was
either by questionnaire or by a clinical examination. The
outcomes were categorized according to regions. In addition,
carpal tunnel syndrome (CTS) and lumbar radiculopathy were
categorized as neuropathic pain. Search Comparison No cold environment at
workplace
Carpal tunnel syndrome after
clinical examination aNo effects sizes were calculated from this study due to a possible logical flaw in the provided data. of 21 and 24, respectively (Supplementary Table S1). Any
disagreements
between
the
authors
were
resolved
and
concluded in joint sessions including four of the authors. first assessed as titles and abstracts by reviewers ACH, MS, and
TN for eligibility, and the approach from the preferred reporting
items for scoping reviews statement was followed (Tricco et al.,
2018). The number of studies at each stage of the process are
shown in Figure 2. Based on the inclusion criteria, 46 studies were
selected and considered in full text by four of the reviewers in a
plenary session (ACH, MS, TN, and EHF). Any disagreement
concerning eligibility for inclusion was resolved through joint
discussion. Of the 46 titles, 17 were selected for inclusion
(Figure 2). No studies were identified from other sources like
reference lists of included studies. Search Comparison Self-reported working in cold
environments ≥25% of the time Prospective
Self-reported working in cold
environments ≥25% of the time
Self-reported working in
cold environments <25%
of the time
Musculoskeletal com
(MSC) lasting 3 mo
more
Cross-
sectional
Working in −20°C to −30°C
Working outdoors or in
office
Musculoskeletal pai
from the Nordic qu
Prospective
Winter
Summer
Clinically verified A
tendinosis or parate
Cross-
sectional
Ambient air temperature 2°C,
measured at workplace
Ambient air temperature
11°C, measured at
workplace
Self-reported, Nord
questionnaire
Cross-
sectional
Self-reported cold environment
at workplace
No cold environment at
workplace
Self-reported catego
Yes/No
gdom
Cross-
sectional
Self-reported sometimes or
always exposed to cold at work
Never exposed to cold at
work
Self-reported, shoul
lasting 24 h or more
Cross-
sectional
Self-reported cold exposure by
hours/week exposed in past
winter
Self-reported, repea
believed to be cause
Cross-
sectional
Self-reported
Not working in <10°C ≥
20 h a week
Self-reported low ba
Nordic questionnair
Temperature at
workplace <10°C ≥20 h a week
Not wet clothes >5 h a
week
Wet clothes >5 h a week
Cross-
sectional
Perceiving slight/some/
extensive cooling experience of
body parts
Perceiving none cooling
experience of body parts
Self-reported pain a
activities of daily lif
Cross-
sectional
Exposure to outdoor or cold
environment at work reported
on NRS from 0 (Do not agree) to
10 (Fully agree). Categorized
into tertiles
First tertile
Self-reported neck p
pain, and lumbar ra
Cross-
sectional
Exposure to outdoor or cold
environment in leisure time
reported on NRS from 0 (Do not
agree) to 10 (Fully agree). Categorized into tertiles
First tertile
Self-reported neck p
pain, and lumbar ra Ambient air temperature
11°C, measured at
workplace
No cold environment at
workplace
Never exposed to cold at
work Self-reported pain affecting
activities of daily life
Self-reported neck pain, back
pain, and lumbar radiculopathy Self-reported neck pain, back
pain, and lumbar radiculopathy Frontiers in Physiology 04 frontiersin.org Farbu et al. 10.3389/fphys.2022.934163 TABLE 1 (Continued) The included studies in alphabetical order. Study
Population
Country
Design
Exposure
Comparison
Outcome
Yagev et al. (2007)
Carpal tunnel
syndrome patients
scheduled for
operation
Israel
Case-
control
Self-reported cold environment
at workplace
No cold environment at
workplace
Carpal tunnel syndrome after
clinical examination
aNo effects sizes were calculated from this study due to a possible logical flaw in the provided data. TABLE 1 (Continued) The included studies in alphabetical order. Measurement of exposure To make it easier for the reader to judge the validity of the
estimates provided we included an assessment of the included
studies. The studies were assessed by the reviewers using a score
system developed for this review (Supplementary Table S1). Sub-
scores for method, exposure, and outcome were added up to a
total score, a higher score meaning less risk of bias. Maximum
points for the outcome sub-score were different for regional pain
and neuropathic pain, leading to a potential maximum total score There were two main categories of exposure measure:
temperature and self-reported exposure to cold conditions. Seven studies used temperature as a measure of cold exposure. The measures were heterogenous, ranging from categories based
on contact with frozen items (Chiang et al., 1990), to categories
based on seasons (Milgrom et al., 2003), ambient air temperature
measured at the workplace (Piedrahı´;ta et al., 2004; Dovrat and Frontiers in Physiology frontiersin.org 05 Farbu et al. 10.3389/fphys.2022.934163 TABLE 2 Effect estimates sorted after type and type of assessment of neuropathic conditions. TABLE 2 Effect estimates sorted after type and type of assessment of neuropathic conditions. Outcome
Exposure
Effect
estimate
(95% CI)
Percent
having
outcome
of total
sample
Numbers of
participants
Risk
of
bias
Study
Exposed
Total
Clinical
examination
Carpal tunnel
syndrome
Handling frozen food
(−12 to −15°C)
Both
genders
OR 9.39
(2.37–37–19)
30
121
207
16
Chiang et al. (1990)
Self-reported cold
environment at workplace
Both
genders
OR 3.52
(1.08–11.47)
n.a
Not
reported
229
15
Yagev et al. (2007)
Self-reported
Lumbar
radiculopathy
Self-reported occupational
exposure. Lowest tertile as
reference medium (2. tertile)
high (3. tertile)
Both
genders
OR 1.29
(1.02–1.62)
6.2
2093
8,740
6
Stjernbrandt
and Farbu
(2022)
OR 1.36
(1.07–1.73)
1958
Self-reported leisure-time
exposure. Categorized into
tertiles. Highest compared to
lowest
Both
genders
OR 1.15
(0.91–1.44)
Not reported
Not
reported
12 627
6
Stjernbrandt
and Farbu
(2022)
OR: Odds Ratio CI: confidence interval. Outcome
Exposure OR: Odds Ratio CI: confidence interval. Katz-Leurer, 2007; Ghani et al., 2020), self-reported number of
years worked in an environment below 9°C (Altuntas and
Cankaya, 2020), or different regions by latitude, implying
different mean temperatures between the regions (Burstrom
et al., 2013). Regarding level of exposure, one of the ten
studies using self-reported exposure used weekly average time
of cold exposure (Raatikka et al., 2007), while many self-reported
cold exposures were specified with a certain minimum time of
cold exposure per day, or week. Measurement of exposure For example, cold environment
at the workplace >4 h pr day (Bodin et al., 2012), temperature at
workplace <10°C ≥20 h a week or wearing wet clothes >5 h a
week (Skandfer et al., 2014), working ≥25% of the time in cold
environment (Farbu et al., 2019; Farbu et al., 2021a). One study
used never, occasional or always exposed to cold environment at
work (Pope et al., 1997), and two used self-reported cold
environment at the workplace without assessing the duration
or frequency of exposure (Yagev et al., 2007; Pinar et al., 2013). Another study assessed self-reported exposure to outdoor
or cold environment and participants reported on a
numerical rating scale from 0 (Do not agree) to 10 (Fully
agree) for both occupational exposure and leisure time
exposure. Only one study specified cold environment
with a temperature threshold (Skandfer et al., 2014),
while in one study participants were not asked about the
environment, but the perceived cooling of different body
parts (Sormunen et al., 2009). Most of the studies focused
on
occupational
exposure. However,
in
one
study
participants were asked specifically about leisure time
cold
exposure
as
well
as
occupational
exposure
(Stjernbrandt and Farbu, 2022), and one used total time
exposed to cold during a week (Raatikka et al., 2007). The
studies using season or geographical regions also include differences in leisure time exposure (Milgrom et al., 2003;
Burstrom et al., 2013). Assessments of outcome Most of the studies used several different outcomes. The
outcome measures could roughly be divided into neuropathic
(Table 2) or musculoskeletal pain (Table 3). The neuropathic
outcomes were clinically verified carpal tunnel syndrome in two
studies (Chiang et al., 1990; Yagev et al., 2007), and self-reported
lumbar radiculopathy in one (Stjernbrandt and Farbu, 2022). 15 of the studies included musculoskeletal pain as outcome. Two
of these studies included a diagnosis verified by clinical
examination; one used achilles paratendinitis and another
rotator cuff syndrome (RCS) (Milgrom et al., 2003; Bodin
et
al.,
2012). A
total
of
14
studies
used
self-reported
musculoskeletal pain, either pain in general or at different
sites. Six
studies
used
questions
from
the
Nordic
Questionnaire to assess the 12-months prevalence of pain,
either at multiple sites (Piedrahı´;ta et al., 2004; Altuntas and
Cankaya, 2020; Ghani et al., 2020), or at specific sites such as low
back (Dovrat and Katz-Leurer, 2007; Skandfer et al., 2014) or
shoulder (Bodin et al., 2012). In some of the studies participants
were asked about pain that reduced work ability (Burstrom et al.,
2013; Altuntas and Cankaya, 2020) or caused a disadvantage in
daily activities (Sormunen et al., 2009). In one study participants
were asked about shoulder pain lasting more than 24 h during the
last month (Pope et al., 1997), and two studies specified that the
pain should have lasted 3 months or more (Farbu et al., 2019;
Farbu et al., 2021a), thereby more specifically assessing chronic
pain. In one study participants were asked to report repeated Frontiers in Physiology frontiersin.org 06 Effect estimate
(95% CI)
Percent having
outcome of
total sample
Numbers of participants
Risk of bias
Study
Exposed
Total
Men
Not reported
7.0
149
2,161
11
Bodin et al. (2012)
Women
OR 1.3 (0.5–3.5)
9.3
71
1,548
Men
RR 2.62 (1.71–4.01)a
6.8
697
1,500
10
Milgrom et al. (2003)
Both genders
IRR 1.15 (1.03–1.29)
55.7
258
2,347
12
Farbu et al. (2021a)
IRR 0.95 (0.60–1.48)
8.4
IRR 1.11 (0.83–1.49)
17.6
ent
Both genders
OR 1.84 (1.37–2.47)
39.3
323
955
8
Pinar et al. (2013)
Men
OR 1.13 (1.03–1.22)
30.2
n.a
2,332
8
Raatikka et al. (2007)
Women
OR 1.35 (1.14–1.58)
27.2
2,840
Both genders
OR 0.95 (0.73–1.24)
16.3
623
5493
8
Farbu et al. Assessments of outcome (2019)
OR 1.57 (1.23–2.01)
18.7
666
5657
C
Men
OR 2.6 (1.03–6.5)
32.8
64
122
10
Dovrat and Katz-Leurer (2007)
Men
OR 1.19 (1.14–1.24)
24.3
23 514
118 258
10
Burstrom et al. (2013)
OR 1.09 (1.05–1.13)
57 148
Men
OR 1.54 (1.31–1.81)
51
1,196
3,530
9
Skandfer et al. (2014)
OR 1.81 (1.54–2.14)
1,668
Men
PR 4.48 (1.61–12.4)
9.3
50
112
9
Piedrahí;ta et al. (2004)
Both genders
OR 3.22 (2.28–4.55)
70
195
1,117
9
Sormunen et al. (2009)
OR 6.08 (4.01–9.24)
175
OR 7.36 (3.99–13.58)
78
Men
OR 1.17 (1.04–1.30)
6.7
n.a
2,267
8
Raatikka et al. (2007)
Women
OR 1.41 (1.04–1.87)
5.0
2,785
Both genders
OR 1.18 (0.91–1.52)
14.3
779
6,553
8
Farbu et al. (2019)
Both genders
OR 1.10 (0.94–1.29)
14.9
2093
8740
7
Stjernbrandt and Farbu (2022)
OR 1.38 (1.17–1.63)
1958
Both genders
OR 1.01 (0.86–1.18)
Not reported
Not reported
12 627
7
Stjernbrandt and Farbu (2022)
Men
RR 4.11 (2.66–6.34)
Not reported
100
200
6
Ghani et al. (2020)
Men
PR 2.24 (0.32–15.45)
2.5
50
112
9
Piedrahí;ta et al. (2004)
Men
RR 21.00 (6.82–64.65)
Not reported
100
200
6
Ghani et al. (2020)
(Continued on following page) re
Men
OR 1.12 (0.98–1.26)
10
n.a
2,293
8
Raatikka et al. (2007)
Women
OR 1.09 (0.86–1.37)
10.8
2,821
ork
Both genders
OR 2.77 (1.99–3.85)
65
177
1,117
9
Sormunen et al. (2009)
OR 6.05 (4.03–9.10)
161
OR 11.28 (4.69–27.15)
45
to 8–12°C
Men
PR 4.48 (0.85–23.6)
3.7
50
112
9
Piedrahí;ta et al. (2004)
re
Men
OR 1.31 (1.12–1.51)
4.9
n.a
2,288
8
Raatikka et al. (2007)
Women
OR 1.16 (0.81–1.98)
4.7
2,812
>4 h/day
Men
Not reported
28.0
149
2,161
8
Bodin et al. (2012)
Women
OR 2.2 (1.3–3.8)
31.1
71
1,548
Men
RR 1.8 (0.6–5.4)
1
29
113
8
Pope et al. (1997)
RR 6.4 (1.5–27)
4.2
4
Women
RR 1.4 (0.4–5.1)
18.7
14
123
RR 1.1 (0.2–8−5)
5
ld
time
Both genders
OR 1.39 (1.08–1.78)
13.7
779
6,553
8
Farbu et al. (2019)
(Continued on following page) of assessment of pain or regional pain. Effect estimate
(95% CI)
Percent having
outcome of
total sample
Numbers of participants
Risk of bias
Study
Exposed
Total
Men
OR 1.57 (1.47–1.67)
8.6
23 514
118 258
10
Burstrom et al. (2013)
OR 1.18 (1.12–1.25)
57 148
C
Men
PR 11.2 (1.34–93.4)
3.7
50
112
9
Piedrahí;ta et al. Assessments of outcome (2004)
Both genders
OR 1.46 (1.13–1.89)
13.7
779
6,553
8
Farbu et al. (2019)
Both genders
OR 1.15 (0.99–1.34)
17.5
2093
8,740
Stjernbrandt and Farbu (2022)
OR 1.36 (1.16–1.59)
1958
Both genders
OR 1.10 (0.95–1.28)
Not reported
Not reported
12 627
7
Stjernbrandt and Farbu (2022)
Men
RR 15.00 (6.33–35.51)
Not reported
100
200
6
Ghani et al. (2020)
Both genders
OR 2.28 (1.48–3.50)
84
202
1,117
9
Sormunen et al. (2009)
OR 3.88 (2.49–6.05)
283
OR 10.33 (4.81–22.19)
163
Men
OR 1.12 (0.98–1.26)
10
n.a
2,293
8
Raatikka et al. (2007)
Women
OR 1.09 (0.86–1.37)
10.8
2,821
Both genders
OR 2.77 (1.99–3.85)
65
177
1,117
9
Sormunen et al. (2009)
OR 6.05 (4.03–9.10)
161
OR 11.28 (4.69–27.15)
45
C
Men
PR 4.48 (0.85–23.6)
3.7
50
112
9
Piedrahí;ta et al. (2004)
Men
OR 1.31 (1.12–1.51)
4.9
n.a
2,288
8
Raatikka et al. (2007)
Women
OR 1.16 (0.81–1.98)
4.7
2,812
y
Men
Not reported
28.0
149
2,161
8
Bodin et al. (2012)
Women
OR 2.2 (1.3–3.8)
31.1
71
1,548
Men
RR 1.8 (0.6–5.4)
1
29
113
8
Pope et al. (1997)
RR 6.4 (1.5–27)
4.2
4
Women
RR 1.4 (0.4–5.1)
18.7
14
123
RR 1.1 (0.2–8−5)
5
Both genders
OR 1.39 (1.08–1.78)
13.7
779
6,553
8
Farbu et al. (2019)
(Continued on following page) re. ype of assessment of pain or regional pain. Effect estimate
(95% CI)
Percent having
outcome of
total sample
Numbers of participants
Risk of bias
Study
Exposed
Total
Men
RR 151.00 (9.48–2,403.28)
Not reported
100
200
6
Ghani et al. (2020)
Men
PR 2.24 (0.14–35.1)
1.2
50
112
9
Piedrahí;ta et al. (2004)
Men Women
OR 1.15 (0.94–1.45)
2.1
n.a
2,269
8
Raatikka et al. (2007)
OR 1.30 (0.79–1.98)
2.1
2,800
e
Both genders
OR 1.34 (0.98–1.83)
8.4
779
6,553
8
Farbu et al. (2019)
compared to normal
Men
RR 10.40 (4.33–2,434.82)
Not reported
100
200
6
Ghani et al. (2020)
Both genders
OR 2.74 (1.78−4–22)
70
54
1,117
9
Sormunen et al. (2009)
OR 6.11 (4.01–9.31)
72 90
OR 20.12 (11.29–35.85)
Men
PR 2.24 (0.58–8.6)
4.9
50
112
9
Piedrahí;ta et al. (2004)
Men Women
OR 1.11 (0.94–1.34)
5.6
n.a
2,268
8
Raatikka et al. (2007)
OR 1.51 (1.18–1.91)
6.9
2,795
e
Both genders
OR 1.16 (0.79–1.71)
6
779
6,553
8
Farbu et al. (2019)
Men Women
OR 1.11 (0.95-1–27)
18.4
n.a
2,309
8
Raatikka et al. Assessments of outcome (2007)
OR 1.34 (1.11–1.60)
16.6
2,815
Men
RR 23.33 (7.59–71.64)
Not reported
100
200
6
Ghani et al. (2020)
Men
PR 1.49 (0.26–8.66)
3.1
50
112
9
Piedrahí;ta et al. (2004)
e
Both genders
OR 1.26 (0.90–1.75)
8.9
779
6,553
8
Farbu et al. (2019)
Men Women
OR 1.06 (0.90–1.23)
6.8
n.a
2,268
8
Raatikka et al. (2007)
OR 1.13 (0.85–1.46)
7.6
2,793
e
Both genders
OR 1.47 (1.10–1.96)
10
779
6,553
8
Farbu et al. (2019)
Men
RR 111.00 (6.95–1772.51)
Not reported
100
200
6
Ghani et al. (2020)
Men
RR 6.87 (3.45–13.67)
Not reported
100
200
6
Ghani et al. (2020)
Men
PR 2.24 (0.14–35.1)
1.2
50
112
9
Piedrahí;ta et al. (2004)
Men Women
OR 1.16 (1.03–1.30)
11
n.a
2,278
8
Raatikka et al. (2007)
OR 1.34 (1.08–1.64)
11.6
2,808
e
Both genders
OR 0.8 (0.54–2.04)
6.6
779
6,553
8
Farbu et al. (2019)
−30°C)
Men
RR 3.53 (2.13–5.83)
Not reported
100
200
6
Ghani et al. (2020)
ratio; CI, Confidence interval aCalculated by the authors of this review. 10.3389/fphys.2022.934163 Farbu et al. lasting ≥3 months (Farbu et al., 2019) were not statistically significant. One study did not find any association between leisure time cold
exposure and back pain (Stjernbrandt and Farbu, 2022) musculoskeletal pain believed to be caused by cold temperature
(Raatikka et al., 2007). Six of the seven studies using neck pain as an outcome found
statistically significant associations between cold exposure and
neck pain. The associations were found using self-reported
exposure,
subjective
experience,
geographical
regions
and
measured temperature at the workplace (Table 3). Three of
the studies consisted of reasonable large sample sizes from
6,533 to 118 258. Even though one study found a statistically
significant association for occupational cold exposure, the
association for leisure time cold exposure was not significant
(Stjernbrandt and Farbu, 2022). Another study that did not find a
statistically significant association had asked participants for
head/neck pain the participants believed to be caused by cold
exposure (Raatikka et al., 2007). Results in the included studies There were several different outcomes in many of the studies,
and consequently a total of 85 effect estimates were extracted
from the included studies (Tables 2, 3). Most of the estimates in
the included studies showed an association between cold
exposure and pain, either neuropathic or musculoskeletal pain
(Tables 2, 3). However, not all were statistically significant, many
of the estimates had wide confidence intervals and the estimates
between studies varied. Four studies included pain in elbows, forearms, or arms as
outcomes (Piedrahı´;ta et al., 2004; Raatikka et al., 2007; Farbu
et al., 2019; Ghani et al., 2020). Even though all four studies found
increased odds for cold exposure, only one was statistically
significant. Studies of pain in the wrist, hands or fingers also
consistently found increased odds for pain for cold exposure, but
not all associations were statistically significant (Table 3). One
study found significantly higher odds for pain in wrist or fingers
believed to be caused by cold with increasing cold exposure
among women, but not among men (Raatikka et al., 2007). The two studies on CTS found increased odds for cold
exposure, either measured as self-reported cold environment
at work (Yagev et al., 2007), or as handling frozen food
(Chiang et al., 1990). In one study, the highest tertile of
occupational cold exposure had significantly increased odds
for lumbar radiculopathy, but not the highest tertile of leisure
time cold exposure (Table 2) (Stjernbrandt and Farbu, 2022). Among the studies using pain in general as an outcome, a
prospective cohort study found a significantly increased risk of having
any musculoskeletal complaints after 7–8 years (Farbu et al., 2021a). However, there were no significantly increased risks of severe or
widespread musculoskeletal complaints. This contrasts the earlier
findings from a cross-sectional analysis from the same authors
showing a significant association between cold exposure and pain
at ≥3 sites, but not for pain at 1–2 sites. A significant association
between cold exposure and general musculoskeletal pain was also
found in a sample of workers from a Turkish ammunition factory
(Pinar et al., 2013), as well as between cold exposure and pain believed
to be caused by cold exposure in a general population in Finland
(Raatikka et al., 2007). Critical appraisal of individual sources of
evidence Two other studies found a
significant association for men, but not for women (Pope et al.,
1997; Raatikka et al., 2007). One study found that those working
in cold environment ≥25% of the time had significantly increased
odds for shoulder pain lasting ≥3 months (Farbu et al., 2019). Critical appraisal of individual sources of
evidence The risk of bias in the included studies was assessed by a
scoring system developed for this review (Supplementary Table
S1). The total score is presented in Tables 2, 3. The sub scores on
exposure, outcome and method are presented in Supplementary
Table S2. Most of the studies had less than half of the possible
maximum scores, indicating a higher risk of bias. Fourteen of the
studies had cross-sectional design, two were cohort studies
(Milgrom et al., 2003; Farbu et al., 2021a) and one was a case-
control study (Yagev et al., 2007). A total of seven studies included shoulder pain as outcome. One study of French employees showed that being exposed to
cold >4 h per day was positively associated with self-reported
shoulder pain in women, but not with clinically diagnosed RCS. The results for men were not reported because the authors did
not find a statistically significant higher prevalence of shoulder
pain with or without RCS among the 149 men exposed to cold
environment (Bodin et al., 2012). Two other studies found a
significant association for men, but not for women (Pope et al.,
1997; Raatikka et al., 2007). One study found that those working
in cold environment ≥25% of the time had significantly increased
odds for shoulder pain lasting ≥3 months (Farbu et al., 2019). Four studies included pain in elbows, forearms, or arms as
outcomes (Piedrahı´;ta et al., 2004; Raatikka et al., 2007; Farbu
et al., 2019; Ghani et al., 2020). Even though all four studies found
increased odds for cold exposure, only one was statistically
significant. Studies of pain in the wrist, hands or fingers also
consistently found increased odds for pain for cold exposure, but
not all associations were statistically significant (Table 3). One
study found significantly higher odds for pain in wrist or fingers
believed to be caused by cold with increasing cold exposure
among women, but not among men (Raatikka et al., 2007). A total of seven studies included shoulder pain as outcome. One study of French employees showed that being exposed to
cold >4 h per day was positively associated with self-reported
shoulder pain in women, but not with clinically diagnosed RCS. The results for men were not reported because the authors did
not find a statistically significant higher prevalence of shoulder
pain with or without RCS among the 149 men exposed to cold
environment (Bodin et al., 2012). Discussion Taken together, the limited evidence available indicates that cold
exposure increases the risk of musculoskeletal conditions. The studies
on cold exposure as a risk factor for CTS had the least risk of bias and
found strong associations (OR of 3.52 and 9.39), but they were limited
in size. Associations between cold exposure and back, neck and
shoulder pain were found in several studies with reasonable large
sample sizes. However, the studies included in this review are in
general at risk of being biased. The majority was cross-sectional and
only two studies were prospective, in addition to one case-control
study. One of the prospective studies attained the outcome 7–8 years
after the measurement of exposure and did not include information
about the status of exposure at follow-up. Further, most included
studies use self-reported exposures or outcomes, and were done in
specific populations. Measuring cold exposure is difficult. One used definition of
cold environment at work is an ambient temperature <10°C
(International Organization of Standardisation, 2008). However,
contact with cold surfaces, cold liquids, draughts, humidity, and
amount of clothing also affects the heat loss of an individual. The
level of physical activity, and consequently heat production, might
lessen the need for protection against the cold. The lack of an
association for leisure time exposure might indicate that the setting
of the exposure is of importance. The possibility to mitigate the
negative effects of cold exposure by wearing more clothes or
terminate the exposure might be higher when it is a leisure
time activity. Another possibility is that cold exposure might
not be a risk factor for musculoskeletal conditions, and that the
association between occupational exposure and pain could be
confounded by other risk factors. Workers that are exposed to
cold might be more exposed to other known concurrent risk
factors for pain like heavy lifting, vibration, and awkward working
positions. Some of the studies try to take other occupational risk
factors into account, but good measurement of all possible
confounders and the statistical strength to satisfactorily adjust
for these is difficult to achieve. Occupation is also considered as a
marker of socioeconomic position, which again is found to be
related to pain and other risk factors for pain such as level of
physical activity, smoking, obesity, and poor health (Poleshuck and
Green, 2008; Mills et al., 2019). Results in the included studies On the
other side, the experience of pain and the environment, as well as
having pain, could very well be closely connected (Sundstrup
et al., 2015; Farbu et al., 2021b). The central sensitization
associated with pain could increase the risk of feeling cold or
cooling, and the likelihood of reporting a cold environment. Consequently, the cross-sectional studies using self-reported
exposure could be vulnerable to reverse causation. association between cold exposure and pain in the foot
lasting ≥3 months (Farbu et al., 2019). Results in the included studies Among the five studies using hip, knees, thighs, calves or leg,
two studies found associations: one between working in cold
environments
≥25%
of
the
time
and
pain
in
the
leg
lasting ≥3 months or more (Farbu et al., 2019), and one found
significant associations between cold store work and pain in the
hip/thighs and knees (Ghani et al., 2020). There were four studies using ankle and/or foot as outcomes
(Piedrahı´;ta et al., 2004; Raatikka et al., 2007; Farbu et al., 2019;
Ghani et al., 2020). One of the studies with the highest score in
the critical appraisal found a higher incidence of achilles
paratendinitis
among
military
recruits
trained
in
winter
compared to summer (Milgrom et al., 2003), and one study
reported
an association
between hours
exposed
to cold
environment and pain in ankles or feet believed to be caused
by cold exposure (Raatikka et al., 2007). One study found no Most studies found cold exposure to be significantly associated
with back pain (Table 3). The associations were found using self-
reported exposure, subjective experience, geographical regions, and
measured temperature at the workplace (Table 3). The results for
upper back (including 4 cases) (Piedrahı´;ta et al., 2004) and back pain Frontiers in Physiology frontiersin.org 10 Farbu et al. 10.3389/fphys.2022.934163 exposure increases the risk for musculoskeletal conditions. Another finding that strengthens the hypothesis is that some
of the included studies found a dose-response relationship
between cold exposure and pain (Pope et al., 1997; Raatikka
et al., 2007; Sormunen et al., 2009; Burstrom et al., 2013;
Stjernbrandt and Farbu, 2022). One study also found a dose-
response relationship for frequency of feeling cold and chronic
pain (Farbu et al., 2019). Two of these studies use a subjective
measure of cold exposure, i.e., feeling cold or cooling, and the
self-reported exposure in several of the other studies could be
influenced by the subjective experience. A study of workers in the
fishing industry could not find any simple relationship between
the ambient temperature and the frequency of feeling cold and
feeling cold often was associated with musculoskeletal pain (Bang
et al., 2005). Thus, the subjective experience of the environment
might be an important marker for the cold exposures’ effect on
the body, but a poor marker of the thermal environment. Discussion Consequently, some studies could
be confounded, and even studies with a comprehensive adjustment
for other risk factors could be vulnerable to residual confounding. h d
d h
f The limited evidence indicating that cold exposure is a risk
factor for pain or chronic pain identified in this review could be
supported by literature that did not meet our inclusion criteria. One study noted that a colder temperature at the working place
was the only notable difference between two groups with
different incidence of tenosynovitis in food-processing workers
(Kurppa et al., 1991), and other studies have found associations
between
different
environmental
complaints
and
pain
(Hildebrandt et al., 2002; Magnavita et al., 2011; Sundstrup
et al., 2015). A case-control study found an association
between
working
in
a
cold
environment
and
having
rheumatoid arthritis (Zeng et al., 2017). Further, several
experimental studies have found that cold temperature might
affect neuromuscular function. For example, cold temperature
caused decreased muscle power and contraction velocity
(Racinais and Oksa, 2010), it increased stiffness of tendons
(Alegre et al., 2016), as well as decreased nerve conductivity
(Racinais and Oksa, 2010). A more direct association is found in
individuals experiencing non-freezing cold injury, a neuropathic
condition causing pain and sensory disturbances (Vale et al.,
2017). The possible nerve swelling caused by cooling could be
part of the explanation of the association between cold exposure
and CTS (Ulaşli et al., 2014). These findings could indicate that
cold exposure cause structural changes which increases the risk of
pain. In addition, many report that their pain condition is
aggravated by weather (Timmermans et al., 2014) and that cold
temperature causes pain. Further, the findings that weather affects
pain tolerance indicates that the climatic environment can be of
importance for how and when we feel pain (Farbu et al., 2021b). The discussed heterogenous measurements of exposure, as
well as for outcomes, impedes any meta-analysis. The estimated
effect size would not be interpretable. On the other side,
associations were found using many different measures of
cold
exposure
and
in
many
different
countries. These
convergent
results
strengthen
the
hypothesis
that
cold Frontiers in Physiology frontiersin.org 11 Farbu et al. Farbu et al. 10.3389/fphys.2022.934163 Pain is one of the most prominent symptoms of musculoskeletal
conditions, and pain is an experience and a feeling, it is subjective in
nature, and it seems to vary over time (Glette et al., 2020; Farbu et al.,
2021b). Publisher’s note All claims expressed in this article are solely those of
the
authors
and
do
not
necessarily
represent
those
of
their affiliated organizations, or those of the publisher, the
editors
and
the
reviewers. Any
product
that
may
be
evaluated in this article, or claim that may be made by its
manufacturer, is not guaranteed or endorsed by the publisher. Another limitation is that many of the studies included questions
on aches, stiffness, or trouble in certain regions of the body. Thus,
even though we have used the term regional pain in this review, some
participants might have had other symptoms than pain. Further, our
list of musculoskeletal conditions was not exhaustive. Although the evidence for cold exposure per se as a causal
factor for musculoskeletal conditions is uncertain, it is in many
instances relatively easy and cheap to prevent by means of better
or more clothing. The association between perceived climatic
environment and pain indicates that the person’s own experience
of the climate might be a good indicator of cold exposure and the Discussion Measuring it is therefore inherently difficult. In addition, the
reportingof pain seemsto be affected bythe societytheindividuallives
in (Zimmer et al., 2021). To further complicate it, the correlation
between clinical findings and pain can often be poor (Dieppe and
Lohmander, 2005), meaning a clinically verified diagnosis is not a
good measure of an individual’s pain. Thus, the best measure of
outcome might be dependent on the causal hypothesis about the
association: does cold exposure increase the risk of experiencing pain
or does it cause structural changes which again increases the risk of
pain? need for protective measures. There is a need for prospective
studies, and future research should clearly state their aim and
hypothesis, restrain from testing multiple hypotheses at the same
time, be sufficiently powered, and try to incorporate both
objective and subjective measures of the environment. Future
studies could also include an assessment of important aspects of
pain like intensity and duration. Funding This work was supported by Helse Nord Grant HNF1345-17. The funder had no role in the design and conduct of the study;
collection, management, analysis, and interpretation of the data;
preparation, review, or approval of the manuscript; and decision
to submit the manuscript for publication. Some of the included studies had few participants and did not
have sufficient power. For example, one study calculated a prevalence
ratio based on only 2 cases (Piedrahı´;ta et al., 2004), and another
study found an RR of 151 with 100 participants in each group (Ghani
et al., 2020). This estimate is only possible if the number of events in
one group is less than one or higher than 100. Nevertheless, many
studies calculated many estimates. A total of 85 estimates were
extracted from 16 studies, but none of the included studies
adjusted for multiple testing. Author contributions ER, ACH, MS, TN, and EHF worked out the search-terms and
inclusion criteria. ER refined and carried out the search, produced the
figure presenting the full electronic search strategy and revised the
methodology together with TN and EHF from PRISMA SR to
PRISMA ScR. ACH, MS, and TN read titles and abstracts. ACH,
MS,TN and EHF read all full texts considered for inclusion and jointly
identified design and measurements of exposure and outcome. EHF
extracted the effect estimates. TN, MS, and EHF wrote the first draft. All authors revised the manuscript. The results presented in this review are vulnerable to possible
publication bias. Some of the included studies do not have a
specific aim of investigating cold exposure as a risk factor. Other
studies that also included cold exposure as one of many risk
factors may have chosen to not report the estimates due to non-
significant results. To which extent this is present is not known. Another limitation is the use of odds ratios as an effect
measure,
as
it
has
no
intuitive
interpretation. OR
is
“overestimated” if interpreted as relative risk, and this bias
increases with increasing prevalence (Szklo and Nieto, 2018). The prevalence of the outcomes in the included studies ranged
from under 10% to over 80%, and direct comparison of the ORs
between studies is precarious. These problems also complicate the
communication of the results, and the translation into the clinic. Conflict of interest The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could
be construed as a potential conflict of interest. The results in some of the included studies might also be
affected by the healthy-worker effect (Hernán et al., 2004). Many
studies use occupational exposure, and it might very well be that
those vulnerable to cold exposure or developing pain already
have
changed
their
occupation. This
could
lead
to
an
underestimation of the effect. References J. Med. Sci. Sports 20 (Suppl. 3), 1–18. doi:10.1111/j.1600-0838.2010.01204.x Chiang, H. C., Chen, S. S., Yu, H. S., and Ko, Y. C. (1990). The occurrence of
carpal tunnel syndrome in frozen food factory employees. Gaoxiong Yi Xue Ke Xue
Za Zhi 6 (2), 73–80. Skandfer, M., Talykova, L., Brenn, T., Nilsson, T., and Vaktskjold, A. (2014). Low
back pain among mineworkers in relation to driving, cold environment and
ergonomics. Ergonomics 57 (10), 1541–1548. doi:10.1080/00140139.2014.904005 Dieppe, P. A., and Lohmander, L. S. (2005). Pathogenesis and management of pain
in osteoarthritis. Lancet 365 (9463), 965–973. doi:10.1016/s0140-6736(05)71086-2 Sormunen, E., Remes, J., Hassi, J., Pienimaki, T., and Rintamaki, H. (2009). Factors associated with self-estimated work ability and musculoskeletal symptoms
among male and female workers in cooled food-processing facilities. Ind. Health 47
(3), 271–282. doi:10.2486/indhealth.47.271 Dovrat, E., and Katz-Leurer, M. (2007). Cold exposure and low back pain in store
workers in Israel. Am. J. Ind. Med. 50 (8), 626–631. doi:10.1002/ajim.20488 Farbu, E. H., Höper, A. C., Brenn, T., and Skandfer, M. (2021a). Is
working
in
a
cold
environment
associated
with
musculoskeletal
complaints 7–8 years later? A longitudinal analysis from the tromsø
study. Int. Arch. Occup. Environ. Health 94 (4), 611–619. doi:10.1007/
s00420-020-01606-6 Standardisation, I. O. (2008). International organization of standardisation ISO
15743:2008 ergonomics of the thermal environment - cold workplaces- risk
assessment and managment. Geneva. Stjernbrandt, A., and Farbu, E. H. (2022). Occupational cold exposure is
associated
with
neck
pain,
low
back
pain,
and
lumbar
radiculopathy. Ergonomics 24, 1–10. doi:10.1080/00140139.2022.2027030 Farbu, E. H., Rypdal, M., Skandfer, M., Steingrímsdóttir Ó, A., Brenn, T., Stubhaug,
A., et al. (2021b). To tolerate weather and to tolerate pain: Two sides of the same coin? Tromsø Study 7, 878–886. Pain. doi:10.1097/j.pain.0000000000002437 Sundstrup, E., Jakobsen, M. D., Brandt, M., Jay, K., Persson, R., Andersen, L. L.,
et al. (2015). Central sensitization and perceived indoor climate among workers
with chronic upper-limb pain: Cross-sectional study. Pain Res. Treat. 2015, 793750. doi:10.1155/2015/793750 Farbu, E. H., Skandfer, M., Nielsen, C., Brenn, T., Stubhaug, A., Höper, A. C., et al. (2019). Working in a cold environment, feeling cold at work and chronic pain: A
cross-sectional analysis of the tromsø study. BMJ Open 9 (11), e031248. doi:10. 1136/bmjopen-2019-031248 Szklo, M., and Nieto, F. J. (2018). Epidemiology: Beyond the basics. Sudbury:
Sudbury: Jones & Bartlett Learning, LLC. Ghani, N., Tariq, F., Javed, H., Nisar, N., and Tahir, A. (2020). Supplementary Materials The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fphys. 2022.934163/full#supplementary-material Frontiers in Physiology frontiersin.org 12 Farbu et al. 10.3389/fphys.2022.934163 References Low-temperature
health hazards among workers of cold storage facilities in Lahore, Pakistan. Med. Pr. 71 (1), 1–7. doi:10.13075/mp.5893.00857 Timmermans, E. J., van der Pas, S., Schaap, L. A., Sánchez-Martínez, M., Zambon, S.,
Peter, R., et al. (2014). Self-perceived weather sensitivity and joint pain in older people with
osteoarthritis in six European countries: Results from the European project on
OSteoArthritis (EPOSA). BMC Musculoskelet. Disord. 15, 66. doi:10.1186/1471-2474-15-66 Glette, M., Stiles, T. C., Borchgrevink, P. C., and Landmark, T. (2020). The natural
course of chronic pain in a general population: Stability and change in an
eight–wave longitudinal study over four years (the HUNT pain study). J. Pain
21 (5), 689–699. doi:10.1016/j.jpain.2019.10.008 Treede, R.-D., Rief, W., Barke, A., Aziz, Q., Bennett, M. I., Benoliel, R., et al. (2015). A classification of chronic pain for ICD-11. Pain 156 (6), 1003–1007. doi:10. 1097/j.pain.0000000000000160 Hernán, M. A., Hernández-Díaz, S., and Robins, J. M. (2004). A structural approach to
selection bias. Epidemiology 15 (5), 615–625. doi:10.1097/01.ede.0000135174.63482.43 Tricco, A. C., Lillie, E., Zarin, W., O’Brien, K. K., Colquhoun, H., Levac, D., et al. (2018). PRISMA extension for scoping reviews (PRISMA-ScR): Checklist and
explanation. Ann. Intern. Med. 169 (7), 467–473. doi:10.7326/m18-0850 Hildebrandt, V. H., Bongers, P. M., van Dijk, F. J., Kemper, H. C., and Dul, J. (2002). The influence of climatic factors on non-specific back and neck-shoulder
disease. Ergonomics 45 (1), 32–48. doi:10.1080/00140130110110629 Ulaşli, A. M., Tok, F., Karaman, A., Yaman, F., Dikici, O., Oruç, S., et al. (2014). Nerve enlargement after cold exposure: A pilot study with ultrasound imaging. Muscle Nerve 49 (4), 502–505. doi:10.1002/mus.23947 Hoy, D. G., Smith, E., Cross, M., Sanchez-Riera, L., Blyth, F. M., Buchbinder, R.,
et al. (2015). Reflecting on the global burden of musculoskeletal conditions: Lessons
learnt from the global burden of disease 2010 study and the next steps forward. Ann. Rheum. Dis. 74 (1), 4–7. doi:10.1136/annrheumdis-2014-205393 Vale, T. A., Symmonds, M., Polydefkis, M., Byrnes, K., Rice, A. S. C., Themistocleous, A. C., et al. (2017). Chronic non-freezing cold injury results in neuropathic pain due to a
sensory neuropathy. Brain 140 (10), 2557–2569. doi:10.1093/brain/awx215 International
Organization
of
Standardisation
(2008). ISO
15743:2008
Ergonomics of the thermal environment-Cold workplaces-Risk assessment and
managment. Geneva: International Organization of Standardisation. Visser, B., and van Dieën, J. H. (2006). Pathophysiology of upper extremity muscle
disorders. J. Electromyogr. Kinesiol. 16 (1), 1–16. doi:10.1016/j.jelekin.2005.06.005 Kurppa, K., Viikari-Juntura, E., Kuosma, E., Huuskonen, M., and Kivi, P. (1991). References NIOSH (1997). “A critical review of epidemiologic evidence for work-related
musculoskeletal disorders of the neck, upper extremity, and low back,” in
Musculoskeletal disorders and workplace factors. Editor B. P. Bernard (NIOSH). Alegre, L. M., Hasler, M., Wenger, S., Nachbauer, W., and Csapo, R. (2016). Does
knee joint cooling change in vivo patellar tendon mechanical properties? Eur. J. Appl. Physiol. 116 (10), 1921–1929. doi:10.1007/s00421-016-3444-5 Altuntas, Y. D., and Cankaya, T. (2020). Effects of working years in cold
environment on the musculoskeletal system and carpal tunnel symptoms. Agri
Derg. 32 (3), 120–127. doi:10.14744/agri.2020.35651 Piedrahı´ta, H., Punnett, L., and Shahnavaz, H. (2004). Musculoskeletal
symptoms in cold exposed and non-cold exposed workers. Int. J. Industrial
Ergonomics 34 (4), 271–278. doi:10.1016/j.ergon.2004.04.008 Bang, B. E., Aasmoe, L., Aardal, L., Andorsen, G. S., Bjornbakk, A. K., Egeness, C., et al. (2005). Feeling cold at work increases the risk of symptoms from muscles, skin, and airways
in seafood industry workers. Am. J. Ind. Med. 47 (1), 65–71. doi:10.1002/ajim.20109 Pinar, T., Cakmak, Z. A., Saygun, M., Akdur, R., Ulu, N., Keles, I., et al. (2013). Symptoms of musculoskeletal disorders among ammunition factory workers in Turkey. Arch. Environ. Occup. Health 68 (1), 13–21. doi:10.1080/19338244.2011.627893 Blyth, F. M., Briggs, A. M., Schneider, C. H., Hoy, D. G., and March, L. M. (2019). The global burden of musculoskeletal pain-where to from here? Am. J. Public Health
109 (1), 35–40. doi:10.2105/ajph.2018.304747 Poleshuck, E. L., and Green, C. R. (2008). Socioeconomic disadvantage and pain. Pain 136 (3), 235–238. doi:10.1016/j.pain.2008.04.003 Pope, D. P., Croft, P. R., Pritchard, C. M., Silman, A. J., and Macfarlane, G. J. (1997). Occupational factors related to shoulder pain and disability. Occup. Environ. Med. 54 (5), 316–321. doi:10.1136/oem.54.5.316 Bodin, J., Ha, C., Chastang, J. F., Descatha, A., Leclerc, A., Goldberg, M., et al. (2012). Comparison of risk factors for shoulder pain and rotator cuff syndrome in
the working population. Am. J. Ind. Med. 55 (7), 605–615. doi:10.1002/ajim.22002 Raatikka, V. P., Rytkonen, M., Nayha, S., and Hassi, J. (2007). Prevalence of cold-
related complaints, symptoms and injuries in the general population: The FINRISK
2002 cold substudy. Int. J. Biometeorol. 51 (5), 441–448. doi:10.1007/s00484-006-0076-1 Burstrom, L., Jarvholm, B., Nilsson, T., and Wahlstrom, J. (2013). Back and neck
pain due to working in a cold environment: A cross-sectional study of male
construction workers. Int. Arch. Occup. Environ. Health 86 (7), 809–813. doi:10. 1007/s00420-012-0818-9 Racinais, S., and Oksa, J. (2010). Temperature and neuromuscular function. Scand. References Incidence of tenosynovitis or peritendinitis and epicondylitis in a meat-processing
factory. Scand. J. Work Environ. Health 17 (1), 32–37. doi:10.5271/sjweh.1737 Woolf, A. D., and Pfleger, B. (2003). Burden of major musculoskeletal conditions. Bull. World Health Organ. 81 (9), 646–656. Yagev, Y., Gringolds, M., Karakis, I., and Carel, R. S. (2007). Carpal tunnel
syndrome: Under-recognition of occupational risk factors by clinicians. Ind. Health
45 (6), 820–822. doi:10.2486/indhealth.45.820 Magnavita, N., Elovainio, M., De Nardis, I., Heponiemi, T., and Bergamaschi, A. (2011). Environmental discomfort and musculoskeletal disorders. Occup. Med. 61
(3), 196–201. doi:10.1093/occmed/kqr024 Zeng, P., Bengtsson, C., Klareskog, L., and Alfredsson, L. (2017). Working in cold
environment and risk of developing rheumatoid arthritis: Results from the Swedish EIRA
case–control study. RMD Open 3 (2), e000488. doi:10.1136/rmdopen-2017-000488 Milgrom, C., Finestone, A., Zin, D., Mandel, D., and Novack, V. (2003). Cold
weather training: A risk factor for achilles paratendinitis among recruits. Foot Ankle
Int. 24 (5), 398–401. doi:10.1177/107110070302400504 Zimmer, Z., Fraser, K., Grol-Prokopczyk, H., and Zajacova, A. (2021). A global
study of pain prevalence across 52 countries: Examining the role of country-level
contextual
factors. Pain
[online
ahead
of
print]. doi:10.1097/j.pain. 0000000000002557 Mills, S. E. E., Nicolson, K. P., and Smith, B. H. (2019). Chronic pain: A review of
its epidemiology and associated factors in population-based studies. Br. J. Anaesth. 123 (2), e273–e283. doi:10.1016/j.bja.2019.03.023 Frontiers in Physiology frontiersin.org 13
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One of the Nine Doublet Microtubules of Eukaryotic Flagella Exhibits Unique and Partially Conserved Structures
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Introduction Motile cilia and flagella are important organelles that propel
cells or generate fluid flow across tissues, e.g., for mucus clearance
in airways. Defects in the assembly or function of these organelles
have been linked to a number of human diseases, called
ciliopathies, such as primary ciliary dyskinesia and male infertility
[1,2]. Motile flagella are also essential for successful host infection
by pathogenic organisms such as Trypanosoma brucei, the cause of
sleeping sickness [3,4], making their flagella potential drug targets. Motile cilia and flagella are important organelles that propel
cells or generate fluid flow across tissues, e.g., for mucus clearance
in airways. Defects in the assembly or function of these organelles
have been linked to a number of human diseases, called
ciliopathies, such as primary ciliary dyskinesia and male infertility
[1,2]. Motile flagella are also essential for successful host infection
by pathogenic organisms such as Trypanosoma brucei, the cause of
sleeping sickness [3,4], making their flagella potential drug targets. Most motile cilia and flagella share a highly conserved 9+2
arrangement of microtubules in the axonemal core structure [5,6];
here, nine doublet microtubules (DMTs) surround two central
singlet microtubules of the central pair complex (CPC) and attach
to the CPC through radial spokes (Figure 1). In general, two
distinct numbering systems are used for designating DMTs of 9+2
cilia and flagella. Historically, numbering was based on the relative
position of each DMT with regard to the plane of the CPC [7,8],
which has been adopted for the cilia and flagella of many animals,
including sea urchin and mammalian spermatozoa, which have
a fixed CPC [7–13]. A later numbering system, based on the
direction of beating, was proposed for Chlamydomonas, given its
rotating CPC [14–16]. Dynein motor proteins are arranged in two g
g
The generation of typical beating patterns for cilia and flagella,
such as the quasi-planar motility of many 9+2 cilia and flagella,
requires spatial control of dynein-driven inter-doublet sliding
around the axoneme [19–21]. In vitro DMT sliding studies using
axonemes from Chlamydomonas, a unicellular green alga, and from
sea urchin sperm have indicated that inter-doublet sliding occurs
predominantly between specific subsets of DMTs [12,14,22]. Significant progress has been made in dissecting axonemal
complexes that regulate dynein activity in general, such as the
CPC [23–27], the radial spokes [13,27–30], the I1 inner dynein
[31–34], the N-DRC [35–38], and the calmodulin and spoke
associated complex (CSC) [39,40]. Abstract The axonemal core of motile cilia and flagella consists of nine doublet microtubules surrounding two central single
microtubules. Attached to the doublets are thousands of dynein motors that produce sliding between neighboring
doublets, which in turn causes flagellar bending. Although many structural features of the axoneme have been described,
structures that are unique to specific doublets remain largely uncharacterized. These doublet-specific structures introduce
asymmetry into the axoneme and are likely important for the spatial control of local microtubule sliding. Here, we used
cryo-electron tomography and doublet-specific averaging to determine the 3D structures of individual doublets in the
flagella of two evolutionarily distant organisms, the protist Chlamydomonas and the sea urchin Strongylocentrotus. We
demonstrate that, in both organisms, one of the nine doublets exhibits unique structural features. Some of these features
are highly conserved, such as the inter-doublet link i-SUB5-6, which connects this doublet to its neighbor with a periodicity
of 96 nm. We also show that the previously described inter-doublet links attached to this doublet, the o-SUB5-6 in
Strongylocentrotus and the proximal 1–2 bridge in Chlamydomonas, are likely not homologous features. The presence of
inter-doublet links and reduction of dynein arms indicate that inter-doublet sliding of this unique doublet against its
neighbor is limited, providing a rigid plane perpendicular to the flagellar bending plane. These doublet-specific features and
the non-sliding nature of these connected doublets suggest a structural basis for the asymmetric distribution of dynein
activity and inter-doublet sliding, resulting in quasi-planar waveforms typical of 9+2 cilia and flagella. Citation: Lin J, Heuser T, Song K, Fu X, Nicastro D (2012) One of the Nine Doublet Microtubules of Eukaryotic Flagella Exhibits Unique and Partially Conserved
Structures. PLoS ONE 7(10): e46494. doi:10.1371/journal.pone.0046494 Editor: Yanmin Yang, Stanford University School of Medicine, United States of America Editor: Yanmin Yang, Stanford University School of Medicine, United States of America Received June 7, 2012; Accepted September 5, 2012; Published October 10, 2012 Received June 7, 2012; Accepted September 5, 2012; Published October 10, 2012 Copyright: 2012 Lin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by funding from the National Institutes of Health (GM083122; http://www.nigms.nih.gov), from the W. M. One of the Nine Doublet Microtubules of Eukaryotic
Flagella Exhibits Unique and Partially Conserved
Structures Jianfeng Lin, Thomas Heuser, Kangkang Song, Xiaofeng Fu, Daniela Nicastro*
Biology Department, Rosenstiel Center, Brandeis University, Waltham, Massachusetts, United States of America Jianfeng Lin, Thomas Heuser, Kangkang Song, Xiaofeng Fu, Daniela Nicastro*
Biology Department, Rosenstiel Center, Brandeis University, Waltham, Massachusetts, United States of America Jianfeng Lin, Thomas Heuser, Kangkang Song, Xiaofeng Fu, Daniela
Biology Department, Rosenstiel Center, Brandeis University, Waltham, Massachusetts, United States of America iology Department, Rosenstiel Center, Brandeis University, Waltham, Massachusetts, United States of America Abstract Keck Foundation
(http://www.wmkeck.org), the National Science Foundation (DMR-MRSEC-0820492 supporting TH; http://mrsec.org) and by a Pew Biomedical Scholars Award (to
DN; http://www.pewtrusts.org). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: nicastro@brandeis.edu rows, the outer and inner dynein arms (ODA and IDA), along the
length of the DMT A-tubule. Driven by ATP hydrolysis, dyneins
transmit forces along the B-tubule of the neighboring DMT,
causing sliding between adjacent DMTs [17,18]. Inter-doublet
links connecting neighboring DMTs, such as the nexin-dynein
regulatory complex (N-DRC), are thought to restrict this sliding
displacement between DMTs and thus convert the inter-doublet
sliding into a bending motion of the axoneme. October 2012 | Volume 7 | Issue 10 | e46494 Citation: Lin J, Heuser T, Song K, Fu X, Nicastro D (2012) One of the Nine Doublet Microtubules of Eukaryotic Flagella Exhibits Unique and Partially Conserved
Structures. PLoS ONE 7(10): e46494. doi:10.1371/journal.pone.0046494 Introduction The flagella from both organisms
have a highly conserved cylindrical arrangement of nine DMTs (red boxes). Each DMT is built from many copies of a 96-nm long unit that repeats
along the DMT length. The axonemes are shown in cross-sectional views from the flagellar base (proximal) towards the tip (distal). The locations of
the previously described 5–6 bridge (left) and proximal 1–2 bridge (right) are indicated. (B) For both organisms, schematic representations of a 96-nm
repeat are shown in longitudinal and cross-sectional views; orientations of the 96-nm repeat are maintained in all following figures unless stated
otherwise. Other labels: A-tubule (At), B-tubule (Bt), inner dynein arms (IDA 1a, 1b, 2–6 and x; rose) [43], intermediate chain/light chain complex (ICLC),
nexin-dynein regulatory complex (N-DRC, yellow) [37], outer dynein arm (ODA, purple), radial spoke (RS, gray) [13,30]. doi:10.1371/journal.pone.0046494.g001 DMT sliding through radial spokes [26]. However, the CPC/
radial spoke system cannot be the only source of asymmetric
signaling, because ATP-induced DMT sliding results in the
selective extrusion of specific DMTs even in radial spoke-defective
Chlamydomonas mutants [14,26]. This suggests that, besides radial
spoke signaling, local differences in the composition and/or
arrangement of axonemal structures also contribute to the selective
activity of dynein and the sliding between DMT subsets. DMT sliding through radial spokes [26]. However, the CPC/
radial spoke system cannot be the only source of asymmetric
signaling, because ATP-induced DMT sliding results in the
selective extrusion of specific DMTs even in radial spoke-defective
Chlamydomonas mutants [14,26]. This suggests that, besides radial
spoke signaling, local differences in the composition and/or
arrangement of axonemal structures also contribute to the selective
activity of dynein and the sliding between DMT subsets. Recent cryo-electron tomography (cryo-ET) studies have de-
scribed two new doublet-specific structures and have provided
remarkable new details of some of the above-mentioned doublet-
specific features; these include dynein arm IAX [16,43] and the
radial spoke joist (RSJ) in sea urchin flagella [13], as well as new
details regarding the proximal 1–2 bridge [16] and the beak
[16,43–45]. Still lacking is an understanding of how these
structural differences contribute to the coordination of dynein
activity on different DMTs, the associated formation of axonemal
bending, different bending patterns, or changes in waveforms in
response to environmental stimuli. Previous classical electron microscopy(EM) studies have observed
some doublet-specific structures in motile cilia and flagella. Introduction However, the mechanism for
inducing sliding only between specific subsets of DMTs is not well
understood. Previous reports have suggested that the CPC
distributes asymmetric regulation signals to define localized Most motile cilia and flagella share a highly conserved 9+2
arrangement of microtubules in the axonemal core structure [5,6];
here, nine doublet microtubules (DMTs) surround two central
singlet microtubules of the central pair complex (CPC) and attach
to the CPC through radial spokes (Figure 1). In general, two
distinct numbering systems are used for designating DMTs of 9+2
cilia and flagella. Historically, numbering was based on the relative
position of each DMT with regard to the plane of the CPC [7,8],
which has been adopted for the cilia and flagella of many animals,
including sea urchin and mammalian spermatozoa, which have
a fixed CPC [7–13]. A later numbering system, based on the
direction of beating, was proposed for Chlamydomonas, given its
rotating CPC [14–16]. Dynein motor proteins are arranged in two October 2012 | Volume 7 | Issue 10 | e46494 1 PLOS ONE | www.plosone.org Unique Doublet Microtubule Figure 1. Schematic models of the axoneme organization in the sea urchin Strongylocentrotus and the green alga Chlamydomonas. (A)
Two numbering systems for designating the DMTs in 9+2 cilia and flagella are currently in use (left [7], right [15]). The flagella from both organisms
have a highly conserved cylindrical arrangement of nine DMTs (red boxes). Each DMT is built from many copies of a 96-nm long unit that repeats
along the DMT length. The axonemes are shown in cross-sectional views from the flagellar base (proximal) towards the tip (distal). The locations of
the previously described 5–6 bridge (left) and proximal 1–2 bridge (right) are indicated. (B) For both organisms, schematic representations of a 96-nm
repeat are shown in longitudinal and cross-sectional views; orientations of the 96-nm repeat are maintained in all following figures unless stated
otherwise. Other labels: A-tubule (At), B-tubule (Bt), inner dynein arms (IDA 1a, 1b, 2–6 and x; rose) [43], intermediate chain/light chain complex (ICLC),
nexin-dynein regulatory complex (N-DRC, yellow) [37], outer dynein arm (ODA, purple), radial spoke (RS, gray) [13,30]. doi:10.1371/journal.pone.0046494.g001 Figure 1. Schematic models of the axoneme organization in the sea urchin Strongylocentrotus and the green alga Chlamydomonas. (A)
Two numbering systems for designating the DMTs in 9+2 cilia and flagella are currently in use (left [7], right [15]). Image Processing 3D tomograms were calculated using the IMOD software
package [50] with gold fiducial marker alignment and weighted
back projection. Only tomograms of intact and non- or mildly
compressed flagella and axonemes were used for further image
processing. Some of the tomograms were previously utilized for
the analysis of other axonemal complexes [13,30,37,51] but with
different image processing methods. We used several well-
established criteria for determining the polarity of the axonemes
in the tomograms, such as the sequence of axonemal structures
along the length of the A-tubule (I1 dynein proximal of the N-
DRC), and the clockwise sequence of A-tubules connecting to
neighboring B-tubules when the flagellum is viewed in cross-
section from the proximal end. Doublet-specific averaging was
carried
out
using
PEET
(Particle
Estimation
for
Electron
Tomography) software [51]. Subtomograms containing the 96-
nm axonemal repeats were extracted from tomograms (summa-
rized in Table 1), aligned, and separately averaged in 3D for each
DMT. The DMT numbers were identified based on unique Quantifoil holey carbon grids (Quantifoil Micro Tools GmbH,
Jena, Germany) were glow discharged at 240 mA for 30 sec and
then coated with 10-nm colloidal gold (Sigma-Aldrich, St. Louis,
MO). Intact and actively beating Strongylocentrotus sperm cells (i.e. ATP inherently present in the flagella) or quiescent Chlamydomonas
axonemes (no ATP present in the buffer) were applied to the grid
together
with
a
tenfold-concentrated
10-nm
colloidal
gold
solution. After blotting from the front side with Whatman #1 Table 1. Chlamydomonas strains used in this study and presence of discussed features. Table 1. Chlamydomonas strains used in this study and presence of discussed features. Number of Tomograms
Strain
Swimming
Reference
analyzed
i-SUB5-6
proximal 1–2 bridge
phenotype
present
present
absent
WT (CC-125, 137c)
WT waveform, fast swimming
51, 77
19
19
5
14
pWT (pf2-4::PF2-GFPa, ida6::IDA6-GFPb,
ida6::IDA6-SNAPc)
WT waveform, fast swimming
34, 37
11
11
3
8
I1 mutant (pf9-3, CC-3913)
Slow swimming
51, 55
7
7
1
6
N-DRC mutants (sup-pf-3,
CC-1399, pf2)
Abnormal waveform, slow
swimming
36, 37
8
8
2
6
N-DRC mutant also lacking IA4
(ida6, CC-3090)
Abnormal waveform, slow
swimming
78
6
6
2
4
Some of the tomograms from these strains were previously used with different image processing methods to analyze other axonemal complexes [30,37,51]. aStrain provided by Raqual Bower and Mary E. Porter (University of Minnesota). The N-DRC defective pf2 mutant strain [36] was rescued with a GFP-tagged WT PF2 gene. Specimen Preparation Live sea urchins (Strongylocentrotus purpuratus) were purchased
from Monterey Abalone Co. (Monterey, CA). Spawning was
induced by injecting 1 to 2 ml of 0.5 M KCl into the perivisceral
cavity [46], and sperm were collected and kept on ice without
dilution in artificial seawater to prevent sperm activation [47]. Chlamydomonas axonemes were isolated from several C. reinhardtii
strains (Table 1) as previously described [30]. Briefly, cells growing
in liquid Tris-acetate-phosphate medium [48] were harvested,
washed, and resuspended in a minimal medium containing
10 mM
4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid
(HEPES), pH 7.4, 1 mM SrCl2, 4% sucrose, and 1 mM dithio-
threitol. The flagella were detached from the cells using the pH-
shock method [42]. After adding 5 mM MgSO4, 1 mM ethylene
glycol tetraacetic acid (EGTA), 0.1 mM EDTA, and 100 mg/ml
aprotinin, pepstatin, and leupeptin to the solution, the flagella
were separated from the cell bodies by centrifuging for 10 min at
1,800 6 g and 4uC and purified by two additional centrifugation
steps at 2,400 6g for 10 min at 4uC with a 20% sucrose cushion. Purified flagella were demembranated with 0.1% Nonidet P-40
(Sigma-Aldrich, St. Louis, MO) or 1% IGEPAL CA-630 (Sigma-
Aldrich). The axonemes were collected by centrifugation at 35,000
6 g for 1 h at 4uC, and were resuspended in 10 mM HEPES,
pH 7.4,
25 mM NaCl,
4 mM MgSO4,
1 mM EGTA,
and
0.1 mM EDTA. Cryo-ET Single axis tilt-series of images were taken using a Tecnai F30
transmission electron microscope (Tecnai F30; FEI, Hillsboro,
OR) equipped with a post-column energy filter (Gatan, Pleasan-
ton, CA). Cryo samples were transferred to the microscope using
a cryo holder (Gatan) and imaged at 300 keV, at 26 or 28 mm
defocus, under low dose conditions, and in the zero-loss mode of
the energy filter (20 eV slit width). Tilt-series of images were
acquired using the SerialEM image acquisition software [49] and
collected from approximately 265 to +65u in steps of 1.5–2.5u. The cumulative electron dose per tilt series was limited to
,100 e/A˚ 2. All images were recorded on a 2 k62 k charge-
coupled device camera (Gatan) at a nominal magnification of
13,500, resulting in a pixel size of ,1 nm. Introduction Most
notablearetwointer-doubletbridges;thefirstdoublet-specificbridge
discoveredwasthetwo-part5–6bridgethatlinksDMTs5and6(using
the numbering system that is based on the CPC position) in the
axonemes of many animal cilia and flagella, e.g., sea urchin sperm
flagella [7,12] and mollusk paddle and gill cilia [9,10,41] (Figure 1A). However, the 3D structure of the 5–6 bridge remains unknown. A
second doublet-specific bridge was described as a similar two-part
bridge in the proximal quarter of Chlamydomonas flagella, but was
named the 1–2 bridge, because it connects DMTs 1 and 2 (using the
numberingsystemthatisbasedonthedirectionoftheprincipalbend)
(Figure 1A). The evolutionary relationship between these inter-
doublet bridges remains unknown. Another well-known doublet-
specific structure is the beak (B-tubule projection) that is only
observed in the B-tubules of DMTs 1, 5 and 6 in the proximal half of
Chlamydomonas flagella [15,42]. In this study, we used cryo-ET and image processing techniques
that
preserve
doublet-specific
differences
in
subtomogram
averages, to individually analyze each of the nine DMTs from
the flagella of two evolutionarily distant organisms, Chlamydomonas
and Strongylocentrotus. The flagella of both organisms exhibit quasi-
planar bending direction under normal conditions; however,
Chlamydomonas exhibits an asymmetric ‘‘breast stroke’’ often
referred to as a cilia-like waveform, whereas the sea urchin sperm
flagellum generates a sinusoidal waveform. Our results provide the
3D structures of all nine DMTs from both organisms, revealing
that Chlamydomonas DMT1 and Strongylocentrotus DMT5 exhibit
unique structural features that differ from those of the other
DMTs. Although some of these features appear to be non-
homologous, others are conserved, suggesting a unifying number- October 2012 | Volume 7 | Issue 10 | e46494 2 October 2012 | Volume 7 | Issue 10 | e46494 PLOS ONE | www.plosone.org Unique Doublet Microtubule filter paper (Whatman, Piscataway, NJ) for ,2 s, the grid was
immediately plunge-frozen in liquid ethane using a home-made
plunge-freezing device. The vitrified sample was then stored in
liquid nitrogen until examined by EM. ing system for indexing DMTs in eukaryotic cilia and flagella. Our
observations provide new insights into the role of doublet-specific
structures for coordinating the asymmetric distribution of DMT
sliding during bend formation. Conserved Structural Features Observed for DMT1 of the
Chlamydomonas axoneme Although motile cilia and flagella share a highly conserved 9+2
axoneme core structure in general, structural differences have
been reported across different organisms [13,29,45]. To better
understand the common mechanism of 9+2 ciliary and flagellar
motility, our study further examined similarities and diversity in
axonemal structures among the two evolutionarily distant flagella
model organisms Strongylocentrotus and Chlamydomonas. One advan-
tage of studying Chlamydomonas is the large arsenal of available
motility mutants, allowing for a correlation of structural features
with phenotypes and functions. Here, we analyzed doublet-specific
differences in wild-type Chlamydomonas (WT), several mutants, and
several drc-mutant rescue strains with the WT phenotype, named
pseudo wild-type (pWT). Details about the strains used and the
features examined are summarized in Table 1. The pWT strains
have been studied extensively and are biochemically, structurally,
and phenotypically indistinguishable from WT [30,34,36,37,44]. Using the same doublet-specific averaging techniques described
for Strongylocentrotus above, we calculated subtomographic averages
of the 96-nm axonemal repeats of each individual DMT of the
axonemes of all investigated Chlamydomonas strains (Figures 3, 4, 5). After determining the doublet identity based on doublet-specific
features, such as the presence of a beak or missing ODAs,
corresponding DMTs were combined from multiple tomograms. Axonemal averages of the WT and pWT strains provided nearly
identical 3D structures (for comparison, see Figure S3), but greater
detail was revealed by the averages of the pWT strains due to their
higher resolution. g
)
Attached to the A-tubule of DMT5 are structures previously
observed in cross-sectional EM views, which are collectively called
the 5–6 bridge, connecting DMTs 5 and 6 in many flagella [7,9–
12,41], including sea urchin flagella (Figure 2, left column). Our
3D doublet-specific average of DMT5 revealed that the two parts
of this sea urchin bridge between DMTs 5 and 6 (SUB5-6) look
markedly different. In the cross-sectional view (Figure 2A, C), both
parts of SUB5-6 together have a diamond-shaped outline,
consistent with previous studies [7,9–12,41]. The outer part, o-
SUB5-6, substitutes the outer dyneins in the distal 7/8th of DMT5
(Figure 4H), while regular ODAs are present in the proximal 1/8th
(Figure 4G). o-SUB5-6 extends from protofilaments A6, A7, and
A8 (protofilament numbering according to Linck and Stephens
[53]) of the A-tubule of DMT5 and attaches to B6 and B7 of the
B-tubule of DMT6. DMT5 is Distinct from the Other DMTs in the
Strongylocentrotus axoneme gy
DMT sliding is the driving mechanism of ciliary and flagellar
motility. However, to generate bending, this sliding must occur in
a well-orchestrated fashion between selected DMT pairs. To
explore the structural differences that could be involved in defining
which DMTs slide and/or which do not, we performed cryo-ET
and doublet-specific subtomogram averaging of Strongylocentrotus
purpuratus sperm flagella. We separately examined thirteen cryo-
tomographic reconstructions of sea urchin flagella and individually
averaged the 96-nm axonemal repeats from each DMT. We
observed several distinct features that allowed us to identify and
combine
corresponding
DMT
from
different
flagella. The
combined doublet-specific averages of each DMT provide un-
precedented 3D structural detail and reveal the unique structural
features of DMT5 that differ from those of the other DMTs
(Figure 2, compare DMTs 5/6 in the left column to DMTs 6/7
(for example) in the right column; Figure S1; Movie S1). Since the
sea urchin sperm cells were frozen intact with heads, we were able
to determine the location of every tomogram based on its relative
distance to the sperm head. Therefore, we also calculated doublet-
specific averages for specific flagellar regions. We found that the
most proximal 1/8th of DMT5 (5 tomograms) lacks one of the
differences that distinguishes the remaining 7/8th of DMT5 (8
tomograms) from all other doublets. Image Processing The resultant strain, pf2-4::PF2-GFP, is structurally and phenotypically indistinguishable from WT. bStrain provided by Douglas Tritschler and Mary E. Porter (University of Minnesota). The N-DRC defective ida6 mutant strain [78] was rescued with a GFP-tagged WT
IDA6 gene and the resulting ida6::IDA6-GFP strain has the same motility and structure as WT. cStrain provided by Douglas Tritschler and Mary E. Porter (University of Minnesota). The N-DRC defective ida6 mutant strain [78] was rescued with a SNAP-tagged WT
IDA6 gene. The resultant strain, ida6::IDA6-SNAP, is structurally and phenotypically indistinguishable from WT. doi:10.1371/journal.pone.0046494.t001 Some of the tomograms from these strains were previously used with different image processing methods to analyze other axonemal complexes [30,37,51]. aStrain provided by Raqual Bower and Mary E. Porter (University of Minnesota). The N-DRC defective pf2 mutant strain [36] was rescued with a GFP-tagged WT PF2 gene. The resultant strain, pf2-4::PF2-GFP, is structurally and phenotypically indistinguishable from WT. bStrain provided by Douglas Tritschler and Mary E. Porter (University of Minnesota). The N-DRC defective ida6 mutant strain [78] was rescued with a GFP-tagged WT
IDA6 gene and the resulting ida6::IDA6-GFP strain has the same motility and structure as WT. cStrain provided by Douglas Tritschler and Mary E. Porter (University of Minnesota). The N-DRC defective ida6 mutant strain [78] was rescued with a SNAP-tagged WT
IDA6 gene. The resultant strain, ida6::IDA6-SNAP, is structurally and phenotypically indistinguishable from WT. doi:10.1371/journal.pone.0046494.t001 October 2012 | Volume 7 | Issue 10 | e46494 3 3 PLOS ONE | www.plosone.org Unique Doublet Microtubule structures such as the 5–6 bridge in sea urchin [7] or the 1–2
bridge and missing ODAs in Chlamydomonas [15,16]. The DMT
region in which each tomogram was recorded could be de-
termined either directly using the relative location to the sperm
head of the sea urchin sperm flagellum, or indirectly by well-
established
structural
markers
of
particular
regions
of
the
Chlamydomonas flagellum [15]. All axonemal repeats of the same
DMT that share the same structural features were combined and
averaged from all tomograms of the same strain. The IMOD
software package [50] and the UCSF Chimera package [52] were
used for visualization of the tomographic slices and 3D visualiza-
tion by isosurface rendering, respectively (Figures 2, 3, 4, 5;
Figures S1, S2, S3; Movies S1, S2, S3). Image Processing structures display a unique morphology (compare Figure 2A, C, E,
G to Figure 2B, D, F, H; Movie S2), including a large density that
is connected to DMT6 rather than the narrow connections of the
ODAs to the neighboring DMT through their thin coiled-coil
stalks (Figure S2A, B). In addition to linking DMTs 5 and 6, both
i-SUB5-6 and o-SUB5-6 connect to the I1 dynein and the N-
DRC. In each axonemal repeat, one o-SUB5-6 complex per
axonemal repeat connects to the I1 dynein through the proximal
outer-inner-dynein (OID) linker, and the most distal o-SUB5-6
complex links to the N-DRC via the distal OID linker. In contrast,
only one i-SUB5-6 complex is present per repeat, which connects
proximal to the I1 dynein and distal to the N-DRC (Figure 2E, I). There are two more obvious structural differences present along
the entire length of DMT5 in comparison to the other eight
DMTs. Typically, the inner row of dynein arms of eukaryotic
axonemes contains the double-headed I1 complex (dynein f) and
five to six single-headed dyneins (IA2-6 and IAX; dyneins a-e, g)
(Figure 2F, J) [13,16,43,45,51,54]. Here, we show that in
Strongylocentrotus axonemes, DMT5 lacks 4 of these single-headed
dyneins: IA2-4 and IAX (Figures 2E, I, 4G). Among these IA2-4
are only missing from the unique DMT5, while IAX is also
missing from DMTs 1 and 9 (Table 2; Figure S1). The N-DRC on
DMT5 appears to form a more substantial connection to the
neighboring DMT than the N-DRC of the other DMTs (compare
Figure 2I and 2J). Conserved Structural Features Observed for DMT1 of the
Chlamydomonas axoneme The dotted circle in (E) outlines the i-SUB5-6 complex that is missing from other DMTs, as
shown for DMT6 (F). (G–J) Details of the outer and inner SUB5-6 structures. The o-SUB5-6 complexes (green arrowheads) are morphologically distinct
from the ODAs, but share the same 24-nm periodicity (compare G and H); i-SUB5-6 (orange) has a 96-nm periodicity and makes clear connections
with I1 dynein and N-DRC while linking DMT5 to DMT6. Other labels: A-tubule (At), B-tubule (Bt), inner dynein arms (IDA 1a, 1b, 2–6, x; rose), nexin-
dynein regulatory complex (N-DRC, yellow), outer dynein arm (ODA, purple), radial spoke (RS, dark-gray). Cross-sections are viewed from the proximal,
and in longitudinal views proximal is on the left. The sea urchin DMT numbers are according to [7]. The protofilaments are numbered after [53]. Scale
bar (H): 25 nm. doi:10.1371/journal.pone.0046494.g002 Figure 2. Distinct structural features in the distal 7/8th of Strongylocentrotus DMT5 revealed by cryo-ET. Isosurface renderings (A, B, I,
and J) and tomographic slices (C–H) show unique structural features present only on DMT5 (left column) in cross-sectional (A–D) and longitudinal
views from the front (E and F) and the bottom (G–J). The other eight DMTs are similar to each other but distinct from DMT5. For comparison, the
averages of DMT6 are presented in the right column (see also Figure S1 and Movie S1). (A–D) Cross-sectional views of SUB5-6, which is present only
on DMT5. SUB5-6 consists of two parts: the outer o-SUB5-6 (green) and the inner i-SUB5-6 (orange). The red dashed lines in (C and D) indicate the
locations of the tomographic slices shown in (E–H). Note that the center of axonemal repeats shifts longitudinally between neighboring doublets;
therefore the longitudinal positions of the slices shown in (A and C) and (B and D) are not identical. (E and F) Several IDAs (IA2, 3, 4, IAX) are missing
from DMT5, and the ODAs are substituted by o-SUB5-6. The dotted circle in (E) outlines the i-SUB5-6 complex that is missing from other DMTs, as
shown for DMT6 (F). (G–J) Details of the outer and inner SUB5-6 structures. The o-SUB5-6 complexes (green arrowheads) are morphologically distinct
from the ODAs, but share the same 24-nm periodicity (compare G and H); i-SUB5-6 (orange) has a 96-nm periodicity and makes clear connections
with I1 dynein and N-DRC while linking DMT5 to DMT6. Conserved Structural Features Observed for DMT1 of the
Chlamydomonas axoneme In contrast, the inner part, i-SUB5-6, is
present along the entire length of DMT5, extending from
protofilaments A4 and A5 of DMT5 to B8 and B9 of DMT6
(Figure 2A; Movie S1). Another significant difference between the
two SUB5-6 parts is that the i-SUB5-6 structure repeats with
a periodicity of 96 nm along the length of the doublet (Figure 2E,
I), whereas the o-SUB5-6 structure repeats with a periodicity of
24 nm (Figure 2E, G). Although the periodicity and location of o-
SUB5-6 resemble those of ODAs (Figure 2F, H), o-SUB5-6 Similar to the Strongylocentrotus axoneme, the Chlamydomonas
axoneme also contains one DMT that exhibits unique structural
features similar to sea urchin DMT5 but that differentiate it from the
remaining eight DMTs. Following the current DMT numbering
system for Chlamydomonas flagella, this specialized doublet is termed
DMT1 (Figures 3, 4; Figure S1; Movie S3). In contrast to sea urchin
spermthatwerefrozenintact,Chlamydomonasaxonemeswereisolated,
making the determination of the region in which tomograms were
recorded not as easy. Therefore, we utilized previously established October 2012 | Volume 7 | Issue 10 | e46494 October 2012 | Volume 7 | Issue 10 | e46494 PLOS ONE | www.plosone.org 4 Unique Doublet Microtubule structural markers to identify specific regions of the Chlamydomonas
Strongylocentrotus and consistent with previous reports, regional
Figure 2. Distinct structural features in the distal 7/8th of Strongylocentrotus DMT5 revealed by cryo-ET. Isosurface renderings (A, B, I,
and J) and tomographic slices (C–H) show unique structural features present only on DMT5 (left column) in cross-sectional (A–D) and longitudinal
views from the front (E and F) and the bottom (G–J). The other eight DMTs are similar to each other but distinct from DMT5. For comparison, the
averages of DMT6 are presented in the right column (see also Figure S1 and Movie S1). (A–D) Cross-sectional views of SUB5-6, which is present only
on DMT5. SUB5-6 consists of two parts: the outer o-SUB5-6 (green) and the inner i-SUB5-6 (orange). The red dashed lines in (C and D) indicate the
locations of the tomographic slices shown in (E–H). Note that the center of axonemal repeats shifts longitudinally between neighboring doublets;
therefore the longitudinal positions of the slices shown in (A and C) and (B and D) are not identical. (E and F) Several IDAs (IA2, 3, 4, IAX) are missing
from DMT5, and the ODAs are substituted by o-SUB5-6. October 2012 | Volume 7 | Issue 10 | e46494 Conserved Structural Features Observed for DMT1 of the
Chlamydomonas axoneme Other labels: A-tubule (At), B-tubule (Bt), inner dynein arms (IDA 1a, 1b, 2–6, x; rose), nexin-
dynein regulatory complex (N-DRC, yellow), outer dynein arm (ODA, purple), radial spoke (RS, dark-gray). Cross-sections are viewed from the proximal,
and in longitudinal views proximal is on the left. The sea urchin DMT numbers are according to [7]. The protofilaments are numbered after [53]. Scale
bar (H): 25 nm. doi:10.1371/journal.pone.0046494.g002 doi:10.1371/journal.pone.0046494.g002 Strongylocentrotus and consistent with previous reports, regional
doublet-specific average confirmed that Chlamydomonas axonemes
exhibitstructuralheterogeneityalongtheflagellarlength,i.e.,DMT1 structural markers to identify specific regions of the Chlamydomonas
flagellum, such as the 1–2 bridge in the proximal quarter and the B-
tubule beak in the proximal half of the flagellum [15]. Similar to October 2012 | Volume 7 | Issue 10 | e46494 October 2012 | Volume 7 | Issue 10 | e46494 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 5 Unique Doublet Microtubule Figure 3. Distinct structural features in the distal three quarters of Chlamydomonas DMT1. Isosurface renderings (A, B, I, and J) and
tomographic slices (C–H) show i-SUB5-6 and the missing IDAs on Chlamydomonas DMT1 (left column) in cross-sectional (A–D) and longitudinal views
from the front (E and F) and the bottom (G–J). The structure of DMT1 is unique and different from DMTs 2–9, which have a similar structure; DMT2 is
shown for comparison in the right column (see also Figure S1 and Movie S3). (A–D) Cross-sectional views of the i-SUB5-6 complex (orange), which is
present on DMT1 only and links DMT1 to DMT2. The ODAs are missing from DMT1 but are present on all other DMTs. The red dashed lines in (C and
D) indicate the locations of the tomographic slices shown in (E–H). Note that the beak structure in the B-tubule of DMT1 in (A and C) has previously
been reported to be present in the proximal half of the flagellum [15], and is therefore still visible in this average of the distal three quarters of the
axoneme. (E and F) Several IDAs (IA3, 4, and IAX) are absent on DMT1 (E) but present on DMT2 (F). The orange dotted circle in (E) outlines the i-SUB5-
6 complex that is present on DMT1, but absent from DMT2 (F). (G–J) Details of the i-SUB5-6 structure (orange) demonstrate that it has a periodicity of
96 nm and makes clear connections with both I1 dynein and N-DRC while linking DMT1 to DMT2. Conserved Structural Features Observed for DMT1 of the
Chlamydomonas axoneme Other labels: A-tubule (At), B-tubule (Bt), inner
dynein arms (IDA 1a, 1b, 2–6 and x; rose), nexin-dynein regulatory complex (N-DRC, yellow), outer dynein arm (ODA, purple), radial spoke (RS, dark-
gray). The Chlamydomonas DMT numbers are according to Hoops and Witman [15]. The protofilaments are numbered after Linck and Stephens [53]. Scale bar (H): 25 nm. doi:10.1371/journal.pone.0046494.g003 Figure 3. Distinct structural features in the distal three quarters of Chlamydomonas DMT1. Isosurface renderings (A, B, I, and J) and
tomographic slices (C–H) show i-SUB5-6 and the missing IDAs on Chlamydomonas DMT1 (left column) in cross-sectional (A–D) and longitudinal views
from the front (E and F) and the bottom (G–J). The structure of DMT1 is unique and different from DMTs 2–9, which have a similar structure; DMT2 is
shown for comparison in the right column (see also Figure S1 and Movie S3). (A–D) Cross-sectional views of the i-SUB5-6 complex (orange), which is
present on DMT1 only and links DMT1 to DMT2. The ODAs are missing from DMT1 but are present on all other DMTs. The red dashed lines in (C and
D) indicate the locations of the tomographic slices shown in (E–H). Note that the beak structure in the B-tubule of DMT1 in (A and C) has previously
been reported to be present in the proximal half of the flagellum [15], and is therefore still visible in this average of the distal three quarters of the
axoneme. (E and F) Several IDAs (IA3, 4, and IAX) are absent on DMT1 (E) but present on DMT2 (F). The orange dotted circle in (E) outlines the i-SUB5-
6 complex that is present on DMT1, but absent from DMT2 (F). (G–J) Details of the i-SUB5-6 structure (orange) demonstrate that it has a periodicity of
96 nm and makes clear connections with both I1 dynein and N-DRC while linking DMT1 to DMT2. Other labels: A-tubule (At), B-tubule (Bt), inner
dynein arms (IDA 1a, 1b, 2–6 and x; rose), nexin-dynein regulatory complex (N-DRC, yellow), outer dynein arm (ODA, purple), radial spoke (RS, dark-
gray). The Chlamydomonas DMT numbers are according to Hoops and Witman [15]. The protofilaments are numbered after Linck and Stephens [53]. Scale bar (H): 25 nm. doi 10 1371/journal pone 0046494 g003 doi:10.1371/journal.pone.0046494.g003 doi:10.1371/journal.pone.0046494.g003 appear to be conserved between Chlamydomonas DMT1 and
Strongylocentrotus DMT5. appear to be conserved between Chlamydomonas DMT1 and
Strongylocentrotus DMT5. Conserved Structural Features Observed for DMT1 of the
Chlamydomonas axoneme contains structural features only observed in the proximal region of
theaxoneme(Figures3,4).Somebutnotalloftheseuniquestructures October 2012 | Volume 7 | Issue 10 | e46494 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 6 Unique Doublet Microtubule The i-SUB5-6 structure observed for Strongylocentrotus DMT5 is
i-SUB5-6 is similar to that of Strongylocentrotus (compare Figures 2,
gure 4. Distinct structural features in specific regions of Chlamydomonas DMT1 and Strongylocentrotus DMT5. (A–E) The 1–2 bridge in
e proximal quarter of Chlamydomonas DMT1. Tomographic slices show the proximal 1–2 bridge between DMTs 1 and 2 in Chlamydomonas pWT in
oss-sectional (A and B) and longitudinal views from the front (C and E) and the bottom (D). Red dashed lines in (A) indicate the locations of
mographic slices shown in (C–E). The 1–2 bridge consists of two parts that link DMTs 1 and 2; each part consists of a row of periodic densities (blue
cles in A, and C–E). The connection of the top row to DMT2 is highlighted (yellow arrowheads in A and D). The red circles in (A and C) indicate an
ditional row of periodic but shorter densities that do not reach DMT2. Note that the I1 dynein is absent from the proximal quarter of DMT1 and
ly a small neighboring density, the I1 tether [34], is visible (C and E). (F) A tomographic slice from the distal part of DMT1 is shown for direct
mparison with the proximal slice in (E). Note that the distal region of Chlamydomonas DMT1 lacks the 1–2 bridge, while the I1 dynein is present. (G
d H) Tomographic slices in front view show a comparison between the proximal 1/8th (G) and distal 7/8th (H) of the Strongylocentrotus DMT5. While
the very proximal region regular ODAs are present, they are substituted by o-SUB5-6 structures in the distal 7/8th of the flagellum. Note that along
e entire length of DMT5 i-SUB5-6 is consistently present, while several IDAs (IA 2–4, IAX) are lacking. Scale bars are 25 nm (scale bar in B valid for A
d B; scale bar in F valid for C–H). i:10.1371/journal.pone.0046494.g004 Figure 4. Distinct structural features in specific regions of Chlamydomonas DMT1 and Strongylocentrotus DMT5. (A–E) The 1–2 bridge in
the proximal quarter of Chlamydomonas DMT1. October 2012 | Volume 7 | Issue 10 | e46494 Conserved Structural Features Observed for DMT1 of the
Chlamydomonas axoneme Tomographic slices show the proximal 1–2 bridge between DMTs 1 and 2 in Chlamydomonas pWT in
cross-sectional (A and B) and longitudinal views from the front (C and E) and the bottom (D). Red dashed lines in (A) indicate the locations of
tomographic slices shown in (C–E). The 1–2 bridge consists of two parts that link DMTs 1 and 2; each part consists of a row of periodic densities (blue
circles in A, and C–E). The connection of the top row to DMT2 is highlighted (yellow arrowheads in A and D). The red circles in (A and C) indicate an
additional row of periodic but shorter densities that do not reach DMT2. Note that the I1 dynein is absent from the proximal quarter of DMT1 and
only a small neighboring density, the I1 tether [34], is visible (C and E). (F) A tomographic slice from the distal part of DMT1 is shown for direct
comparison with the proximal slice in (E). Note that the distal region of Chlamydomonas DMT1 lacks the 1–2 bridge, while the I1 dynein is present. (G
and H) Tomographic slices in front view show a comparison between the proximal 1/8th (G) and distal 7/8th (H) of the Strongylocentrotus DMT5. While
in the very proximal region regular ODAs are present, they are substituted by o-SUB5-6 structures in the distal 7/8th of the flagellum. Note that along
the entire length of DMT5 i-SUB5-6 is consistently present, while several IDAs (IA 2–4, IAX) are lacking. Scale bars are 25 nm (scale bar in B valid for A
and B; scale bar in F valid for C–H). doi:10.1371/journal.pone.0046494.g004 i-SUB5-6 is similar to that of Strongylocentrotus (compare Figures 2,
3), including the connection between DMTs 1 and 2 (Figure 3A,
C, G, I), as well as to the I1 dynein and N-DRC through its
proximal and distal regions (Figure 3I; Movie S3). Another The i-SUB5-6 structure observed for Strongylocentrotus DMT5 is
also present along the entire length of the Chlamydomonas DMT1
(Figures 3, 4; Table 1). The location of this structure between the
I1 dynein and N-DRC, and the overall structure of Chlamydomonas PLOS ONE | www.plosone.org October 2012 | Volume 7 | Issue 10 | e46494 7 Unique Doublet Microtubule Figure 5. i-SUB5-6 is present in Chlamydomonas I1 mutants and N-DRC mutants. Conserved Structural Features Observed for DMT1 of the
Chlamydomonas axoneme (A–H) A comparison of tomographic slices from averages
of the distal part of DMT1 from Chlamydomonas pWT (A and B), I1-dynein-lacking mutant pf9-3 (C and D), a combined average of the N-DRC mutants
sup-pf-3 and pf2 (E and F), and the dynein-IA4-lacking mutant ida6 (G–I) indicates that the lack of I1 dynein or IA4 (dynein e) or a dramatic reduction in
the N-DRC has little effect on the i-SUB5-6 structure (orange arrowheads). The tomographic slices were viewed from the front (left column) and the
bottom (right column) and show the same locations of the axonemal repeat as shown in Figure 3E and 3G, respectively. The orientation of the
tomographic slice shown in (B) is indicated by red dashed lines in (A). (I and J) Tomographic slices from combined averages of DMT2-9 show the
absence of IA4 in the ida6 axoneme (I) compared to pWT (J). Scale bar (B): 10 nm. doi:10.1371/journal.pone.0046494.g005 Figure 5. i-SUB5-6 is present in Chlamydomonas I1 mutants and N-DRC mutants. (A–H) A comparison of tomographic slices from averages
of the distal part of DMT1 from Chlamydomonas pWT (A and B), I1-dynein-lacking mutant pf9-3 (C and D), a combined average of the N-DRC mutants
sup-pf-3 and pf2 (E and F), and the dynein-IA4-lacking mutant ida6 (G–I) indicates that the lack of I1 dynein or IA4 (dynein e) or a dramatic reduction in
the N-DRC has little effect on the i-SUB5-6 structure (orange arrowheads). The tomographic slices were viewed from the front (left column) and the
bottom (right column) and show the same locations of the axonemal repeat as shown in Figure 3E and 3G, respectively. The orientation of the
tomographic slice shown in (B) is indicated by red dashed lines in (A). (I and J) Tomographic slices from combined averages of DMT2-9 show the
absence of IA4 in the ida6 axoneme (I) compared to pWT (J). Scale bar (B): 10 nm. doi:10.1371/journal.pone.0046494.g005 Another difference from sea urchin is that in the proximal region,
where the proximal 1–2 bridge is present, another IDA is lacking:
the double-headed I1 dynein complex (Figure 4E). similarity to sea urchin flagella is the absence of ODAs and several
single-headed
IDAs
from
the
entire
length
of
DMT1
in
Chlamydomonas axonemes. However, rather than missing four
IDAs, Chlamydomonas DMT1 lacks only three: IA3, 4, and IAX;
IA2 remains present (Figures 3E, I, 4E). Conserved Structural Features Observed for DMT1 of the
Chlamydomonas axoneme As previously reported
[16,43] and similar to sea urchin flagella, IAX is also missing from
Chlamydomonas DMT9 (Table 2; Figure S1); in addition, we found
that IAX is greatly reduced from DMT5. A significant difference
from Strongylocentrotus DMT5 is that Chlamydomonas DMT1 does not
contain o-SUB5-6 structures in place of regular ODAs. The ODA
region of Chlamydomonas DMT1 is either completely vacant in most
examined tomograms (Figure 3A, C, E; Movie S3) or, in the
proximal region of the axoneme [15], the ODA site is occupied by
the structures of the proximal 1–2 bridge, which shares no
similarities with o-SUB5-6 (Figure 4, see below for details). The Proximal 1–2 Bridge on DMT1 of the
Chlamydomonas axoneme [16] reported that in Chlamydomonas IAX (dynein b/g) is clearly missing from DMT9, and maybe missing from DMT1– consistent with the data presented here;
however, the authors did not report that IAX is greatly reduced at DMT5, as shown here. bHeuser et al. [34]: in Chlamydomonas axonemes the intermediate chain and light chain complex (ICLC) of the I1 dynein contains a doublet-specific protrusion only
found on DMTs 3 and 4, but not on the other seven DMT. doi:10.1371/journal.pone.0046494.t002 however, several Chlamydomonas mutants have been shown to
display specific structural defects in I1 dynein or N-DRC; for
example, in the pf9-3 axoneme, the entire I1 dynein is missing
[34,51,55]. Similarly, in many drc-mutant axonemes, the majority
of the N-DRC density is missing, particularly that for the nexin
linker and the portion that connects to i-SUB5-6 [35,37]. In
addition, some of these drc-mutants also lack dynein IA4 (dynein e),
which has been hypothesized to be part of the density here
identified as i-SUB5-6 [16]. included the proximal 1–2 bridge into a proximal axonemal
average (Figure 4A, B, C, D, E) and combined the remaining eight
tomograms into a distal axonemal average that shows no
structures in the ODA region (Figures 3A, C, E, 4F). The
proximal axonemal average of Chlamydomonas DMT1 provides
a detailed 3D structure of the proximal 1–2 bridge (Figure 4A, B,
C, D, E). Consistent with previous studies [15,16], we found that the
proximal 1–2 bridge is composed of two straight linker parts
(Figure 4A, B, blue circles), shown in cross-sectional views, which
make substantial connections to the B-tubule of DMT2 (Figure 4A,
D, yellow arrowheads). Both the outer and inner portions of the
proximal 1–2 bridge exhibit an 8-nm periodicity of rungs along the
flagellar axis, but the inner portion has a 32-nm-long discontinuity
in the region of the N-DRC (Figure 4C, E). This high
repetitiveness makes the 1–2 bridge clearly visible in 10-nm- and
100-nm-thick cross-sectional slices through the tomographic
average (Figure 4A, B). i-SUB5-6 is also present in the proximal
quarter of Chlamydomonas DMT1; however, due to its lower
periodicity (96 nm) compared to the proximal 1–2 bridge, i-SUB5-
6 is difficult to distinguish in the 100-nm tomographic slice
(Figure 4B). This explains why only the two-part 1–2 bridge has
been reported for Chlamydomonas axonemes. Discussion Most motile cilia and flagella have a constant bending plane and
generate quasi-planar waveforms, which result in faster swimming
than helical waveforms [56]. Variations from a quasi-planar to
a helical waveform sometimes occur under certain conditions, e.g.,
in response to light [57] or increased viscosity of the surrounding
medium [58]. The DMT pairs DMT5-6 in Strongylocentrotus and
DMT1-2 in Chlamydomonas are located at a plane
almost
perpendicular to the bending plane (Figure 1) and are known to
show little or no inter-doublet sliding [12,14,22,59,60]. In
Chlamydomonas, DMT1 is a continuation of the basal body triplet
1, which is part of the three triplets contacted by the distal striated
fibers that are important for controlling the rotational orientation The Proximal 1–2 Bridge on DMT1 of the
Chlamydomonas axoneme Although Strongylocentrotus o-SUB5-6 is not present in Chlamydo-
monas DMT1, we observed another inter-doublet link, the
proximal 1–2 bridge, in approximately one quarter of our
Chlamydomonas tomograms (Table 1). This is consistent with
previous findings of classical EM and cryo-ET studies regarding
the presence of a 1–2 bridge in the proximal quarter of the
Chlamydomonas flagellum [15,16]. We separately calculated dou-
blet-specific averages of DMT1 for eleven tomograms of pWT
strains; then, we combined the three DMT1 averages that October 2012 | Volume 7 | Issue 10 | e46494 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 8 Unique Doublet Microtubule Table 2. Correlation of DMT numbering systems and doublet-specific features. Reference
Doublet number
DMT numbering system
Unified numbering
this study
D-I
D-II
D-III
D-IV
D-V
D-VI
D-VII
D-VIII
D-IX
Sea urchin (S.p.)
7
1
2
3
4
5
6
7
8
9
Chlamydomonas (C.r.)
15
6
7
8
9
1
2
3
4
5
Doublet-specific features
C.r. & S.p. i-SUB5-6,
S.p.o-SUB5-6, C.r.1–2
bridge
this study; 7, 15
2
2
2
2
2
2
2
2
2
S.p. dynein IA2,3,4
this study
+
+
+
+
2
+
+
+
+
C.r. dynein IA3,4
this study; 16
+
+
+
+
2
+
+
+
+
S.p. dynein IAX
this study;
2
+
+
+
2
+
+
+
2
C.r. dynein IAX
this study; 16a
+
+
+
2
2
+
+
+
2
C.r. beak
42
+
2
2
2
+
2
2
2
+
C.r. I1 dynein
modification
34b
2
2
2
2
2
2
+
+
2
S.p. RS joist
13
2
2
+
+
2
2
+
+
+
S.p. RS spur
13
2
+
2
2
2
2
2
2
2
aBui et al. [16] reported that in Chlamydomonas IAX (dynein b/g) is clearly missing from DMT9, and maybe missing from DMT1– consistent with the data presented here;
however, the authors did not report that IAX is greatly reduced at DMT5, as shown here. bHeuser et al. [34]: in Chlamydomonas axonemes the intermediate chain and light chain complex (ICLC) of the I1 dynein contains a doublet-specific protrusion only
found on DMTs 3 and 4, but not on the other seven DMT. doi:10.1371/journal.pone.0046494.t002 aBui et al. The Proximal 1–2 Bridge on DMT1 of the
Chlamydomonas axoneme Above the two linker
parts of the proximal 1–2 bridge, we found a third row of densities
with the same 8-nm periodicity (red circles in Figure 4A, C). However, these rungs extend for only approximately 10 nm
towards the neighboring DMT and are thus much shorter than the
densities in the two rows linking DMTs 1 and 2 (Figure 4A, B). To determine whether the assembly of i-SUB5-6 is dependent
on N-DRC, I1 dynein, or dynein IA4, we compared the axonemal
averages of the available I1 and N-DRC mutants in regards to
possible affects on the i-SUB5-6 structure (Figure 5). Our results
indicate that although I1 dynein is completely missing in the pf9-3
axoneme, i-SUB5-6 is still present throughout the entire length of
DMT1 and exhibits no obvious structural defects (Figure 5C, D). Similarly, in N-DRC mutants, the lack of the N-DRC regions that
would usually directly link to i-SUB5-6 does not disrupt the
assembly of i-SUB5-6 on DMT1 (Figure 5E, F). In addition, drc-
mutant axonemes that are known to lack dynein IA4 [36,37,44]
show that an absence of IA4 has little effect on the assembly of i-
SUB5-6 (Figure 5G, H). The Unique DMT in axonemes Exhibits a Significantly
Reduced Number of Dynein Arms y
Axonemal dyneins are arranged in two rows on the DMTs, the
ODAs and IDAs, and ATP hydrolysis-driven dynein motility
powers sliding between adjacent DMTs [17,18]. ODAs repeat
every 24 nm along the length of the A-tubule of DMTs and
contribute more than two-thirds of the sliding force [67]. The
IDAs are more diverse, with seven distinct isoforms (structurally
known as dyneins IA1-6 and IAX; biochemically known as
dyneins a-g), and they are arranged along the A-tubule of DMTs
with a 96-nm periodicity [43,51,54,68]. Our cryo-ET analysis
revealed clear structural differences on the unique DMT in the
region in which axonemal dyneins are found on all other DMTs. These differences were conserved between Strongylocentrotus and
Chlamydomonas. First, our doublet-specific averages revealed that
the ODAs were replaced by o-SUB5-6 over almost the entire
length of the Strongylocentrotus DMT5 and were either simply
missing from Chlamydomonas DMT1 or replaced by the proximal 1–
2 bridge. Despite exhibiting the same 24-nm periodicities, the
observed 3D structure of sea urchin o-SUB5-6 shows no
indications that they are modified ODAs (Figure 2; Movie S2). This feature is consistent with previous studies demonstrating that
the o-SUB5-6 structures can not be decorated with antibodies
against ODAs [69,70]. Previous studies have also observed that
ODAs are almost completely missing from Chlamydomonas DMT1
[15,16,71]. Second, several IDAs were also missing from the
specialized DMT: dynein IA2-4 and IAX for Strongylocentrotus, and
IA3, 4 and IAX for Chlamydomonas. In addition, the I1 dynein is
also absent from the proximal quarter of Chlamydomonas DMT1,
where the proximal 1–2 bridge is present. Thus far, none of the
proteins forming i-SUB5-6, o-SUB5-6 or the proximal 1–2 bridge
have been identified, and no evidence suggests that these inter-
doublet linkers are constituted by the axonemal dyneins present in
these regions on all other DMTs. Intriguingly, we found that the inner part of the 5–6 bridge, i-
SUB5-6, is present along the entire length of a specialized DMT in
both Strongylocentrotus and Chlamydomonas flagella (Figures 2, 3, 4, 6),
suggesting that i-SUB5-6 is a highly conserved inter-doublet link. The i-SUB5-6 has the same periodicity as N-DRC, and in cross-
sectional views, its location is also similar to the position of the N-
DRC linker, making it difficult to distinguish i-SUB5-6 from N-
DRC using classical EM. Comparison with Mutants Shows that Defects in the N-
DRC and I1 Dynein Structures have Little Effect on the i-
SUB5-6 Structure As mentioned above, i-SUB5-6 and its connections to two
known regulators of dynein activity, I1 dynein and N-DRC, are
consistently observed on Chlamydomonas DMT1 and Strongylocen-
trotus DMT5. No i-SUB5-6 mutant has been identified thus far; PLOS ONE | www.plosone.org October 2012 | Volume 7 | Issue 10 | e46494 9 Unique Doublet Microtubule of the flagella [15,61,62]. Using cryo-ET and doublet-specific
subtomogram averaging, we investigated the 3D structures of the
nine DMTs from Strongylocentrotus and Chlamydomonas axonemes,
revealing that Strongylocentrotus DMT5 and Chlamydomonas DMT1
display unique structural features, some of which are conserved
between the two organisms. Many of these features are consistent
with the provision of robust connections between adjacent DMTs
and the prevention of inter-doublet sliding. These features suggest
that the functional roles of specific doublets during flagellar
motility are structurally pre-determined at the level of the DMTs
and associated complexes rather than completely relying only on
regulatory signals provided by the CPC/radial spoke system. doublets, i-SUB5-6 is only present on a single doublet, and o-
SUB5-6 and the 1–2 bridge are even more restricted to sub-
regions of this unique doublet. In addition, o-SUB5-6 and the 1–2
bridge form connections with higher periodicities and larger
interfaces with the neighboring DMTs than N-DRC. Therefore, it
is likely that the two SUB5-6 bridges and the 1–2 bridge contribute
significant constraints to inter-doublet sliding in an asymmetric
fashion. As a result, these connections might dramatically increase
the stiffness of the connected DMT pair. Quasi-planar motility in
9+2 cilia and flagella is facilitated by restricting any off-axis
bending, i.e., any bending not along the bending plane [20]. Increasing the stiffness of a DMT pair could efficiently resist lateral
shear forces generated by the other DMT pairs and therefore
define a bending plane perpendicular to the linked DMT pair. The different distributions of the sea urchin o-SUB5-6 and the
Chlamydomonas 1–2 bridge might partly account for the asymmet-
ric, cilia-like waveform of Chlamydomonas flagella (‘‘breast stroke’’),
in contrast to the sinusoidal beating pattern of sea urchin flagella. Additional Inter-doublet Links Present on one Unique
DMT Suggest a Robust Connection Intended to Resist
Inter-doublet Sliding g
N-DRC is a well-known inter-doublet link connecting each of
the 9 DMTs of an axoneme to its neighboring DMT. It is thought
to function in the regulation of dynein activity and the conversion
of inter-doublet sliding into axonemal bending [35,37,63,64]. In
addition to N-DRC, additional links, such as the 5–6 bridge in the
axonemes of echinoderms and mollusks or the 1–2 bridge in
Chlamydomonas, have previously been observed by classical EM
[7,10,12,15,41]. A recent cryo-ET study reported the first 3D
structural details of the Chlamydomonas proximal 1–2 bridge [16]. However, 3D details of the 5–6 bridge in echinoderms and
mollusks have not been available; therefore, the relationship
between these two bridge structures has remained unclear. Here,
we observed that the sea urchin 5–6 bridge (SUB5-6) and the
Chlamydomonas 1–2 bridge exhibit markedly different morphologies
and periodicities from each other, and therefore, they most likely
form two distinct and non-homologous inter-doublet links. The Unique DMT in axonemes Exhibits a Significantly
Reduced Number of Dynein Arms Schematic models of the axonemes and doublets D-V from Strongylocentrotus and
diagrams of the axonemes viewed in cross-section from proximal correlate the DMTs from Strongylocen
numbering convention for the DMTs in eukaryotic 9+2 axonemes is used, based on the observed conserv
facilitates comparisons of DMTs among species and assigns the same doublet number to corresponding
systems (Arabic numerals in B-tubules) are also depicted for comparison. Under the new numbering system
both species described in this study, are present only on doublet D-V (some of these features connect to D
flagellar beating plane that runs through doublet D-I and between D-V/D-VI that are connected by i-SUB5-
longitudinal front views that summarize the conserved structural features. The i-SUB5-6 complex (orange) is Figure 6. Schematic models of the axonemes and doublets D-V from Strongylocentrotus and Chlamydomonas. (A and C) Simplified
diagrams of the axonemes viewed in cross-section from proximal correlate the DMTs from Strongylocentrotus and Chlamydomonas. A unifying
numbering convention for the DMTs in eukaryotic 9+2 axonemes is used, based on the observed conserved structural features. This convention
facilitates comparisons of DMTs among species and assigns the same doublet number to corresponding DMTs. The previously used numbering
systems (Arabic numerals in B-tubules) are also depicted for comparison. Under the new numbering system, the conserved inter-doublet linkers of
both species described in this study, are present only on doublet D-V (some of these features connect to D-VI). Double-headed arrows indicate the
flagellar beating plane that runs through doublet D-I and between D-V/D-VI that are connected by i-SUB5-6. (B and D) The diagrams of D-V show
longitudinal front views that summarize the conserved structural features. The i-SUB5-6 complex (orange) is present in every 96-nm repeat along the
unique DMT D-V and links to the neighboring DMT D-VI. ODAs are absent from the distal 7/8th of Strongylocentrotus DMT D-V, and almost from the
entire length of Chlamydomonas DMT D-V. However, additional inter-doublet links such as the Strongylocentrotus o-SUB5-6 complexes (green) or the
Chlamydomonas proximal 1–2 bridge (blue) are present on DMT D-V, while several IDAs (IA2, 3, 4, and IAX in Strongylocentrotus; IA3, 4, and IAX in
Chlamydomonas) are missing from this DMT. doi:10.1371/journal.pone.0046494.g006 Figure 6. Schematic models of the axonemes and doublets D-V from Strongylocentrotus and Chlamydomonas. (A and C) Simplified
diagrams of the axonemes viewed in cross-section from proximal correlate the DMTs from Strongylocentrotus and Chlamydomonas. The Unique DMT in axonemes Exhibits a Significantly
Reduced Number of Dynein Arms This likely explains why the i-SUB5-6
structure has not been described by classical EM studies of
Chlamydomonas flagella, and the same could be true for many cilia
and flagella studies in general. A recent cryo-ET study has also
observed the i-SUB5-6 structure (termed IDL3) in Chlamydomonas
DMT1 [16]. We expect that future cryo-ET studies of 9+2 cilia
and flagella of other organisms will also reveal i-SUB5-6 as
a conserved feature of one specialized DMT. Previous classical EM studies did not report any changes of the
5–6 bridge along the length of mussel gill cilia [11]. Our data,
however, revealed that the outer part of the 5–6 bridge, o-SUB5-6,
is not present in the very proximal region of the flagellum. This is
in contrast to the Chlamydomonas flagellum, where the proximal 1–2
bridge is limited to only the proximal quarter of the flagellum
(Figure 6). Although our data revealed that sea urchin o-SUB5-6
and the Chlamydomonas proximal 1–2 bridge are structurally very
different and most likely non-homologous, the function of these
two massive inter-doublet connections between two adjacent
DMTs could still be analogous. The nexin link of N-DRC is
thought to either form permanent and elastic connections to the
neighboring doublet or connections that translocate when inter-
doublet sliding occurs [64–66]. All three bridging structures
described here, i-SUB5-6, o-SUB5-6 and the 1–2 bridge, could
similarly be permanent or transient inter-doublet connections. However, in contrast to the N-DRC, which is present on all This significantly reduced number of dynein arms along the
entire length of the specialized DMT of Chlamydomonas and sea
urchin flagella should result in a dramatic reduction of the sliding
force that could be produced between the unique DMT and its
neighboring DMT. Therefore, the loss of dynein arms from the
same particular DMT is consistent with the interpretation that the
addition of inter-doublet links to the unique DMT prevents or
limits sliding of Strongylocentrotus DMTs 5 and 6 or Chlamydomonas
DMTs 1 and 2 against each other. This interpretation is also
consistent with previous reports that DMT5 is permanently
connected to DMT6 in mussel gill cilia [9,11,17]. Considering that October 2012 | Volume 7 | Issue 10 | e46494 PLOS ONE | www.plosone.org 10 Unique Doublet Microtubule Figure 6. The Unique DMT in axonemes Exhibits a Significantly
Reduced Number of Dynein Arms A unifying
numbering convention for the DMTs in eukaryotic 9+2 axonemes is used, based on the observed conserved structural features. This convention
facilitates comparisons of DMTs among species and assigns the same doublet number to corresponding DMTs. The previously used numbering
systems (Arabic numerals in B-tubules) are also depicted for comparison. Under the new numbering system, the conserved inter-doublet linkers of
both species described in this study, are present only on doublet D-V (some of these features connect to D-VI). Double-headed arrows indicate the
flagellar beating plane that runs through doublet D-I and between D-V/D-VI that are connected by i-SUB5-6. (B and D) The diagrams of D-V show
longitudinal front views that summarize the conserved structural features. The i-SUB5-6 complex (orange) is present in every 96-nm repeat along the
unique DMT D-V and links to the neighboring DMT D-VI. ODAs are absent from the distal 7/8th of Strongylocentrotus DMT D-V, and almost from the
entire length of Chlamydomonas DMT D-V. However, additional inter-doublet links such as the Strongylocentrotus o-SUB5-6 complexes (green) or the
Chlamydomonas proximal 1–2 bridge (blue) are present on DMT D-V, while several IDAs (IA2, 3, 4, and IAX in Strongylocentrotus; IA3, 4, and IAX in
Chlamydomonas) are missing from this DMT. doi:10.1371/journal.pone.0046494.g006 11 October 2012 | Volume 7 | Issue 10 | e46494 PLOS ONE | www.plosone.org Unique Doublet Microtubule spoke would be absent from Strongylocentrotus DMTs D-I, D-II, D-
V, and D-VI [13] and the beaks would be located in the B-tubules
of Chlamydomonas DMTs D-I, D-V, and D-IX [15,42]. Table 2
correlates the different DMT numbering systems and summarizes
known and newly described doublet-specific features. these DMTs are located in a plane almost perpendicular to the
bending plane, the limitation or prevention of sliding between
these DMTs is expected to be critical for ciliary and flagellar quasi-
planar
waveforms. Thus,
the
unique
features
observed
in
Strongylocentrotus DMT5 and Chlamydomonas DMT1 are likely to
be important for ciliary and flagellar quasi-planar motility. Previous studies also reported that in mammalian sperm, DMTs
5 and 6 do not exhibit inter-doublet sliding [72,73]. These
observations suggest that inter-doublet linkers similar to i-SUB5-6,
o-SUB5-6 and/or the proximal 1–2 bridge are also present on
DMT5 of motile mammalian cilia and flagella, indicating that the
permanent linkage between these DMTs is a common feature of
9+2 cilia and flagella. Supporting Information Figure S1
Comparison among all nine individual DMTs
in the distal region of the Strongylocentrotus flagellum
and
Chlamydomonas
axoneme. After
determining
the
doublet identities using doublet-specific axonemal markers (e.g.,
the 5–6 bridge in Strongylocentrotus or the B-tubule projection in
Chlamydomonas), doublet-specific averages for DMTs 1–9 were
generated by combining the axonemal repeats for each individual
DMT from all tomograms of a particular strain. The structures of
eight of the doublets, namely DMTs 1–4 and 6–9 in Strongylocen-
trotus and DMTs 2–9 in Chlamydomonas, look very similar at this
resolution, whereas DMT5 in Strongylocentrotus and DMT1 in
Chlamydomonas have unique structural features. The sea urchin
DMT numbering is according to Afzelius [7] whereas the
Chlamydomonas DMT numbering follows Hoops and Witman
[15]. The tomographic slices are viewed from the front and show
the same locations of the axonemal repeat as displayed in
Figures 2E (for Strongylocentrotus) and 3E (for Chlamydomonas). Dynein IAX (dynein b/g) is absent from three DMTs in
Strongylocentrotus and Chlamydomonas, namely DMTs 1, 5, and 9
(blue arrowheads); however, note that these DMTs are not
homologous between the two species (see summary in Table 2). Scale bar (A): 25 nm. g
,
Considering that much of our knowledge about 9+2 cilia and
flagella stems from studies of sea urchin sperm and Chlamydomonas
flagella, it is important to correlate the axonemal organization,
including the identity of the DMTs, between the two organisms. Historically, the second Chlamydomonas numbering system was
based on the assumption that the directions of the principal/
reversed bends are consistently related to corresponding DMTs. However, this assumption was challenged by a later study showing
that sea urchin DMT1 is on the inside edge of the principal bend
[12] and not of the reverse bend as in mussel gill cilia. Therefore,
here, we unified the DMT numbering for 9+2 axonemes based on
the conserved structural features (Figure 6), consistent with the
original convention for most 9+2 axonemes, including sea urchin
and mammalian flagella. Figure S2
The ODAs connect to the adjacent DMT
through narrow dynein stalks. (A–D) Tomographic slices
show the narrow connections of the small dynein coiled-coil stalks
of ODAs in the sea urchin Strongylocentrotus (A and B) and the green
alga Chlamydomonas (C and D) in cross-sectional (A and C) and
longitudinal views from the bottom (B and D) (looking from the
central pair towards the DMT). The Unique DMT in axonemes Exhibits a Significantly
Reduced Number of Dynein Arms In conclusion, we have visualized the 3D structures of individual
DMTs in the flagella of two evolutionarily distant organisms,
Chlamydomonas and Strongylocentrotus. In both organisms, one of the
nine DMTs exhibits unique structural features, which provide
a structural basis for restricting the inter-doublet sliding between
this unique DMT and the adjacent DMT, and can therefore
define the bending plane in quasi-planar waveforms typical of 9+2
cilia and flagella. Revealing structural and functional differences
among the nine doublets is an important step towards the long-
term goal of understanding the inner workings of ciliary and
flagellar motility. Conserved Structures Suggest a Unifying Numbering
Convention for DMTs in Eukaryotic axonemes y
At present, two opposing numbering systems are used for
designating the DMTs of 9+2 cilia and flagella. Historically, the
numbering of DMTs was based on the relative position of each
DMT with respect to the plane of the CPC [7,8]) and/or the
specific morphologies of mammalian outer dense fibers [74]. This
convention was adopted for the cilia and flagella of many animals
with a fixed (i.e., non-rotating) CPC [7,9–13,74], including sea
urchin and mammalian spermatozoa. In this convention, the
bending direction is perpendicular to the plane of the CPC, and
runs through DMT1 and between the DMT pair DMT5-6
(Figure 1). In Chlamydomonas flagella, the CPC is not fixed at the
flagellar base and rotates during flagellar beating [25,75]. Therefore, another numbering system was proposed for Chlamy-
domonas based on the direction of the principal and reverse bends
[15]. Because the DMT1 of mussel gill cilia (which have a typical
5–6 bridge) is located on the inside edge of the reverse bend,
Chlamydomonas DMTs were indexed accordingly, such that DMT1
is also on the inside edge of the reverse bend [11,15,76]. Under
these two DMT numbering systems, the unique DMTs in
Strongylocentrotus and Chlamydomonas are indexed as DMT5 and
DMT1, respectively. However, the conserved structural features
identified in the present study strongly suggest that Strongylocentrotus
DMT5 and Chlamydomonas DMT1 are corresponding, if not
homologous, DMTs. References Examination of the distal region of the ciliary
shaft and the role of the filaments in motility. J Cell Biol 26: 805–834. 28. Yang P, Diener DR, Yang C, Kohno T, Pazour GJ, et al. (2006) Radial spoke
proteins of Chlamydomonas flagella. J Cell Sci 119: 1165–1174. 10. Deiner M, Tamm SL, Tamm S (1993) Mechanical properties of ciliary
axonemes and membranes as shown by paddle cilia. J Cell Sci 104 (Pt 4): 1251–
1262. 29. Pigino G, Bui KH, Maheshwari A, Lupetti P, Diener D, et al. (2011)
Cryoelectron tomography of radial spokes in cilia and flagella. J Cell Biol 195:
673–687. 11. Gibbons IR (1961) The relationship between the fine structure and direction of
beat in gill cilia of a lamellibranch mollusc. J Biophys Biochem Cytol. 11: 179–
205. 30. Barber CF, Heuser T, Carbajal-Gonzalez BI, Botchkarev VVJr, Nicastro D
(2012) Three-dimensional structure of the radial spokes reveals heterogeneity
and interactions with dyneins in Chlamydomonas flagella. Mol Biol Cell 23: 111–
120. 12. Sale WS (1986) The axonemal axis and Ca2+-induced asymmetry of active
microtubule sliding in sea urchin sperm tails. J Cell Biol 102: 2042–2052. 13. Lin J, Heuser T, Carbajal-Gonzalez BI, Song K, Nicastro D (2012) The
structural heterogeneity of radial spokes in cilia and flagella is conserved. Cytoskeleton (Hoboken) 69: 88–100. 31. Porter ME, Sale WS (2000) The 9+2 axoneme anchors multiple inner arm
dyneins and a network of kinases and phosphatases that control motility. J Cell
Biol 151: F37–42. 14. Wargo MJ, McPeek MA, Smith EF (2004) Analysis of microtubule sliding
patterns in Chlamydomonas flagellar axonemes reveals dynein activity on specific
doublet microtubules. J Cell Sci 117: 2533–2544. 32. Wirschell M, Hendrickson T, Sale WS (2007) Keeping an eye on I1: I1 dynein as
a model for flagellar dynein assembly and regulation. Cell Motil Cytoskeleton
64: 569–579. 15. Hoops HJ, Witman GB (1983) Outer doublet heterogeneity reveals structural
polarity related to beat direction in Chlamydomonas flagella. J Cell Biol 97: 902–
908. 33. Wirschell M, Yang C, Yang P, Fox L, Yanagisawa HA, et al. (2009) IC97 is
a novel intermediate chain of I1 dynein that interacts with tubulin and regulates
interdoublet sliding. Mol Biol Cell 20: 3044–3054. 16. Bui KH, Sakakibara H, Movassagh T, Oiwa K, Ishikawa T (2009) Asymmetry
of inner dynein arms and inter-doublet links in Chlamydomonas flagella. J Cell Biol
186: 437–446. 34. (MPG) Movie S2
Comparison of the 3D structures of an ODA
and the o-SUB5-6 complex in the flagellum of the sea
urchin Strongylocentrotus. An animated isosurface rendering
shows that the 3D structure of o-SUB5-6 (green), which is only
found on DMT5, is different from the ODA structure (purple)
present on all other DMTs. At the beginning of the movie, the
proximal end of the repeat is on the left side. (MPG) A–D correspond to Figure 4B, 3C, 4E, and 4F, respectively. The
Chlamydomonas DMT numbers are according to Hoops and
Witman [15]. Scale bars are 25 nm (scale bar in B valid for A,
B, E and F; scale bar in D valid for C, D, G, and H). (TIF) Movie S3
Comparison of the 3D structure of DMT1 to
that of the other DMTs in the distal three quarters of the
Chlamydomonas flagellum. An animated 3D visualization of
isosurface renderings shows that the DMT1 structure is distinct
from that of the other DMTs. The i-SUB5-6 structure (orange),
which connects to the neighboring doublet, and the lack of certain
dyneins (e.g., ODAs) are unique features of DMT1. (MPG) Movie S1
Comparison of the 3D structure of DMT5 to
that of the other DMTs in the distal 7/8th of the
Strongylocentrotus flagellum (sea urchin). An animated
3D visualization shows isosurface renderings of the averaged 96-
nm axonemal repeats and reveals that the structure of DMT5 is
distinctly different from that of the other doublets. Note that the
SUB5-6 (sea urchin bridge connecting DMTs 5 and 6) structure
consists of two parts: the inner i-SUB5-6 (orange) and the outer o-
SUB5-6 complex (green). References 21. Brokaw CJ (2002) Computer simulation of flagellar movement VIII: co-
ordination of dynein by local curvature control can generate helical bending
waves. Cell Motil Cytoskeleton 53: 103–124. 1. Afzelius BA (2004) Cilia-related diseases. J Pathol 204: 470–477. 1. Afzelius BA (2004) Cilia-related diseases. J Pathol 204: 470–477. 2. Fliegauf M, Benzing T, Omran H (2007) When cilia go bad: cilia defects and
ciliopathies. Nat Rev Mol Cell Biol 8: 880–893. 22. Nakano I, Kobayashi T, Yoshimura M, Shingyoji C (2003) Central-pair-linked
regulation of microtubule sliding by calcium in flagellar axonemes. J Cell Sci
116: 1627–1636. 3. Broadhead R, Dawe HR, Farr H, Griffiths S, Hart SR, et al. (2006) Flagellar
motility is required for the viability of the bloodstream trypanosome. Nature
440: 224–227. 23. Gibbons BH, Baccetti B, Gibbons IR (1985) Live and reactivated motility in the
9+0 flagellum of Anguilla sperm. Cell Motil 5: 333–350. 4. Ralston KS, Lerner AG, Diener DR, Hill KL (2006) Flagellar motility
contributes to cytokinesis in Trypanosoma brucei and is modulated by an
evolutionarily conserved dynein regulatory system. Eukaryot Cell 5: 696–711. 24. Hosokawa Y, Miki-Noumura T (1987) Bending motion of Chlamydomonas
axonemes after extrusion of central-pair microtubules. J Cell Biol 105: 1297–
1301. 5. Hyams JS, Borisy GG (1975) Flagellar coordination in Chlamydomonas reinhardtii:
isolation and reactivation of the flagellar apparatus. Science 189: 891–893. 25. Omoto CK, Gibbons IR, Kamiya R, Shingyoji C, Takahashi K, et al. (1999)
Rotation of the central pair microtubules in eukaryotic flagella. Mol Biol Cell 10:
1–4. 6. Hyams JS, Borisy GG (1978) Isolated flagellar apparatus of Chlamydomonas:
characterization of forward swimming and alteration of waveform and reversal
of motion by calcium ions in vitro. J Cell Sci 33: 235–253. y
J
7. Afzelius B (1959) Electron microscopy of the sperm tail; results obtained with
a new fixative. J Biophys Biochem Cytol 5: 269–278. 26. Wargo MJ, Smith EF (2003) Asymmetry of the central apparatus defines the
location of active microtubule sliding in Chlamydomonas flagella. Proc Natl Acad
Sci USA 100: 137–142. 8. Bradfield JRG (1955) Fibre patterns in animal flagella and cilia. Symp Soc Exp
Biol 9: 306–334. 27. Smith EF, Yang P (2004) The radial spokes and central apparatus: mechano-
chemical transducers that regulate flagellar motility. Cell Motil Cytoskeleton 57:
8–17. 9. Satir P (1965) Studies on cilia: II. Author Contributions Conceived and designed the experiments: DN. Performed the experiments:
JL TH KS XF. Analyzed the data: JL TH DN. Wrote the paper: JL DN. Conceived and designed the experiments: DN. Performed the experiments:
JL TH KS XF. Analyzed the data: JL TH DN. Wrote the paper: JL DN. Acknowledgments We thank Daniel T.N. Chen (Brandeis University) for providing
Strongylocentrotus purpuratus sperm and Mary Porter, Raqual Bower and
Douglas Tritschler (University of Minnesota, Minneapolis, MN) for
providing several Chlamydomonas strains. We also thank Chen Xu for
providing training and outstanding management of the Brandeis EM
facility. We thank Daniel T.N. Chen (Brandeis University) for providing
Strongylocentrotus purpuratus sperm and Mary Porter, Raqual Bower and
Douglas Tritschler (University of Minnesota, Minneapolis, MN) for
providing several Chlamydomonas strains. We also thank Chen Xu for
providing training and outstanding management of the Brandeis EM
facility. Supporting Information The red dashed lines in (A and C)
indicate the locations of the tomographic slices shown in (B and
D). The yellow arrowheads in (B and D) indicate the dynein stalks
that connect to the adjacent doublet microtubule. Scale bar (A and
B): 25 nm. To avoid confusion between the two prevailing DMT number-
ing conventions, yet to encourage consistency with previous and
future studies, we indexed the DMTs using the convention of the
sea urchin numbering system, but with a ‘‘D’’ (for ‘‘doublet’’)
followed by Roman numerals instead of Arabic numbers. Under
this DMT numbering system, the bending plane passes through
DMT D-I and between DMTs D-V and D-VI. The unique DMT
in Strongylocentrotus and Chlamydomonas is DMT D-V. By mapping
previously described doublet-specific features onto this DMT
numbering system, the doublet-specific RSJ of the third radial Figure S3
Comparison of DMT1 between Chlamydomo-
nas pWT and WT. (A–H) Tomographic slices from the averaged
repeats of pWT (A–D) and WT (E–H) axonemes show the i-
SUB5-6 complex in cross-sectional (A, B, E, and F) and
longitudinal views from the front (C, D, G, and H). The averaged
pWT and WT structures are highly similar and show almost
identical i-SUB5-6 and proximal 1–2 bridge structures. The red
dashed lines in (A, B, E, and F) indicate the locations of the
tomographic slices shown in (C, D, G, and H), respectively. Panels October 2012 | Volume 7 | Issue 10 | e46494 PLOS ONE | www.plosone.org 12 Unique Doublet Microtubule A–D correspond to Figure 4B, 3C, 4E, and 4F, respectively. The
Chlamydomonas DMT numbers are according to Hoops and
Witman [15]. Scale bars are 25 nm (scale bar in B valid for A,
B, E and F; scale bar in D valid for C, D, G, and H). (TIF) Unique Doublet Microtubule 38. Lin J, Tritschler D, Song K, Barber CF, Cobb JS, et al. (2011) Building blocks of
the nexin-dynein regulatory complex in Chlamydomonas flagella. J Biol Chem 286:
29175–29191. 58. Smith DJ, Gaffney EA, Gadelha H, Kapur N, Kirkman-Brown JC (2009) Bend
propagation in the flagella of migrating human sperm, and its modulation by
viscosity. Cell Motil Cytoskeleton 66: 220–236. y
y
59. Shingyoji C, Murakami A, Takahashi K (1977) Local reactivation of Triton-
extracted flagella by iontophoretic application of ATP. Nature 265: 269–270. 39. Dymek EE, Smith EF (2007) A conserved CaM- and radial spoke associated
complex mediates regulation of flagellar dynein activity. J Cell Biol 179: 515–
526. g
y
p
pp
60. Lindemann CB, Lesich KA (2010) Flagellar and ciliary beating: the proven and
the possible. J Cell Sci 123: 519–528. 40. Dymek EE, Heuser T, Nicastro D, Smith EF (2011) The CSC is required for
complete radial spoke assembly and wild-type ciliary motility. Mol Biol Cell 22:
2520–2531. 61. Geimer S, Melkonian M (2004) The ultrastructure of the Chlamydomonas reinhardtii
basal apparatus: identification of an early marker of radial asymmetry inherent
in the basal body. J Cell Sci 117: 2663–2674. 41. Warner FD (1976) Ciliary inter-microtubule bridges. J Cell Sci 20: 101–114. y J
62. Hoops HJ, Wright RL, Jarvik JW, Witman GB (1984) Flagellar waveform and
rotational orientation in a Chlamydomonas mutant lacking normal striated fibers. J Cell Biol 98: 818–24. 42. Witman GB, Carlson K, Berliner J, Rosenbaum JL (1972) Chlamydomonas flagella. I. Isolation and electrophoretic analysis of microtubules, matrix, membranes,
and mastigonemes. J Cell Biol 54: 507–539. J
63. Gibbons IR (1963) Studies on the Protein Components of Cilia from Tetrahymena
pyriformis. Proc Natl Acad Sci USA 50: 1002–1010. g
J
43. Nicastro D (2009) Cryo-electron microscope tomography to study axonemal
organization. Methods Cell Biol 91: 1–39. 64. Minoura I, Yagi T, Kamiya R (1999) Direct measurement of inter-doublet
elasticity in flagellar axonemes. Cell Struct Funct 24: 27–33. 44. Nicastro D, Fu X, Heuser T, Tso A, Porter ME, et al. (2011) Cryo-electron
tomography reveals conserved features of doublet microtubules in flagella. Proc
Natl Acad Sci USA 108: E845–853. 65. Bozkurt HH, Woolley DM (1993) Morphology of nexin links in relation to
interdoublet sliding in the sperm flagellum. Cell Motil Cytoskeleton 24: 109–
118. 45. Pigino G, Maheshwari A, Bui KH, Shingyoji C, Kamimura S, et al. Unique Doublet Microtubule (2012)
Comparative structural analysis of eukaryotic flagella and cilia from Chlamydo-
monas, Tetrahymena, and sea urchins. J Struct Biol 178: 199–206. 66. Lindemann CB, Macauley LJ, Lesich KA (2005) The counterbend phenomenon
in dynein-disabled rat sperm flagella and what it reveals about the interdoublet
elasticity. Biophys J 89: 1165–1174. y
J
46. Gibbons IR (1982) Sliding and bending in sea urchin sperm flagella. Symp Soc
Exp Biol 35: 225–287. y
p y J
67. Kamiya R (1995) Exploring the function of inner and outer dynein arms with
Chlamydomonas mutants. Cell Motil Cytoskeleton 32: 98–102. 47. Gatti JL, Christen R (1985) Regulation of internal pH of sea urchin sperm. A
role for the Na/K pump. J Biol Chem 260: 7599–7602. y
y
68. Kagami O, Kamiya R (1992) Translocation and rotation of microtubules caused
by multiple species of Chlamydomonas inner-arm dynein. J Cell Sci 103: 653–664. 48. Gorman DS, Levine RP (1965) Cytochrome f and plastocyanin: their sequence
in the photosynthetic electron transport chain of Chlamydomonas reinhardtii. Proc
Natl Acad Sci USA 54: 1665–1669. y
p
p
y
y
J
69. Ogawa K, Mohri T, Mohri H (1977) Identification of dynein as the outer arms
of sea urchin sperm axonemes. Proc Natl Acad Sci USA 74: 5006–5010. 49. Mastronarde DN (2005) Automated electron microscope tomography using
robust prediction of specimen movements. J Struct Biol 152: 36–51. 70. Ogawa K, Negishi S, Obika M (1982) Immunological dissimilarity in protein
component (dynein 1) between outer and inner arms within sea urchin sperm
axonemes. J Cell Biol 92: 706–713. p
p
50. Kremer JR, Mastronarde DN, McIntosh JR (1996) Computer visualization of
three-dimensional image data using IMOD. J Struct Biol 116: 71–76. three-dimensional image data using IMOD. J Struct Biol 116: 71–76 71. Huang B, Piperno G, Luck DJ (1979) Paralyzed flagella mutants of
Chlamydomonas reinhardtii. Defective for axonemal doublet microtubule arms. J Biol Chem 254: 3091–3099. 51. Nicastro D, Schwartz C, Pierson J, Gaudette R, Porter ME, et al. (2006) The
molecular architecture of axonemes revealed by cryoelectron tomography. Science 313: 944–948. 72. Lindemann CB, Orlando A, Kanous KS (1992) The flagellar beat of rat sperm is
organized by the interaction of two functionally distinct populations of dynein
bridges with a stable central axonemal partition. J Cell Sci 102 (Pt 2): 249–260. 52. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, et al. References Heuser T, Barber CF, Lin J, Krell J, Rebesco M, et al. (2012) Cryo-electron
tomography reveals doublet specific structures and unique interactions in the I1
dynein. Proc Natl Acad Sci USA 109: E2067–2076. 17. Satir P (1968) Studies on cilia. III. Further studies on the cilium tip and a ‘‘sliding
filament’’ model of ciliary motility. J Cell Biol 39: 77–94. dynein. Proc Natl Acad Sci USA 109: E2067–2076. 35. Huang B, Ramanis Z, Luck DJ (1982) Suppressor mutations in Chlamydomonas
reveal a regulatory mechanism for Flagellar function. Cell 28: 115–124. 18. Summers KE, Gibbons IR (1971) Adenosine triphosphate-induced sliding of
tubules in trypsin-treated flagella of sea-urchin sperm. Proc Natl Acad Sci USA
68: 3092–3096. 36. Rupp G, Porter ME (2003) A subunit of the dynein regulatory complex in
Chlamydomonas is a homologue of a growth arrest-specific gene product. J Cell
Biol 162: 47–57. 19. Costello DP (1973a) A new theory on the mechanics of ciliary and flagellar
motility. I. Supporting observations. Biol Bull 145: 279–291. 37. Heuser T, Raytchev M, Krell J, Porter ME, Nicastro D (2009) The dynein
regulatory complex is the nexin link and a major regulatory node in cilia and
flagella. J Cell Biol 187: 921–933. 20. Costello DP (1973b) A new theory on the mechanics of ciliary and flagellar
motility. II. Theoretical considerations. Biol Bull 145: 292–309. October 2012 | Volume 7 | Issue 10 | e46494 October 2012 | Volume 7 | Issue 10 | e46494 13 PLOS ONE | www.plosone.org Unique Doublet Microtubule Unique Doublet Microtubule (2004) UCSF Chimera–a visualization system for exploratory research and
analysis. J Comput Chem 25: 1605–1612. g
p
J
(
)
73. Lesich KA, Kelsch CB, Ponichter KL, Dionne BJ, Dang L, et al (2012) The
calcium response of mouse sperm flagella: role of calcium ions in the regulation
of dynein activity. Biol Reprod 86: 105. y
J
p
53. Linck RW, Stephens RE (2007) Functional protofilament numbering of ciliary,
flagellar, and centriolar microtubules. Cell Motil Cytoskeleton 64: 489–495. 54. Bui KH, Sakakibara H, Movassagh T, Oiwa K, Ishikawa T (2008) Molecular
architecture of inner dynein arms in situ in Chlamydomonas reinhardtii flagella. J Cell
Biol 183: 923–932. y
y
p
74. Fawcett DW (1975) The mammalian spermatozoon. Dev Biol 44: 394–436. 75. Omoto CK, Witman GB (1981) Functionally significant central-pair rotation in
a primitive eukaryotic flagellum. Nature 290: 708–710. 55. Myster SH, Knott JA, O’Toole E, Porter ME (1997) The Chlamydomonas Dhc1
gene encodes a dynein heavy chain subunit required for assembly of the I1 inner
arm complex. Mol Biol Cell 8: 607–620. p
y
g
76. Gibbons IR (1981) Cilia and flagella of eukaryotes. J Cell Biol 91: 107s–124s. 77. Harris EH (2009) The genus Chlamydomonas. In: Harris EH, editor. The
Chlamydomonas Sourcebook: Introduction to Chlamydomonas and its laboratory use. Oxford: Academic Press. 1–24. 56. Chwang AT, Wu TY (1971) A note on the helical movement of micro-
organisms. Proc R Soc Lond B Biol Sci 178: 327–346. 78. Kato T, Kagami O, Yagi T, Kamiya R (1993) Isolation of two species of
Chlamydomonas reinhardtii flagellar mutants, ida5 and ida6, that lack a newly
identified heavy chain of the inner dynein arm. Cell Struct Funct 18: 371–377. 57. Sineshchekov OA, Govorunova EV (1999) Rhodopsin-mediated photosensing in
green flagellated algae. Trends Plant Sci 4: 201. PLOS ONE | www.plosone.org October 2012 | Volume 7 | Issue 10 | e46494 14
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Cancer Feature Selection and Classification Using a Binary Quantum-Behaved Particle Swarm Optimization and Support Vector Machine
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Computational and mathematical methods in medicine
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Hindawi Publishing Corporation
Computational and Mathematical Methods in Medicine
Volume 2016, Article ID 3572705, 9 pages
http://dx.doi.org/10.1155/2016/3572705
Research Article
Cancer Feature Selection and Classification Using a Binary
Quantum-Behaved Particle Swarm Optimization and Support
Vector Machine
Maolong Xi,1 Jun Sun,2 Li Liu,3 Fangyun Fan,2 and Xiaojun Wu2
1
Department of Control Technology, Wuxi Institute of Technology, Wuxi, Jiangsu 214121, China
Department of Computer Science, Jiangnan University, Wuxi, Jiangsu 214122, China
3
Affiliated Hospital of Jiangnan University, Wuxi, Jiangsu 214062, China
2
Correspondence should be addressed to Maolong Xi; wx xml@hotmail.com
Received 28 January 2016; Accepted 27 July 2016
Academic Editor: Yuhai Zhao
Copyright © 2016 Maolong Xi et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
This paper focuses on the feature gene selection for cancer classification, which employs an optimization algorithm to select a subset
of the genes. We propose a binary quantum-behaved particle swarm optimization (BQPSO) for cancer feature gene selection,
coupling support vector machine (SVM) for cancer classification. First, the proposed BQPSO algorithm is described, which is
a discretized version of original QPSO for binary 0-1 optimization problems. Then, we present the principle and procedure for
cancer feature gene selection and cancer classification based on BQPSO and SVM with leave-one-out cross validation (LOOCV).
Finally, the BQPSO coupling SVM (BQPSO/SVM), binary PSO coupling SVM (BPSO/SVM), and genetic algorithm coupling SVM
(GA/SVM) are tested for feature gene selection and cancer classification on five microarray data sets, namely, Leukemia, Prostate,
Colon, Lung, and Lymphoma. The experimental results show that BQPSO/SVM has significant advantages in accuracy, robustness,
and the number of feature genes selected compared with the other two algorithms.
1. Introduction
Nowadays, cancer has been one of the most common lethal
factors for human beings. Missed and mistaken diagnosis
sometimes makes people lose the best chance for appropriate treatments. Therefore, more auxiliary measurements are
needed to promote the accuracy of cancer diagnosis and
clinical test combined with medical ways [1–4]. With the
rapid development of information sciences and molecular
biological sciences, gene microarray technology brings people large amount of high-throughput gene profiles which are
widely used in cancer diagnosis, clinical inspection, and other
aspects. However, microarray expression data are highly
redundant and noisy, and most genes are uninformative with
respect to studied classes, as only a fraction of genes may
present distinct profiles for different classes of samples. As
such, effective methods of selecting feature genes for cancer
are critically necessary. These methods should be able to
robustly identify a subset of informative genes embedded
out of a large data set which is contaminated with high
dimensional noise.
It was Golub et al. who first employed gene expression
data for cancer classification [5]. They proposed to use gene
expression data of acute leukemia for cancer classification
by adopting “SNR” index to calibrate the contribution of
genes to the cancer classification and by using a weighted
voting mechanism to distinguish cancer types [5]. This
study demonstrated that the use of gene expression data to
determine cancer types for the auxiliary medical diagnosis
is an effective measure. Afterwards, an increasing number of
researchers in the fields of biology and information sciences
have proposed many effective feature gene selection methods,
so that the research in this discipline is becoming one of the
hotspots in bioinformatics.
Currently, there are two categories of the methods of
obtaining feature genes for cancer classification based on gene
2
expression data, namely, feature transformation methods and
feature selection methods. By definition, feature transformation refers to a way of transforming the original feature
attributes into a new set of features that represent the original
features to the greatest extent but reduce the dimension
as much as possible in order to achieve the purpose of
dimension reduction. This means that the new features are
low-dimensional features with similar classification abilities.
Feature transformation methods for cancer classification by
using gene expression data include principal component
analysis (PCA) [6], kernel PCA [7], independent component
analysis (ICA) values [8], locally linear embedding (LLE) [9],
partial least squares (PLS) [10], the maximum margin criterion (MMC) [11], and linear discriminant analysis (LDA) [12].
Conde et al. [13, 14] proposed a feature transform method
based on clustering. This approach uses self-organizing tree
algorithm to carry out gene clustering and calculates the
average gene expression level for each category, which is then
accepted as a new feature to establish the cancer classification
model. Kan et al. [15] employed PCA to make transformation
of the gene expression data of children small round blue
cell tumors and then used artificial neural network for
classification.
Feature transformation methods can indeed reduce the
dimension for gene expression data and can eliminate the
“curse of dimensionality” phenomenon due to large number
redundant genes so that they can help to establish effective
cancer classification models. However, the new features
obtained by feature transform property no longer have the
original biological meaning; that is, the methods destroy the
biological information of the original gene expression data,
which makes it impossible to determine the target genes
associated with the cancer. For this reason, feature gene
selection methods have attracted more attention.
The feature gene selection uses an optimization algorithm
to select a subset of the genes, which has the most classification information, from the original gene microarray data.
The most commonly used feature gene selection methods can
be divided in to filter, wrapper, and embedded ones. Filter
algorithm is independent of the subsequent learning algorithm but uses some criteria for scoring gene subsets, which
measure the contribution of the genes to classification. Such
methods generally use SNR [5], 𝑡 test [16], the correlation
coefficient [17], mutual information [18], relief [19], information gain [20], or Fisher discrimination [21]. Obviously, filter
methods have advantages such as simplicity, fast calculation,
and independence of classification algorithms. However, they
evaluate a single gene with some criteria but ignore the
correlation between genes, which resulted in a large amount
of redundant information contained in candidate genes.
Different from filter methods, wrapper methods combine
gene selection and classification method and use training
accuracy of the learning algorithm to assess the subset
of features to guide gene selection. Such methods include
the sequential random search heuristics [22], random forest method [23], and PKLR [24]. In the cancer feature
gene selection, a typical wrapper feature selection method
combines support vector machine (SVM) and a recursive
feature selection method [25]. In this method, support vector
Computational and Mathematical Methods in Medicine
machines are used to classify the data set, then each gene is
excluded in turn, and the performance change of the SVM
after exclusion of the gene is calculated, and afterwards, the
gene with the least absolute value of the association weight
is removed from the training set until the training set is
empty. The gene sets deleted together in the last step are the
optimal subset. Li et al. [26] adopted genetic algorithm (GA)
to select feature genes of cancer. Zhang et al. [27] coupled a
binary particle swarm optimization (BPSO) and the SVM for
classification of Colon data set.
Embedded methods are extension of wrapper approaches
and undertake feature selection in the process of classifier
training, without dividing the data set into a training set and a
validation set. Typical embedded algorithms include decision
tree [28] and artificial neural networks [29].
In this work, we propose a new method, which couples a binary quantum-behaved particle swarm optimization
with SVM approach, to select feature gene subset from
cancer microarray data. In order to prove the advantages
of BQPSO/SVM, we also implement two other algorithms,
BPSO/SVM and GA/SVM. The BPSO and GA used in this
work are both the original version. These two algorithms or
improved ones were used in this case by other scholars early
in [30–32]. All these three approaches are experimentally
assessed on five well-known cancer data sets (Leukemia,
Colon, Prostate, Lung, and Lymphoma).
This paper is structured as follows. In Section 2, we review
the BQPSO algorithm, and in Section 3 the SVM technique
is described and our BQPSO/SVM method is proposed. In
Section 4, the five microarray data sets used in this work are
described. Experimental results are presented in Section 5,
including biological descriptions of several obtained genes.
Finally, the paper is concluded in Section 6.
2. Binary Encoded Quantum-Behaved Particle
Swarm Optimization (BQPSO)
PSO algorithm is a population-based evolutionary search
technique, which was firstly proposed in [33]. Social behavior
of animals such as bird flocking and fish schooling and swarm
theory is the underlying motivation for the development
of PSO. Inspired by the quantum theory, Sun et al. [34]
developed a novel variant of PSO called Quantum-behaved
Particle Swarm Optimization (QPSO), where a strategy based
on a quantum 𝛿 potential well is employed to sample around
the personal best points and then introduced the mean best
position into the algorithm [35–37].
Based on our previous work in [38], in this paper, we
further proposed a discrete binary version of QPSO (BQPSO)
as a search algorithm coupling SVM for gene selection
based on cancer gene expression data. In the proposed
BQPSO, the position of the particle is represented as a binary
string. For instance, in Figure 1 𝑋1 (1011001010) is the first
particle and 𝑋2 (0010010110) is the second one; they all have
two substrings (two decision variables), and the distance is
defined as the Hamming distance between two binary strings;
namely,
(1)
𝑋1 − 𝑋2 = 𝑑𝐻 (𝑋1 , 𝑋2 ) ,
Computational and Mathematical Methods in Medicine
X1
X11
1
0
1
1
0
0
0
1
X12
0
1
X2
X21
0
0
3
0
1
0
1
0
X22
1
0
1
Figure 1: Binary coding of particle’s position.
where 𝑑𝐻(⋅) is the function to get Hamming distance between
𝑋1 and 𝑋2 , which is the count of bits different in the two
strings; the distance is seven in Figure 1. In the BQPSO, the
dimension is defined as the number of decision variables, so
that a particle can have more than one decision variable. For
example, particle 𝑖 is represented as 𝑋𝑖 = (𝑋𝑖1 , 𝑋𝑖2 , . . . , 𝑋𝑖𝐷),
and it has 𝐷 decision variables, and 𝑋𝑖𝑑 refers to the 𝑑th
substring (𝑑th decision variable) of the position of the 𝑖th
particle. Given that the lengths of 𝑋𝑖𝑑 and 𝑋𝑖 are 𝑙𝑑 and 𝑙,
respectively, then we can get equation as follows:
𝑑
𝑙 = ∑𝑙𝑑 , 𝑑 = 1, 2, . . . , 𝐷.
(2)
Get mbest(pbest)
for 𝑗 = 1 to 𝑙 (the length of binary string)
sum = 0;
for each particle i
sum = sum + pbest[𝑖][𝑗];
endfor
avg = sum/𝑀;
if avg > 0.5 mbest[𝑗] = 1; endif
if avg < 0.5 mbest[𝑗] = 0; endif
if avg = 0.5
if rand() < 0.5 mbest[𝑗] = 0;
else mbest[𝑗] = 1;
endif
endif
endfor
Return mbest
Pseudocode 1: Pseudocode for obtaining mbest.
obtained by the mutation of 𝑃𝑖 with the probability computed
by
𝑏
{
{
{𝑙 ,
𝑃𝑟 = { 𝑑
{
{1,
{
𝑖=1
In the BQPSO, the mean best (𝑚𝑏𝑒𝑠𝑡) position of all
particles is determined by the states of the bits of all particles’
𝑝𝑏𝑒𝑠𝑡. In detail, for 𝑗th bit of the 𝑚𝑏𝑒𝑠𝑡, if 1 appears more
often than 0 at the 𝑗th bit of all 𝑝𝑏𝑒𝑠𝑡, the 𝑗th bit of 𝑚𝑏𝑒𝑠𝑡
will be 1; otherwise the bit will be 0. However, if 1 and
0 have the same frequency of occurrence, the 𝑗th bit of
𝑚𝑏𝑒𝑠𝑡 will be set randomly to be 1 or 0, with probability 0.5
for either state. The function for obtaining 𝑚𝑏𝑒𝑠𝑡 is called
𝑚𝑏𝑒𝑠𝑡 = Get 𝑚𝑏𝑒𝑠𝑡 (𝑝𝑏𝑒𝑠𝑡). The pseudocode of the function
for obtaining 𝑚𝑏𝑒𝑠𝑡 is given in Pseudocode 1.
𝑃𝑖 is the coordinate of local attractor for particle 𝑖. In
the continuous version of QPSO, the coordinate 𝑃𝑖𝑑 of 𝑃𝑖
lies between 𝑝𝑏𝑒𝑠𝑡𝑖𝑑 and 𝑔𝑏𝑒𝑠𝑡𝑑 . In the BQPSO, the point
𝑃𝑖 is generated through one-point or multipoint crossover
operation of 𝑝𝑏𝑒𝑠𝑡𝑖 and 𝑔𝑏𝑒𝑠𝑡 like that used in genetic
algorithm (GA), and this definitely make 𝑃𝑖 lay between
𝑝𝑏𝑒𝑠𝑡𝑖𝑑 and 𝑔𝑏𝑒𝑠𝑡𝑑 as well. The function getting 𝑚𝑏𝑒𝑠𝑡 in
BQPSO is called 𝑃𝑖 = Get 𝑃 (𝑝𝑏𝑒𝑠𝑡𝑖 , 𝑔𝑏𝑒𝑠𝑡).
Update equation of the particle position in the original
QPSO is given by
1
𝑋𝑖𝑑 − 𝑃𝑖𝑑 = 𝛼 𝑚𝑏𝑒𝑠𝑡𝑑 − 𝑋𝑖𝑑 ln ( ) , 𝜇 = rand () . (3)
𝜇
In the BQPSO, (4) can be written again as follows:
𝑑𝐻 (𝑋𝑖𝑑 , 𝑃𝑖𝑑 ) = ⌈𝑏⌉ ,
(4)
where
1
𝑏 = 𝛼 ∗ 𝑑𝐻 (𝑋𝑖𝑑 , 𝑚𝑏𝑒𝑠𝑡𝑑 ) ∗ ln ( ) ,
𝜇
(5)
Because 𝑑𝐻(⋅) is Hamming distance, 𝑏 must be an integer,
which is the reason for the use of function ⌈⋅⌉. New string 𝑋𝑖 is
𝑏
> 1.
𝑙𝑑
(6)
In [35], here 𝑙𝑑 is the length of substring 𝑋𝑖𝑑 . Function
getting 𝑋𝑖𝑑 is denoted as 𝑋𝑖𝑑 = Transf (𝑃𝑖𝑑 , 𝑃𝑟 ). The transformation of Transf (𝑃𝑖𝑑 , 𝑃𝑟 ) is described in Pseudocode 2.
The BQPSO can be summarized as Get 𝑚𝑏𝑒𝑠𝑡 (𝑝𝑏𝑒𝑠𝑡),
Get 𝑃 (𝑝𝑏𝑒𝑠𝑡𝑖 , 𝑔𝑏𝑒𝑠𝑡), and Transf (𝑃𝑖𝑑 , 𝑃𝑟 ).
3. Gene Selection and Classification by
BQPSO/SVM
3.1. The SVM Classifier. Support vector machine proposed in
[39] is a technique derived from statistical learning theory. It
is widely used to classify points by assigning them to one of
two disjoint half spaces [40, 41]. That is to say SVM carries
out mainly a 2-class classification. For linearly separable
data, SVM gets the hyperplane which maximizes the margin
between the training samples and the class boundary. For
nonlinearly separable cases, samples are mapped to a high
dimensional space. In this space, such a separating hyperplane can be found. The assignment is conducted by way of a
mechanism called the kernel function.
Theoretically, SVM is able to correctly classify any linearly
separable data. Consider the data with two classes, which can
be expressed as
(𝑥𝑖 , 𝑦𝑖 ) ,
𝜇 = rand () .
if
𝑖 = 1, 2, . . . , 𝑙, 𝑥 ∈ 𝑅𝑛 , 𝑦 ∈ {±1} ,
(7)
and then the hyperplane that separated the two classes of the
data is given by
(𝑤 ⋅ 𝑥) + 𝑏 = 0.
(8)
4
Computational and Mathematical Methods in Medicine
and the optimal classification function is
Transf(𝑃𝑖𝑑 , 𝑃𝑟 )
for each bit in the substring 𝑃𝑖𝑑 ;
if rand() < 𝑃𝑟
if the state of the bit is 1
Set its state to 0;
else set its sate to 1;
endif
endif
endfor
𝑋𝑖𝑑 = 𝑃𝑖𝑑 ;
Return 𝑋𝑖𝑑
𝑓 (𝑥) = sgn {(𝑤∗ ⋅ 𝑥) + 𝑏∗ }
(15)
}
{ 𝑙
= sgn {( ∑ 𝑎𝑗∗ 𝑦𝑗 (𝑥𝑗 ⋅ 𝑥𝑖 )) + 𝑏∗ } , 𝑥 ∈ 𝑅𝑛 .
}
{ 𝑗=1
Pseudocode 2: Pseudocode of the transformation.
In order to guarantee that the data can be correctly classified
and the distance between the classes is as large as possible, the
hyperplane must satisfy
𝑦𝑖 [(𝑤 ⋅ 𝑥𝑖 ) + 𝑏] ≥ 1,
𝑖 = 1, 2, . . . , 𝑙,
(9)
by which the distance is obtained as 2/‖𝑤‖ so that the problem
of constructing the hyperplane is converted to the following
optimization problem:
min 𝜙 (𝑤) =
1
1
‖𝑤‖2 = (𝑤 ⋅ 𝑤)
2
2
(10)
with (9) being the constraint. By introducing the following
Lagrange function to solve problem (10):
𝐿 (𝑤, 𝑏, 𝑎) =
1
‖𝑤‖ − 𝑎 (𝑦 ((𝑤 ⋅ 𝑥) + 𝑏) − 1) ,
2
(11)
where 𝑎 > 0 is known as the Lagrange coefficient. Solving
the Lagrangian dual of the problem, one obtains a simplified
problem:
𝑙
max
𝑄 (𝑎) = ∑ 𝑎𝑗 −
𝑗=1
1 𝑙 𝑙
∑ ∑ 𝑎 𝑎 𝑦 𝑦 (𝑥 ⋅ 𝑥 )
2 𝑖=1 𝑗=1 𝑖 𝑗 𝑖 𝑗 𝑖 𝑗
(12)
𝑙
s.t.
∑ 𝑎𝑗 𝑦𝑗 = 0 𝑗 = 1, 2, . . . , 𝑙, 𝑎𝑗 ≥ 0.
𝑗=1
Solving the problem in (12), we can get
3.2. The Proposed BQPSO/SVM Approach. In many bioinformatics problems the number of features is significantly larger
than the number of samples. In order to improve the classification or to help to recognize interesting features in noisy
environments, tools for reducing the number of features are
indispensable. The hybrid BQPSO/SVM approach proposed
in the following contributes especially in this sense.
First of all, the data should be preprocessed. Normalization of data must be conducted so as to eliminate the impact
of the dimensionless on the classification. Then we need to
take traditional 𝑡-test on the data, order the genes by 𝑝 value
ascending, and get 50 top-ranked genes from all. Afterwards,
most of the noisy data have been removed. These 50 genes
comprise the whole search space of the BQPSO algorithm for
gene selection.
For the BQPSO used in this work, the swarm sizes for
the BQPSO and BPSO were set to be 20 and the population
size for GA was also 20. Each particle has just one decision
variable, and thus the dimension of the particle is just one.
The length of the particle is 50, so every particle is a binary
string with length of 50, and 1 represents that this gene
is chosen and 0 is not. Feature gene selection and cancer
classification based on hybrid BQPSO/SVM algorithm can be
described as the procedure in Pseudocode 3.
3.3. Evaluation Function. Since a particle 𝑋𝑖 is a binary string
representing a gene subset in BQPSO/SVM, the evaluation of
each particle is executed by the SVM classifier to assess the
quality of the represented gene subset. The fitness of a particle
𝑋𝑖 is calculated employing a leave-one-out cross validation
(LOOCV) method to calculate the accuracy of SVM trained
with this subset. In leave-one-out cross validation, one of all
samples is evaluated as test data while the others except this
one are used as training data, repeated until all samples have
been used as test data. The classification accuracy of LOOCV
is the average accuracy of 𝑛 times classifying, if the data set
has 𝑛 samples. The evaluation function is described in
𝑇
𝑎∗ = (𝑎1∗ , 𝑎2∗ , . . . , 𝑎𝑙∗ ) ,
∗
𝑤 =
fitness (𝑋𝑖 ) = 𝛼 ∗ accuracy + 𝛽 ∗
𝑙
∑ 𝑎𝑗∗ 𝑦𝑗 𝑥𝑗 ,
𝑗=1
(13)
𝑙
𝑏∗ = 𝑦𝑖 − ∑ 𝑦𝑗 𝑎𝑗∗ (𝑥𝑗 ⋅ 𝑥𝑖 ) , 𝑗 ∈ {𝑗 | 𝑎𝑗∗ ≻ 0} ,
𝑗=1
by which the hyperplane is obtained as
(𝑤∗ ⋅ 𝑥) + 𝑏∗ = 0
(14)
50
,
feature number
(16)
where 𝛼 and 𝛽 are weight values and set to 0.6 and 0.4,
respectively, for the purpose of controlling that the accuracy
value takes precedence over the subset size, since high accuracy is preferred when leading the search process. The target
here consists of maximizing the accuracy and minimizing
the number of genes (feature number). For convenience
(only maximum of fitness), the second factor is presented as
50/feature number.
Computational and Mathematical Methods in Medicine
5
Processing of data set;
Initialize the current positions and the pbest positions of all particles which are binary bits with each representing whether the
corresponding gene is selected or not;
do
Determine the mean best position among the particles by mbest = Get mbest(pbest), select a suitable value for 𝛽;
for 𝑖 = 1 to population size 𝑀
Call the LIBSVM tool box to construct the SVM classifier and get the classification accuracy for the data;
With the classification accuracy and the number of selected genes (i.e. the number of features given by the number of bits
with value 1), evaluate the objective function value 𝑓(𝑋𝑖 ) according to Section 3.3;
Update 𝑃𝑖 , and 𝑃𝑔 , it means
if 𝑓(𝑋𝑖 ) < 𝑓(𝑃𝑖 ) then 𝑃𝑖 = 𝑋𝑖 ;
and 𝑃𝑔 = arg min𝑃𝑖 𝑓(𝑃𝑖 ), 1 ≤ 𝑖 ≤ 𝑀;
then get a stochastic position by 𝑝𝑖 = Get P (𝑝𝑏𝑒𝑠𝑡𝑖 , 𝑔best)
for 𝑗 = 1 to dimensionality 𝑁
Compute the mutation probability 𝑃𝑟 ;
Generate the new substring 𝑋𝑖𝑑 by 𝑋𝑖𝑑 = Transf(𝑃𝑖𝑑 , 𝑃𝑟 );
and get the new position 𝑋𝑖 by combining all new substring 𝑋𝑖𝑑 (𝑑 = 1, 2, . . . , 𝐷)
endfor
endfor
until termination criterion is met;
Output the best solution which have been found (𝑔best)
Pseudocode 3: The psudocode the BPSO/SVM.
4. The Data Sets
Table 1: Description for the test databases.
There are several DNA microarray data sets from published
cancer gene expression studies. Five of them were used in
this paper. They are Leukemia data set, Prostate data set,
Colon data set, Lung data set, and Lymphoma data set. All of
them were taken from the BRB-ArrayTools in [42] with URL
http://linus.nci.nih.gov/∼brb/DataArchive New.html. More
details of these five data sets are showed in Tables 1 and 2.
The value in parenthesis in Table 3 is the number of examples
of class 1 or class 2 involved in that data set.
Number Name of data
set
1
Leukemia
2
Prostate
3
Colon
4
Lung
5
Lymphoma
5. Experimental Results and
Performance Comparison
Number Name of data set
BQPSO/SVM approach was implemented on MATLAB,
along with BPSO/SVM and GA/SVM. The SVM classifier
used in these three approaches is based on the LIBSVM
library in [43]. For the SVM configuration, since we were
considering the performance of the search algorithm in the
work, rather than the influence of parameters in SVM to
classification, we used the default parameters of LIBSVM.
And the default kernel function was configured as radial basis
function. The fitness function in this work is the classification
accuracy of leave-one-out cross validation (LOOCV).
All experiments were carried out using a PC with
Windows OS and a Pentium Dual-Core 2.60 GHz CPU,
with 2 G of RAM. BQPSO/SVM, BPSO/SVM, and GA/SVM
algorithms on five cancer related microarray data sets were
independent executed 25 times over each data set, in order
to have statistically meaningful conclusions as these three
algorithms are stochastic search methods.
Number of
examples
Number of
genes
Classes
72
102
62
181
77
7129
12600
2000
12533
7129
2
2
2
2
2
Table 2: Description for the test databases.
1
2
3
4
5
Leukemia
Prostate
Colon
Lung
Lymphoma
Class 1
(quantity)
Class 2
(quantity)
AML (25)
N1 (50)
N3 (22)
MPM5 (31)
DLBCL7 (58)
ALL (47)
PC2 (52)
CC4 (40)
ADCA6 (150)
FL8 (19)
1: normal, 2: prostate cancer, 3: normal, 4: colon cancer, 5: malignant pleural
mesothelioma, 6: adenocarcinoma, 7: diffuse large B-cell lymphoma, and 8:
follicular lymphoma.
5.1. Parameter Settings. The parameters used in BQPSO,
BPSO, and GA algorithms are shown in Table 3. These
parameters were selected after several test evaluations of
each algorithm and data set instance until reaching the best
configuration in terms of the overall quality of solutions.
5.2. Discussion and Analysis. Depending on the results of
the experiments, we made analysis of results focusing on
6
Computational and Mathematical Methods in Medicine
30
BQPSO
25
Swarm size
Iteration
Dimension of particle
𝛽
20
100
1
1
BPSO
Swarm size
Iteration
Maximum of velocity
(𝑤, 𝑐1, 𝑐2)
20
100
6
(0.5, 2, 2)
Number of genes
Table 3: BQPSO, BPSO, and GA parameters for gene subset selection and classification.
20
15
10
5
GA
Swarm size
Iteration
Probability of crossover
Probability of mutation
20
100
0.9
0.04
the performance and robustness, as well as the quality of
the obtained solutions providing a biological description
of most significant ones. We conducted the experiments
for BPSO/SVM and GA/SVM in order to demonstrate the
advantage of the proposed BQPSO/SVM without any other
factors affecting, since in our work all these three algorithms
are operated in exactly the same hardware and software
environment and with the same data sets and parameters.
5.2.1. Performance Analysis. Next, we compare BQPSO/SVM
with BPSO/SVM and GA/SVM. Since these three algorithms
are running in the same environment, parameters, and data
sets, the results are absolutely comparable. Table 4 lists the
highest LOOCV accuracy in 25 independent executions of
each method for each data set. The mean columns contain
the average of the LOOCV accuracy obtained from 25
independent executions.
The performance comparison shows that, compared to
BPSO/SVM and GA/SVM, BQPSO/SVM has an obvious
advantage. In terms of the correct rate, the search capability
of BQPSO/SVM is stronger than the other two competitors.
The purpose of feature selection in our work is to find
small subsets with high classification accuracy. In Figure 2,
the number of genes is the mean size of subsets from 25
executions. Obviously, the proposed BQPSO/SVM provided
smaller subsets of genes than the other two methods.
5.2.2. Algorithm Robustness. Besides the quality of the algorithm, its ability to generate similar or identical results when
executed several times is also important. One of the most
important norms in assessing any proposed algorithm is
robustness. It is particularly important for metaheuristics
which are employed in this work. The standard deviation
(std. dev.) in Table 5 denotes the standard deviation of
accuracy from 25 independent executions. As it can be seen
from the standard deviation, the robustness of the proposed
algorithm is significantly better than GA/SVM. Compared
0
Leukemia
Prostate
Colon
Lung
Lymphoma
Datasets
BQPSO/SVM
BPSO/SVM
GA/SVM
Figure 2: The average number of genes selected by BQPSO/SVM,
BPSO/SVM, and GA/SVM, respectively.
with BPSO/SVM, our proposed algorithm obtained smaller
standard deviation with Prostate data set and Colon data
set but found much better solutions which led to a larger
standard deviation. Overall, from Table 5, it is shown that
BQPSO/SVM has an obvious advantage over the other two
approaches in terms of robustness.
5.2.3. Brief Biological Analysis of Selected Genes. Finally, the
best subsets of genes were found for each data set. We add up
all subsets having the highest accuracy and list the selected
genes. For Colon data set, the top 5 genes with the highest
selection frequency of each microarray data are presented in
Table 6.
(i) Among the genes listed in Table 5, two of them were
also selected by [44]. The first gene is uroguanylin
precursor Z50753. It was shown that a reduction
of uroguanylin might be an indication of colon
tumors in [45, 46] which reported that treatment with
uroguanylin has a positive therapeutic significance to
the reduction in precancerous colon ploys.
(ii) The second selected gene of colon data set is R87126
(myosin heavy chain, nonmuscle). The isoform B
of R87126 serves as a tumor suppressor and is well
known as a component of the cytoskeletal network
[47].
6. Conclusion
In this paper, a hybrid technique for gene selection and
classification of high dimensional DNA Microarray data
was presented and compared. This technique is based on a
metaheuristic algorithm BQPSO used for feature selection
Computational and Mathematical Methods in Medicine
7
Table 4: Comparison of accuracy with the proposed algorithm, BPSO/SVM, and GA/SVM.
BQPSO/SVM
Data set
Best
100
100
93.55
100
100
Leukemia
Prostate
Colon
Lung
Lymphoma
BPSO/SVM
Mean
100
99.25
92.52
99.96
99.79
Best
100
99.02
91.94
100
100
GA/SVM
Mean
100
99.02
91.94
99.96
99.74
Best
100
98.04
91.94
100
98.70
Mean
99.61
96.00
88.65
99.87
98.18
Table 5: Comparison in terms of statistical results of BQPSO/SVM, BPSO/SVM, and GA/SVM.
Data set
Leukemia
Prostate
Colon
Lung
Lymphoma
Best
100
100
93.55
100
100
BQPSO/SVM
Std. dev.
0
0.43
0.79
0.15
0.49
Best
100
99.02
91.94
100
100
BPSO/SVM
Std. dev.
0
0
0
0.15
0.53
GA/SVM
Best
100
98.04
91.94
100
98.70
Std. dev.
0.64
1.20
1.89
0.24
0.75
Table 6: Top 5 genes with the highest selection frequency of colon data set.
Data set
Colon
Accession number
Z50753
R87126
X63629
M76378
X53586
using the SVM classifier to identify potentially good gene
subsets and is compared with the BPSO and GA. In addition,
genes selected are validated by an accurate leave-one-out
cross validation method to improve the actual classification.
All three approaches were experimentally assessed on five
well-known cancer data sets. Results of 100% classification
rate and less than average 11 genes are obtained in most of
our executions. The use of preprocessing method has shown
a great influence on the performance of proposed algorithm,
since it introduces an early set of acceptable solutions in
their evolution process. Continuing the line of this work,
we are interested in optimization of BQPSO/SVM in order
to discover new and better subsets of genes using specific
Microarray data sets.
Competing Interests
The authors declare that they have no financial and personal
relationships with other people or organizations that can
inappropriately influence their work; there is no professional
or other personal interest of any nature or kind in any
product, service, and/or company that could be construed as
influencing the position presented in, or the review of, this
paper.
Acknowledgments
The research work was supported by the National Natural Science Foundation of China (Projects nos. 61373055
Gene description
H. sapiens mRNA for GCAP-II/uroguanylin precursor
Myosin heavy chain, nonmuscle (Gallus gallus)
H. sapiens mRNA for p cadherin
Human cysteine-rich protein (CRP) gene, exons 5 and 6
Human mRNA for integrin alpha 6
and 61300150), Natural Science Foundation for College and
Universities in Jiangsu Province (Project no. 16KJB520051),
and the Qing Lan Project of Jiangsu and Wuxi Institute of
Technology.
References
[1] A. C. Pease, D. Solas, E. J. Sullivan, M. T. Cronin, C. P. Holmes,
and S. P. A. Fodor, “Light-generated oligonucleotide arrays
for rapid DNA sequence analysis,” Proceedings of the National
Academy of Sciences of the United States of America, vol. 91, no.
11, pp. 5022–5026, 1994.
[2] Y. Zhao, G. Wang, X. Zhang, J. X. Yu, and Z. Wang, “Learning
phenotype structure using sequence model,” IEEE Transactions
on Knowledge and Data Engineering, vol. 26, no. 3, pp. 667–681,
2014.
[3] Y. Zhao, G. Wang, Y. Li, and Z. Wang, “Finding novel diagnostic
gene patterns based on interesting non-redundant contrast
sequence rules,” in Proceedings of the 11th IEEE International
Conference on Data Mining (ICDM ’11), pp. 972–981, IEEE,
Vancouver, Canada, December 2011.
[4] Y. Zhao, J. X. Yu, G. Wang, L. Chen, B. Wang, and G.
Yu, “Maximal subspace co-regulated gene clustering,” IEEE
Transactions on Knowledge and Data Engineering, vol. 20, no.
1, pp. 83–98, 2008.
[5] T. R. Golub, D. K. Slonim, P. Tamayo et al., “Molecular classification of cancer: class discovery and class prediction by gene
expression monitoring,” Science, vol. 286, no. 5439, pp. 531–537,
1999.
8
[6] H. Abdi and L. J. Williams, “Principal component analysis,”
Wiley Interdisciplinary Reviews: Computational Statistics, vol. 2,
no. 4, pp. 433–459, 2010.
[7] Z. Liu, D. Chen, and H. Bensmail, “Gene expression data classification with kernel principal component analysis,” Journal of
Biomedicine and Biotechnology, vol. 2005, no. 2, pp. 155–159,
2005.
[8] C.-H. Zheng, D.-S. Huang, X.-Z. Kong, and X.-M. Zhao, “Gene
expression data classification using consensus independent
component analysis,” Genomics, Proteomics and Bioinformatics,
vol. 6, no. 2, pp. 74–82, 2008.
[9] S. Chao and C. Lihui, “Feature dimension reduction for microarray data analysis using locally linear embedding,” in
Proceedings of the 3rd Asia-Pacific Bioinformatics Conference,
Singapore, January 2005.
[10] D. V. Nguyen and D. M. Rocke, “Tumor classification by partial
least squares using microarray gene expression data,” Bioinformatics, vol. 18, no. 1, pp. 39–50, 2002.
[11] S. Zhang and R. Jing, “Dimension reduction based on modified
maximum margin criterion for tumor classification,” in Proceedings of the 4th International Conference on Information and
Computing (ICIC ’11), pp. 552–554, Phuket Island, China, April
2011.
[12] Y. Guo, T. Hastie, and R. Tibshirani, “Regularized linear discriminant analysis and its application in microarrays,” Biostatistics, vol. 8, no. 1, pp. 86–100, 2007.
[13] L. Conde, A. Mateos, J. Herrero, and J. Dopazo, “Unsupervised
reduction of the dimensionality followed by supervised learning
with a perceptron improves the classification of conditions in
DNA microarray gene expression data,” in Proceedings of the
12th IEEE Workshop on Neural Networks for Signal Processing
(NNSP ’02), pp. 77–86, Martigny, Switzerland, 2002.
[14] A. Mateos, J. Herrero, J. Tamames et al., “Supervised neural
networks for clustering conditions in DNA array data after
reducing noise by clustering gene expression profiles,” in Methods of Microarray Data Analysis II, pp. 91–103, Springer, Berlin,
Germany, 2002.
[15] T. Kan, Y. Shimada, F. Sato et al., “Gene expression profiling in
human esophageal cancers using cDNA microarray,” Biochemical and Biophysical Research Communications, vol. 286, no. 4,
pp. 792–801, 2001.
[16] P. Jafari and F. Azuaje, “An assessment of recently published
gene expression data analyses: reporting experimental design
and statistical factors,” BMC Medical Informatics and Decision
Making, vol. 6, no. 1, article 27, 2006.
[17] M. A. Hall, Correlation-Based Feature Selection for Machine
Learning, The University of Waikato, Hamilton, New Zealand,
1999.
[18] Y. Wang, I. V. Tetko, M. A. Hall et al., “Gene selection from
microarray data for cancer classification—a machine learning
approach,” Computational Biology and Chemistry, vol. 29, no. 1,
pp. 37–46, 2005.
[19] J. Zhang, S. Liu, and Y. Wang, “Gene association study with
SVM, MLP and cross-validation for the diagnosis of diseases,”
Progress in Natural Science, vol. 18, no. 6, pp. 741–750, 2008.
[20] Y.-X. Li, Q.-J. Liu, and X.-G. Ruan, “A method for extracting
knowledge from tumor gene expression data,” Acta Electronica
Sinica, vol. 32, no. 9, pp. 1479–1482, 2004.
[21] Y. Lai, B. Wu, L. Chen, and H. Zhao, “A statistical metho for
identifying differential gene-gene co-expression patterns,”
Bioinformatics, vol. 20, no. 17, pp. 3146–3155, 2004.
Computational and Mathematical Methods in Medicine
[22] I. Guyon and A. Elisseeff, “An introduction to variable and
feature selection,” The Journal of Machine Learning Research, vol.
3, no. 11, pp. 57–82, 2003.
[23] R. Dı́az-Uriarte and S. A. de Andrés, “Gene selection and classification of microarray data using random forest,” BMC Bioinformatics, vol. 7, no. 1, article 3, 2006.
[24] J. Zhu and T. Hastie, “Classification of gene microarrays by
penalized logistic regression,” Biostatistics, vol. 5, no. 3, pp. 427–
443, 2004.
[25] I. Guyon, J. Weston, S. Barnhill, and V. Vapnik, “Gene selection for cancer classification using support vector machines,”
Machine Learning, vol. 46, no. 1–3, pp. 389–422, 2002.
[26] L. Li, T. A. Darden, C. R. Weinberg, A. J. Levine, and L.
G. Pedersen, “Gene assessment and sample classification for
gene expression data using a genetic algorithm/k-nearest neighbor method,” Combinatorial Chemistry and High Throughput
Screening, vol. 4, no. 8, pp. 727–739, 2001.
[27] H. Zhang, X. Song, and H. Wang, “Feature gene selection based
on binary particle swarm optimization and support vector
machine,” Computers and Applied Chemistry, vol. 24, no. 9, pp.
1159–1162, 2007.
[28] L.-Y. Du, “Analysis of decision tree algorithm based on data
mining,” Journal of Jilin Institute of Architecture & Civil Engineering, no. 5, pp. 48–50, 2014.
[29] B. Liu, Q. Cui, T. Jiang, and S. Ma, “A combinational feature
selection and ensemble neural network method for classification of gene expression data,” BMC Bioinformatics, vol. 5, article
136, 2004.
[30] H. Liu and H. Motoda, Feature Extraction, Construction and
Selection: A Data Mining Perspective, Springer, New York, NY,
USA, 1998.
[31] E. Alba, J. Garcı́a-Nieto, L. Jourdan, and E.-G. Talbi, “Gene
selection in cancer classification using PSO/SVM and GA/SVM
hybrid algorithms,” in Proceedings of the IEEE Congress on
Evolutionary Computation (CEC ’07), pp. 284–290, IEEE, Singapore, September 2007.
[32] E. B. Huerta, B. Duval, and J.-K. Hao, “A hybrid GA/SVM
approach for gene selection and classification of microarray
data,” in Applications of Evolutionary Computing, vol. 3907 of
Lecture Notes in Computer Science, pp. 34–44, Springer, Berlin,
Germany, 2006.
[33] J. Kennedy and R. Eberhart, “Particle swarm optimization,” in
Proceedings of the 1995 IEEE International Conference on Neural
Networks, pp. 1942–1948, December 1995.
[34] J. Sun, B. Feng, and W. Xu, “Particle swarm optimization
with particles having quantum behavior,” in Proceedings of the
Congress on Evolutionary Computation (CEC ’04), pp. 325–331,
June 2004.
[35] J. Sun, W. Xu, and B. Feng, “A global search strategy of
Quantum-behaved Particle Swarm Optimization,” in Proceedings of the IEEE Conference on Cybernetics and Intelligent
Systems, vol. 1, pp. 111–116, Singapore, December 2004.
[36] J. Sun, W. Fang, X. Wu, V. Palade, and W. Xu, “Quantumbehaved particle swarm optimization: analysis of individual
particle behavior and parameter selection,” Evolutionary Computation, vol. 20, no. 3, pp. 349–393, 2012.
[37] J. Sun, X. Wu, V. Palade, W. Fang, C.-H. Lai, and W. Xu,
“Convergence analysis and improvements of quantum-behaved
particle swarm optimization,” Information Sciences, vol. 193, pp.
81–103, 2012.
Computational and Mathematical Methods in Medicine
[38] M. L. Xi, J. Sun, and Y. Wu, “Quantum-behaved particle swarm
optimization with binary encoding,” Control and Decision, vol.
25, no. 1, pp. 99–104, 2010.
[39] C. Cortes and V. Vapnik, “Support-vector networks,” Machine
Learning, vol. 20, no. 3, pp. 273–297, 1995.
[40] B. Gu, V. S. Sheng, Z. Wang, D. Ho, S. Osman, and S. Li,
“Incremental learning for ]-Support Vector Regression,” Neural
Networks, vol. 67, pp. 140–150, 2015.
[41] B. Gu and V. S. Sheng, “A robust regularization path algorithm
for ]-support vector classification,” IEEE Transactions on Neural
Networks and Learning Systems, 2016.
[42] B. Efron, T. Hastie, I. Johnstone, and R. Tibshirani, “Least angle
regression,” The Annals of Statistics, vol. 32, no. 2, pp. 407–499,
2004.
[43] C.-C. Chang and C.-J. Lin, “LIBSVM: a Library for support vector machines,” ACM Transactions on Intelligent Systems and
Technology (TIST), vol. 2, no. 3, article 27, 2011.
[44] A.-J. Yang and X.-Y. Song, “Bayesian variable selection for disease classification using gene expression data,” Bioinformatics,
vol. 26, no. 2, pp. 215–222, 2010.
[45] D. A. Notterman, U. Alon, A. J. Sierk, and A. J. Levine, “Transcriptional gene expression profiles of colorectal adenoma, adenocarcinoma, and normal tissue examined by oligonucleotide
arrays,” Cancer Research, vol. 61, no. 7, pp. 3124–3130, 2001.
[46] K. Shailubhai, H. H. Yu, K. Karunanandaa et al., “Uroguanylin
treatment suppresses polyp formation in the Apc(Min/+)
mouse and induces apoptosis in human colon adenocarcinoma
cells via cyclic GMP,” Cancer Research, vol. 60, no. 18, pp. 5151–
5157, 2000.
[47] J. W. P. Yam, K. W. Chan, and W.-L. W. Hsiao, “Suppression
of the tumorigenicity of mutant p53-transformed rat embryo
fibroblasts through expression of a newly cloned rat nonmuscle
myosin heavy chain-B,” Oncogene, vol. 20, no. 1, pp. 58–68, 2001.
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Long non-coding RNA BCYRN1 exerts an oncogenic role in colorectal cancer through regulating miR-204-3p/KRAS axis
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Yang et al. Cancer Cell Int (2020) 20:453
https://doi.org/10.1186/s12935-020-01543-x Yang et al. Cancer Cell Int (2020) 20:453
https://doi.org/10.1186/s12935-020-01543-x Cancer Cell International Cancer Cell International Open Access Long non‑coding RNA BCYRN1 exerts
an oncogenic role in colorectal cancer
by regulating the miR‑204‑3p/KRAS axis Liu Yang1, Yinan Zhang1, Jun Bao2* and Ji‑Feng Feng2* Liu Yang1, Yinan Zhang1, Jun Bao2* and Ji‑Feng Feng2* © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing,
adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and
the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material
in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material
is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the
permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativeco
mmons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/
zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract r findings suggest that BCYRN1 plays a carcinogenic role in CRC by regulating the miR-204-3p/KRAS axis Conclusions: Our findings suggest that BCYRN1 plays a carcinogenic role in CRC by regulating the m *Correspondence: baojun70s@126.com; fengjifeng_jsch@sohu.com
2 Department of Chemotherapy, The Affiliated Cancer Hospital of Nanjing
Medical University & Jiangsu Cancer Hospital & Jiangsu Institute of Cancer
Research, No. 42 Baiziting, Nanjing, China
Full list of author information is available at the end of the article *Correspondence: baojun70s@126.com; fengjifeng_jsch@sohu.com
2 Department of Chemotherapy, The Affiliated Cancer Hospital of Nanjing
Medical University & Jiangsu Cancer Hospital & Jiangsu Institute of Cancer
Research, No. 42 Baiziting, Nanjing, China
Full list of author information is available at the end of the article Abstract Background: It has been well documented that long non-coding RNAs (lncRNAs) regulate numerous characteristics
of cancer, including proliferation, migration, metastasis, apoptosis, and even metabolism. LncRNA BCYRN1 (BCYRN1)
is a newly identified brain cytoplasmic lncRNA with 200 nucleotides that was discovered to be highly expressed in
tumour tissues, including those of hepatocellular carcinoma, gastric cancer and lung cancer. However, the roles of
BCYRN1 in colorectal cancer (CRC) remain obscure. This study was designed to reveal the role of BCYRN1 in the occur‑
rence and progression of CRC. Methods: RT-PCR was used to detect the expression level of BCYRN1 in tumour tissues and CRC cell lines. BCYRN1
was knocked down in CRC cells, and cell proliferation changes were evaluated by cell counting kit-8 (CCK-8),
5-ethynyl-2′-deoxyuridine (EdU), and Ki-67 and proliferating cell nuclear antigen (PCNA) expression assays. Cell
migration and invasion changes were evaluated by wound healing, Transwell and invasion-related protein expres‑
sion assays. Flow cytometry analysis was used to assess whether BCYRN1 regulates the apoptosis of CRC cells. The
dual luciferase reporter gene detects the competitive binding of BCYRN1 to miR-204-3p. In vivo experiments were
performed to evaluate the effect of BCYRN1 on tumour development. TargetScan analysis and dual luciferase reporter
gene assays were applied to detect the target gene of miR-204-3p. Rescue experiments verified that BCYRN1 affects
CRC by regulating the effect of miR-204-3p on KRAS. Results: We found that compared with normal tissues and human intestinal epithelial cells (HIECs), CRC tumour
tissues and cell lines had significantly increased BCYRN1 levels. We further determined that knockdown of BCYRN1
inhibited the proliferation, migration, and invasion and promoted the apoptosis of CRC cells. In addition, bioinfor‑
matics analysis and dual luciferase reporter assay showed that BCYRN1 served as a competitive endogenous RNA
(ceRNA) to regulate the development of CRC through competitively binding to miR-204-3p. Further studies proved
that overexpression of miR-204-3p reversed the effects of BCYRN1 on CRC. Next, TargetScan analysis and dual lucif‑
erase reporter assay indicated that KRAS is a target gene of miR-204-3p and is negatively regulated by miR-204-3p. A
series of rescue experiments showed that BCYRN1 affected the occurrence and development of CRC by regulating
the effects of miR-204-3p on KRAS. In addition, tumorigenesis experiments in a CRC mouse model confirmed that
BCYRN1 downregulation effectively inhibited tumour growth. Experimental human tissues and animalsh Recently, epigenetic changes have been reported to
play important roles in the occurrence and progression
of malignancies [6, 7]. LncRNAs are a class of transcripts
with lengths greater than 200 nucleotides and have lim-
ited or non-protein coding potential [8–10]. Various
studies have demonstrated that lncRNAs participate in
the multilevel regulation of gene expression by target-
ing microRNAs (miRNAs), mRNAs, and even proteins
[11, 12]. Furthermore, growing evidence has proven that
lncRNAs regulate multiple characteristics of cancer, such
as proliferation, migration, apoptosis, and even metas-
tasis [13, 14]. BCYRN1 is a brain cytoplasmic lncRNA
that is newly identified and activated by c-MYC [15]. It
has been reported that BCYRN1 is highly expressed in
various tumour tissues [16], such as hepatocellular carci-
noma [17], gastric cancer [18], and lung cancer [15] but is
expressed at lower levels in healthy control (HC) tissues. In addition, BCYRN1 plays an important role in inhibit-
ing smooth muscle differentiation and vascular devel-
opment in the cardiovascular system [19, 20]. However,
little is known about the role of BCYRN1 in CRC, which
aroused our great interest. The research tissues of CRC patients and healthy controls
were obtained from The Affiliated Cancer Hospital of
Nanjing Medical University, and all patients signed writ-
ten consent forms. Tissue debris was immediately frozen
in liquid nitrogen after surgery and stored at − 80 °C. All animal experiments were designed according to the
standards of the Guide for the Care and Use of Labora-
tory Animals (NIH, 8th edition, 2011). The Ethics Com-
mittee of The Affiliated Cancer Hospital of Nanjing
Medical University approved the present research. Keywords: lncRNA BCYRN1, miR-204-3p, KRAS, Colorectal cancer Keywords: lncRNA BCYRN1, miR-204-3p, KRAS, Colorectal cancer other hand, lncRNAs competitively bind to miRNAs as
ceRNAs, resulting in the inability of the miRNAs to com-
bine with their corresponding mRNAs, thus regulating
gene expression and function [11, 26, 27].h Background Colorectal cancer (CRC) has a high incidence and mor-
tality worldwide; specifically, it ranks third in cancer
morbidity and second in cancer mortality globally [1, 2]. CRC has become one of the most common and danger-
ous malignancies in China [3]. The mainstay treatment
for CRC is surgery, chemotherapy, radiotherapy and
other comprehensive therapies, but the 5-year survival
rate has not changed significantly in recent decades and is
still poor [4, 5]. Therefore, delivering high-efficiency CRC
diagnosis and treatment methods should be the aim of
all physicians in internal medicine research. The present
study aims to provide an up-to-date molecular mecha-
nism of CRC occurrence. The present study aimed to determine the function and
underlying mechanism of BCYRN1 in CRC progression. Our research determined that BCYRN1 plays an onco-
genic role in CRC by regulating the miR-204-3p/KRAS
axis, revealing a novel mechanism for the occurrence and
development of CRC and providing a novel therapeutic
and prognostic target for CRC. Materials and methods Experimental human tissues and animalsh Cell culture Four human CRC cell lines, LoVo, HCT116, SW480,
and SW620 and HIECs, were purchased from American
Type Culture Collection (ATCC; Manassas, VA, USA)
and cultured in DMEM (Gibco, Grand Island, NY, USA)
containing 10% foetal bovine serum (FBS; Gibco, Grand
Island, NY, USA), 100 IU/mL penicillin and 1 × 105 μg/
mL streptomycin in a humidified atmosphere containing
5% CO2 at 37 °C. MiRNAs are a group of small, non-coding RNAs with
nucleotides of approximately 22 and are identified as
negative regulators [21, 22]. miRNAs can act as targets of
lncRNAs and participate in the regulation of gene expres-
sion. Specifically, miRNA is involved in gene expression
by binding to the complementary sequence location of
the 3′-untranslated region (3′-UTR) of target mRNA to
disturb the structural stability and translation of mRNAs
[23, 24]. Increasing evidence has demonstrated that miR-
NAs play crucial roles in cancer, cardiovascular disease
and other diseases [22, 25] by regulating relative cell pro-
liferation, migration, invasion and apoptosis [23]. On the © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing,
adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and
the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material
in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material
is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the
permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativeco
mmons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/
zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Yang et al. Cancer Cell Int (2020) 20:453 Page 2 of 13 Flow cytometric analysis Flow cytometric analysis was designed to evaluate
whether BCYRN1 regulated the apoptosis of CRC cells. Consistently, cells were seeded into a 6-well plate. The
cells were collected and stained with annexin V-FITC
(Beyotime Biotechnology, Shanghai, China) and propid-
ium iodide (PI) reagent after 48 h transfection according
to the manufacturer’s instructions. Then, cell apoptosis
was determined by flow cytometry (BD FACSCalibur
cytometer, Becton Dickinson, San Jose, CA, USA). Cell transfection
hRNA BCYRN shRNA-BCYRN1 (sh-BCYRN1), shRNA-KRAS (sh-
KRAS) and corresponding shRNA negative control (sh-
NC) transfection were performed using Lipofectamine
3000 reagent (Invitrogen, Carlsbad, CA, USA) according
to the manufacturer’s instructions. In addition, RNAifec-
tin™ transfection reagent, NC, miR-204-3p mimic and
miR-204-3p inhibitor were obtained from Applied Bio-
logical Materials Inc. (Richmond, BC, Canada). HIECs
and CRC cells were cultured in 6-well plates for transfec-
tion, and the efficiency of transfection was determined by
qRT-PCR. Yang et al. Cancer Cell Int (2020) 20:453 Yang et al. Cancer Cell Int (2020) 20:453 Page 3 of 13 In vivo experiments
Th
d
i The nude mice were injected subcutaneously with CRC
cells (approximately 106 cells) into the right flanks. Then,
sh-NC or sh-BCYRN1 was directly injected into the mice
to knockdown BCYRN1. Tumour size was measured
every 5 days for a total of 6 measurements. At the end
of the experiments, mice were humanely sacrificed with
overdose anaesthesia for the collection of tumours. Each
tumour was weighed to evaluate the effects of BCYRN1
on the development of tumours. Evaluation of cell migration and invasion g
Cell migration and invasion were assessed with the
wound healing assay, Transwell assay and invasion-
related protein expression. For the wound healing assay,
cells were seeded into a 6-well plate. A sterile 1-mL
pipette tip was used to scratch the bottom of the plate to
form a gap. The images of cell migration were captured
at 0 h and 48 h after transfection, respectively, with an
inverted microscope (Axio Vert. A1, Zeiss, Oberkochen,
Germany). The scratch distance at 48 h was subtracted
from the scratch distance at 0 h to calculate the per-
centage closure of the wound [29]. The average distance
of migration reflects the cell migration capacity. For the
Transwell assay, cells were plated into the no FBS medium
in the upper chamber of a 12-well Transwell with an Dual luciferase reporter assayh p
y
The partial sequences of BCYRN1 and the 3′-untrans-
lated region (3′-UTR) of KRAS containing the puta-
tive binding sites of miR-204-3p were synthesized and
inserted into a luciferase reporter vector plasmid (GeneP-
harma, Shanghai, China). According to the instructions,
firefly luciferase reporter plasmids and equal amounts of
miR-204-3p mimics or NC mimics were transfected into
cells. Then, the relative luciferase activities were detected
by the Dual-Luciferase Reporter Assay System (Promega,
Madison, WI, USA) on a Luminometer 20/20n (Turmer
Biosystems, Sunnyvale, CA, USA) after transfection for
48 h. Renilla luciferase activity was employed as an inter-
nal control for cellular density and transfection efficiency. RNA extraction and qRT‑PCR 8-μm pore size (Merck kGaA, Darmstadt, Germany). FBS
medium (10%) was added to the lower chamber. After
24 h, the cells that migrated to the submembrane sur-
face were stained with crystal violet. Stained cells were
counted in 6 randomly selected regions. Total RNA was extracted from human tissues, mouse
tissues and cells with TRIzol reagent (Life Technologies,
Gaithersburg, MD, USA). Reverse transcription experi-
ments were performed with PrimeScript® RT reagent
Kits (Takara, Otsu, Shiga, Japan) and a StepOnePlus™
Real-Time PCR System (Applied Biosystems, Foster
City, CA, USA). Next, quantitative reactions were per-
formed with SYBR Green RT-PCR (Takara Biotechnol-
ogy Co., Ltd., Tokyo, Japan) using the StepOnePlus™
system. GAPDH and U6 snRNA were used as the internal
controls. Evaluation of cell proliferation p
Cell proliferation was detected by cell counting kit-8
(CCK-8) assay, 5-ethynyl-2′-deoxyuridine (EdU) incor-
poration test, and nucleoprotein Ki-67 and proliferating
cell nuclear antigen (PCNA) expression analysis. First, for
the CCK-8 experiment, cells were seeded into a 96-well
plate with 10% FBS DMEM for 24 h. Then, the medium
was changed with serum-free DMEM for CCK-8 kit
(Beyotime Biotechnology, Shanghai, China) detection. A microplate reader (Model ELX800, BioTek, Vermont,
USA) was used to determine the absorbance at 450 nm,
which reflects cell viability and proliferation. Second,
cells were seeded into a 24-well plate for EdU assessment. After 48 h of transfection, DNA synthesis was examined
with an EdU incorporation assay (Guangzhou RiboBio,
Guangzhou, China). The proportion of EdU-positive
cells was analysed by fluorescence microscopy (DP70,
Olympus Optical, Tokyo, Japan). Finally, cell prolifera-
tion was evaluated by Ki-67 and PCNA expression. Ki-67
is a nucleoprotein that is a marker of tumour prolifera-
tion [28]. PCNA acts on chromatin and participates in all
aspects of the DNA replication chain [25]. The expres-
sion levels of Ki-67 and PCNA can be used to evaluate
the status of cell proliferation. Western blot analysis Cyclooxygenase-2 (Cox-2) is an enzyme complex that
plays significant roles in metastasis and invasion of malig-
nancies [30]. The matrix metalloproteinase (MMP) fam-
ily, especially the gelatinases MMP-2 and MMP-9, are
recognized as markers of tumour migration and invasion
[31]. The expression of Cox-2, MMP-2 and MMP-9 can
reflect the status of cell migration and invasion. The pro-
tein expression levels of Ki-67, PCNA, Cox-2, MMP-2,
MMP-9, Bax, Bcl-2, cleaved caspase-3, cleaved caspase-9
and KRAS were detected by Western blot analysis. Total
protein was extracted, and equal amounts of protein
(30 μg) were separated by SDS-PAGE and then trans-
ferred to PVDF membranes. Protein bands were visual-
ized with the Enhanced Chemiluminescence Detection
Kit (Thermo Fisher Scientific, Rockford, IL, USA). All
primary antibodies were obtained from Abcam (Cam-
bridge, MA, USA). The dilution of PCNA antibody was
1:5,000, and the dilution ratio of other primary antibodies
was 1:1,000. BCYRN1 is highly expressed in the tissues and cell lines
of CRC In our preliminary study, we noted that BCYRN1 was
highly expressed in CRC tissues (Fig. 1a). Then, we exam-
ined the expression levels of BCYRN1 in HIECs and four
types of human CRC cell lines. Consistently, BCYRN1
expression was significantly increased in CRC cell lines
(Fig. 1b). These data aroused our interest, as they sug-
gested that BCYRN1 levels may be closely related to the
development of CRC. Statistical analysis Haematoxylin–eosin (HE) staining and terminal deoxy-
nucleotidyl transferase (TdT)-mediated dUTP nick-end
labelling (TUNEL) staining were performed to detect
cancer cell apoptosis in mouse tumour tissues. Briefly,
mouse tumour tissues were prefixed, and the paraffin-
embedded sections were stained with haematoxylin–
eosin and TUNEL (containing 2 μL TdT enzyme, 48 μL
Fluorescent labelling solution and 50 μL TUNEL detec-
tion solution) according to the manufacturer’s instruc-
tions. The images were obtained with an Olympus BX51
microscope (Olympus, Tokyo, Japan) coupled with an
Olympus DP70 digital camera. Experiments were conducted in a randomized, double-
blinded situation. Data are expressed as the mean ± SD. Student’s unpaired t-test was used to compare the differ-
ences between two groups. One-way or two-way ANOVA
followed by post hoc Bonferroni test was employed for
multiple comparisons. Statistical significance was consid-
ered as *P < 0.05, **P < 0.01, and ***P < 0.001. Immunohistochemistry Tumours were obtained, fixed in 4% paraformaldehyde,
embedded in paraffin, and then cut into 4 μm sections. After deparaffinization and blocking, the sections were
incubated with primary anti-Ki-67 (1:100; Abcam; Cam-
bridge, MA, USA) overnight at 4 °C. The immunofluo-
rescence images were captured with an Olympus BX51
microscope (Olympus, Tokyo, Japan) after incubat-
ing horseradish peroxidase-conjugated goat anti-rabbit
antibody. Yang et al. Cancer Cell Int (2020) 20:453 Yang et al. Cancer Cell Int (2020) 20:453 Page 4 of 13 Page 4 of 13 Effects of miR‑204‑3p on CRC cell proliferation, migration,
invasion and apoptosis We then studied the role of miR-204-3p in the regula-
tion of CRC progression. First, we used miR-204-3p
mimics to treat LoVo and SW480 cells. The overex-
pression efficiency of miR-204-3p is shown in Fig. 5a. Then, we found that miR-204-3p upregulation appar-
ently inhibited cell proliferation, as demonstrated by
CCK-8 and EdU assays (Fig. 5b, c). In addition, flow
cytometry analysis revealed that miR-204-3p overex-
pression accelerated cell apoptosis (Fig. 5d). Consist-
ently, CRC cell migration was obviously decreased by
miR-204-3p mimics (Fig. 5e). These results concluded
that miR-204-3p upregulation inhibits CRC cell prolif-
eration, migration and invasion while promoting CRC
cell apoptosis. (See figure on next page.)
Fig. 2 The effects of BCYRN1 knockdown on CRC cell proliferation and apoptosis. a BCYRN1 levels in LoVo and SW480 cells. b CRC cell proliferation
was detected with CCK-8 assay. c The fluorescence images and data statistics chart of the EdU assay. d The expression of PCNA and Ki-67. e Original
images and histogram of the flow cytometry assay. f The expression of apoptosis-related proteins in LoVo cells and SW480 cells. Values are the
mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. sh-NC. n = 3 per group Knockdown of BCYRN1 inhibits the proliferation
and promotes the apoptosis of CRC cells To further illuminate the roles of BCYRN1 in CRC, a
series of experiments were designed in LoVo and SW480
cells. First, we used sh-BCYRN1 to downregulate endog-
enous BCYRN1, and the knockdown efficiency is shown
in Fig. 2a. CCK-8 and EdU assays showed that downregu-
lation of BCYRN1 evidently decreased the proliferation
of LoVo and SW480 cells (Fig. 2b, c). Furthermore, knock-
down of BCYRN1 obviously inhibited the expression of
proliferation-related proteins, such as PCNA and Ki-67
(Fig. 2d). Subsequently, we detected cell apoptosis by flow
cytometry analysis. The results showed that knockdown
of BCYRN1 significantly increased apoptosis of LoVo and Fig. 1 LncRNA BCYRN1 is highly expressed in the tissues and cell lines of CRC. a BCYRN1 levels in HC tissues and CRC tumour tissues. Data from 15
HC people and 15 GC patients. b BCYRN1 levels in HIECs and CRC cell lines. Values are the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. HCs or
HIECs. n = 3 per group Yang et al. Cancer Cell Int (2020) 20:453 Yang et al. Cancer Cell Int (2020) 20:453 Page 5 of 13 These data proved that miR-204-3p is a target gene of
BCYRN1 and is negatively regulated by BCYRN1. These data proved that miR-204-3p is a target gene of
BCYRN1 and is negatively regulated by BCYRN1. SW480 cells (Fig. 2e). In addition, several pro-apoptosis
proteins, including Bax, cleaved caspase-3 and cleaved
caspase-9 [32, 33], were highly expressed in LoVo and
SW480 cells when they were treated with sh-BCYRN1. In contrast, the expression of Bcl-2, an inhibitor of cell
apoptosis [33], was abolished in the sh-BCYRN1 group
(Fig. 2f). These results indicated that BCYRN1 knock-
down reduced proliferation while promoting the apopto-
sis of CRC cells. KRAS is a target gene of miR‑204‑3p
f
h
l
d h
d
l We further explored the underlying mechanism of miR-
204-3p in CRC. TargetScan, a miRNA target prediction
database, was used to predict the downstream gene of
miR-204-3p. We found that KRAS was a possible target
gene of miR-204-3p, and the predicted position of the
combination was in the 310–317 region of the KRAS
3′-UTR (Fig. 6a). It is generally known that KRAS is a
crucial carcinogenic factor [34]. Moreover, the results of
dual luciferase reporter analysis confirmed that KRAS
was indeed the target gene of miR-204-3p (Fig. 6b). More
significantly, overexpression of miR-204-3p inhibited
the expression of KRAS mRNA and protein in LoVo and
SW480 cells (Fig. 6c, d). We next investigated the rela-
tionship between KRAS levels and CRC progression. We
noted that KRAS was highly expressed in CRC tumour
tissues and cell lines (Fig. 6e, f). These results indicated
that KRAS is a target gene of miR-204-3p and is nega-
tively regulated by miR-204-3p. Downregulation of BCYRN1 decreases the migration
and invasion of CRC cellsf Next, we investigated the effects of BCYRN1 expression
on CRC cell migration and invasion. Both wound heal-
ing and Transwell assays revealed that the migration and
invasion of LoVo and SW480 cells were effectively sup-
pressed by sh-BCYRN1 transfection (Fig. 3a, b). Con-
sistently, migration- and invasion-related proteins, such
as Cox-2, MMP-2 and MMP-9, were inhibited in LoVo
and SW480 cells treated with sh-BCYRN1 (Fig. 3c). These results elucidated that BCYRN1 levels were closely
related to the migration and invasion properties of CRC
cells. miR‑204‑3p is a target gene of BCYRN1 A recent report showed that lncRNAs function as ceR-
NAs to competitively bind to miRNAs [11]. To illumi-
nate the potential regulatory mechanism through which
BCYRN1 affects CRC progression, we performed bioin-
formatic analysis with LncBase Predicted V.2 and found
that BCYRN1 possesses a putative binding site for miR-
204-3p (Fig. 4a). We further retrieved potential targets
of BCYRN1 in different databases. To determine the
interaction of BCYRN1 and miR-204-3p, we performed
dual luciferase reporter analysis and found a signifi-
cant reduction in the luciferase activity of BCYRN1
WT when LoVo or SW480 cells were transfected with
miR-204-3p mimics. However, there was no notable
difference in the luciferase activity of BCYRN1 Mut
between the miR-204-3p mimic group and the negative
control (NC) group (Fig. 4b). Accordingly, downregula-
tion of BCYRN1 distinctly increased the expression of
miR-204-3p in LoVo and SW480 cells (Fig. 4c). In addi-
tion, qRT-PCR results revealed that miR-204-3p was
significantly reduced in CRC tumour tissues and cell
lines, especially in LoVo and SW480 cells (Fig. 4d, e). Effects of the BCYRN1/miR‑204‑3p/KRAS axis on CRC cell
proliferation, migration, invasion and apoptosis Effects of the BCYRN1/miR‑204‑3p/KRAS axis on CRC cell
proliferation, migration, invasion and apoptosis
To understand the effects of the BCYRN1/miR-204-3p/
KRAS axis on CRC progression, we designed a series proliferation, migration, invasion and apoptosis
To understand the effects of the BCYRN1/miR-204-3p/
KRAS axis on CRC progression, we designed a series (i
g
p g )
Fig. 2 The effects of BCYRN1 knockdown on CRC cell proliferation and apoptosis. a BCYRN1 levels in LoVo and SW480 cells. b CRC cell proliferation
was detected with CCK-8 assay. c The fluorescence images and data statistics chart of the EdU assay. d The expression of PCNA and Ki-67. e Original
images and histogram of the flow cytometry assay. f The expression of apoptosis-related proteins in LoVo cells and SW480 cells. Values are the
mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. sh-NC. n = 3 per group Yang et al. Cancer Cell Int (2020) 20:453 Page 6 of 13 Yang et al. Cancer Cell Int (2020) 20:453 Page 7 of 13 Fig. 3 The effects of BCYRN1 knockdown on CRC cell migration and invasion. a The wound-healing assay showing the migration of CRC cells. b
Images of the Transwell assay and histogram. c The expression of migration-related proteins determined by Western blot analysis. miR‑204‑3p is a target gene of BCYRN1 Values are the
mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. sh-NC. n = 3 per group Fig. 3 The effects of BCYRN1 knockdown on CRC cell migration and invasion. a The wound-healing assay showing the migration of CRC cells. b
Images of the Transwell assay and histogram. c The expression of migration-related proteins determined by Western blot analysis. Values are the
mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. sh-NC. n = 3 per group Fig. 3 The effects of BCYRN1 knockdown on CRC cell migration and invasion. a The wound-healing assay showing the migration of CRC cells. b
Images of the Transwell assay and histogram. c The expression of migration-related proteins determined by Western blot analysis. Values are the
mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. sh-NC. n = 3 per group data indicated that BCYRN1 worsens CRC progression
at least in part by inhibiting miR-204-3p levels and pro-
moting KRAS expression. of rescue experiments using miR-204-3p inhibitor, sh-
KRAS and sh-BCYRN1 in LoVo and SW480 cells. The
knockdown efficiency of miR-204-3p and KRAS in
CRC cells treated with sh-BCYRN1 is shown in Fig. 7a. Then, we found that the miR-204-3p inhibitor signifi-
cantly facilitated the proliferation of LoVo and SW480
cells treated with sh-BCYRN1, whereas sh-KRAS effec-
tively reversed these effects (Fig. 7b, c). Furthermore,
the miR-204-3p inhibitor abolished the apoptosis of sh-
BCYRN1-treated CRC cells, whereas sh-KRAS relieved
these phenomena (Fig. 7d). In addition, the migra-
tion of sh-BCYRN1-treated CRC cells was evidently
increased in the miR-204-3p inhibitor-treated group,
while sh-KRAS reversed these roles (Fig. 7e). These of rescue experiments using miR-204-3p inhibitor, sh-
KRAS and sh-BCYRN1 in LoVo and SW480 cells. The
knockdown efficiency of miR-204-3p and KRAS in
CRC cells treated with sh-BCYRN1 is shown in Fig. 7a. Then, we found that the miR-204-3p inhibitor signifi-
cantly facilitated the proliferation of LoVo and SW480
cells treated with sh-BCYRN1, whereas sh-KRAS effec-
tively reversed these effects (Fig. 7b, c). Furthermore,
the miR-204-3p inhibitor abolished the apoptosis of sh-
BCYRN1-treated CRC cells, whereas sh-KRAS relieved
these phenomena (Fig. 7d). In addition, the migra-
tion of sh-BCYRN1-treated CRC cells was evidently
increased in the miR-204-3p inhibitor-treated group,
while sh-KRAS reversed these roles (Fig. 7e). These Knockdown of BCYRN1 inhibits tumour growth To evaluate the therapeutic potential of BCYRN1 in CRC
patients, we performed BCYRN1 knockdown by using
sh-BCYRN1 in CRC model mice. Encouragingly, knock-
down of BCYRN1 significantly decreased the tumour
volume and weight of CRC model mice (Fig. 8a–c). HE
staining, immunohistochemical staining and TUNEL
staining showed that downregulation of BCYRN1 inhib-
ited Ki-67 expression and promoted cancer cell apopto-
sis (Fig. 8d). More importantly, sh-BCYRN1 treatment Yang et al. Cancer Cell Int (2020) 20:453 Page 8 of 13 Fig. 4 The target relationship between BCYRN1 and miR-204-3p. a The predicted location of the miR-204-3p combination according to LncBase
Predicted V.2 analysis. b The results of the dual luciferase reporter assay. c The effects of BCYRN1 knockdown on miR-204-3p expression in LoVo and
SW480 cells. d miR-204-3p levels in CRC tumour tissues and HC tissues. Data from 15 HC people and 15 GC patients. e miR-204-3p levels in HIECs
and CRC cell lines. Values are the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. NC mimics, HC group, HIECs group, or sh-NC. n = 3 per group Fig. 4 The target relationship between BCYRN1 and miR-204-3p. a The predicted location of the miR-204-3p combination according to LncBase
Predicted V.2 analysis. b The results of the dual luciferase reporter assay. c The effects of BCYRN1 knockdown on miR-204-3p expression in LoVo and
SW480 cells. d miR-204-3p levels in CRC tumour tissues and HC tissues. Data from 15 HC people and 15 GC patients. e miR-204-3p levels in HIECs
and CRC cell lines. Values are the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. NC mimics, HC group, HIECs group, or sh-NC. n = 3 per group obviously promoted the expression of miR-204-3p and
decreased the expression of KRAS (Fig. 8e). These results
revealed that knockdown of BCYRN1 inhibited the
development of CRC. CRC needs to be explored urgently. Our current study
revealed an important new mechanism of the occur-
rence of CRC and provided a novel idea for the effective
treatment of CRC, briefly, to control CRC progression by
blocking BCYRN1. LncRNAs have been shown to play vital roles in the
regulation of cancer progression [13, 14], and the main
functions of lncRNAs are recruiting chromatin modi-
fication complexes for transcriptional regulation [38]
and interacting with miRNAs, mRNAs, and/or proteins
for posttranscriptional regulation [11, 12]. Knockdown of BCYRN1 inhibits tumour growth It has been
proven that one of most common and crucial roles of
lncRNA is their functions as a “miRNA sponges” [27,
39]. This means that lncRNA can be used as a bait to
bind with a specific miRNA, resulting in the inability of
the miRNA to combine with its corresponding mRNA. BCYRN1 is a newly identified lncRNA with similar char-
acteristics [15]. Previous studies have revealed that miR-
NAs, such as miR-125-5p, miR-149 and miR-490-3p,
act as targets of BCYRN1 in hepatocellular carcinoma, Discussion Cancer mortality remains an enormous public health
challenge on a global scale [35]. Although new advances
have been made in the genomic changes in most differ-
ent cancers [36], the molecular mechanisms underlying
the development of CRC are poorly understood. Moreo-
ver, compared with patients with other malignancies, a
large number of CRC patients have distant metastasis at
the early stage of diagnosis, which leads to a low 5-year
survival rate of approximately 10%-20% [37]. In addi-
tion, therapeutic methods are very limited in advanced
CRC patients and mainly involve combinations of toxic
drugs, such as 5-fluorouracil, irinotecan and/or oxalipl-
atin, for chemotherapy. Therefore, the molecular mech-
anism involved in the occurrence and development of Yang et al. Cancer Cell Int (2020) 20:453 Page 9 of 13 Fig. 5 The effects of miR-204-3p overexpression on CRC cell proliferation, migration, invasion and apoptosis. a miR-204-3p expression in LoVo and
SW480 cells. b CRC cell proliferation was evaluated with CCK-8 assay and EdU assay (c). d LoVo and SW480 cell apoptosis was determined with
flow cytometry assay. e Original images and histogram of the Transwell assay. Values are the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. NC
mimics group. n = 3 per group Fig. 5 The effects of miR-204-3p overexpression on CRC cell proliferation, migration, invasion and apoptosis. a miR-204-3p expression in LoVo and
SW480 cells. b CRC cell proliferation was evaluated with CCK-8 assay and EdU assay (c). d LoVo and SW480 cell apoptosis was determined with
flow cytometry assay. e Original images and histogram of the Transwell assay. Values are the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. NC
mimics group. n = 3 per group Fig. 5 The effects of miR-204-3p overexpression on CRC cell proliferation, migration, invasion and apoptosis. a miR-204-3p expression in LoVo and
SW480 cells. b CRC cell proliferation was evaluated with CCK-8 assay and EdU assay (c). d LoVo and SW480 cell apoptosis was determined with
flow cytometry assay. e Original images and histogram of the Transwell assay. Values are the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. NC
mimics group. n = 3 per group human glioma and lung cancer, respectively [17, 40, 41]. Recently, the relationship between BCYRN1 and CRC
has been gradually observed. Gu et al. Discussion first found that
BCYRN1 levels were closely related to the occurrence,
development and prognosis of CRC, and microarray
bioinformatics analysis confirmed that the possible tar-
get of BCYRN1 was the NRP3 gene [42]. Moreover, Yu
JH and Chen Y proved that BCYRN1 levels were signifi-
cantly increased in tumour tissues and cell lines (SW620)
of CRC by a series of well-designed in vitro experiments
[43]. However, it remains obscure whether BCYRN1 can control miRNAs to regulate the progression of CRC. Our
present study indicated that BCYRN1 is involved in the
occurrence and development of CRC through specific
binding to a new target miRNA: miR-204-3p. We found
that BCYRN1 was highly expressed, while the expression
levels of miR-204-3p were significantly reduced in the tis-
sues and cell lines of CRC compared to those of controls. In addition, knockdown of BCYRN1 distinctly increased
miR-204-3p expression. These findings provide new
insights into the mechanism of CRC progression and the
treatment of CRC. Yang et al. Cancer Cell Int (2020) 20:453 Page 10 of 13 Fig. 6 KRAS is a target gene of miR-204-3p. a The predicted location of the KRAS combination according to TargetScan. b The results of the dual
luciferase reporter assay. c, d The effects of miR-204-3p overexpression on KRAS mRNA and protein expression in LoVo and SW480 cells. e KRAS
mRNA levels in tumour tissues and HC tissues. Data from 15 HCs and 15 GC patients. f KRAS mRNA levels in HIECs and CRC cell lines. Values are the
mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. NC mimics group, HC group or HIECs group. n = 3 per group Fig. 6 KRAS is a target gene of miR-204-3p. a The predicted location of the KRAS combination according to TargetScan. b The results of the dual
luciferase reporter assay. c, d The effects of miR-204-3p overexpression on KRAS mRNA and protein expression in LoVo and SW480 cells. e KRAS
mRNA levels in tumour tissues and HC tissues. Data from 15 HCs and 15 GC patients. f KRAS mRNA levels in HIECs and CRC cell lines. Values are the
mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. NC mimics group, HC group or HIECs group. n = 3 per group Fig. 7 The effects of the BCYRN1/miR-204-3p/KRAS axis on CRC progression in vitro. Discussion a miR-204-3p expression in LoVo and SW480 cells with BCYRN1
knockdown. b, c The effects of proliferation determined by CCK-8 assay and EdU analysis. d Original images and histogram of the flow cytometry
assay. e Cell migration detected by Transwell assay. Values are the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. NC inhibitor group. #P < 0.05,
##P < 0.01, and ###P < 0.001 vs. the miR-204-3p inhibitor group. n = 6 per group Fig. 7 The effects of the BCYRN1/miR-204-3p/KRAS axis on CRC progression in vitro. a miR-204-3p expression in LoVo and SW480 cells with BCYRN1
knockdown. b, c The effects of proliferation determined by CCK-8 assay and EdU analysis. d Original images and histogram of the flow cytometry
assay. e Cell migration detected by Transwell assay. Values are the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. NC inhibitor group. #P < 0.05,
##P < 0.01, and ###P < 0.001 vs. the miR-204-3p inhibitor group. n = 6 per group Fig. 7 The effects of the BCYRN1/miR-204-3p/KRAS axis on CRC progression in vitro. a miR-204-3p expression in LoVo and SW480 cells with BCYRN1
knockdown. b, c The effects of proliferation determined by CCK-8 assay and EdU analysis. d Original images and histogram of the flow cytometry
assay. e Cell migration detected by Transwell assay. Values are the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. NC inhibitor group. #P < 0.05,
##P < 0.01, and ###P < 0.001 vs. the miR-204-3p inhibitor group. n = 6 per group Yang et al. Cancer Cell Int (2020) 20:453 Page 11 of 13 Fig. 8 Effects of BCYRN1 knockdown on tumour development in vivo. a Phenotype images of tumours. b The line graph of tumour volume. c
The bar chart of tumour weight. d Representative images and histograms of HE staining, immunohistochemical staining and TUNEL fluorescence
staining. e RNA expression levels of BCYRN1, miR-204-3p and KRAS in mouse tumour tissues. Values are the mean ± SD. *P < 0.05, **P < 0.01, and
***P < 0.001 vs. sh-NC. n = 3 per group Fig. 8 Effects of BCYRN1 knockdown on tumour development in vivo. a Phenotype images of tumours. b The line graph of tumour volume. c
The bar chart of tumour weight. Discussion d Representative images and histograms of HE staining, immunohistochemical staining and TUNEL fluorescence
staining. e RNA expression levels of BCYRN1, miR-204-3p and KRAS in mouse tumour tissues. Values are the mean ± SD. *P < 0.05, **P < 0.01, and
***P < 0.001 vs. sh-NC. n = 3 per group As mentioned above, miRNAs are involved in the regu-
lation of cancer [22, 23]. Interestingly, miRNAs can act
as tumour suppressor genes [44] or oncogenes [45] to
regulate the biological characteristics of cancer. Cui et al. reported that miR-204-3p acts on its potential target gene
fibronectin 1 (FN1) and inhibits its expression, thereby
decreasing the growth of hepatocellular carcinoma
tumour endothelial cells [46]. Recently, several studies
have reported the roles of miR-204-3p in ovarian cancer
[47], lung adenocarcinoma [48] and bladder cancer [49],
but the functions of miR-204-3p in CRC remain obscure. Our current research showed that miR-204-3p is a poten-
tial target gene of BCYRN1 and is negatively regulated by
BCYRN1. We further investigated whether miR-204-3p
overexpression inhibited CRC cell proliferation, migra-
tion and invasion while exacerbating CRC cell apoptosis. In addition, bioinformatics analysis and dual luciferase
reporter assay proved that KRAS was a potential target
gene of miR-204-3p. KRAS is an important oncogene
and a key tumour maintenance gene in many carcinomas
[34]. Then, we showed that KRAS was highly expressed
in CRC tissues and cell lines, and overexpression of miR-
204-3p eliminated this effect. These results indicated that
overexpression of miR-204-3p and downregulation of KRAS can be considered new approaches to the treat-
ment of CRC. In summary, these findings demonstrated that
BCYRN1 participates in the occurrence and develop-
ment of CRC. In terms of functional regulation, BCYRN1
promotes CRC progression at least in part by regulat-
ing the miR-204-3p/KRAS axis. Our study broadens the
current understanding of BCYRN1 function in CRC and
provides a novel therapeutic target for CRC. Availability of data and materials
Yes. 19. Wang YC, Chuang YH, Shao Q, et al. Brain cytoplasmic RNA 1 suppresses
smooth muscle differentiation and vascular development in mice. J Biol
Chem. 2018;293(15):5668–78. Acknowledgements
None. Acknowledgements
None. 12. Wang P, Xue Y, Han Y, et al. The STAT3-binding long noncoding
RNA lnc-DC controls human dendritic cell differentiation. Science. 2014;344(6181):310–3. Consent for publication 21. Ghildiyal M, Zamore PD. Small silencing RNAs: an expanding universe. Nat
Genet. 2009;10:94–108. All authors agree to publish. Ambros V. The functions of animal microRNAs. Nature. 2004;431:350 Author details
1 1 Department of Colorectal Surgery, The Affiliated Cancer Hospital of Nanjing
Medical University & Jiangsu Cancer Hospital & Jiangsu Institute of Cancer
Research, Nanjing, China. 2 Department of Chemotherapy, The Affiliated Can‑
cer Hospital of Nanjing Medical University & Jiangsu Cancer Hospital & Jiangsu
Institute of Cancer Research, No. 42 Baiziting, Nanjing, China. 25. Ren XS, Tong Y, Qiu Y, et al. MiR155-5p in adventitial fibroblasts-derived
extracellular vesicles inhibits vascular smooth muscle cell proliferation
via suppressing angiotensin-converting enzyme expression. J Extracell
Vesicles. 2019;9(1):1698795. 26. Necsulea A, Soumillon M, Warnefors M, et al. The evolution of
lncRNA repertoires and expression patterns in tetrapods. Nature. 2014;505(7485):635–40. Received: 4 June 2020 Accepted: 5 September 2020 Received: 4 June 2020 Accepted: 5 September 2020 27. Qu L, Ding J, Chen C, et al. Exosome-transmitted lncARSR promotes suni‑
tinib resiistance in renal cancer by acting as a competing endogenous
RNA. Cancer Cell. 2016;29(5):653–68. Competing interests 23. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004;116:281–97. The authors declare that they have no competing interests. 24. Lim LP, Lau NC, Garrett-Engele P, et al. Microarray analysis shows that
some microRNAs downregulate large numbers of target mRNAs. Nature. 2005;433:769–73. Authors’ contributions 13. Prensner JR, Iyer MK, Sahu A, et al. The long noncoding RNA SChLAP1
promotes aggressive prostate cancer and antagonizes the SWI/SNF
complex. Nat Genet. 2013;45:1392–8. Liu Yang conceived and designed the study. Liu Yang, Yinan Zhang, Jun Bao,
and Ji-Feng Feng performed the literature search and data extraction. Liu Yang
analysed the data and the manuscript. All authors read and approved the final
manuscript. 14. Hung CL, Wang LY, Yu YL, et al. A long noncoding RNA connects c-Myc to
tumor metabolism. Proc Natl Acad Sci USA. 2014;111(52):18697–702. 15. Hu T, Lu Y. BCYRN1, a c-MYC-activated long non-coding RNA, regulates
cell metastasis of non-small-cell lung cancer. Cancer Cell Int. 2015;15:36. Ethics approval and consent to participate This study was carried out based on the Ethics Committee of The Affiliated
Cancer Hospital of Nanjing Medical University. The clinical analysis was per‑
formed according to the principles of the Helsinki Declaration. 20. Zhang XY, Tang XY, Ma LJ, et al. Schisandrin B down-regulated lncRNA
BCYRN1 expression of airway smooth muscle cells by improving miR-150
expression to inhibit the proliferation and migration of ASMC in asth‑
matic rats. Cell Prolif. 2017;50(6):e12382. Funding 16. Booy EP, McRae EK, Koul A, et al. The long non-coding RNA BC200
(BCYRN1) is critical for cancer cell survival and proliferation. Mol Cancer. 2017;16(1):109. This study was funded by the National Natural Science Foundation of China
(81602145 to LY), Jiangsu Provincial Natural Science Foundation (BK20171509
to LY), Jiangsu Provincial Medical Youth Talent, The Project of Invigorating
Health Care through Science, Technology Education (QNRC2016649 to LY),
the China Postdoctoral Science Foundation (2018M632265 to LY), the Talents
Program of Jiangsu Cancer Hospital (YC201812 to LY), and the “333 Talents”
Program of Jiangsu Province (BRA2020390 to LY). 17. Ding S, Jin Y, Hao Q, et al. LncRNA BCYRN1/miR-490-3p/POU3F2, served
as a ceRNA network, is connected with worse survival rate of hepatocel‑
lular carcinoma patients and promotes tumor cell growth and metastasis. Cancer Cell Int. 2020;20:6. 18. Zhai H, Li Y. BCYRN1 is correlated with progression and prognosis in
gastric cancer. Biosci Rep. 2019. https://doi.org/10.1042/BSR20190505. 18. Zhai H, Li Y. BCYRN1 is correlated with progression and prognosis in
gastric cancer. Biosci Rep. 2019. https://doi.org/10.1042/BSR20190505. Conclusion 1. BCYRN1 is highly expressed in CRC tissues and cell
lines compared to normal controls. 2. Downregulation of BCYRN1 decreases the migration
and invasion of CRC cells. Knockdown of BCYRN1
promotes the apoptosis of CRC cells.f 3. miR-204-3p is a target gene of BCYRN1 and affects
CRC cell proliferation, migration, invasion and apop-
tosis. 4. KRAS is a target gene of miR-204-3p, and the
BCYRN1/miR-204-3p/KRAS axis affects the prolifer-
ation, migration, invasion and apoptosis of CRC cells. 5. Knockdown of BCYRN1 inhibits tumour growth. Page 12 of 13 Page 12 of 13 Page 12 of 13 Yang et al. Cancer Cell Int (2020) 20:453 Yang et al. Cancer Cell Int (2020) 20:453 References 28. Gates EDH, Lin JS, Weinberg JS, et al. Guiding the first biopsy in glioma
patients using estimated Ki-67 maps derived from MRI: conventional
versus advanced imaging. Neuro-Oncology. 2019;21(4):527–36. 1. Bray F, Ferlay J, Soerjomataram I, et al. Global cancer statistics 2018: GLO‑
BOCAN estimates of incidence and mortality worldwide for 36 cancers in
185 countries. CA Cancer J Clin. 2018;68(6):394–424. 1. Bray F, Ferlay J, Soerjomataram I, et al. Global cancer statistics 2018: GLO‑
BOCAN estimates of incidence and mortality worldwide for 36 cancers in
185 countries. CA Cancer J Clin. 2018;68(6):394–424. 29. Pan Z, Cai J, Lin J, et al. A novel protein encoded by circFNDC3B inhibits
tumor progression and EMT through regulating Snail in colon cancer. Mol
Cancer. 2020. https://doi.org/10.1186/s12943-020-01179-5. 185 countries. CA Cancer J Clin. 2018;68(6):394–424. 2. Arnold M, Sierra MS, Laversanne M, et al. Global patterns and trends in
colorectal cancer incidence and mortality. Gut. 2017;66(4):683–91. 2. Arnold M, Sierra MS, Laversanne M, et al. Global patterns and trends in
colorectal cancer incidence and mortality. Gut. 2017;66(4):683–91. 30. Nagaraju GP, EI-Rayes BF. Cyclooxygenase-2 in gastrointestinal malignan‑
cies. Cancer. 2019;125(8):1221–7. 3. Brenner H, Kloor M, Pox CP. Colorectal cancer. Lancet. 2014;383(9927):1490–502. 3. Brenner H, Kloor M, Pox CP. Colorectal cancer. Lancet. 31. La Rocca G, Pucci-Minafra I, Marrazzo A, et al. Zymographic detection
and clinical correlations of MMP-2 and MMP-9 in breast cancer sera. Br J
Cancer. 2004;90(7):1414–21. 4. Dekker E, Rex DK. Advances in CRC prevention: screening and surveil‑
lance. Gastroenterology. 2018;154(7):1970–84. 4. Dekker E, Rex DK. Advances in CRC prevention: screening and surveil‑
lance. Gastroenterology. 2018;154(7):1970–84. 5. Schreuders EH, Ruco A, Rabeneck L, et al. Colorectal cancer screening: a
global overview of existing programmes. Gut. 2015;64(10):1637–49. 5. Schreuders EH, Ruco A, Rabeneck L, et al. Colorectal cancer screening: a
global overview of existing programmes. Gut. 2015;64(10):1637–49. 32. Hu L, Chen M, Chen X, et al. Chemotherapy-induced pyroptosis is medi‑
ated by BAK/BAX-caspase-3-GSDME pathway and inhibited by 2-bromo‑
palmitate. Cell Death Dis. 2020;11(4):281. 6. Neureiter D, Jäger T, Ocker M, et al. Epigenetics and pancreatic cancer:
pathophysiology and novel treatment aspects. World J Gastroenterol. 2014;20:7830–48. 6. Neureiter D, Jäger T, Ocker M, et al. Epigenetics and pancreatic cancer:
pathophysiology and novel treatment aspects. World J Gastroenterol. 2014;20:7830–48. 33. Walensky LD. Targeting BAX to drug death directly. Nat Chem Biol. 2019;15:657–65. 7. Michalak EM, Burr ML, Bannister AJ, et al. References Long non-coding RNA BCYRN1 promotes
glycolysis and tumor progression by regulating the miR-149/PKM2 axis in
non-small-cell lung cancer. Mol Med Rep. 2020;21(3):1509–16. 49. Guo J, Zhao P, Liu Z, et al. MiR-2014-3p inhibited the proliferation of
bladder cancer cells via modulating lactate dehydrogenase-mediated
glycolysis. Front Oncol. 2019;9:1242. 42. Gu L, Lu L, Zhou D, et al. Long noncoding RNA BCYRN1 promotes the
proliferation of colorectal cancer cells via up-regulating NPR3 expression. Cell Physiol Biochem. 2018;48(6):2337–499. 43. Yu JH, Chen Y. Clinical significance of lncRNA BCYRN1 in colorectal
cancer and its role in cell metastasis. Eur Rev Med Pharmacol Sci. 2019;23(21):9371–8. 43. Yu JH, Chen Y. Clinical significance of lncRNA BCYRN1 in colorectal
cancer and its role in cell metastasis. Eur Rev Med Pharmacol Sci. 2019;23(21):9371–8. References The roles of DNA, RNA and
histone methylation in ageing and cancer. Nat Rev Mol Cell Biol. 2019;20(10):573–89. 7. Michalak EM, Burr ML, Bannister AJ, et al. The roles of DNA, RNA and
histone methylation in ageing and cancer. Nat Rev Mol Cell Biol. 2019;20(10):573–89. 34. Terhune PG, Phifer DM, Tosteson TD, et al. K-ras mutations in focal prolif‑
eration lesions of human pancreas. Cancer Epidemiol Biomarkers Prev. 1998;7(6):515–21. 8. Cech TR, Steitz JA. The noncoding RNA revolution-trashing old rules to
forge new ones. Cell. 2014;157:77–94. 35. Siegel RL, Miller KD, Jemal A, et al. Cancer statistics. CA Cancer J Clin. 2017;67:7–30. 9. Tay Y, Rinn J, Pandolfi PP. The multilayered complexity of ceRNA crosstalk
and competition. Nature. 2014;505:344–52. 36. Punt CJA, Koopman M, Vermeulen L, et al. From tumour heterogeneity to
advances in precision treatment of colorectal cancer. Nat Rev Clin Oncol. 2017;14:235–46. 10. Marchese FP, Huarte M. Long non-coding RNAs and chromatin modifiers:
their place in the epigenetic code. Epigenetics. 2014;9(1):21–6. 37. Kuipers EJ, Grady WM, Lieberman D, et al. Colorectal cancer. Nat Rev Dis
Primers. 2015;1:15065. 11. Thomson DW, Dinger ME. Endogenous microRNA sponges: evidence and
controversy. Nat Rev Genet. 2016;17(5):272–83. Page 13 of 13 Yang et al. Cancer Cell Int (2020) 20:453 Yang et al. Cancer Cell Int (2020) 20:453 38. Yang L, Lin C, Jin C, et al. lncRNA-dependent mechanisms of androgen-
receptor-regulated gene activation programs. Nature. 2013;500:598–602. 46. Cui ZH, Shen SQ, Chen ZB, et al. Growth inhibition of hepatocellular carci‑
noma tumor endothelial cells by miR-204-3p and underlying mechanism. World J Gastroenterol. 2014;20(18):5493–504. 38. Yang L, Lin C, Jin C, et al. lncRNA-dependent mechanisms of androgen-
receptor-regulated gene activation programs. Nature. 2013;500:598–602. 39. Lorenzen J, Thum T. Long noncoding RNAs in kidney and cardiovascular
diseases. Nat Rev Nephrol. 2016;12:360–73. 39. Lorenzen J, Thum T. Long noncoding RNAs in kidney and cardiovascular
diseases. Nat Rev Nephrol. 2016;12:360–73. 47. Yuan D, Qian H, Guo T, et al. LncRNA-ATB promotes the tumorigen‑
esis of ovarian cancer via targeting miR-204-3p. Onco Targets Ther. 2020;13:573–83. 40. Yu W, Xiang D, Jia H, et al. The lncRNA BCYRN1 functions as an oncogene
in human glioma by downregulating miR-125a-5p in vitro. Cancer Manag
Res. 2020;12:1151–61. 48. Yang S, Liu T, Sun Y, et al. The long noncoding RNA LINC00483 promotes
lung adenocarcinoma progression by sponging miR-204-3p. Cell Mol Biol
Lett. 2019;24:70. 41. Lang N, Wang C, Zhao J, et al. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub‑
lished maps and institutional affiliations. 44. Suzuki HI, Miyazono K. Emerging complexity of microRNA generation
cascades. J Biochem. 2011;149:15–25. 44. Suzuki HI, Miyazono K. Emerging complexity of microRNA generation
cascades. J Biochem. 2011;149:15–25. 45. Newman MA, Hammond SM. Emerging paradigms of regulated micro‑
RNA processing. Genes Dev. 2010;24:1086–92. 45. Newman MA, Hammond SM. Emerging paradigms of regulated micro‑
RNA processing. Genes Dev. 2010;24:1086–92. •
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opinion & debate Psychiatric Bulletin (2006), 30, 43^45 R O GE R PA X T ON, P E T E R K E N N EDY A ND J OH N C A R P E N T ER
Research in the real world{ A new approach to research and development (R&D) for
mental health services is developing in the North-East,
Yorkshire and Humber. It derives from experience with
service redesign using the ‘collaborative’ approach devel-
oped by the US Institute for Health Improvement. Kennedy & Griffiths (2003) described such an approach
involving 37 mental health trusts, each with a multidisci-
plinary team, with the aim of improving acute in-patient
wards. After studying the patient’s journey through care
they agreed a set of improvement targets that all teams
would work to achieve. Progress towards targets was
measured and reported by all teams, who met periodi-
cally to compare performance and learn from each other. Remarkable energy to achieve objectives was released
among front-line staff involved. Focus was sustained on
what most concerned and benefited patients. Good ideas
and results quickly spread to all these services affecting
thousands of patients. The task of the researchers was to propose meth-
odologies by which the research questions might be
answered. At a follow-up meeting, the appropriateness
and feasibility of the methods were debated by the clin-
ical teams, modified and accepted. The methods included
structured interviews about team organisation, qualita-
tive enquiry into principles and practice, and a population
survey of assertive outreach service users. A key concern
was to benchmark services across the region in relation
to what previous research had shown to work and to
national policy guidance (Department of Health, 2001). Variations according to the urban/rural mix found in this
region of England were also of importance to this study. This programme of research is being managed and
coordinated by a partnership of clinical and academic
staff experienced in health and social services research. The leading academics are from the Universities of
Durham and Hull. Meetings to report back on the find-
ings have had full attendance of front-line staff and
managers, and user and carer involvement is growing. Teams are using the results. A Delphi study (Linstone &
Muny, 1975) has been completed in order to agree what
additional clinical measures will be used to obtain more
detailed information on outcomes. {See pp. 45-46
and pp. 46-47,
this issue. Paxton et al Research in the real world Paxton et al Research in the real world Paxton et al Research in the real world Helpful tactics The above are fine ideas perhaps, but making them work
in practice is another matter. Practice-based research
requires the kinds of service and academic partnerships
specified in the guidance of the PNF funding stream, but
making such partnerships work is challenging. The chal-
lenges include workload, financial pressures, differing
priorities between the NHS and universities, and the
belief of some NHS staff and organisations that R&D is a
frill rather than a worthwhile NHS activity. Despite continuing financial and workload pressures, and
the substantial task of coordinating and sustaining such a
large and diverse group, service and academic partners
remain engaged and enthusiastic about the further
potential of the partnership. Factors that seem to have
helped are: . the pursuit of mutual benefits from projects:
people - whether academic, service provider,
patient or manager - will not give their time, energy
and ideas unless there is a pay-off that each values; Considerable attention needs to be given to
supporting and developing partnership working if large
and diverse groupings are to collaborate successfully on a
continuing basis in order to deliver the research the NHS
most needs. Our assertive outreach example has
successfully engaged 12 trusts with other service organi-
sations, and 4 universities (Newcastle, Durham, Hull,
Bradford) over 4 years to date. It is part of a wider colla-
boration working on service development and research
and evaluation of the new mental health service com-
ponents: assertive outreach, crisis resolution/home
treatment and early intervention. Some features of the
programme are that it: . close engagement with and support from the
National Institute for Mental Health regional
development centre; . establishing programme coordination as a function
that all participants recognise explicitly as a main
ingredient for success that has to be adequately
funded; . establishing the programme coordinator (both per-
sonally and on an organisational basis) as a neutral
honest broker, whose requirements from each parti-
cipant organisation must be met to deliver the bene-
fits to all members of the partnership; . aims to carry out research on priorities and problems
identified by practitioners, their patients and their
provider organisations; . all partnership organisations‘buying in’to multi-
centre projects with a (usually small) resource contri-
bution in the form of a local project manager, some of
whose time is spent on liaison with the overall pro-
gramme coordinator and data handling. . Helpful tactics promotes wide involvement in designing and carrying
out the research, and ensures that service personnel
understand and are committed to answering the
research questions; . the constant endeavour to make it easy for organisa-
tions to participate, but having explicit entry criteria
that participating organisations must meet; . is flexible in helping to develop high-quality services
and building research capacity; . values and uses diverse research methodologies; . building trust through openness, even-handedness,
and fair acknowledgement of personal contributions. . begins by describing services as they actually are, stu-
dies and learns from existing service differences, and
moves on to evaluating changes generated within
services, including comparative evaluation across the
partnership. Where next? The National Institute for Mental Health regional devel-
opment centres have a natural interest in helping to
connect research and practice. We need to strengthen
and extend this partnership with the help of our regional
development centre. In particular, we wish to look to the
centre for the administrative leadership and central costs
of coordinating the partnership. We also hope that
through the establishment of a north-east hub of the
Mental Health Research Network we will be able to
contribute more to the further development of research
which is grounded in the real world. Progress has been substantial. Reports analysing the
characteristics and circumstances of over 800 users of
the assertive outreach teams have been produced, with
comparisons to the users of community mental health
teams. This has enabled clinicians and managers to
benchmark their work (Schneider et al, 2006; Slade et al,
2006). Detailed descriptions of team functions and
structures have been developed, compared, and fed
back. Several service development networks have
merged into the partnership, adding strength and
avoiding duplication. Tools to measure the fidelity of the
new services to the policy implementation guidance
models (Department of Health, 2001) have been
produced and used. The mental health minimum data-set
is being used to investigate wider service impacts. At a
regional conference in December 2003, it was clear that
engagement in the partnership, and receipt of research
information produced by the partnership, were highly
valued by both clinical and management staff. Following a
delay in funding for a researcher in 2004, the project has
been re-energised and a follow-up survey of teams and
their case-loads is underway. Research with service development This acute mental health collaborative was a powerful
and practical approach to achieving some of the changes
called for in the NHS Plan and the National Service
Framework for Mental Health (Department of Health,
1999, 2000). The same emphasis on collaboration, with a
more rigorous approach to collection and standardisation
of data, is now being applied to promote wide engage-
ment in service-based research. A network of 29 asser-
tive outreach teams in 12 trusts is taking part in multi-site
evaluation. We think this approach illustrates how health
services research can become dynamic and influence
practice across large populations, and help deliver the
intended objective of the NHS Priorities and Needs R&D
Funding Stream (PNF) - research should make a differ-
ence to clinical practice. This is in line with a number of
recent papers which have advocated and described
practice-based research (Audin et al, 2001; Gilbody et al,
2002; Slade, 2002; Proctor et al, 2004). This is research
that uses real-world patient populations in ordinary
service settings, rather than highly selected patient
groups in university clinics. Practice-based research can
deliver randomised controlled trials (RCTs) and other
research methods that are needed at different stages. This approach is most likely to yield results that are
immediately and directly incorporated into practice. To
tackle some questions about complex interventions there
is a need for rigorous qualitative as well as quantitative
research (Campbell et al, 2000; Medical Research
Council, 2000), and in mental health research a need to
span both the natural and social sciences (Slade & Priebe,
2001). The first step in developing this collaboration was
the convening of a large workshop involving over 80
assertive outreach staff and managers at which partici-
pants identified and debated a range of questions which
might be answered by empirical evidence. The underpin-
ning philosophy was the utilisation-focused approach,
described by Patton (1997). Here, stakeholders, working
in large and small groups, are encouraged not only to
formulate questions but to identify who specifically
would be interested in the findings and, most impor-
tantly, how the findings could be used to improve
services. Only when audiences and uses were established
were the research questions agreed. 43 https://doi.org/10.1192/pb.30.2.43 Published online by Cambridge University Press Paxton et al Research in the real world Acknowledgements We are grateful to the many clinical, management and
academic colleagues and, of course, service users and
carers, who have contributed to the work outlined here. opinion
& debate References London: Department of Health. Psychiatric Bulletin (2006), 30, 45^46 Psychiatric Bulletin (2006), 30, 45^46 {See pp. 43-45
and pp. 46-47,
this issue. S T EFA N P R I E BE
The world of real research. Commentary on . . .
Research in the real world{ example, the often restrictive inclusion criteria and
drop-out rates in research studies are a problem, but
quality improvement initiatives will also need inclusion
criteria and encounter patients who are unable to give
informed consent, refuse to be interviewed or drop out
of follow-up assessments. Paxton et al describe an approach for involving different
stakeholders in processes to improve the quality of
mental health services. Such initiatives can surely be
powerful tools to change clinical practice and have been
successfully applied in different healthcare systems. The
approach has been developed within the context of
quality improvement, but the authors have relabelled it as
research in their paper. The world of academic research has been changing
rapidly over the last 10 years. Funding depends on
success in an increasingly globalised competition arena
and requires researchers who are more or less dedicated
to full-time research. Researchers who spend much of
their time on local quality improvement initiatives may
struggle to generate the necessary income to continue
with their work - an implication that one may regard as
problematic but is nevertheless very ‘real’. ‘Real-world’ research Paxton et al place their processes in ‘the real world’,
implicitly and explicitly suggesting that other service
research does not happen in the same real world. I am
not sure what type of research they refer to. There are
numerous publications every week reporting service
research studies that were conducted in the real world,
that is, interviewing real patients in real services with real
outcomes after real treatments. I am not aware of service
research that happens in laboratories, ‘in vitro’ or with
fictitious patients. Also, I wonder which ‘university clinics’
Paxton et al refer to in their paper as carrying out mental
health service research. The UK does not have specific
‘university clinics’ and (unless it is an experimental study
on an innovative treatment) research is exclusively
conducted in ‘ordinary services’, something that Paxton et
al claim as specific to their approach. The fact that many
rigorous studies have shortcomings that limit the gener-
alisability of the findings is certainly true, but will not be
totally overcome by the approach of Paxton et al. For Declaration of interest None. 44 https://doi.org/10.1192/pb.30.2.43 Published online by Cambridge University Press https://doi.org/10.1192/pb.30.2.43 Published online by Cambridge University Press Paxton et al Research in the real world SLADE, J., SCHNEIDER,J. & BRANDON,T. (2006) Assertive outreach team and
caseload survey. Mental Health and
Learning Disabilities Research and
Practice, inpress. PATTON, Q. M. (1997) Utilization
Focused Evaluation.The New Century
Text (3rd edn). London: Sage. GILBODY, S. M., HOUSE, A. O. &
SHELDON,T. A. (2002) Outcomes
research inmentalhealth. Systematic
review. BritishJournal of Psychiatry,
181, 8-16. *Roger Paxton
Director of Research and Clinical Effectiveness, Newcastle,
NorthTyneside andNorthumberland Mental HealthNHSTrust, StNicholas Hospital,
Jubilee Road, Newcastle NE3 3XT, e-mail: roger.paxton@nmht.nhs.uk,
Peter
Kennedy
North East,Yorkshire and Humber, NIMHE Regional Development
Centre,
John Carpenter
Professor of SocialWork and Applied Social
Sciences, University of Bristol References GILBODY, S. M., HOUSE, A. O. &
SHELDON,T. A. (2002) Outcomes
research inmentalhealth. Systematic
review. BritishJournal of Psychiatry,
181, 8-16. AUDIN, K., MELLOR-CLARK, J.,
BARKHAM, M., et al (2001) Practice
research networks for effective
psychological therapies. Journal of
Mental Health, 10, 241-251. PROCTOR, S. E., MITFORD, E., PAXTON,
R. (2004) First episode psychosis: A
novel methodology reveals higher than
expected incidence: A reality based
population profile in Northumberland. Journal of Evaluationin Clinical Practice,
10, 539-547. PROCTOR, S. E., MITFORD, E., PAXTON,
R. (2004) First episode psychosis: A
novel methodology reveals higher than
expected incidence: A reality based
population profile in Northumberland. Journal of Evaluationin Clinical Practice,
10, 539-547. SLADE, M. (2002) Editorial: Routine
outcome assessment inmentalhealth
services. Psychological Medicine, 32,
1339-1343. SLADE, M. (2002) Editorial: Routine
outcome assessment inmentalhealth
services. Psychological Medicine, 32,
1339-1343. CAMPBELL, M., FITZPATRICK, R.,
HAINES, A., et al (2000) Framework for
design and evaluation of complex
interventions to improve health. BMJ,
321, 694-696. CAMPBELL, M., FITZPATRICK, R.,
HAINES, A., et al (2000) Framework for
design and evaluation of complex
interventions to improve health. BMJ,
321, 694-696. KENNEDY, P. & GRIFFITHS, H. (2003)
Mental health‘collaborative’challenges
care culture. Psychiatric Bulletin, 27,
164-166. SLADE, M. & PRIEBE, S. (2001) Are
randomised controlled trials the only
gold that glitters? BritishJournal of
Psychiatry,179, 286-287. SLADE, M. & PRIEBE, S. (2001) Are
randomised controlled trials the only
gold that glitters? BritishJournal of
Psychiatry,179, 286-287. SCHNEIDER, J., BRANDON,T.,WOOFF,
D., et al (2006) Assertive outreach:
policy andreality. Psychiatric Bulletin, in
press. SCHNEIDER, J., BRANDON,T.,WOOFF,
D., et al (2006) Assertive outreach:
policy andreality Psychiatric Bulletin in SCHNEIDER, J., BRANDON,T.,WOOFF,
D., et al (2006) Assertive outreach: SCHNEIDER, J., BRANDON,T.,WOOFF,
D., et al (2006) Assertive outreach: policy andreality. Psychiatric Bulletin, in
press. LINSTONE, H. & MUNY,T. (1975) The
Delphi Method:Techniques and
Applications. Boston: Addison
Wesley. DEPARTMENT OF HEALTH (1999)
National Service Framework for Mental
Health. London: Department of Health. DEPARTMENTOFHEALTH (2000)The
NHSPlan:APlanforInvestment,aPlanfor
Reform.London:DepartmentofHealth. *Roger Paxton
Director of Research and Clinical Effectiveness, Newcastle,
NorthTyneside andNorthumberland Mental HealthNHSTrust, StNicholas Hospital,
Jubilee Road, Newcastle NE3 3XT, e-mail: roger.paxton@nmht.nhs.uk,
Peter
Kennedy
North East,Yorkshire and Humber, NIMHE Regional Development
Centre,
John Carpenter
Professor of SocialWork and Applied Social
Sciences, University of Bristol MEDICALRESEARCHCOUNCIL(2000) A
Framework for Development and
Evaluation of RCTs for Complex
Interventions to Improve Health. London: MRC. DEPARTMENT OF HEALTH (2001)
Mental Health Policy Implementation
Guide. London: Department of Health. DEPARTMENT OF HEALTH (2001)
Mental Health Policy Implementation
Guide. Globalisation of research The globalisation of research also means that most infor-
mation on studies is available worldwide. Researchers
need to keep up to date on what other researchers in the
world are doing (for example to avoid unethical duplica-
tion of similar studies). It is hard to imagine how clinicians
and other stakeholders can remain fully aware of the
research literature so that they can competently decide
on the most relevant and timely research question. Their
involvement in such decisions is certainly welcome, and
who would not want that ‘the research questions are fully
understood and owned by service personnel as well as
the academics’? Yet, service personnel are likely to expect 45 https://doi.org/10.1192/pb.30.2.43 Published online by Cambridge University Press
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https://openalex.org/W2190535555
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https://www.frontiersin.org/articles/10.3389/fcvm.2015.00034/pdf
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English
| null |
Quality of Life in Patients with an Implantable Cardioverter Defibrillator: A Systematic Review
|
Frontiers in cardiovascular medicine
| 2,015
|
cc-by
| 6,690
|
Edited by:
Mehdi Namdar,
Hôpitaux Universitaires de Genève,
Switzerland Reviewed by:
Moisés Rodríguez-Mañero,
University Hospital Santiago de
Compostela, Spain
Danilo Ricciardi,
Campus Bio-Medico University of
Rome, Italy Reviewed by:
Moisés Rodríguez-Mañero,
University Hospital Santiago de
Compostela, Spain
Danilo Ricciardi,
Campus Bio-Medico University of
Rome, Italy *Correspondence:
Thomas Reinhold
thomas.reinhold@charite.de Keywords: implantable defibrillator, quality of life, life style, anxiety, depression Quality of life in patients with an
implantable cardioverter defibrillator:
a systematic review
Juliane Tomzik1 , Katharina C. Koltermann1 , Markus Zabel2 , Stefan N. Willich1 Juliane Tomzik1 , Katharina C. Koltermann1 , Markus Zabel2 , Stefan N. Willich1
and Thomas Reinhold1* 1 Institute for Social Medicine, Epidemiology and Health Economics, Charité – Universitätsmedizin Berlin, Berlin, Germany,
2 Department of Cardiology and Pneumology, University of Göttingen, Göttingen, Germany Despite the indisputable mortality advantages of implantable cardioverter defibrillators
(ICDs), no consensus exists regarding their impact on quality of life (QoL). This system-
atic review investigates differences in QoL between patients with ICDs and controls. We systematically searched the MEDLINE, EMBASE, Cochrane, Web of Science, and
PsychINFO databases. Articles were included if they were published after the year 2000
and reported on original studies with a control group. Five randomized controlled trials
with a total of 5,138 patients and 10 observational studies with a total of 1,513 patients
met the inclusion criteria. Nine studies found comparable QoL for ICD recipients and
patients in the control groups, three studies found an increased QoL for ICD patients,
and three studies found a decreased QoL for ICD patients. The question of whether QoL
relates to ICD therapy cannot be answered conclusively due to the heterogeneity of the
existing studies. Lower QoL was apparent among patients with an ICD who experienced
several device discharges. Medical staff should be particularly aware of the signs of both
psychological and physical disorders in these patients. Further investigations on QoL in
ICD patients are desirable, but ethical reasons restrict the conduct of randomized trials. INTRODUCTION Specialty section:
This article was submitted to
Cardiac Rhythmology,
a section of the journal
Frontiers in Cardiovascular Medicine
Received: 04 September 2015
Accepted: 20 October 2015
Published: 03 November 2015 Specialty section:
This article was submitted to
Cardiac Rhythmology,
a section of the journal
Frontiers in Cardiovascular Medicine Since the first implantation of an implantable cardioverter defibrillator (ICD) in a human being in
1980 (1), multiple clinical trials have assessed its impact on mortality and observed beneficial effects
among ICD patients (2–9). Consequently, ICD therapy has become a widespread treatment option
for patients who are at risk of sudden cardiac death (10, 11). In addition to its impact on survival, the
influence of ICD implantation on patients’ health-related quality of life (QoL) has become increas-
ingly important. Improved QoL in cardiac patients after ICD placement might be explained by the
reassurance and protection afforded to these patients by their device. Additionally, the ICD may
reduce patients’ health concerns and enable them to return to an autonomous and vital lifestyle (12,
13). However, living with an implanted device anticipating or recalling unpredictable and painful
shocks may result in feelings of dependence, psychological distress, or fear. Possible consequences
include anxiety, depression, or avoidance behaviors, such as self-imposed limitations on physical
activities, employment, or driving (14–16). These reactions and aesthetic aspects may result in a
reduced QoL among patients with an ICD. Received: 04 September 2015
Accepted: 20 October 2015
Published: 03 November 2015 Review
published: 03 November 2015
doi: 10.3389/fcvm.2015.00034 Review published: 03 November 2015
doi: 10.3389/fcvm.2015.00034 Definition of Search Strategyh The methods used in our systematic review are based on the
Cochrane Handbook for Systematic Reviews of Interventions
(17). Table 1 includes a complete list of search terms. We searched
the MEDLINE (accessed via PubMed), EMBASE (accessed via
Ovid), Cochrane, Web of Science, and PsychINFO (accessed
by Ebsco Host) databases, including both broad bibliographic
databases and subject-specific databases. Details on the searched
fields within the databases are included in Table 2. The initial
search was not limited by any constraints concerning the article
type, the language, the date of publication, or the electronic avail-
ability of the abstracts. Citation: Tomzik J, Koltermann KC, Zabel M,
Willich SN and Reinhold T (2015)
Quality of life in patients with an
implantable cardioverter defibrillator:
a systematic review. Front. Cardiovasc. Med. 2:34. doi: 10.3389/fcvm.2015.00034 Front. Cardiovasc. Med. 2:34. doi: 10.3389/fcvm.2015.00034 November 2015 | Volume 2 | Article 34 1 Frontiers in Cardiovascular Medicine | www.frontiersin.org Quality of life after ICD-implantation Tomzik et al. Because the implantation of an ICD is often a prophylactic
therapy, not all individual patients who receive such a device will
experience lifesaving shocks and derive a survival benefit from it. This fact and the divergent arguments supporting either a reduced
or an increased QoL among patients with an ICD warrant an explo-
ration of patients’ actual perceptions of their QoL following ICD
implantation. A variety of cardiac patients exist who are alternately
treated with an ICD and could potentially serve as a control group. We represent this variety in our systematic review to provide an
extensive overview. The primary objective of our systematic review
was to investigate whether the health-related QoL of patients with
ICDs differs from that of patients who have received medical treat-
ment and from that of patients who have undergone pacemaker
implantation or received no intervention. If they were reported
in the studies, we also considered ICD shocks or patients’ age as
secondary objectives because they are linked to QoL. Case studies, cost-effectiveness analyses, systematic reviews,
comments, letters, and overview texts were excluded, as were
studies without a clearly defined control group without ICD. To
provide a broad overview, we did not further specify the type
of comparison, though the comparison between single studies
became more difficult as a consequence. Possible therapeutic alter-
natives included no intervention, pacemaker implantation, and
medical treatment. We also did not include studies in the review
that considered the effects of shocks or device recalls on QoL as
their primary objective or studies on resynchronization therapy. Selection Procedure Searches were conducted in all databases on January 13, 2014. Identified records were exported to EndNote X7 and filtered
automatically for duplicates. The selection of relevant articles was
supervised by a clinical cardiologist. The first level of selection
included an independent screening of titles by two reviewers, a
health economist and a mathematician. The reviewers’ relative
distance from cardiology supported the neutral selection of
articles via the application of the defined inclusion and exclusion
criteria. The titles of the identified articles were jointly screened,
and a decision regarding each article’s inclusion was reached fol-
lowing discussion and the achievement of consensus. The second
step of screening entailed the consideration of the abstracts of the
remaining articles in a manner similar to that of the first level of
selection. The third step entailed full-text screening. The references
of the resulting studies, as well as those of each of the systematic
reviews identified via the abstract screening, were subsequently
hand-searched by one reviewer for further relevant studies. Study Selection From the initial search results, we included studies in the review
if they evaluated the influence of an ICD on QoL, were original
studies, and were published in either German or English after
the year 2000. Since 2000, all ICD devices have incorporated
high-grade technology that is intended to reliably discriminate
between supraventricular and ventricular rhythms (1). To be
included, patients’ QoL or a closely related endpoint had to be the
primary outcome of the study in question. Studies were incorpo-
rated irrespective of how QoL was measured and whether they
assessed primary or secondary device implantation. One reviewer assessed the level of evidence of the studies that were
included using the Oxford Centre for Evidence-Based Medicine
Levels of Evidence (18). Additionally, the study quality was
evaluated by the consideration of the risk for bias and confounding
and for the existence of a comprehensive and complete report of
methodological details and results (++/+/− = high/good/poor
methodological quality).if For the aggregation of studies, one reviewer defined different
subcategories of QoL (e.g., physical and mental health) based on TABLE 1 | Search terms in the databases. Frontiers in Cardiovascular Medicine | www.frontiersin.org TABLE 1 | Search terms in the databases. TABLE 2 | The databases and the field search settings used. Database
Field search
MEDLINE (accessed by PubMed)
Title/abstract, MeSH terms
Cochrane
Title, abstract, keywords
Expander: word variations have
been searched
EMBASE (Embase Classic + Embase)
(accessed by OvidSP)
Title, abstract, subject heading
Web of Science
Title, abstract, author keyword,
keywords plus®
PsychINFO (accessed by Ebsco Host)
All fields
Expander: apply related words
November 2015 | Volume 2 | Article 34 TABLE 2 | The databases and the field search settings used. (implantable OR internal)
AND
(cardioverter OR defibrillator OR ICD)
AND
(quality of life OR QOL OR adaptation OR acceptance OR attitude to health
OR health status OR health state OR psychological OR psychologic OR
emotional OR mental OR mood disorder OR mental disorder OR psychiatric
disorder OR anxiety OR depression OR depressive OR panic OR fear OR
worry OR anger OR frustration OR sadness OR self-doubt OR distress
OR stress OR lability OR uncertainty OR concern OR helplessness OR
dependence OR hypervigilance OR welfare OR well-being OR wellbeing OR
protective OR comfort OR relief OR safety OR independence OR physical
OR mobility OR pain OR vitality) (quality of life OR QOL OR adaptation OR acceptance OR attitude to health
OR health status OR health state OR psychological OR psychologic OR
emotional OR mental OR mood disorder OR mental disorder OR psychiatric
disorder OR anxiety OR depression OR depressive OR panic OR fear OR
worry OR anger OR frustration OR sadness OR self-doubt OR distress
OR stress OR lability OR uncertainty OR concern OR helplessness OR
dependence OR hypervigilance OR welfare OR well-being OR wellbeing OR
protective OR comfort OR relief OR safety OR independence OR physical
OR mobility OR pain OR vitality) November 2015 | Volume 2 | Article 34 Frontiers in Cardiovascular Medicine | www.frontiersin.org Frontiers in Cardiovascular Medicine | www.frontiersin.org 2 Quality of life after ICD-implantation Tomzik et al. each of the applied questionnaires and their subscales. For each
study, a thorough inspection of the questions contained in the
respective questionnaires was conducted to determine which of
the defined subcategories would be examined by a particular study. each of the applied questionnaires and their subscales. For each
study, a thorough inspection of the questions contained in the
respective questionnaires was conducted to determine which of
the defined subcategories would be examined by a particular study. TABLE 1 | Search terms in the databases. of which we finally included 15 studies as follows: 5 randomized
controlled trials (RCTs) (19–23), 3 observational studies (24–26),
and 7 cross-sectional studies (27–33). An overview of study
characteristics, findings, and quality assessment is included in
Table 3.h ythmias
nce, not able to read
r a live-threatening
ac arrest or VT or VF
ed VT o VF in clinical RESULTS The studies included in our review varied with respect to the
control groups and inclusion and exclusion criteria, as well as the
demographic characteristics of the study. For example, one study
investigated QoL in children (31), and another study investigated The selection process for the studies included in this review is
illustrated in Figure 1. The database search yielded 9,904 records, The selection process for the studies included in this review is
illustrated in Figure 1. The database search yielded 9,904 records,
demographic characteristics of the study. For examp
investigated QoL in children (31), and another study
FIGURE 1 | Li
h fl
h FIGURE 1 | Literature search flowchart. November 2015 | Volume 2 | Article 34 Frontiers in Cardiovascular Medicine | www.frontiersin.org OE/
udy
alityc
b++
b++ study (ICD group,
control group),
enrollment (E),
follow-up (FU)
compared to control group
study
qualit
Worse
Neutral
Better
ngestive heart failure
LVEF ≤ 35%
Med. (830 patients:
conventional medical
therapy plus amiodarone,
and 833 patients:
conventional medical
therapy plus amiodarone
placebo)
2479
(816, 1663)
E: 1997–2001
FU: 30 months
✓
2b++
“In a large primary prevention population
with moderately symptomatic heart failure,
single lead ICD therapy was not associated
with any detectable adverse quality-of-life
effects over 30 months of follow-up”
a, patients from study
Med. (conventional medical
treatment)
1089
(658, 431)
E: 1997–2001
FU: maximum
48 months
✓
2b++
“strong evidence […] [that] the ICD provides
little or no quality of life benefits”
tory of symptomatic
mature ventricular
Med. (conventional medical
therapy)
453
(227, 226)
E: 1998–2002
FU: maximum
63 months
✓
2b++
“HRQL (health-related QoL) was not
affected by ICD implantation in patients
in the defibrillators in Non-ischemic
Cardiomyopathy Treatment Evaluation
study”
ythmias
ance, not able to read
Med. (amiodarone)
317
(157, 160)
E: 1990–1997
FU: 12 months
✓
2b+
“Quality of life is better with ICD therapy than
with amiodarone therapy”
or a live-threatening
ac arrest or VT or VF
ed VT or VF in clinical
fter acute MI, non-
d reversible cause of
alance), <18 years,
entia, life expectancy
e but home, no
Med. (91 patients:
amiodarone, and 79
patients: antiarrhythmic
medications other than
amiodarone)
264
(94, 179)
E: 1995–1998
FU: 24 months
✓
2b+
“QOL improves more after ICD than after
amiodarone therapy”
(Continue ythmias
nce, not able to read
r a live-threatening
ac arrest or VT or VF
ed VT or VF in clinical , stable congestive heart failure
ic causes, LVEF ≤ 35%
ne QoL data, patients from stu
y CAD), history of symptomatic
or ≥10 premature ventricular control group),
enrollment (E),
follow-up (FU)
quality
Worse
Neutral
Better
VT (including sustained VT resulting in
he setting of LVEF ≤ 40% and clinically
odynamic compromise), survival of at least
Med. (antiarrhythmic drugs)
800
(416, 384)
E: n.r. FU: 12 months
✓
2b−
“ICD and AAD (antiarrhythmic drugs) therapy
are associated with similar alterations in self-
perceived QoL over 1-year follow-up”
ng in Iceland at the beginning of 2002
ete questionnaires due to mental or
d by caring physician)
PM
108
(41, 67)
E: 2002–2003
FU: n.a. ✓
4+
“ICD patients had a comparable QoL with
pacemaker recipients and were not more
likely to suffer from anxiety, depression, or
general psychiatric distress”
comprehend English
sant medications for pre-existing
PM
95
(46, 49)
E: 2005
FU: n.a. ✓
4+
“Quality-of-life scores were normal for all
ICD patients with respect to both mental
and physical component scores, and not
different from the pacemaker group”
8 and 18 and their parents
PM
173
(40, 133)
E: 2004–2008
FU: n.a. ✓
4−
“Patient- and parent-proxy-reported QOL
is significantly affected by the presence of
cardiac rhythm devices and is worsened in
those patients with CHD (congenital heart
disease) and ICD systems as opposed to
pacing systems”
pectoral implantation of ICD or PM
PM
152
(76, 76)
E: 1993–1999
FU: n.a. ✓
4−
“There was no difference between the
three groups (ICD with experienced shock,
ICD without experienced shock, PM), with
respect to scores on any aspect of the HAD
and SF-36”
between April 2004 and March 2007 after
PM & Oth. (49 patients: PM, 50
patients: angioplasty,
and 50 patients: catheter
ablation for drug-resistant
atrial fibrillation)
249
(100, 149)
E: 2010
FU: n.a. ✓
4+
“Mean scores for each assessment were
similar for each group”
(Continued) Controlb
No. of patients in
study (ICD group,
control group),
enrollment (E),
follow-up (FU)
Conclusion: ICD patients’ QoL
compared to control group
Worse
Neutral
Bette
e to VT,
Oth. (antiarrhythmic drugs,
angioplasty, or surgical
revascularization)
168
(133, 35)
E: n.r. FU: 12 months
✓
“In general, OT (other treatment) patients
achieved a better quality of life than ICD
patients”
fter a sodium
Oth. (asymptomatic patients
without an ICD)
190
(138, 52)
E: n.r. FU: n.a. ✓
“BrS (Brugada Syndrome) patients have
a good quality of life with no difference
between implanted and non-implanted
patients”
Oth. (ToF patients without an
ICD)
54
(26, 28)
E: n.r. FU: n.a. Relationship Between QoL and
Shocks in ICD Patients All but two studies investigated whether a correlation exists
between the perceived QoL of patients with an ICD and deliv-
ered shocks. Four cross-sectional studies and one cohort study
did not observe any differences in health-related QoL between
patients with an ICD who had or had not experienced a shock
(26–29, 31). In the SCD-HeFT trial, Mark et al. (20) also did not
observe a difference in QoL, with the exception of those patients
who were shocked within a month of their QoL assessment. This
finding suggests that the negative influence of ICD shocks on QoL
decreases with time. Due to the small number of patients whose
QoL was measured within a month of ICD shock, this finding
remains to be reproduced. f
Pacemaker patients served as control subjects in five cross-
sectional studies (27–31). Due to various methodological
limitations, these studies were assessed to be of moderate to low
quality. All studies except the one by Czosek et al. (31) yielded
no differences regarding QoL between pacemaker patients and
patients with an ICD. Czosek et al. considered pediatric patients
and revealed a lower QoL for children with an ICD than for
children implanted with a pacemaker. Two RCTs reported that QoL of ICD patients depended on the
number of shocks received. Irvine et al. (19) and Passman et al. (22) found a reduced mental well-being only for patients who
experienced five or more shocks.h Redhead et al. (30) compared ICD patients with three
groups of patients undergoing other typical cardiac procedures:
pacemaker patients, patients who underwent angioplasty, and
patients treated via catheter ablation for drug-resistant atrial
fibrillation. The remaining four studies (24, 25, 32, 33) selected
control subjects from patients receiving antiarrhythmic drugs,
patients who underwent angioplasty or patients who underwent
surgical revascularization, or had control groups that received an
unspecified intervention. Two of these studies found decreased
QoL for ICD patients, whereas the other two revealed comparable
QoL in ICD and control groups. However, these studies had
major methodological limitations. The heterogeneous results
must therefore be considered with caution. Three cross-sectional studies (30, 32, 33) revealed a poorer
psychological well-being for ICD patients following shocks,
including psychosocial problems, anxiety, and concerns
regarding complications. Kamphuis et al. (25) demonstrated
decreased physical functioning among patients who had received
a shock. Schron et al. DISCUSSION In general, we could not ascertain a uniform trend regarding the
QoL of patients who received an ICD. The majority of studies
concluded that there was no difference between the ICD groups
and the control groups. For the subgroup of patients who
experience ICD shocks, the data suggest unchanged or poorer
QoL compared to ICD patients without ICD discharge. A high
number of shocks and the recency of shocks appear to correlate
with reduced QoL. However, an underlying causality cannot be
deduced from this correlation. It is possible that repeated shock
experiences will influence a patient’s perception of his health in
a negative way. It is also possible that sicker patients with poorer
QoL are increasingly affected by shocks.f QoL of ICD Patients Compared with
that of Distinct Control Groupsii All five of the identified RCTs (19–23) and one cohort study
(26) compared patients who underwent ICD implantation with
patients who received medical treatment. Medical treatment
ranged from conventional medical therapy to treatment with
amiodarone. Each of the RCTs assessed all-cause mortality as the
primary endpoint and QoL as a secondary endpoint. The number
of study participants and the quality of study was intermediate to
high. Two RCTs reported improved QoL for patients with an ICD
compared to patients with medical treatment. The remaining four
studies noted no QoL differences among the groups.i Relationship Between QoL and
Shocks in ICD Patients (23) observed lower physical and mental
scores among the patients of the AVID trial with a history of ICD
discharge. Relationship Between QoL and
Age in ICD Patientsh Relationship Between QoL and
Age in ICD Patientsh QoL in patients with Tetralogy of Fallot (ToF), with a mean age
of 44 years (33). The mean age of the patients who received an
ICD in the other studies ranged between 50 and 69 years of age. The quality assessment revealed frequent methodological
limitations, such as an insufficient description of the statistical
analyses, the recruitment process, and the existence of missing
data and their handling. Furthermore, the problem of multiple
testing and confounding variables was often not incorporated
into the statistical analyses. QoL in patients with Tetralogy of Fallot (ToF), with a mean age
of 44 years (33). The mean age of the patients who received an
ICD in the other studies ranged between 50 and 69 years of age.h The cross-sectional study by Czosek et al. (31) and the RCT by
Passman et al. (22) found no correlation between QoL and patients’
ages. Opic et al. (33) reported worse psychosocial functioning in
younger patients. Poor QoL was noted among elderly patients by
Hsu et al. (26). Kamphuis et al. (25) reported improved vitality but
poorer health perception among elderly ICD patients. Due to the
heterogeneity of these conclusions, it was not possible to elucidate
any trends regarding the relationship between age and QoL in
patients with an ICD or to determine whether this relationship dif-
fered among ICD recipients compared with the general population. The quality assessment revealed frequent methodological
limitations, such as an insufficient description of the statistical
analyses, the recruitment process, and the existence of missing
data and their handling. Furthermore, the problem of multiple
testing and confounding variables was often not incorporated
into the statistical analyses. ✓
“ToF patients with an ICD show less
favorable psychosocial functioning
compared to ToF patients without ICD”
ome
Oth. (patients with CAD)
60
(30, 30)
E: n.r. FU: 14 days
✓
“The purpose of this study was to compare
sleep patterns between CAD and ICD
patients […]. The primary and surprising
finding was that CAD patients had poorer
sleep compared with ICD patients in terms
of sleep efficiency and total sleep time”
sification; LVEF, left ventricular ejection fraction; MI, myocardial infarction; CAD, coronary artery disease; VT, ventricular tachyc
ified. t study; 4, case-series and cross-sectional studies; ++/+/−, high/good/poor methodological quality. Bet
patients
an ICD
nts have
ference
planted st due to VT,
or after a sodiu
yndrome
Classification; LV
specified. ohort study; 4, c or after a
yndrome
Classificat
pecified. ohort stud Tomzik et al. Quality of life after ICD-implantation Frontiers in Cardiovascular Medicine | www.frontiersin.org Different Subcategories of
the ICD Patients’ QoLh The studies included in our review were arranged according to
characteristics that contribute to QoL. These include physical
and mental health, e.g., physical limitations, bodily pain, social
functioning, anxiety, and depression.f Table 4 shows different subcategories of QoL and the stud-
ies that examined these dimensions. Studies were categorized
depending on whether improvement or impairment was
observed for ICD patients or whether no significant difference
was observed. The majority of studies did not show a difference
in QoL between ICD patients and controls. f
Due to differences in control groups, study designs, and
inclusion and exclusion criteria, there was limited comparability Frontiers in Cardiovascular Medicine | www.frontiersin.org November 2015 | Volume 2 | Article 34 7 stinct subcategories of QoL. man
Irvine
(19)
Hsu
(26)
Schron
(23)
Leosdottir
(28)
Newall
(29)
Czosek
(31)
Duru
(27)
Redhead
(30)
Kamphuis
(25)
Probst
(32)
Opic
(33)
n.s. n.s. n.s. n.s. +
n.s. n.s. n.s. n.s. n.s. −
n.s. n.s. n.s. n.s. n.s. n.s. −
n.s. n.s. +
n.s. n.s. n.s. −
n.s. +
n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. +
n.s. n.s. n.s. n.s. n.s. −
n.s. n.s. n.s. n.s. n.s. n.s. n.s. +
n.s. n.s. n.s. n.s. n.s. n.s. n.s. +
n.s. n.s. n.s. n.s. fields, not investigated or not reported. Quality of life after ICD-implantation Tomzik et al. and heterogeneous methodological quality among the studies
reviewed. The three studies that were assessed to be of high
quality and to have a high level of evidence were the SCD-HeFT
(20), the MADIT II (21), and the DEFINITE (22) studies, which
included a total of 4,021 patients. These three RCTs found similar
QoL between ICD patients and controls under medical treat-
ment. Comparable results were also published in further studies,
but the reader should keep in mind the frequent differences
especially in the underlying patient structures. For example,
Probst et al. (32) detected no relevant differences of Qol in ICD
patients and controls as well, but compared to SCD-HeFT (20),
MADIT II (21), and DEFINITE (22), this investigation is focused
on patients suffering from Brugada syndrome. These patients had
a relevant lower age (50–54 years) compared with the mean age
of ICD patients in SCD-HeFT, MADIT, and DEFINITE (ranging
from 59 to 64 years). Additionally, Brugada patients are less suf-
fering from comorbidities and are more frequently professionally
active (32). and acceptance of ICD therapy may not have been comparable. Finally, our review did not include systematic reviews and
meta-analyses, which were, however, used later to check the
thoroughness of our review. Previous reviews and meta-analyses on QoL in ICD patients
drew opposing conclusions. McCready et al. (34) concluded that
ICD implantation was superior to medical therapy with respect
to QoL, based on eight studies published between 1995 and 2002. Groeneveld et al. (16), who included 27 studies published between
1995 and 2005, came to the same conclusion. Additionally, they
emphasized that changes in QoL were strongly dependent on
the comparison groups. Compared with the general public or
pacemaker patients, ICD recipients had a lower QoL. Unchanged
or improved QoL was noted for ICD patients based on pre- and
post-implant comparisons by Shea (15), who reviewed three trials
conducted in the 1990s. Francis et al. (35) included 30 studies
that were published between 1993 and 2004. Five randomized
trials suggested either an unchanged or an improved QoL among
the patients who underwent an ICD implantation. The 16 non-
randomized trials under study showed a balanced result. In their
meta-analysis, Burke et al. (36) concluded either unchanged or
poorer QoL for patients who received an ICD. They considered 20
publications, each of which was published before the year 2000,
using various comparisons, including pre- and post-implantation
comparisons and comparisons with cardiac patients who did not
receive therapy and patients who received antiarrhythmic drug
therapy. Because several RCTs have demonstrated that ICDs exert
a survival advantage over medical treatment (2–9), the alloca-
tion of ICD therapy and medical treatment via randomization
is ethically questionable. Therefore, the RCTs identified by our
systematic review were conducted over 10 years ago. The more
current studies included in our review were observational stud-
ies and were characterized by a lower evidence level. Their small
patient numbers and their single-center and cross-sectional
designs further diminished the informative value of these studies. The longitudinal studies included in our review had short follow-
up (FU) periods. Three of the six longitudinal studies followed
patients for only 12 months (19, 23, 25). The FU periods of the
remaining studies lasted as long as 30, 48, or 63 months (20–22). Because QoL is particularly influenced by incisive experiences,
including device implantation, and habituation, a FU period of
several years may be more appropriate. Our systematic review confirmed the trend observed by
McCready et al. (34) and Groeneveld et al. (16), as we deter-
mined an either unchanged or improved QoL for ICD patients
compared with patients under optimal medical treatment;
however, either unchanged or poorer QoL was noted among
ICD patients compared with pacemaker recipients. However,
due to both the small number of studies and various limitations
concerning both methodology and implementation, we do not
view this trend as firm but rather as a finding that remains to
be reproduced.h y
y
pp
p
Two additional limitations of the studies that assessed the
relationship between ICD implantation and various outcomes
are the impossibility of blinding and the definition of a suitable
control group. Unblinded cardiologists do not ensure the equal
treatment of patients who are receiving distinct therapies. Hence,
there exists a high risk of bias. This is particularly important when
assessing an outcome such as QoL, which is often influenced by
a patient’s contact with medical staff. The selection of a control
group for an ICD population often requires relatively large
compromises. Patients with an implanted pacemaker, for instance,
often differ in both their demographic and clinical characteristics. They are usually older than patients with an ICD and are more
likely to be women. The value of pre- and post-implantation QoL comparisons
appears to be limited. We expect an imminent implantation to
strongly influence patients’ QoL and their psychological well-
being in particular. QoL may decrease due to worries about the
surgical operation or increase due to the expected improvement
of state of health owing to the ICD. Therefore, we focused on the
comparison between patients who received an ICD and a non-
ICD control group in our systematic review.h The heterogeneous results of the studies available for our
review did not permit a definitive answer to the question of
whether health-related QoL differs between patients with an
ICD and respective controls. One may remain critical of whether
patients with pacemakers or patients who have received medi-
cal treatment are representative as control patients. Lower QoL
was apparent among ICD patients who experienced several
device discharges. Medical staff should be particularly aware
of psychological and physical effects in these patients. Future
research on these open QoL questions is difficult because it is
currently not ethical to randomize patients to ICD or control
treatment. As a procedural limitation of the development of our review,
the screening of titles as an initial selection step must be
mentioned. This step is more prone to missing relevant articles
than screening of abstracts. An additional issue is the inclusion
of newer studies that were published only following the year
2000. Frontiers in Cardiovascular Medicine | www.frontiersin.org REFERENCES 15. Shea JB. Quality of life issues in patients with implantable cardioverter
defibrillators: driving, occupation, and recreation. AACN Clin Issues (2004)
15(3):478–89. doi:10.1097/00044067-200407000-00013 1. Klein HU, Inama G. Implantable defibrillators: 30 years of history. G Ital
Cardiol (2010) 11(10 Suppl 1):48s–52s. 1. Klein HU, Inama G. Implantable defibrillators: 30 years of history. G Ital
Cardiol (2010) 11(10 Suppl 1):48s–52s. 16. Groeneveld PW, Matta MA, Suh JJ, Heidenreich PA, Shea JA. Costs and
quality-of-life effects of implantable cardioverter-defibrillators. Am J Cardiol
(2006) 98(10):1409–15. doi:10.1016/j.amjcard.2006.06.041 2. Moss AJ, Hall WJ, Cannom DS, Daubert JP, Higgins SL, Klein H, et al. Improved survival with an implanted defibrillator in patients with coronary
disease at high risk for ventricular arrhythmia. Multicenter automatic defibril-
lator implantation trial investigators. N Engl J Med (1996) 335(26):1933–40. doi:10.1056/nejm199612263352601 hi 2. Moss AJ, Hall WJ, Cannom DS, Daubert JP, Higgins SL, Klein H, et al. Improved survival with an implanted defibrillator in patients with coronary
disease at high risk for ventricular arrhythmia. Multicenter automatic defibril-
lator implantation trial investigators. N Engl J Med (1996) 335(26):1933–40. doi:10.1056/nejm199612263352601 17. Higgins JPT, Green, S, editors. Cochrane Handbook for Systematic Reviews of
Interventions Version 5.1.0 [updated March 2011] [Internet] (2011) [cited 2013
Dec 18]. Available from: http://www.cochrane-handbook.org 18. Phillips B, Ball C, Sackett D, Badenoch D, Straus S, Haynes B, et al. Levels
of Evidence (March 2009) (2009) [cited 2014 July 22]. Available from: http://
www.cebm.net 3. The Antiarrhythmics versus Implantable Defibrillators (AVID) Investigators. A comparison of antiarrhythmic-drug therapy with implantable defibrillators
in patients resuscitated from near-fatal ventricular arrhythmias. N Engl J Med
(1997) 337(22):1576–83. doi:10.1056/nejm199711273372202 3. The Antiarrhythmics versus Implantable Defibrillators (AVID) Investigators. A comparison of antiarrhythmic-drug therapy with implantable defibrillators
in patients resuscitated from near-fatal ventricular arrhythmias. N Engl J Med
(1997) 337(22):1576–83. doi:10.1056/nejm199711273372202 19. Irvine J, Dorian P, Baker B, O’Brien BJ, Roberts R, Gent M, et al. Quality of
life in the Canadian implantable defibrillator study (CIDS). Am Heart J (2002)
144(2):282–9. doi:10.1067/mjh.2002.124049 4. Connolly SJ, Gent M, Roberts RS, Dorian P, Roy D, Sheldon RS, et al. Canadian implantable defibrillator study (CIDS): a randomized trial of the
implantable cardioverter defibrillator against amiodarone. Circulation (2000)
101(11):1297–302. doi:10.1161/01.CIR.101.11.1297 4. Connolly SJ, Gent M, Roberts RS, Dorian P, Roy D, Sheldon RS, et al. Canadian implantable defibrillator study (CIDS): a randomized trial of the
implantable cardioverter defibrillator against amiodarone. Circulation (2000)
101(11):1297–302. doi:10.1161/01.CIR.101.11.1297 20. REFERENCES Mark DB, Anstrom KJ, Sun JL, Clapp-Channing NE, Tsiatis AA, Davidson-
Ray L, et al. Quality of life with defibrillator therapy or amiodarone in heart
failure. N Engl J Med (2008) 359(10):999–1008. doi:10.1056/NEJMoa0706719 5. Kuck KH, Cappato R, Siebels J, Ruppel R. Randomized comparison of antiar-
rhythmic drug therapy with implantable defibrillators in patients resuscitated
from cardiac arrest: the cardiac arrest study Hamburg (CASH). Circulation
(2000) 102(7):748–54. doi:10.1161/01.CIR.102.7.748 21. Noyes K, Corona E, Zwanziger J, Hall J, Zhao HW, Wang HK, et al. Health-
related quality of life consequences of implantable cardioverter defibrilla-
tors – results from MADIT II. Med Care (2007) 45(5):377–85. doi:10.1097/01. mlr.0000257142.12600.c1 6. Moss AJ, Zareba W, Hall WJ, Klein H, Wilber DJ, Cannom DS, et al. Prophylactic implantation of a defibrillator in patients with myocardial
infarction and reduced ejection fraction. N Engl J Med (2002) 346(12):877–83. doi:10.1056/NEJMoa013474 22. Passman R, Subacius H, Ruo B, Schaechter A, Howard A, Sears SF, et al. Implantable cardioverter defibrillators and quality of life: results from the
defibrillators in nonischemic cardiomyopathy treatment evaluation study. Arch Intern Med (2007) 167(20):2226–32. doi:10.1001/archinte.167.20.2226 7. Bristow MR, Saxon LA, Boehmer J, Krueger S, Kass DA, De Marco T, et al. Cardiac-resynchronization therapy with or without an implantable defibrilla-
tor in advanced chronic heart failure. N Engl J Med (2004) 350(21):2140–50. doi:10.1056/NEJMoa032423 23. Schron EB, Exner DV, Yao Q, Jenkins LS, Steinberg JS, Cook JR, et al. Quality
of life in the antiarrhythmics versus implantable defibrillators trial: impact
of therapy and influence of adverse symptoms and defibrillator shocks. Circulation (2002) 105(5):589–94. doi:10.1161/hc0502.103330 8. Kadish A, Dyer A, Daubert JP, Quigg R, Estes NA, Anderson KP, et al. Prophylactic defibrillator implantation in patients with nonischemic
dilated cardiomyopathy. N Engl J Med (2004) 350(21):2151–8. doi:10.1056/
NEJMoa033088 24. Cross NJ, McCrae CS, Smith KM, Conti JB, Sears SF. Comparison of acti-
graphic and subjective measures of sleep in implantable cardioverter defibril-
lator and coronary artery disease patients. Clin Cardiol (2010) 33(12):753–9. doi:10.1002/clc.20827 9. Bardy GH, Lee KL, Mark DB, Poole JE, Packer DL, Boineau R, et al. Amiodarone or an implantable cardioverter-defibrillator for conges-
tive heart failure. N Engl J Med (2005) 352(3):225–37. doi:10.1056/
NEJMoa043399 25. Kamphuis HC, De Leeuw JR, Derksen R, Hauer R, Winnubst JA. A 12-month
quality of life assessment of cardiac arrest survivors treated with or without
an implantable cardioverter defibrillator. Europace (2002) 4(4):417–25. doi:10.1053/eupc.2002.0258 10. Eucomed. Statistics for Cardiac Rhythm Management products, 2005-2014
[Internet]. In some cases, there existed a time lag of several years
between the completion of the study and the publication of the
results. Hence, there were studies that investigated patients much
earlier than the publication date (19, 27). Consequently, the
technology of the implanted devices and the patients’ awareness November 2015 | Volume 2 | Article 34 Frontiers in Cardiovascular Medicine | www.frontiersin.org 9 Quality of life after ICD-implantation Tomzik et al. FUNDING The research leading to these results has received fund-
ing from the European Community’s Seventh Framework
Programme (FP7/2007-2013) under grant agreement no. 602299
(EU-CERT-ICD). The research leading to these results has received fund-
ing from the European Community’s Seventh Framework
Programme (FP7/2007-2013) under grant agreement no. 602299
(EU-CERT-ICD). The research leading to these results has received fund-
ing from the European Community’s Seventh Framework
Programme (FP7/2007-2013) under grant agreement no. 602299
(EU-CERT-ICD). AUTHOR CONTRIBUTIONS design, data collection, screening of literature, summarizing and
interpreting literature, critical revision of the article, and approval
of the article. JT contributed to concept and design, data collection, screening
of literature, summarizing and interpreting literature, drafting
article, critical revision of the article, and approval of the article. KK contributed to concept and design, critical revision of the
article, and approval of the article. MZ contributed to supervi-
sion of screening of literature, critical revision of the article, and
approval of the article. SW contributed to critical revision of the
article and approval of the article. TR contributed to concept and REFERENCES Brussels [cited 2015 Aug 8]. Available from: http://www.eucomed. org/uploads/_medical_technology/facts_figures/CRM_Graphs_2015.pdfh 26. Hsu J, Uratsu C, Truman A, Quesenberry C, McDonald KM, Hlatky MA,
et al. Life after a ventricular arrhythmia. Am Heart J (2002) 144(3):404–12. doi:10.1067/mhj.2002.125497 11. Mond HG, Proclemer A. The 11th world survey of cardiac pacing and
implantable cardioverter-defibrillators: calendar year 2009 – a world society
of arrhythmia’s project. Pacing Clin Electrophysiol (2011) 34(8):1013–27. doi:10.1111/j.1540-8159.2011.03150.x 27. Duru F, Buchi S, Klaghofer R, Mattmann H, Sensky T, Buddeberg C, et al. How different from pacemaker patients are recipients of implantable car-
dioverter-defibrillators with respect to psychosocial adaptation, affective
disorders, and quality of life? Heart (2001) 85(4):375–9. doi:10.1136/
heart.85.4.375 12. Rahman B, Macciocca I, Sahhar M, Kamberi S, Connell V, Duncan
RE. Adolescents with implantable cardioverter defibrillators: a patient
and parent perspective. Pacing Clin Electrophysiol (2012) 35(1):62–72. doi:10.1111/j.1540-8159.2011.03229.x 28. Leosdottir M, Sigurdsson E, Reimarsdottir G, Gottskalksson G, Torfason B,
Vigfusdottir M, et al. Health-related quality of life of patients with implant-
able cardioverter defibrillators compared with that of pacemaker recipients. Europace (2006) 8(3):168–74. doi:10.1093/europace/euj052 13. Tagney J, James JE, Albarran JW. Exploring the patient’s experiences of
learning to live with an implantable cardioverter defibrillator (ICD) from one
UK centre: a qualitative study. Eur J Cardiovasc Nurs (2003) 2(3):195–203. doi:10.1016/S1474-5151(03)00061-6 29. Newall EG, Lever NA, Prasad S, Hornabrook C, Larsen PD. Psychological
implications of ICD implantation in a New Zealand population. Europace
(2007) 9(1):20–4. doi:10.1093/europace/eul142 14. Sears SF Jr, Todaro JF, Lewis TS, Sotile W, Conti JB. Examining the psychoso-
cial impact of implantable cardioverter defibrillators: a literature review. Clin
Cardiol (1999) 22(7):481–9. doi:10.1002/clc.4960220709 30. Redhead AP, Turkington D, Rao S, Tynan MM, Bourke JP. Psychopathology
in postinfarction patients implanted with cardioverter-defibrillators for November 2015 | Volume 2 | Article 34 Frontiers in Cardiovascular Medicine | www.frontiersin.org 10 Tomzik et al. Quality of life after ICD-implantation secondary prevention. A cross-sectional, case-controlled study. J Psychosom
Res (2010) 69(6):555–63. doi:10.1016/j.jpsychores.2010.06.002 36. Burke JL, Hallas CN, Clark-Carter D, White D, Connelly D. The psychosocial
impact of the implantable cardioverter defibrillator: a meta-analytic review. Br J Health Psychol (2003) 8(Pt 2):165–78. doi:10.1348/135910703321649141 31. Czosek RJ, Bonney WJ, Cassedy A, Mah DY, Tanel RE, Imundo JR, et al. Impact of cardiac devices on the quality of life in pediatric patients. Circ
Arrhythm
Electrophysiol
(2012)
5(6):1064–72. doi:10.1161/
circep.112.973032 Conflict of Interest Statement: Juliane Tomzik: none. Katharina C. Frontiers in Cardiovascular Medicine | www.frontiersin.org REFERENCES Koltermann:
consultancies as part-time manager for Boston Healthcare Associates, external sci-
entific consulting services for Takeda, CGS Clinical Guideline Services, Keimzelle
Medical Ventures, and Scenarium Group GmbH. Markus Zabel: consultancies,
research and travel grants: Biotronik, Medtronic, Boston Scientific. Stefan N. Willich: none. Thomas Reinhold: received fees from ICD manufacturer Biotronik
for external scientific consulting services. 32. Probst V, Plassard-Kerdoncuf D, Mansourati J, Mabo P, Sacher F, Fruchet
C, et al. The psychological impact of implantable cardioverter defibrillator
implantation on Brugada syndrome patients. Europace (2011) 13(7):1034–9. doi:10.1093/europace/eur060 33. Opic P, Utens EM, Moons P, Theuns DA, van Dijk AP, Hoendermis ES, et al. Psychosocial impact of implantable cardioverter defibrillators (ICD) in
young adults with Tetralogy of Fallot. Clin Res Cardiol (2012) 101(7):509–19. doi:10.1007/s00392-012-0420-x Copyright © 2015 Tomzik, Koltermann, Zabel, Willich and Reinhold. This is
an open-access article distributed under the terms of the Creative Commons
Attribution License (CC BY). The use, distribution or reproduction in other forums
is permitted, provided the original author(s) or licensor are credited and that the
original publication in this journal is cited, in accordance with accepted academic
practice. No use, distribution or reproduction is permitted which does not comply
with these terms. 34. McCready MJ, Exner DV. Quality of life and psychological impact of implant-
able cardioverter defibrillators: focus on randomized controlled trial data. Card Electrophysiol Rev (2003) 7(1):63–70. doi:10.1023/A:1023699225221 35. Francis J, Johnson B, Niehaus M. Quality of life in patients with implantable
cardioverter defibrillators. Indian Pacing Electrophysiol J (2006) 6(3):173–81. 35. Francis J, Johnson B, Niehaus M. Quality of life in patients with implantable
cardioverter defibrillators. Indian Pacing Electrophysiol J (2006) 6(3):173–81. November 2015 | Volume 2 | Article 34 Frontiers in Cardiovascular Medicine | www.frontiersin.org 11
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FAIR and open multilingual clinical trials in
Wikidata and Wikipedia Lane Rasberry , Daniel Mietchen
‡
‡ ‡ School of Data Science, University of Virginia, Charlottesville, United States of America Reviewable
v 1
Corresponding author: Lane Rasberry (lr2ua@virginia.edu),
Daniel Mietchen (daniel.mietchen@virginia.edu)
Received: 25 Mar 2021 | Published: 25 Mar 2021
Citation: Rasberry L, Mietchen D (2021) FAIR and open multilingual clinical trials in Wikidata and Wikipedia. Research Ideas and Outcomes 7: e66490. https://doi.org/10.3897/rio.7.e66490 Received: 25 Mar 2021 | Published: 25 Mar 2021
Citation: Rasberry L, Mietchen D (2021) FAIR and open multilingual clinical trials in Wikidata and Wikipedia. Research Ideas and Outcomes 7: e66490. https://doi.org/10.3897/rio.7.e66490 Research Ideas and Outcomes 7: e66490
doi: 10.3897/rio.7.e66490 © Rasberry L, Mietchen D. This is an open access article distributed under the terms of the Creative Commons Attribution License
(CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source
are credited. Research Ideas and Outcomes 7: e66490
doi: 10.3897/rio.7.e66490 Research Ideas and Outcomes 7: e66490
doi: 10.3897/rio.7.e66490 About The School of Data Science at the University of Virginia received funding through an open
call from the Wellcome Trust for this research and development proposal titled "FAIR and
open multilingual clinical trials in Wikidata and Wikipedia". This document combines the
concept note, full application, and supporting details in that application. Our intent in
publishing this is to make the proposal available with free and open copyright licensing for
other researchers to reuse and remix as they wish. The project objective is to import clinical trials metadata from ClinicalTrials.gov into the
Wikidata platform, and to curate the data using Wikidata workflows, and also to present the
overall process as a model for using the Wikipedia ecosystem to share and remix data. If
the project is successful to the extent of our wishes then our hope is that all sorts of
researchers and the public will access and use clinical trials data for both traditional and
new purposes. Although we believe that researchers who currently use clinical trials data will find benefit
from its curation through the Wikipedia platform development process, we also seek to
promote access to this medical information to new audiences both within conventional
analysis about clinical trials and in new and unexpected contexts. The new audiences
which we anticipate are those whom we already know to browse Wikipedia's medical
content, including researchers who prefer to access information outside of English
language and non-researchers including students, journalists, and policy makers who
previously would not have considered seeking this data were it not accessible through the
familiar Wikipedia. By making the data much more accessible and also available for
Wikipedia's style of crowdsourcing, we hope that others will develop and reuse this data
including by linking trials to papers, people, and organizations; visualizing the trials with
charts and maps; general curation of trials by keyword tagging or concept disambiguation;
and language translation of technical terms in structured data collections such as Wikidata. We are sharing this text in alignment with Wikipedia community values of openness and in
a contemporary social context where sharing proposals is uncommon but which we wish
were more routine. At the time of publishing this proposal we have developed the project
but have not yet completed it. This document only presents the proposal, and we will
publish our methods, results, and the overall model in a later paper. medical information, clinical trials, Wikipedia, Wikidata, translation, India, East Africa, Hindi,
Bangla, Bengali, Swahili medical information, clinical trials, Wikipedia, Wikidata, translation, India, East Africa, Hindi,
Bangla, Bengali, Swahili Abstract This project seeks to conduct language translation on metadata labels for research
publications, attribution data, and clinical trials information to make data about medical
research queriable in underserved languages through Wikidata and the Linked Open Web. This project has the benefit of distributing content through Wikipedia and Wikidata, which
already have an annual userbase of a billion users and which already have established
actionable standards to practice diversity, inclusion, openness, FAIRness, and
transparency about program development. The impact will be localized access to basic
research information in various Global South languages to integrate with existing
community efforts for establishing the same. Although Wikidata development in this
direction seems inevitable, the cultural and social exchange required to establish global
multilingual research partnerships could begin now with support rather than later as a
second phase effort for including the developing world. Wikipedia and Wikidata are
established forums with an existing active userbase for multilingual research collaboration,
but the research practices there still are immature. By applying metadata expertise through
this project, we will elevate the current amateur development with more stable Linked
Open Data compatibility to English language databases. Using the wiki distribution and
discussion platform to develop the global conversation about data sharing will set good
precedents for the trend of global research collaboration. Rasberry L, Mietchen D 2 Methodology 1. By default, adopt the established Wikipedia and Wikidata publishing and
engagement practices for open, FAIR, documentation, receiving feedback in
permanent public forums, and collaboration 1. By default, adopt the established Wikipedia and Wikidata publishing and
engagement practices for open, FAIR, documentation, receiving feedback in
permanent public forums, and collaboration 1. By default, adopt the established Wikipedia and Wikidata publishing and
engagement practices for open, FAIR, documentation, receiving feedback in
permanent public forums, and collaboration 2. Contribute to the documentation about the position of Wikipedia and Wikidata in the
Linked Open Data ecosystem, particularly emphasizing university participation in
import and export of research metadata in the Wikimedia platform and collecting
impact metrics for doing so. 3. Within Wikidata, contribute to the WikiCite project which seeks to enrich data
around citations and metadata, including PubMed research papers, and subsets of
CrossRef, ORCID, and ClinicalTrials.gov. Explore and document possibilities to
ingest non-United States clinical trial databases. 4. Identify the set of terms and concepts which are necessary to perform and visualize
queries of medical research data, for example, "clinical research sites in a given
country with the highest trial completion rates in infectious disease research" 5. Translate those terms to languages including Hindi, Bengali, and Swahili to the
level of quality which is established as a norm by existing local community
participants in Wikipedia and Wikidata 5. Translate those terms to languages including Hindi, Bengali, and Swahili to the
level of quality which is established as a norm by existing local community
participants in Wikipedia and Wikidata 6. Use Wikipedia and Wikidata's native metrics reporting processes to measure the
impact to users and the engagement of peer reviewers FAIR and open multilingual clinical trials in Wikidata and Wikipedia FAIR and open multilingual clinical trials in Wikidata and Wikipedia 3 About The term of the project
is extended due to COVID-19, and we changed some of the project focus from the original
proposal in response to the pandemic. The text here does not account for our response to
COVID. FAIR and open multilingual clinical trials in Wikidata and Wikipedia Introduction The School of Data Science at the University of Virginia received funding through an open
call from the Wellcome Trust for this research and development proposal titled "FAIR and
open multilingual clinical trials in Wikidata and Wikipedia". This document combines the
concept note, full application, and supporting details in that application. Our intent in
publishing this is to make the proposal available with free and open copyright licensing for
other researchers to reuse and remix as they wish. The project objective is to import clinical trials metadata from ClinicalTrials.gov into the
Wikidata platform, and to curate the data using Wikidata workflows, and also to present the
overall process as a model for using the Wikipedia ecosystem to share and remix data. If
the project is successful to the extent of our wishes then our hope is that all sorts of
researchers and the public will access and use clinical trials data for both traditional and
new purposes. Although we believe that researchers who currently use clinical trials data will find benefit
from its curation through the Wikipedia platform development process, we also seek to
promote access to this medical information to new audiences both within conventional
analysis about clinical trials and in new and unexpected contexts. The new audiences
which we anticipate are those whom we already know to browse Wikipedia's medical
content, including researchers who prefer to access information outside of English Rasberry L, Mietchen D 4 4 language and non-researchers including students, journalists, and policy makers who
previously would not have considered seeking this data were it not accessible through the
familiar Wikipedia. By making the data much more accessible and also available for
Wikipedia's style of crowdsourcing, we hope that others will develop and reuse this data
including by linking trials to papers, people, and organizations; visualizing the trials with
charts and maps; general curation of trials by keyword tagging or concept disambiguation;
and language translation of technical terms in structured data collections such as Wikidata. We are sharing this text in alignment with Wikipedia community values of openness and in
a contemporary social context where sharing proposals is uncommon but which we wish
were more routine. At the time of publishing this proposal we have developed the project
but have not yet completed it. This document only presents the proposal, and we will
publish our methods, results, and the overall model in a later paper. Who is the project coordinator? Name: Lane Rasberry
Organization: University of Virginia
Department: School of Data Science
Division: Center for Ethics and Justice
Email: rasberry@virginia.edu
ORCID: 0000-0002-9485-6146 Introduction The term of the project
is extended due to COVID-19, and we changed some of the project focus from the original
proposal in response to the pandemic. The text here does not account for our response to
COVID. How will you evaluate the success of your activities? This project will use Wikipedia's own established processes for monitoring and evaluation
of university projects to develop and publish general reference information in Wikipedia
and Wikidata. We will evaluate this project in these established ways: 1. Content metrics - Report the standard publishing metrics as measured by
Wikimedia's own native metrics suite for publishers 2. Diversity and inclusion - Partner with established Wikimedia community
organizations; confirm their oversight and approval 3. Impact metrics – Report audience readership as measured by Wikipedia's own
native metrics suite for users 4. Quality review - university student researchers will evaluate and publish an
evaluation of the source research metadata and the translation process 5. Bias evaluation – we will subjectively publish our opinions on bias we identify and
its cause. One obvious source of bias will be availability of open data, as much
research indexed in PubMed and ClinicalTrials.gov is not compliant with FAIR and open multilingual clinical trials in Wikidata and Wikipedia FAIR and open multilingual clinical trials in Wikidata and Wikipedia 5 recommended metadata standards. This project favors institutions which apply
FAIR principles, and we will identify these practices. Wikipedia as a publishing, technology, and community platform continually introduces
processes for content development and evaluation. In 2012, with the establishment of the
Wiki Education Foundation, there was a major cultural shift to make Wikipedia compatible
with university education and research. Today, that precedent has developed into a suite of
open evaluation tools for measuring audience size, levels of engagement, use of fact-
checking processes, and a culture applying metrics to perform critical review of Wikipedia's
quality. These measurements and processes establish a precedent for this project to follow
in doing publishing and content development, operationalizing ethics in digital governance,
and publicly demonstrating community conversation in seeking feedback on this project's
activities in the context of global Wikimedia content development. List research projects of the project coordinator. 1. Case studies of the student experience of publishing medical information in
Wikipedia
◦
Improving the Quality of Consumer Health Information on Wikipedia: Case
Series (Weiner et al. 2019) 1. Case studies of the student experience of publishing medical information in
Wikipedia
◦
Improving the Quality of Consumer Health Information on Wikipedia: Case
Series (Weiner et al. 2019) Case studies of the student experience of publishing medical information in
Wikipedia ◦
Improving the Quality of Consumer Health Information on Wikipedia: Case
Series (Weiner et al. 2019) Why Medical Schools Should Embrace Wikipedia (Azzam et al. 2016) Why Medical Schools Should Embrace Wikipedia (Azzam et al. 2016) 2. Documentation and programming for the Scholia interface to Wikidata and the
WikiCite project
◦
Robustifying Scholia: paving the way for knowledge discovery and research
assessment through Wikidata (Rasberry et al. 2019) ◦
Robustifying Scholia: paving the way for knowledge discovery and research
assessment through Wikidata (Rasberry et al. 2019) 3. Promotion of diversity, inclusion, and friendly collaboration online
◦
Automatic Detection of Online Abuse and Analysis of Problematic Users in
Wikipedia (Rawat et al. 2019) 3. Promotion of diversity, inclusion, and friendly collaboration online
◦
Automatic Detection of Online Abuse and Analysis of Problematic Users in
Wikipedia (Rawat et al. 2019)
Q
i
Wiki
di
(W
lb
t
l 2015) The project coordinator should describe their related research My name is Lane Rasberry and I am Wikimedian at the School of Data Science at the
University of Virginia. The most important contribution I have made to research has been
my advocacy for free and open media. I particularly do this in the context of Wikimedia
platform publishing where I showcase Wikidata as a FAIR and open repository which is
useful in itself and also a source from which anyone can export information for use in other
platforms. For any other project seeking to be FAIR and open, I present Wikidata as a
model project to emulate as the standard. My usual talking point with Wikipedia is that it is the most requested, published, accessed,
and consulted source of information in English on nearly every topic, including almost
every topic of general interest in medicine. I advocate for institutional partnerships between
expert health organizations, typically medical schools and media sources, and the
Wikimedia platform. Uniformly all expert health organizations have great challenges in
distributing their content to a relevant audience. In comparison, Wikipedia and Wikidata
have the world's best available distribution channels and reach a massive audience
through Internet search, personal assistants and similar mechanisms. My wish is to match
Wikipedia's excellent distribution but lower quality content with the excellent content of
expert organizations which have a relatively poorer distribution and media reach. Wikipedia's media reach is useful for any organization with an educational mission to
distribute high quality information of general interest. I have managed various programs
which integrate information into Wikipedia for the purpose of increased distribution. Along with that distribution, I have designed policy and published documentation on
various ethical considerations of online publishing, including making open media more
accessible to diverse audiences who collaborate in friendly online virtual spaces. Such
documentation and policy creates an environment where more people can participate and
share in the benefits of the FAIR and open global digital commons. Rasberry L, Mietchen D 6 6 How does the project coordinator practice open research? I Lane Rasberry have been a highly active Wikimedia platform contributor since 2008. I
have participated in Wikimedia institutional governance since 2012, including in the
platform organizations Wikimedia Medicine, Wikimedia New York City, and Wikimedia
LGBT+. I am as active as anyone else in the Wikimedia projects for university programs,
the WikiCite project, designing institutional partnerships, and reporting communication
metrics for evidence of impact. The Wikimedia platform opens research literature by being
the most requested, published, accessed, and consulted source of information on all topics
which it covers. I have been a member of the HIV Vaccine Trials Network Community Advisory Board since
2007. In this role I have advocated for public access to medical research information for a
long-running series of clinical trials. Personally I document and publish notes from organizational meetings of all sorts and
publish in the open as a default practice. I advocate for free and open licensing on any
media output which anyone intends for distribution to the public. Who are key collaborators for this project? School of Data Science, University of Virginia
Daniel Mietchen, data scientist
Lane Rasberry, Wikimedian in Residence UVA Global, University of Virginia This is a language department at the university where faculty and classes will translate and
publish structured data into Wikidata, where it will be FAIR and open in the semantic web. Wikimedia community organizationsThese organizations provide community feedback on
publication in Wikipedia and Wikidata and also on the translation process. These
partnerships ensure participation among stakeholders and regional communities of users. FAIR and open multilingual clinical trials in Wikidata and Wikipedia FAIR and open multilingual clinical trials in Wikidata and Wikipedia 7 7 The School of Data Science at the University of Virginia is importing structured data into
Wikidata, evaluating the quality of information in Wikipedia and Wikidata, and documenting
best practices for university partnerships with the Wikimedia platform. This team identifies
the research content for which there is a need in research discovery to be FAIR, available
for query, and translated to promote global collaboration. Daniel Mietchen, data scientist School of Data Science at the University of Virginia,
Principle Investigator of the Scholia / WikiCite project to develop the Wikipedia / Wikimedia
platform based interface for discovering and visualizing scholarly publications in a free and
open system analogous to the popular but closed product Google Scholar. Dr. Mietchen's
primary concern are the academic publications, whereas this proposal to Wellcome would
integrate clinical trial data into this network and also localize the interface for non-English
pilot languages relevant to the developing world. Another way to say this is that Dr. Mietchen operates a Wikipedia-based tool similar to PubMed and Google Scholar, and this
project would collaborate with him to integrate ClinicalTrials.gov data into this and permit
non-English language use. School of Data Science, University of Virginia School of Data Science, University of Virginia FAIR and open multilingual clinical trials in Wikidata and Wikipedia What outcomes will the completed project have? 1. Primary deliverables (Linked Open Data for production) 1. integrate clinical trials cataloging data from sites like ClinicalTrials.gov into
Wikidata 2. translate a limited vocabulary from ClinicalTrials.gov to Hindi, Bengali, and
Swahili Secondary deliverables (cultural products to promote diversity and good ethics) 2. Secondary deliverables (cultural products to promote diversity and good ethics)
1. document Wikidata as a research interface for clinical trials (English, Hindi,
Bengali, and Swahili)
2. publish a general-interest essay on ethical considerations of making
medical research data newly accessible 1. document Wikidata as a research interface for clinical trials (English, Hindi,
Bengali, and Swahili) 2. publish a general-interest essay on ethical considerations of making
medical research data newly accessible Wikidata is currently the central hub of the open Semantic Web. While various sources
such as libraries, repositories, and ClinicalTrials.gov provide "open data", without
integration into a Semantic Web portal like Wikidata, the information is largely inaccessible
even to field experts. By mapping data to Wikidata, the information becomes accessible to
the general public as well as professionals and researchers. Rasberry L, Mietchen D 8 Rasberry L, Mietchen D 8 This project will integrate ClinicalTrials.gov into Wikidata, making it once and for all
generally machine readable and free and open for export to any other platform. Furthermore, we will translate the search interface to three languages, Hindi, Bengali, and
Swahili, bringing this data to those languages for the first time and as a precedent in global
diversity. If we are successful to the limits of our expectations, then all clinical trials data forever after
will be free and open in the Semantic Web. Furthermore, we will set the precedent in this
project that linguistic diversity must be central to open data projects of global interest. Finally, we will publish a case study of this project in advocacy of accessible open data. Vision Our vision for this project is to increase public understanding and global discourse of
medical research by making cataloging data on clinical trials much easier to access, query,
and visualize in aggregate in English and 3 pilot underserved languages. Our aims are to enable the following: 1. through publication in Wikidata, professionals in clinical research will have radically
increased access to routine data about clinical trials, including from
ClinicalTrials.gov and PubMed 1. through publication in Wikidata, professionals in clinical research will have radically
increased access to routine data about clinical trials, including from
ClinicalTrials.gov and PubMed 2. beyond conventional clinical research data, and for the benefit of the general public
and humanities research, through Wikidata we will pilot access to previously
inaccessible social ClinicalTrials.gov data including integration with geolocation
data, grant and funding awards, corporate financing, and demographic data such
as nationality, gender, ethnicity, or socioeconomic status among research
participates 3. after sharing the data, we will document accessibility options for all kinds of people,
including citizen researchers, to use it. While the primary initial userbase will be
people who already use ClinicalTrials.gov, we seek to make this data accessible
and interesting to undergraduate students of all disciplines and pilot data
accessibility in non-English languages including Hindi, Bengali, and Swahili
languages. Monitoring Monitoring is a strength of this proposal which we take for granted as superior, and instead
our focus regarding monitoring will be in good reporting of what monitoring we accomplish
and documenting our monitoring process as a model for others to emulate. and documenting our monitoring process as a model for others to emulate. This project will publish its output into Wikidata, the structured data general
repository which is part of the Wikimedia platform. Since its inception in 2001, W
and the Wikimedia platform have developed a culture and community where an
edit and a mix of humans, human-operated semi-automated tools, and automa
monitor the billions of edits to millions of publications which hundreds of thou
people make every day in hundreds of languages. Our view is that in compariso
other general interest data curation project, Wikidata provides the most openn
transparency to scrutiny and natively provides the most information about how it p
its data collections. The best way to describe the monitoring plan for this project
that we will use the native Wikimedia platform monitoring suite of tools and pro
collect and report metrics including count of edits; count of reported changes or
count of errors identified in the source dataset; count of comments; count
reviewers and volunteer participant editors; and audience communication imp
project has the strength of having a designed monitoring system in place which w
change. Instead, we will make a model report collecting the metrics which are re
this project, and we will document how we collect those metrics from the Wikidata
and how we interpret them, and we will create documentation for anyone else to
for research production into Wikidata and monitor their own projects after our mod
Success indicators include the following:
1. integration of records from 80% of ClinicalTrials.gov trials into Wikidata, w
trial having an average of 10 structured data statements of fact
2. translation of a limited vocabulary for queries and the web interface to m
data accessible in English, Hindi, Bengali, and Swahili
3. published comments - endorsement or other feedback - from a diverse co
of 100 Wikimedia editors This project will publish its output into Wikidata, the structured data general reference
repository which is part of the Wikimedia platform. Open practices Openness and FAIR data integration is a strength of this proposal which we take for
granted as superior, and instead our focus regarding open data practices will be in good
reporting of what content we share and documenting our publication process as a model FAIR and open multilingual clinical trials in Wikidata and Wikipedia 9 for others to emulate. Our publication venue is in Wikidata which has been the most
popular, FAIR, and open cross-domain data repository in the world since at least 2015. This project starts with semi-structured open data in ClinicalTrials.gov which we will map to
Wikidata, thereby making it highly structured, FAIR, and accessible in the Semantic Web
and in multiple languages. Perhaps more significant than our making this data FAIR is our
intent to document our process as a case study to demonstrate how the data was
inaccessible and not FAIR before. Currently, many researchers see ClinicalTrials.gov to be
FAIR and open because they compare it to conventional data management. This project
will demonstrate how much more open this data can be and what networked integration
can accomplish. Monitoring Since its inception in 2001, Wikipedia
and the Wikimedia platform have developed a culture and community where anyone can
edit and a mix of humans, human-operated semi-automated tools, and automated bots
monitor the billions of edits to millions of publications which hundreds of thousands of
people make every day in hundreds of languages. Our view is that in comparison to any
other general interest data curation project, Wikidata provides the most openness and
transparency to scrutiny and natively provides the most information about how it processes
its data collections. The best way to describe the monitoring plan for this project is to say
that we will use the native Wikimedia platform monitoring suite of tools and products to
collect and report metrics including count of edits; count of reported changes or conflicts;
count of errors identified in the source dataset; count of comments; count of active
reviewers and volunteer participant editors; and audience communication impact. This
project has the strength of having a designed monitoring system in place which we will not
change. Instead, we will make a model report collecting the metrics which are relevant to
this project, and we will document how we collect those metrics from the Wikidata platform
and how we interpret them, and we will create documentation for anyone else to post data
for research production into Wikidata and monitor their own projects after our model. Success indicators include the following: 1. integration of records from 80% of ClinicalTrials.gov trials into Wikidata, with each
trial having an average of 10 structured data statements of fact
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2. translation of a limited vocabulary for queries and the web interface to make this
data accessible in English, Hindi, Bengali, and Swahili 3. published comments - endorsement or other feedback - from a diverse community
of 100 Wikimedia editors 3. published comments - endorsement or other feedback - from a diverse community
of 100 Wikimedia editors Rasberry L, Mietchen D 10 4. publication of documentation for anyone to model this project and in advocacy of
FAIR and open data 4. publication of documentation for anyone to model this project and in advocacy of
FAIR and open data What is your plan for managing project deliverables? The short explanation of our output management plan is that we will publish everything into
the Wikimedia platform, and deposit copies into our university institutional repository, and
additionally publish a research paper in an indexed academic journal. All data from this
project will have a Creative Commons Zero (CC0) dedication, and all other media will have
a Creative Commons Attribution 4.0 International (CC BY 4.0) license. We intend for every
part of this project to be open, FAIR, and accessible to a diverse audience of users. There are two kinds of research outputs for this proposal: structured data and prose
documentation. Our primary venue for publishing structured data is Wikidata, because
there it becomes available for production or export in the Semantic Web and keeps
metadata labels in multiple languages for its provenance and open licensing. We will
additionally publish a copy of our data as its own dataset and media product, including in
Zenodo and our university's institutional repository. We will set up a Wikimedia research
project page, as is customary for any project in the Wikimedia platform, and either host or
link out to all media projects from that page in the established way for this platform. This
research page will be a multimedia interface for accessing the data, using the data, and
browsing prose documentation. Prose documentation will include instructions for using this data in Wikidata or exporting it
for use in any other context. We will also publish information about the project to
encourage broad social discourse in diverse academic fields about public access to clinical
trials research. Audiences which we imagine include researchers, non-research
professionals, and citizens with interest in clinical research, health care, public policy,
corporate finance, research funding, public health, and social disparities. While our most
detailed prose explanations will be in English language, as the structured data for this
project is multilingual, we will also publish our portal interface in English, Hindi, Bengali,
and Swahili. Provide an explanation of the budget This project has two salaries - one for a researcher to oversee integration of the content
into Wikidata, and another for a data scientist to provide assistance with refining the
ClinicalTrials.gov data to the Wikidata model. Student researchers will conduct evaluation of data quality, characterization of this project's
dataset within the Open Semantic Web, and critique the proposal for the ethical
considerations which it raises. There are two research projects planned - one for a group of
data science students whose analysis will include machine learning and probably include
entity disambiguation and matching. In the summer project, the students will collaborate FAIR and open multilingual clinical trials in Wikidata and Wikipedia 11 11 with the Center for Ethics and Justice at the university to document the data's accessibility
and utility. The translations will happen fairly early in the project, as they concern the labels and query
terms for accessing the data, and usually not the data itself. This project begins with a
substantial structured data corpus of the target languages and will build from that rather
than originating any new system. Funding program This project was funded by Wellcome Trust via the Open Research Fund 2019 Grant title FAIR and open multilingual clinical trials in Wikidata and Wikipedia Hosting institution School of Data Science, University of Virginia Provide budget details •
Wikidata engagement: 6 weeks FTE
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data science: 6 weeks FTE
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equipment: 0
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graduate student research: 8 weeks FTE, university rates
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undergraduate student research: 8 weeks FTE, university rates
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translation: 4 weeks FTE, market rates through vendors
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other consulting: 4 weeks FTE, market rates through vendors References •
Azzam A, Bresler D, Leon A, Maggio L, Whitaker E, Heilman J, Orlowitz J, Swisher V,
Rasberry L, Otoide K, Trotter F, Ross W, McCue J (2016) Why Medical Schools Should
Embrace Wikipedia. Academic Medicine (1). https://doi.org/10.1097/ACM. 0000000000001381 •
Azzam A, Bresler D, Leon A, Maggio L, Whitaker E, Heilman J, Orlowitz J, Swisher V,
Rasberry L, Otoide K, Trotter F, Ross W, McCue J (2016) Why Medical Schools Should
Embrace Wikipedia. Academic Medicine (1). https://doi.org/10.1097/ACM. 0000000000001381 Rasberry L, Mietchen D 12 •
Rasberry L, Willighagen E, Nielsen F, Mietchen D (2019) Robustifying Scholia: paving
the way for knowledge discovery and research assessment through Wikidata. Research
Ideas and Outcomes 5 https://doi.org/10.3897/rio.5.e35820
•
Rawat C, Sarkar A, Singh S, Alvarado R, Rasberry L (2019) Automatic Detection of
Online Abuse and Analysis of Problematic Users in Wikipedia. 2019 Systems and
Information Engineering Design Symposium (SIEDS) https://doi.org/10.1109/sieds. 2019.8735592
•
Weiner SS, Horbacewicz J, Rasberry L, Bensinger-Brody Y (2019) Improving the
Quality of Consumer Health Information on Wikipedia: Case Series. Journal of Medical
Internet Research 21 (3). https://doi.org/10.2196/12450
•
Wexelbaum R, Herzog K, Rasberry L (2015) Queering Wikipedia. In: Wexelbaum R
(Ed.) Queers Online - LGBT Digital Practices in Libraries, Archives, and Museums. pp. 61–80. Litwin Books, Sacramento, California. [ISBN 978-1936117796]. •
Rasberry L, Willighagen E, Nielsen F, Mietchen D (2019) Robustifying Scholia: paving
the way for knowledge discovery and research assessment through Wikidata. Research
Ideas and Outcomes 5 https://doi.org/10.3897/rio.5.e35820 •
Rawat C, Sarkar A, Singh S, Alvarado R, Rasberry L (2019) Automatic Detection of
Online Abuse and Analysis of Problematic Users in Wikipedia. 2019 Systems and
Information Engineering Design Symposium (SIEDS) https://doi.org/10.1109/sieds. 2019.8735592 •
Weiner SS, Horbacewicz J, Rasberry L, Bensinger-Brody Y (2019) Improving the
Quality of Consumer Health Information on Wikipedia: Case Series. Journal of Medical
Internet Research 21 (3). https://doi.org/10.2196/12450 •
Wexelbaum R, Herzog K, Rasberry L (2015) Queering Wikipedia. In: Wexelbaum R
(Ed.) Queers Online - LGBT Digital Practices in Libraries, Archives, and Museums. pp. 61–80. Litwin Books, Sacramento, California. [ISBN 978-1936117796].
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Novel Method to Remove Bilirubin from the Blood of Jaundiced Rats: A Preliminary Study
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Jie Hao The First Affiliated Hospital of Xi'an Jiaotong University
Li-Fei Yang Introduction Jaundice is the primary symptom of severe liver disease that affects the ability of the liver to metabolise
and secrete bilirubin[1, 2]. Bilirubin is a cytotoxic substance that can cause mitochondrial dysfunction,
lead to organ system dysfunction, and even endanger life[3–6]. Currently, there are several limitations to
jaundice treatment, especially for jaundice caused by liver failure, as existing liver replacement therapy
cannot emulate the full physiological function of the liver[7–9]. In recent years, it has been proven that
the liver can be preserved using machine perfusion to maintain a physiological milieu. Therefore, this
study was designed to verify whether a novel therapy involving cross-circulation between the liver
preserved in vitro and the jaundiced rat can be effective. RESULTS Across different hyperbilirubinemia models, serum total bilirubin level was significantly improved (24.8 ±
1.2 vs. 17.4 ± 1.2 µmol/L, P < 0.05), despite a rise serum transaminase levels after treatment (AST: 4612 ±
382 vs. 5144 ± 390 U/L, P>0.05; ALT: 5051 ± 722 vs. 5488 ± 707 U/L, P<0.05). No necrosis was found in
the preserved liver tissue after treatment. This novel treatment significantly raised the long-term survival
rates of jaundiced rats (P < 0.05). CONCLUSION This novel method could safely and effectively help eliminate bilirubin from the blood of jaundiced rats. METHODS This novel therapy involved cross-circulation of blood between a rat liver preserved in vitro and a rat with
hyperbilirubinemia. All model animals were randomly divided into two groups based on whether they
received the novel therapy. Serum samples were collected at different time points. The levels of serum
transaminase and bilirubin were detected and compared between different groups. Long-term survival
was compared by Kaplan-Meier analysis between different groups. BACKGROUND The removal of bilirubin from serum is the primary method for treating hyperbilirubinemia. However,
currently used treatment methods have several limitations. This study was designed to introduce a novel
method to remove bilirubin from the blood of jaundiced rats. Research Article License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. R
d F ll Li Page 1/12 Animals Forty 6-week-old Sprague-Dawley rats, weighing 230–250 g, housed under a 12-h daylight/darkness cycle
and in an air-conditioned animal room with 50% humidity, were obtained from the Experimental Animal
Center of Xi’an Jiaotong University. These rats were jaundiced due to acute liver failure induced by D-
galactosamine. They were randomised into two groups according to the therapy administered: group T
(therapy, n = 20), rats in this group received the novel therapy; group C (control, n = 20), rats in this group
did not receive any therapy, but underwent carotid artery and jugular vein cannulation. Another 20 rats
were used as donors and underwent partial 50% hepatectomy before their livers were harvested. Forty 6-week-old Sprague-Dawley rats, weighing 230–250 g, housed under a 12-h daylight/darkness cycle
and in an air-conditioned animal room with 50% humidity, were obtained from the Experimental Animal
Center of Xi’an Jiaotong University. These rats were jaundiced due to acute liver failure induced by D-
galactosamine. They were randomised into two groups according to the therapy administered: group T
(therapy, n = 20), rats in this group received the novel therapy; group C (control, n = 20), rats in this group
did not receive any therapy, but underwent carotid artery and jugular vein cannulation. Another 20 rats
were used as donors and underwent partial 50% hepatectomy before their livers were harvested. Buprenorphine (0.05 mg/kg) was injected subcutaneously as an analgesic 1 hour before the operation. All the rats were anaesthetised, with 5% vol isoflurane at a flow rate of 1.5 L/min in a Plexiglas box used
to induce anaesthesia and 2% vol isoflurane at a flow rate of 0.6–0.8 L/min in a cone mask for the
maintenance of anaesthesia. All rats were heparinised by injecting 20 U of heparin diluted in 2 ml of
normal saline solution through the dorsal vein of the penis before treatment. This study was performed
according to the Guide for the Care and Use of Laboratory Animals published by the National Academy of
Sciences. The study design was approved by the Committee on the Ethics of Animal Experiments of Xi'an
Jiaotong University (Permit Number 2010 − 105). Animal model All the rats in group T and group C received an intraperitoneal injection of 1.1 g/kg of D(+)-galactosamine
hydrochloride (Sigma Chemical Corporation, St Louis, Missouri, USA) as a 100 mg/ml solution (pH 6.8) in
normal saline[10] 48 hours before receiving the novel therapy. Materials And Methods
Novel therapy procedure Page 2/12 This novel therapy is designed to stimulate cross-circulation between a liver preserved in vitro and a
jaundiced rat (Fig. 1). First, the jaundiced model rat underwent carotid and jugular vein catheterisation
using an anticoagulant catheter after anaesthesia. Next, a donor's liver was harvested after 30% partial
hepatectomy, and superior-inferior vena cava ligation and biliary intubation were performed. The donor's
liver was then preserved in normal saline at 4 ℃. Third, blood flow was restored by connecting the carotid
artery and jugular vein cannulas to the portal vein and the infrahepatic inferior vena cava of the donor's
liver (Fig. 2). Liver function tests Liver function tests, including serum aspartate aminotransferase (AST), alanine aminotransferase (ALT),
and total bilirubin (TBIL) levels were performed before modelling, at the beginning of treatment in group T
(ending of cannulation in group C), and 2 hours after treatment in group T (2 hours after cannulation in
group C). Rats used for survival analysis were observed for 48 hours after treatment. Animal model A drug-induced liver failure model in rats was successfully established in this study. In group T, serum
AST, ALT, and bilirubin levels increased significantly 24 hours after modelling (AST: 97 ± 2 vs. 4612 ± 382
U/L, P<0.05; ALT: 96 ± 3 vs. 5051 ± 722 U/L, P<0.05; TBIL: 9.7 ± 0.4 vs. 24.8 ± 1.2 mmol/L, P<0.05; see
Fig. 4A-C). The trend of serum bilirubin level change in group C was similar to that in group T (AST: 98 ± 3
vs. 4747 ± 527 U/L, P<0.05; ALT: 96 ± 3 vs. 4369 ± 390 U/L, P<0.05; TBIL: 10.0 ± 0.3 vs. 24.9 ± 1.3 mmol/L,
P<0.05; see Fig. 4D-F). Therapy All the jaundiced rats were treated successfully, and no rats died during the treatment. The donor liver
was perfused well after restoring blood flow, and no ischaemic lesions were found (Fig. 3A). Catheter
obstruction was not observed during the experiment. The donor liver produced bile during the treatment,
and the colour of bile during treatment was darker than that during harvesting (Fig. 3B). Liver function test and histological findings Serum bilirubin concentration in group T decreased significantly after the treatment (24.8 ± 1.2 vs. 17.4 ±
1.2 µmol/L, P<0.05, see Fig. 4C). However, serum AST and ALT levels were elevated after treatment (AST:
4612 ± 382 vs. 5144 ± 390 U/L, P>0.05; ALT: 5051 ± 722 vs. 5488 ± 707 U/L, P<0.05, see Fig. 4A-B). The
serum AST, ALT, and bilirubin levels of rats in group T continued to rise after cannulation (TBIL: 24.9 ± 1.3
vs. 26.0 ± 1.3 mmol/L, P>0.05; AST: 4747 ± 527 vs. 5466 ± 591 U/L, P>0.05; ALT: 4369 ± 390 vs. 5093 ±
380 U/L, P>0.05, see Fig. 4D-F). After treatment, the hepatic lobule structure of the liver in vitro was
normal, and hepatocyte necrosis was not observed on histological examination (Fig. 5). Histological analysis Page 3/12
After treatment, in vitro liver tissue was obtained and fixed in 10% buffered formalin overnight before
embedding in paraffin. Liver sections were cut to 4-µm thick and stained with haematoxylin and eosin. Statistical analysis After treatment, in vitro liver tissue was obtained and fixed in 10% buffered formalin overnight before
embedding in paraffin. Liver sections were cut to 4-µm thick and stained with haematoxylin and eosin. fter treatment, in vitro liver tissue was obtained and fixed in 10% buffered fo
mbedding in paraffin. Liver sections were cut to 4-µm thick and stained with Statistical analysis Page 3/12 Page 3/12 Numerical data were expressed as a mean ± SEM (Standard Error of Mean) and compared using the
Student’s t-test or Mann–Whitney U test. All data were analysed using Prism 6 (GraphPad Software Inc.,
CA, USA). Overall survival was estimated by the Kaplan-Meier method, and differences between groups
were compared via the log-rank test, using IBM SPSS Statistics ver. 22.0 (IBM Co., Armonk, NY, USA). A P-
value less than 0.05 was considered statistically significant. A biomedical statistician reviewed the study
data for statistical correctness. Survival The survival curves for both groups are shown in Fig. 6. Overall, the survival rate of rats in group T was
higher than that in group C within the 48 hours after therapy, and there was a significant statistical
difference between the two groups (P = 0.012). Discussion Page 4/12 Page 4/12 This study details a novel therapeutic strategy for bilirubin elimination from the blood of animal models. This therapy may help improve the prognosis of patients with jaundice. There are several theoretical pathophysiological approaches to filter serum bilirubin in cases of liver
failure. One approach would be to increase liver regeneration or perform liver transplantation. There are several theoretical pathophysiological approaches to filter serum bilirubin in cases of liver
failure. One approach would be to increase liver regeneration or perform liver transplantation. Unfortunately, the current supply of acceptable donor livers is not adequate to meet the high demands of
listed patients awaiting transplantation, resulting in thousands of deaths each year. An extracorporeal
system is another promising feasible approach to liver support. However, evidence suggests that the sole
use of liver support systems to remove bilirubin is not adequate to improve survival in patients with liver
failure[7, 11–13] because these systems can only partially replace the physiological function of the liver. In this study, we proposed a novel therapy to eliminate bilirubin from serum. A liver preserved in vitro was
perfused with the blood of a jaundiced rat through cross-circulation. As the carotid artery pressure is
higher than that of the jugular and portal veins, studies suggested that if the liver was perfused with the
blood at a higher pressure, it might disturb the microcirculation and increase the rate of hepatocellular
apoptosis[14–16]. However, no obvious hepatocyte apoptosis was detected on histological examination
of the liver in vitro after treatment. This might have been because the pressure of the arterial blood
significantly decreased after passing through the catheter. In addition, the liver was well perfused during
the treatment and was able to produce bile, indicating that the liver can play a role in detoxification and
secretion in vitro. Cross-circulation between two humans has been found to be effective for treating uraemia, treatment of
liver failure, provision of large numbers of leukocytes, and cardiopulmonary bypass during cardiac
surgery[17–21]. However, it appears that chance selection of a donor and recipient of divergent
histocompatibility might result in severe and even potentially fatal outcomes, such as immunologic
complications, disease transmission, and compromised donor safety. In this study, cross-circulation was
developed between the organ preserved in vitro and the recipient, which might have helped avoid the
above problems. Discussion In addition to a significant decrease in serum bilirubin, our method also helped reduce
48h survival of the jaundiced rats. Unlike other therapies for removing bilirubin from serum, this therapy
could compensate for all liver functions; this might be the main reason for improved 48h prognosis in
model rats. Although the results of this study are encouraging, several limitations should also be considered. First,
this study only proved that this novel treatment could reduce the serum bilirubin level of an individual
with hyperbilirubinemia and improve the long-term survival in an animal model. The effect of this
treatment on coagulation function and other toxic substances in serum remains unclear. In addition, this
study only verified the feasibility and effectiveness of the novel treatment method in the rat; thus, further
exploration is needed in larger mammals. List Of Abbreviations AST, aspartate aminotransferase Acknowledgements: Not applicable. Acknowledgements: Not applicable. AST, aspartate aminotransferase Page 5/12 ALT, alanine aminotransferase
TBIL, and total bilirubin ALT, alanine aminotransferase ALT, alanine aminotransferase TBIL, and total bilirubin Declarations Ethics approval: This study was performed according to the Guide for the Care and Use of Laboratory
Animals published by the National Academy of Sciences, and the study design was reviewed and
approved by the Committee on the Ethics of Animal Experiments of Xi'an Jiaotong University (Permit
Number 2020-105). The study was carried out in compliance with the ARRIVE guidelines. Consent for publication: Not applicable Availability of data and materials: The datasets used and analysed during the current study are available
from the corresponding author on reasonable request. Competing interests: The authors declare that they have no competing interests ompeting interests: The authors declare that they have no competing intere Competing interests: The authors declare that they have no competing interests Funding: This study was supported by the Shaanxi Province Key R&D Plan University Joint Project-Key
Project (2021GXLH-Z-047) and Natural Science Basic Research Program of Shaanxi (Program No. 2020JQ-528). Authors' contributions: Hao J. and Lu Q. designed the study; Lu Q., Ding H.F., Yang L.F., Wan Y. and Ren L. performed the research and acquired the data; Lu Q., Yang L.F. and Yu J.W. analyzed the data; Lu Q. and
Zhang X.F. drafted the manuscript; Lv Y. and Lu Q. contributed significantly to the revision of the
manuscript. References 7. García Martínez JJ, Bendjelid K: Artificial liver support systems: what is new over the last decade? Annals of intensive care 2018, 8(1):109. 8. Gonzalez HC, Jafri SM, Gordon SC: Management of Acute Hepatotoxicity Including Medical Agents
and Liver Support Systems. Clinics in liver disease 2017, 21(1):163–180. 8. Gonzalez HC, Jafri SM, Gordon SC: Management of Acute Hepatotoxicity Including Medical Agents
and Liver Support Systems. Clinics in liver disease 2017, 21(1):163–180. 9. Wang DW, Yin YM, Yao YM: Advances in the management of acute liver failure. World J
Gastroenterol 2013, 19(41):7069–7077. 9. Wang DW, Yin YM, Yao YM: Advances in the management of acute liver failure. World J
Gastroenterol 2013, 19(41):7069–7077. 10. E C, C L, TM C: Biochemical, hematological and histological changes in a fulminant hepatic failure
rat model for artificial liver assessment. Artificial organs 1979, 3(1):42–46. 10. E C, C L, TM C: Biochemical, hematological and histological changes in a fulminant hepatic failure
rat model for artificial liver assessment. Artificial organs 1979, 3(1):42–46. 11. Palmes D, Qayumi AK, Spiegel HU: Liver bridging techniques in the treatment of acute liver failure. Journal of investigative surgery: the official journal of the Academy of Surgical Research 2000,
13(6):299–311. 11. Palmes D, Qayumi AK, Spiegel HU: Liver bridging techniques in the treatment of acute liver failure. Journal of investigative surgery: the official journal of the Academy of Surgical Research 2000,
13(6):299–311. 12. Rajvanshi P, Larson AM, Kowdley KV: Temporary support for acute liver failure. Journal of clinical
gastroenterology 2002, 35(4):335–344. 12. Rajvanshi P, Larson AM, Kowdley KV: Temporary support for acute liver failure. Journal of clinical
gastroenterology 2002, 35(4):335–344. 13. Larsen FS: Artificial liver support in acute and acute-on-chronic liver failure. Curr Opin Crit Care 2019,
25(2):187–191. 13. Larsen FS: Artificial liver support in acute and acute-on-chronic liver failure. Curr Opin Crit Care 2019,
25(2):187–191. 14. K S, DL S, HU K, S T, R A, C G, T G, AH H, KT B: Portal hyperperfusion causes disturbance of
microcirculation and increased rate of hepatocellular apoptosis: investigations in heterotopic rat liver
transplantation with portal vein arterialization. Transplantation proceedings 2006, 38(3):725–729. 15. YF C, TL H, TY C, A C, LL T, CC W, SH W, CK S, CC L, YW L et al: Liver graft-to-recipient spleen size
ratio as a novel predictor of portal hyperperfusion syndrome in living donor liver transplantation. References Page 6/12
1. JI S, DC R: Diagnosis and evaluation of hyperbilirubinemia. Current opinion in gastroenterology 2017,
33(3):164–170. 2. E S, M J: New insights in bilirubin metabolism and their clinical implications. World journal of
gastroenterology 2013, 19(38):6398–6407. 3. R W: Bilirubin transport and toxicity. Mead Johnson Symposium on Perinatal and Developmental
Medicine 1982(19):25–31. 4. J F: Bilirubin in clinical practice: a review. Liver international: official journal of the International
Association for the Study of the Liver 2008, 28(5):592–605. 5. MM O, O F, H N, MR A, N A, HR M, M H, K M, S A, A J et al: Cholestasis-associated reproductive toxicity
in male and female rats: The fundamental role of mitochondrial impairment and oxidative stress. Toxicology letters 2019, 316:60–72. 1. JI S, DC R: Diagnosis and evaluation of hyperbilirubinemia. Current opinion in gastroenterology 2017,
33(3):164–170. 2. E S, M J: New insights in bilirubin metabolism and their clinical implications. World journal of
gastroenterology 2013, 19(38):6398–6407. 2. E S, M J: New insights in bilirubin metabolism and their clinical implications. World journal of
gastroenterology 2013, 19(38):6398–6407. 3. R W: Bilirubin transport and toxicity. Mead Johnson Symposium on Perinatal and Developmental
Medicine 1982(19):25–31. 3. R W: Bilirubin transport and toxicity. Mead Johnson Symposium on Perinatal and Developmental
Medicine 1982(19):25–31. 4. J F: Bilirubin in clinical practice: a review. Liver international: official journal of the International
Association for the Study of the Liver 2008, 28(5):592–605. 4. J F: Bilirubin in clinical practice: a review. Liver international: official journal of the International
Association for the Study of the Liver 2008, 28(5):592–605. 5. MM O, O F, H N, MR A, N A, HR M, M H, K M, S A, A J et al: Cholestasis-associated reproductive toxicity
in male and female rats: The fundamental role of mitochondrial impairment and oxidative stress. Toxicology letters 2019, 316:60–72. 5. MM O, O F, H N, MR A, N A, HR M, M H, K M, S A, A J et al: Cholestasis-associated reproductive toxicity
in male and female rats: The fundamental role of mitochondrial impairment and oxidative stress. Toxicology letters 2019, 316:60–72. Page 6/12 Page 6/12 6. M AB, RF S, D B: Bilirubin toxicity to human erythrocytes: a review. Clinica chimica acta; international
journal of clinical chemistry 2006, 374:46–56. 7. García Martínez JJ, Bendjelid K: Artificial liver support systems: what is new over the last decade? Annals of intensive care 2018, 8(1):109. References American journal of transplantation: official journal of the American Society of Transplantation and
the American Society of Transplant Surgeons 2006, 6(12):2994–2999. 16. M S, M C, A C, AD P: Liver function impairment in liver transplantation and after extended
hepatectomy. World journal of gastroenterology 2013, 19(44):7922–7929. 17. Di Donna D: [Cross transfusion in therapy of anuria]. Minerva medica 1952, 43(82):640–641. 17. Di Donna D: [Cross transfusion in therapy of anuria]. Minerva medica 1952, 43(82):640–641. 18. Burnell JM, Thomas ED, Ansell JS, Cross HE, Dillard DH, Epstein RB, Eschbach JW, Jr., Hogan R,
Hutchings RH, Motulsky A et al: OBSERVATIONS ON CROSS CIRCULATION IN MAN. The American
journal of medicine 1965, 38:832–841. 18. Burnell JM, Thomas ED, Ansell JS, Cross HE, Dillard DH, Epstein RB, Eschbach JW, Jr., Hogan R,
Hutchings RH, Motulsky A et al: OBSERVATIONS ON CROSS CIRCULATION IN MAN. The American
journal of medicine 1965, 38:832–841. 19. Salisbury PF, Bolomey AA, Miller JH: Cross transfusion. III. clinical experiences with 6 cases. The
American journal of the medical sciences 1952, 223(2):151–167. 19. Salisbury PF, Bolomey AA, Miller JH: Cross transfusion. III. clinical experiences with 6 cases. The
American journal of the medical sciences 1952, 223(2):151–167. 20. Bierman HR, Byron RL, Jr., Kelly KH, Dod KS, Black PM: Studies on cross circulation in man. I. Methods and clinical changes. Blood 1951, 6(6):487–503. 20. Bierman HR, Byron RL, Jr., Kelly KH, Dod KS, Black PM: Studies on cross circulation in man. I. Methods and clinical changes. Blood 1951, 6(6):487–503. 21. Warden HE, Cohen M, Read RC, Lillehei CW: Controlled cross circulation for open intracardiac
surgery: physiologic studies and results of creation and closure of ventricular septal defects. The
Journal of thoracic surgery 1954, 28(3):331–341; discussion, 341 – 333. 21. Warden HE, Cohen M, Read RC, Lillehei CW: Controlled cross circulation for open intracardiac
surgery: physiologic studies and results of creation and closure of ventricular septal defects. The
Journal of thoracic surgery 1954, 28(3):331–341; discussion, 341 – 333. Page 7/12 Page 7/12 Figures Figure 1 Page 8/12
Figure 1
Schematic diagram of the novel treatment method. Figure 1 Figure 1 Figure 1 Schematic diagram of the novel treatment method. Page 8/12 Page 8/12 Page 8/12 Figure 2 The novel method for the treatment of hyperbilirubinemia. A: The liver was perfused with arterial blood
from model animals via the portal vein. The blood then flowed back to the jugular vein of the model
animal through the inferior vena cava. The liver was well perfused and produced bile; B: Carotid and
jugular vein catheterization was performed using an anticoagulant catheter. The novel method for the treatment of hyperbilirubinemia. A: The liver was perfused with arterial blood
from model animals via the portal vein. The blood then flowed back to the jugular vein of the model
animal through the inferior vena cava. The liver was well perfused and produced bile; B: Carotid and
jugular vein catheterization was performed using an anticoagulant catheter. Figure 3
A: The liver preserved in vitro; B: The bile produced during harvesting (*) and treatment (#). Figure 3 A: The liver preserved in vitro; B: The bile produced during harvesting (*) and treatment (#). Page 9/12 Page 9/12 Figure 4
Changes of serum AST, ALT, and TBIL levels in the model animal. Changes of serum AST, ALT, and TBIL levels in the model animal. Page 10/12 Figure 5 Figure 5 Histological examination of the liver in vitro after treatment. Histological examination of the liver in vitro after treatment. Page 11/12 Page 11/12 Figure 6
48-hour survival of rats in different groups Figure 6
48-hour survival of rats in different groups Page 12/12
Figure 6
48-hour survival of rats in different groups Figure 6 48-hour survival of rats in different groups Page 12/12
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Pengaruh Kompensasi dan Pengalaman Kerja Terhadap Produktivitas Kerja Karyawan Departemen Food and Beverage Service
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Jurnal Ilmiah Pariwisata dan Bisnis
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* How to Cite
Ento , F. M., Lestari , D., & Wirawan , P. E. (2023). Pengaruh Kompensasi dan Pengalaman Kerja Terhadap Produktivitas Kerja
Karyawan
Departemen
Food
and
Beverage
Service
.
Jurnal
Ilmiah
Pariwisata
Dan
Bisnis,
2(5),
1101
-.
https://doi.org/10.22334/paris.v2i5.409 Felicia Meydina Ento 1*, Denok Lestari 2, Putu Eka Wirawan 3
1,2,3 D4 Manajemen Perhotelan, Institut Pariwisata dan Bisnis Internasional, Indonesia Felicia Meydina Ento 1*, Denok Lestari 2, Putu Eka Wirawan 3
1,2,3 D4 Manajemen Perhotelan, Institut Pariwisata dan Bisnis Internasional, Indonesia A R T I C L E I N F O Article History:
Submitted 8th Mei 2023
Revised 19th Mei 2023
Accepted 22th Mei 2023
Available online 30th Mei 2023 Kompensasi menjadi pendorong seseorang melaksanakan suatu
kegiatan guna mendapatkan hasil yang terbaik, sedangkan
pengalaman kerja perlu diperhatikan sesuai dengan pengalaman
kerja untuk melaksanakan kerja dengan efisien. Tujuan penelitian
ini untuk mengetahui pengaruh kompensasi dan pengalaman kerja
terhadap produktifitas kerja karyawan departemen food and
beverage service di Hotel Swiss-Belinn Singkawang. Metode yang
digunakan dalam penelitian ini adalah metode kuantitatif. Teknik
pengumpulan data menggunakan kuesioner dengan sampel
sebanyak 20 karyawan departemen Food and BeverageService. Hasil penelitian adalah nilai F hitung sebesar 35,696 dan F tabel 3,59. kompensasi berpengaruh positif dan signifikan secara parsial
terhadap produktifitas kerja dengan koefisien regresi sebesar 0,515
dan nilai t hitung sebesar 2,351 dan sig 0,000. Pengalaman Kerja
berpengaruh positif dan signifikan secara parsial terhadap
produktivitas kerja dengan koefisien regresi sebesar 0,136 dan nilai
t hitung sebesar 0,978 dan sig 0,342. Kompensasi tidak berpengaruh
positif dan tidak signifikan secara parsial terhadap produktivitas
kerja dengan koefisien regresi sebesar 0,978 dan nilai t hitung
sebesar 2,351 dan sig 0,000. Keywords: Compensation; Work Experience;
Work Productivity * Corresponding Author: Felicia Meydina Ento: feliciameyy@gmail.com The Effect Of Compensation And Work Experience On
Work Productivity Of Food And Beverage Service
Department Employees The Effect Of Compensation And Work Experience On
Work Productivity Of Food And Beverage Service
Department Employees The Effect Of Compensation And Work Experience On
Work Productivity Of Food And Beverage Service
Department Employees PARIS (Jurnal Pariwisata dan Bisnis)
Vol 02 No 5, 2023: 1101 – 1100
https://dx.doi.org/10.22334/paris.v2i5
EISSN: 2828-3325
Open Access: https://paris.ipb-intl.ac.id/ PARIS (Jurnal Pariwisata dan Bisnis)
Vol 02 No 5, 2023: 1101 – 1100
https://dx.doi.org/10.22334/paris.v2i5
EISSN: 2828-3325
Open Access: https://paris.ipb-intl.ac.id/ PARIS (Jurnal Pariwisata dan Bisnis)
Vol 02 No 5, 2023: 1101 – 1100
https://dx.doi.org/10.22334/paris.v2i5
EISSN: 2828-3325
Open Access: https://paris.ipb-intl.ac.id/ * Corresponding Author: Felicia Meydina Ento: feliciameyy@gmail.com
1101
* How to Cite
Ento , F. M., Lestari , D., & Wirawan , P. E. (2023). Pengaruh Kompensasi dan Pengalaman Kerja Terhadap Produktivitas Kerja
Karyawan
Departemen
Food
and
Beverage
Service
.
Jurnal
Ilmiah
Pariwisata
Dan
Bisnis,
2(5),
1101
-.
https://doi.org/10.22334/paris.v2i5.409 Pengaruh Kompensasi dan Pengalaman Kerja
Terhadap Produktivitas Kerja Karyawan Departemen
Food and Beverage Service Pengaruh Kompensasi dan Pengalaman Kerja
Terhadap Produktivitas Kerja Karyawan Departemen
Food and Beverage Service The Effect Of Compensation And Work Experience On
Work Productivity Of Food And Beverage Service
Department Employees DOI: https://doi.org/10.22334/paris. v2i5.409 1101 EISSN: 2828-3325 EISSN: 2828-3325 EISSN: 2828-3325 Jurnal Ilmiah Pariwisata dan Bisnis
Vol 02 No 5, 2023: 1101 - 1113
https://dx.doi.org/10.22334/paris.v2i5 A B S T R A C T Compensation becomes a driver for someone to carry out an activity to get the best results, while work
experience needs to be considered in accordance with work experience to carry out work efficiently. The purpose of this study was to find out the effect of compensation and work experience on the work
productivity of employees of the food and beverage service department at Hotel Swiss-Belinn
Singkawang. The method used in this study is a quantitative method. The data collection technique uses
questionnaires with a sample of 20 employees of the Food and Beverage Service department. The results
of the study were the calculated F value of 35,696 and the F table 3.59. Compensation has a positive and
partially significant effect on work productivity with regression coefficients of 0.515 and t-count values
of 2.351 and sig 0.000. Work experience has a positive and partially significant effect on work
productivity with regression coefficients of 0.136 and t-count values of 0.978 and sig 0.342. Compensation had no positive and partially insignificant effect on work productivity with regression
coefficients of 0.978 and t-count values of 2.351 and sig 0.000. https://paris.ipb-intl.ac.id/
Except where otherwise noted, content on this site is licensed under a Creative Commons
Attribution 4.0 International license. (CC BY 4.0) 1. PENDAHULUAN Industri pariwisata seperti hotel dan restoran mengalami perkembangan yang
menuntut agar tamu atau konsumen merasa nyaman dari berbagai aspek untuk
diberikan atau ditawarkan sebagai suatu produk kepada pelanggannya. Secara
umum, produk yang dijual oleh pihak manajemen hotel terdiri dari dua produk utama
yaitu produk nyata (tangible) Komponen produk nyata adalah sesuatu yang dapat
dilihat, disentuh atau diraba, diukur dan dihitung, misalnya: makanan dan minuman,
kamar tidur dan perlengkapan lainnya adalah merupakan komponen-komponen
produk nyata. Sedangkan komponen produk tidak nyata/abstrak (intangible
product). Faktor-faktor produk tidak nyata adalah segala sesuatu yang berkaitan
dengan pelayanan atau service dan citra suatu produk yang dihasilkan oleh hotel yang
hanya dapat dirasakan dan dapat dialami sebagai suatu pengalaman (Putri, 2018:22). Menyadari hal tersebut, saat ini banyak hotel yang menonjolkan bagian Food and
Beverage dalam upaya untuk menarik konsumen untuk mendapatkan kualitas
makanan dan minuman yang baik dan dengan servis yang memuaskan juga untuk
meningkatkan pendapatan. Berbagai upaya dilakukan mulai dari peningkatan rasa
dan kualitas makanan yang ditawarkan, peningkatan pelayanan (service) para waiter
dan waitress, hingga promosi yang gencar untuk memberi informasi kepada para
calon tamu hotel untuk mencoba berbagai tawaran makanan dan minuman yang
dipromosikan. Dengan adanya pemberian kompensasi yang baik dan pengalaman
kerja yang mencukupi, maka akan membantu karyawan tersebut dalam memahami
dan
juga
menyelesaikan
pekerjaannya
dengan
baik. Dalam
menciptakan
produktivitas yang bermutu tinggi, maka dibutuhkan adanya peningkatan
produktivitas yang maksimal dan juga mampu mendaya gunakan potensi sumber
daya manusia yang dimiliki Hotel Swiss-Belinn Singkawang mulai dari seluruh staf
dan karyawan bagian Food and Beverage Service guna menciptakan tujuan organisasi
yang telah direncanakan. Dalam meningkatkan kemampuan produktivitas kerja
karyawan, maka pengalaman kerja juga merupakan salah satu hal yang sangat
diperlukan. Pepatah mengatakan bahwa pengalaman adalah guru yang terbaik. 1102 Jurnal Ilmiah Pariwisata dan Bisnis
Vol 02 No 5, 2023: 1101 - 1113
https://dx.doi.org/10.22334/paris.v2i5 Jurnal Ilmiah Pariwisata dan Bisnis
Vol 02 No 5, 2023: 1101 - 1113
https://dx.doi.org/10.22334/paris.v2i5 EISSN: 2828-3325 EISSN: 2828-3325 Pengalaman kerja seorang karyawan juga menunjukan tingkat penguasaan dan
keterampilan “softskill” karyawan. hal yang sama diungkapkan oleh Sutrisno
(2009:158) berpendapat bahwa pengalaman kerja adalah suatu dasar/acuan seorang
karyawan untuk bisa menempatkan dirinya dengan cepat dalam kondisi yang baru,
berani mengambil resiko dan mampu menghadapi tantangan dengan penuh
tanggungjawab serta mampu berkomunikasi dengan baik terhadap berbagai pihak
untuk tetap menjaga kinerja dan produktivitas serta menghasilkan individu yang
berkompeten dalam bidangnya masing-masing. 1. PENDAHULUAN Kompensasi yang diberikan kepada
karyawan dapat berupa material seperti (Upah, gaji, bonus dan lainnya) dan dapat
berupa non material seperti (Asuransi, pujian, kenikmatan dan lain sebagainya). Kompensasi yang diberikan sesuai dengan prestasi karyawan yang sudah dilakukan
atas pekerjaannya dapat membangkitkan semangat karyawan untuk bekerja lebih
baik lagi, Karyawan bagian Food and Beverage Service Hotel Swis-Bellin Singkawang
mendapatkan kompensasi yang belum memenuhi harapan karyawan, sehingga
memberikan pengaruh negatif bagi peningkatan produktivitas kerja karyawan. Ketidakpuasan karyawan terhadap besarnya kompensasi yang diberikan oleh
perusahaan merupakan elemen utama terciptanya kepuasan kerja. 2. METODE PENELITIAN Lokasi penelitian terletak di Singkawang Grand Mall Area, Jl. Alianyang,
Pasiran, Singkawang Barat, Kota Singkawang, Kalimantan Barat 79123. Waktu
penelitian yang dilakukan peneliti dimulai sejak 18 September 2020 sampai 28
Februari 2021. Peneliti menggunakan keseluruhan populasi yang dimiliki oleh
departemen Food and Beverage Service Hotel Swiss-Belinn Singkawang. data yang
digunakan dalam penelitian ini sebanyak 2 jenis yaitu data kualitatif dan data
kuantitatif. Data kuantitatif yang dimaksud dalam penelitian ini ialah keseluruhan
jawaban kuesioner dan data kualitatif berasal dari sejarah hotel dan alamat hotel. Rumusan Masalah Berdasarkan uraian dari latar belakang di atas, maka dapat dirumuskan bahwa
masalah utama yang melatarbelakangi penelitian ini adalah : 1. Bagaimanakah pengaruh kompensasi terhadap produktivitas kerja karyawan
departemen Food and Bereverage Service Di Hotel Swiss-Belinn Singkawang ? 1. Bagaimanakah pengaruh kompensasi terhadap produktivitas kerja karyawan
departemen Food and Bereverage Service Di Hotel Swiss-Belinn Singkawang ? 2. Bagaimanakah pengaruh pengalaman kerja terhadap produktivitas kerja
kayawan departemen Food and Beverage Service Di Hotel Swiss-Belinn
Singkawang ? 2. Bagaimanakah pengaruh pengalaman kerja terhadap produktivitas kerja
kayawan departemen Food and Beverage Service Di Hotel Swiss-Belinn
Singkawang ? 3. Bagaimanakah pengaruh kompensasi dan pengalaman kerja terhadap
produktivitas kerja departemen Food and Beverage Service Di Hotel Swiss-
Belinn Singkawang ? 3. Bagaimanakah pengaruh kompensasi dan pengalaman kerja terhadap
produktivitas kerja departemen Food and Beverage Service Di Hotel Swiss-
Belinn Singkawang ? 1103 https://paris.ipb-intl.ac.id/
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Attribution 4.0 International license. (CC BY 4.0) Jurnal Ilmiah Pariwisata dan Bisnis
EISSN: 282
Vol 02 No 5, 2023: 1101 - 1113
https://dx.doi.org/10.22334/paris.v2i5
KERANGKA BERFIKIR
Gambar 1. Kerangka Berfikir EISSN: 2828-3325 Jurnal Ilmiah Pariwisata dan Bisnis
Vol 02 No 5, 2023: 1101 - 1113
https://dx.doi.org/10.22334/paris.v2i5 KERANGKA BERFIKIR KERANGKA BERFIKIR Gambar 1. Kerangka Berfikir Berikut hipotesis dari Pengaruh Kompensasi dan Pengalaman Kerja Terhadap
Produktivitas Kerja Hotel Swiss-Belinn Singkawang. a. Ho : Tidak ada pengaruh positif dan signifikan secara simultan dan parsial antara
kompensasi dan pengalaman kerja terhadap produktivitas kerja di Swiss-Belinn
Hotel Singkawang a. Ho : Tidak ada pengaruh positif dan signifikan secara simultan dan parsial antara
kompensasi dan pengalaman kerja terhadap produktivitas kerja di Swiss-Belinn
Hotel Singkawang b. Ha: Ada pengaruh positif dan signifikan secara simultan dan parsial antara
kompensasi dan pengalaman kerja terhadap produktivitas kerja di Swiss-Belinn
Hotel Singkawang b. Ha: Ada pengaruh positif dan signifikan secara simultan dan parsial antara
kompensasi dan pengalaman kerja terhadap produktivitas kerja di Swiss-Belinn
Hotel Singkawang https://paris.ipb-intl.ac.id/
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Attribution 4.0 International license. (CC BY 4.0) Definisi Operasional Produktivitas kerja yang di maksud dalam penelitian ini adalah upaya yang
dilakukan oleh Hotel Swiss-Belinn Singkawang di dalam mempekerjakan karyawan
dengan kemampuan kerja yang baik yang bersifat teoretis, maupun yang bersifat
praktis agar mampu bekerja secara efetif dan efisien. Berdasarkan teori Sedarmayanti
(2001) dan indikator-indikator Simamora (2004:612) menyebutkan produktivitas kerja 1104 Jurnal Ilmiah Pariwisata dan Bisnis
Vol 02 No 5, 2023: 1101 - 1113
https://dx.doi.org/10.22334/paris.v2i5 Jurnal Ilmiah Pariwisata dan Bisnis
Vol 02 No 5, 2023: 1101 - 1113
https://dx.doi.org/10.22334/paris.v2i5 EISSN: 2828-3325 EISSN: 2828-3325 menunjukkan bahwa individu merupakan perbandingan dari efektivitas keluaran
(pencapaian unjuk kerja maksimal) dengan efisiensi salah satu masukan (tenaga kerja)
yang mencangkup kuantitas, kualitas dalam waktu tertentu. Variabel produktivitas
kerja dapat diukur dengan menggunakan indikator yaitu : kuantitas kerja, kualitas
kerja dan ketepatan waktu. Kompensasi yang dimaksud dalam penelitian ini adalah
upaya yang dilakukan oleh Hotel Swiss-Belinn Singkawang untuk menghargai
prestasi karyawan, menjamin keadilan gaji karyawan, mempertahankan karyawan,
memperoleh karyawan yang bermutu dan memenuhi peraturan-peraturan. Menurut
Cahyani (2005:77-78) manajemen kompensasi adalah proses pengembangan dan
penerapan strategi, kebijakan, serta sistem kompensasi organisasi untuk mencapai
sasaranya dengan mendapatkan dan mempertahankan orang yang diperlukan dan
dengan meningkatkan motivasi serta komitmen mereka. Sedangkan indikator
Variabel pengalaman kerja dapat diukur dengan menggunakan indikator yaitu: upah,
gaji, insentif, fasilitas dan tunjangan Mondy dan Noe (2008:374). Untuk mengetahui
pengalaman kerja yang dimiliki oleh karyawan Hotel Swiss-Belinn Singkawang sudah
memenuhi standar yang diperlukan oleh Hotel agar pekerjaan yang dilakukan oleh
karyawan menjadi efisien. Menurut Ranupandojo (1984), mengemukakan bahwa
pengalaman kerja adalah ukuran tentang lama waktu atau masa kerja yang telah
ditempuh seseorang untuk dapat memahami tugas-tugas suatu pekerjaan dan telah
melaksanakan dengan baik.Variabel pengalaman kerja dapat diukur dengan
menggunakan indikator yaitu : Lama waktu/Masa kerja, Tingkat pengetahuan dan
keterampilan yang dimiliki dan penguasaan terhadap pekerjaan dan peralatan Foster
(2001:43.) Analisa Hasil Penelitian Uji Validitas 1105
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Analisa Hasil Penelitian Uji Validitas
Tabel 1. Uji Validitas (Sumber: Data diolah peneliti, 2021) Hasil Penelitian Analisa Hasil Penelitian Uji Validitas Tabel 2. Uji Validitas (Sumber: Data diolah peneliti, 2021) Tabel 2. Uji Validitas (Sumber: Data diolah peneliti, 2021) Tabel 2. Uji Validitas (Sumber: Data diolah peneliti, 2021) Tabel 3. Uji Validitas (Sumber: Data diolah peneliti, 2021) Tabel 3. Uji Validitas (Sumber: Data diolah peneliti, 2021) Uji validitas ini dilakukan dengan menghitung korelasi antara skor masing-
masing butir pertanyaan dengan total skor variabel. Responden dalam penelitian ini
adalah 20 orang, maka r-tabel dengan taraf signifikan 5% : 2 = 2,5% (uji 2 sisi) adalah
0.444. Dari Tabel 1,2 dan 3 terlihat bahwa semua nomor soal semuanya valid. Berdasarkan Tabel 1 Tabel 2 dan Tabel 3 yaitu mengenai hasil analisis instrument
kompensasi, pengalaman kerja dan produktivitas kerja menunjukkan bahwa seluruh
butir soal valid atau layak digunakan. Except where otherwise noted, content on this site is licensed under a Creative Commons
Attribution 4.0 International license. (CC BY 4.0) https://paris.ipb-intl.ac.id/ Tabel 1. Uji Validitas (Sumber: Data diolah peneliti, 2021) Tabel 1. Uji Validitas (Sumber: Data diolah peneliti, 2021) Uji Validitas (Sumber: Data diolah peneliti, 2 1105 https://paris.ipb-intl.ac.id/
Except where otherwise noted, content on this site is licensed under a Creative Commons
Attribution 4.0 International license. (CC BY 4.0) Jurnal Ilmiah Pariwisata dan Bisnis
EISSN: 2828-3325
Vol 02 No 5, 2023: 1101 - 1113
https://dx.doi.org/10.22334/paris.v2i5 Jurnal Ilmiah Pariwisata dan Bisnis
Vol 02 No 5, 2023: 1101 - 1113
https://dx.doi.org/10.22334/paris.v2i5 Jurnal Ilmiah Pariwisata dan Bisnis
Vol 02 No 5, 2023: 1101 - 1113
https://dx.doi.org/10.22334/paris.v2i5 EISSN: 2828-3325 Tabel 2. Uji Validitas (Sumber: Data diolah peneliti, 2021) https://paris.ipb-intl.ac.id/
Except where otherwise noted, content on this site is licensed under a Creative Commons
Attribution 4.0 International license. (CC BY 4.0) https://paris.ipb intl.ac.id/
Except where otherwise noted, content on this site is licensed under a Creative Commons
Attribution 4.0 International license. (CC BY 4.0) https://paris.ipb-intl.ac.id/ Uji Reliabilitas Tabel 4. Uji Reliabilitas (Sumber: Data diolah peneliti, 2021) Tabel 4. Uji Reliabilitas (Sumber: Data diolah peneliti, 2021) Hasil uji reliabilitas masing-masing variabel menunjukkan nilai Cronbach Alpha
diatas 0.60 sehingga dapat disimpulkan bahwa instrument yang digunakan dalam
penelitian ini adalah reliabel, sehingga dapat dipakai untuk analisi lebih lanjut. 1106 Jurnal Ilmiah Pariwisata dan Bisnis
Vol 02 No 5, 2023: 1101 - 1113
https://dx.doi.org/10.22334/paris.v2i5 Jurnal Ilmiah Pariwisata dan Bisnis
Vol 02 No 5, 2023: 1101 - 1113
https://dx.doi.org/10.22334/paris.v2i5 EISSN: 2828-3325 EISSN: 2828-3325 Analisis Regresi Linier Berganda Tabel 5. Analisis Regresi Linier Berganda (Sumber: Data diolah peneliti, 2021)
a. Dependent Variable: Total_Y Tabel 5. Analisis Regresi Linier Berganda (Sumber: Data diolah peneliti, 2021) a. Dependent Variable: Total_Y Except where otherwise noted, content on this site is licensed under a Creative Commons
Attribution 4 0 International license (CC BY 4 0) https://paris.ipb-intl.ac.id/ a.
Dependent Variable: Total_Y Hal ini berarti 80,8% produktivitas kerja operasional di Hotel Swiss-Belinn
Singkawang dipengaruhi oleh kompensasi dan pengalaman kerja. Sedangkan sisanya
sebesar 19,2% dipengaruhi oleh variabel-variabel lain yang tidak diteliti dalam
penelitian ini. Variabel lain yang tidak diteliti dalam penelitian ini yang peneliti dapati
dari hasil wawancara dengan HRD rekruitmen adalah variabel lingkungan kerja dan
motivasi. Uji Analisis Korelasi Berganda dan Analisis Determinasi
T b l 6 A
li i K
l
i B
d (S
b
D t di l h
liti 2021) Uji Analisis Korelasi Berganda dan Analisis Determinasi Tabel 6. Analisis Korelasi Berganda (Sumber: Data diolah peneliti, 2021) Tabel 6. Analisis Korelasi Berganda (Sumber: Data diolah peneliti, 2021) Dari hasil IBM SPSS V.16 terlihat bahwa nilai koefisien korelasi berganda sebesar
0,808 menunjukkan nilai positif dan tinggi. Artinya bahwa apabila kompensasi dan
pengalaman kerja meningkat, maka produktivitas kerja akan semakin baik. Apabila
kompensasi dan pengalaman kerja menurun, maka produktivitas kerja akan semakin
buruk. Hal ini berarti 80,8% produktivitas kerja operasional di Hotel Swiss-Belinn
Singkawang dipengaruhi oleh kompensasi dan pengalaman kerja. Sedangkan sisanya
sebesar 19,2% dipengaruhi oleh variabel-variabel lain yang tidak diteliti dalam
penelitian ini. Variabel lain yang tidak diteliti dalam penelitian ini yang peneliti dapati
dari hasil wawancara dengan HRD rekruitmen adalah variabel lingkungan kerja dan
motivasi. Uji F Uji F
Tabel 7. Uji F (Sumber: Data diolah, 2021)
a. Predictors: (Constant), Total_X2, Total_X1
b. Dependent Variable: Total_Y Uji F
Tabel 7. Uji F (Sumber: Data diolah, 2021)
a. Predictors: (Constant), Total_X2, Total_X1
b. Dependent Variable: Total_Y
Dari data diatas dapat dilihat bahwa nilai F hitung yang didapat sebesar 35.696
dan signifikansinya di bawah 0 05 yaitu 0 000 Hal tersebut berarti F hitung lebih besar Tabel 7. Uji F (Sumber: Data diolah, 2021) a. Predictors: (Constant), Total_X2, Total_X1 https://paris.ipb-intl.ac.id/ https://paris.ipb-intl.ac.id/
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Dependent Variable: Total_Y Dari kedua variabel independen yang dimasukkan dalam regresi, semua
variabel dengan α = 5% : 2 = 2,5% (uji 2 sisi). Dari hal tersebut dapat disimpulkan
bahwa variabel Y dipengaruhi oleh variabel X1 dan X2 dalam bentuk persamaan
regresi sebagai berikut: Y = 6.504+0,136X1 + 0,515X2. Koefisien regresi variabel kompensasi (X1) sebesar 0,136
Koefisien bernilai positif artinya terjadi hubungan positif antara kompensasi dengan
produktivitas kerja. Koefisien regresi variabel pengalaman kerja (X2) sebesar 0,515
Koefisien bernilai positif artinya terjadi hubungan positif antara pengalaman kerja
dengan produktivitas kerja. Oleh karena itu, semakin naik nilai pengalaman kerja
maka semakin meningkat nilai produktivitas kerja. Untuk melihat faktor yang paling
dominan dari kedua variabel independen tersebut dapat dilihat dari besarnya
keofisien regresi. Dari hasil analisis tersebut dapat dilihat bahwa pengalaman kerja
lebih dominan daripada kompensasi. Hal tersebut terlihat dari nilai koefiennya yaitu
sebesar 0,515. 1107 https://paris.ipb-intl.ac.id/
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Attribution 4.0 International license. (CC BY 4.0) Jurnal Ilmiah Pariwisata dan Bisnis
EISSN: 2828-3325
Vol 02 No 5, 2023: 1101 - 1113
https://dx.doi.org/10.22334/paris.v2i5 Jurnal Ilmiah Pariwisata dan Bisnis
Vol 02 No 5, 2023: 1101 - 1113
https://dx.doi.org/10.22334/paris.v2i5 Jurnal Ilmiah Pariwisata dan Bisnis
Vol 02 No 5, 2023: 1101 - 1113
https://dx.doi.org/10.22334/paris.v2i5 EISSN: 2828-3325 https://dx.doi.org/10.22334/paris.v2i5
Uji Analisis Korelasi Berganda dan Analisis Determinasi
Tabel 6. Analisis Korelasi Berganda (Sumber: Data diolah peneliti, 2021)
Dari hasil IBM SPSS V.16 terlihat bahwa nilai koefisien korelasi berganda sebesar
0,808 menunjukkan nilai positif dan tinggi. Artinya bahwa apabila kompensasi dan
pengalaman kerja meningkat, maka produktivitas kerja akan semakin baik. Apabila
kompensasi dan pengalaman kerja menurun, maka produktivitas kerja akan semakin
buruk. Hal ini berarti 80,8% produktivitas kerja operasional di Hotel Swiss-Belinn
Singkawang dipengaruhi oleh kompensasi dan pengalaman kerja. Sedangkan sisanya
sebesar 19,2% dipengaruhi oleh variabel-variabel lain yang tidak diteliti dalam
penelitian ini. Variabel lain yang tidak diteliti dalam penelitian ini yang peneliti dapati
dari hasil wawancara dengan HRD rekruitmen adalah variabel lingkungan kerja dan
motivasi. Uji Analisis Korelasi Berganda dan Analisis Determinasi
Tabel 6. Analisis Korelasi Berganda (Sumber: Data diolah peneliti, 2021)
Dari hasil IBM SPSS V.16 terlihat bahwa nilai koefisien korelasi berganda sebesar
0,808 menunjukkan nilai positif dan tinggi. Artinya bahwa apabila kompensasi dan
pengalaman kerja meningkat, maka produktivitas kerja akan semakin baik. Apabila
kompensasi dan pengalaman kerja menurun, maka produktivitas kerja akan semakin
buruk. Except where otherwise noted, content on this
Attribution 4.0 International license. (CC BY 4.0) 1108
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Except where otherwise noted content on this site is licensed under a Creative Commons p
p
/
here otherwise noted, content on this site is licensed under a Creative Commons
4 0 I t
ti
l li
(CC BY 4 0) a.
Dependent Variable: Total_Y a. Berdasarkan data diatas, nilai t-hitung dari variabel kompensasi sebesar 0,978
kurang dari t-tabel dengan α = 5% : 2 = 2,5% (uji 2 sisi) yaitu 1,734, sehingga dapat
dikatakan bahwa tidak terdapat pengaruh yang signifikan antara variabel kompensasi
dan variabel produktivitas kerja. Sedangkan nilai t-hitung variabel pengalaman kerja
sebesar 2,351 lebih besar daripada t-tabel dengan α = 5% : 2 = 2,5% (uji 2 sisi) yaitu
1,734, sehingga dapat dikatakan bahwa terdapat pengaruh yang signifikan antara
variabel pengalaman kerja terhadap produktivitas kerja. Berdasarkan data diatas, nilai t-hitung dari variabel kompensasi sebesar 0,978
kurang dari t-tabel dengan α = 5% : 2 = 2,5% (uji 2 sisi) yaitu 1,734, sehingga dapat
dikatakan bahwa tidak terdapat pengaruh yang signifikan antara variabel kompensasi
dan variabel produktivitas kerja. Sedangkan nilai t-hitung variabel pengalaman kerja
sebesar 2,351 lebih besar daripada t-tabel dengan α = 5% : 2 = 2,5% (uji 2 sisi) yaitu
1,734, sehingga dapat dikatakan bahwa terdapat pengaruh yang signifikan antara
variabel pengalaman kerja terhadap produktivitas kerja. Berdasarkan data diatas, nilai t-hitung dari variabel kompensasi sebesar 0,978
kurang dari t-tabel dengan α = 5% : 2 = 2,5% (uji 2 sisi) yaitu 1,734, sehingga dapat
dikatakan bahwa tidak terdapat pengaruh yang signifikan antara variabel kompensasi
dan variabel produktivitas kerja. Sedangkan nilai t-hitung variabel pengalaman kerja
sebesar 2,351 lebih besar daripada t-tabel dengan α = 5% : 2 = 2,5% (uji 2 sisi) yaitu
1,734, sehingga dapat dikatakan bahwa terdapat pengaruh yang signifikan antara
variabel pengalaman kerja terhadap produktivitas kerja. https://paris.ipb-intl.ac.id/
Except where otherwise noted, content on this site is licensed under a Creative Commons
Attribution 4.0 International license. (CC BY 4.0) b.
Dependent Variable: Total_Y Dari data diatas dapat dilihat bahwa nilai F hitung yang didapat sebesar 35.696
dan signifikansinya di bawah 0,05 yaitu 0,000. Hal tersebut berarti F hitung lebih besar 1108 Jurnal Ilmiah Pariwisata dan Bisnis
EISSN: 2828-3325
Vol 02 No 5, 2023: 1101 - 1113
https://dx.doi.org/10.22334/paris.v2i5
dari F tabel dengan α = 5% : 2 = 2,5% (uji 2 sisi) yaitu 3,59 sehingga dapat disimpulkan
bahwa Ho ditolak, dengan kata lain ada pengaruh yang signifikan antara variabel
kompensasi dan pengalaman kerja secara simultan terhadap variabel produktivitas
kerja departemen Food and Beverage Service di Hotel Swiss-Belinn Singkawang. Jurnal Ilmiah Pariwisata dan Bisnis
Vol 02 No 5, 2023: 1101 - 1113
https://dx.doi.org/10.22334/paris.v2i5 EISSN: 2828-3325 EISSN: 2828-3325 dari F tabel dengan α = 5% : 2 = 2,5% (uji 2 sisi) yaitu 3,59 sehingga dapat disimpulkan
bahwa Ho ditolak, dengan kata lain ada pengaruh yang signifikan antara variabel
kompensasi dan pengalaman kerja secara simultan terhadap variabel produktivitas
kerja departemen Food and Beverage Service di Hotel Swiss-Belinn Singkawang. Uji T j
Tabel 8. Uji T (Sumber: Data diolah, 2021)
a. Dependent Variable: Total_Y Tabel 8. Uji T (Sumber: Data diolah, 2021)
a. Dependent Variable: Total_Y
Berdasarkan data diatas, nilai t-hitung dari variabel kompensasi sebesar 0,978
kurang dari t-tabel dengan α = 5% : 2 = 2,5% (uji 2 sisi) yaitu 1,734, sehingga dapat
dikatakan bahwa tidak terdapat pengaruh yang signifikan antara variabel kompensasi
dan variabel produktivitas kerja. Sedangkan nilai t-hitung variabel pengalaman kerja
sebesar 2,351 lebih besar daripada t-tabel dengan α = 5% : 2 = 2,5% (uji 2 sisi) yaitu
1,734, sehingga dapat dikatakan bahwa terdapat pengaruh yang signifikan antara
variabel pengalaman kerja terhadap produktivitas kerja. Tabel 8. Uji T (Sumber: Data diolah, 2021)
a. Dependent Variable: Total_Y
Berdasarkan data diatas, nilai t-hitung dari variabel kompensasi sebesar 0,978
kurang dari t-tabel dengan α = 5% : 2 = 2,5% (uji 2 sisi) yaitu 1,734, sehingga dapat
dikatakan bahwa tidak terdapat pengaruh yang signifikan antara variabel kompensasi
dan variabel produktivitas kerja. Sedangkan nilai t-hitung variabel pengalaman kerja
sebesar 2,351 lebih besar daripada t-tabel dengan α = 5% : 2 = 2,5% (uji 2 sisi) yaitu
1,734, sehingga dapat dikatakan bahwa terdapat pengaruh yang signifikan antara
variabel pengalaman kerja terhadap produktivitas kerja. Pembahasan Peneliti melakukan penelitian mengenai pengaruh variabel kompensasi (X1) dan
variabel pengalaman kerja (X2) secara simultan berpengaruh positif dan signifikan
terhadap produktivitas kerja (Y). Hal ini ditunjukan dengan menggunakan uji F sesuai
dengan batasan signifikan > a (0,05) dengan taraf signifikan sebesar (0,000<0,05)
berarti Ha diterima dan sangat signifikan. Hasil Fhitung > Ftabel (35,696 > 3,59) yang
berarti ho ditolak, artinya pada karyawan Food and Beverage Service di Hotel Swiss-
Belinn Singkawang menunjukan bahwa kompensasi dan pengalaman kerja secara
bersama-sama berpengaruh terhadap produktifitas kerja karyawan. Hasil analisis tersebut mengambarkan bahwa kompensasi yang semakin tinggi
diikuti dengan pengalaman kerja yang semakin tinggi pula akan mempengaruhi
kompensasi dan produktivitas kerja. 1109 Jurnal Ilmiah Pariwisata dan Bisnis
Vol 02 No 5, 2023: 1101 - 1113
https://dx.doi.org/10.22334/paris.v2i5 Jurnal Ilmiah Pariwisata dan Bisnis
Vol 02 No 5, 2023: 1101 - 1113
https://dx.doi.org/10.22334/paris.v2i5 EISSN: 2828-3325 EISSN: 2828-3325 Penelitian ini didukung oleh penelitian Rafika (2016) yang berjudul “Pengaruh
Pengalaman Kerja dan Kompensasi Terhadap Produktivitas Kerja Karyawan UD Food
Sukses Makmur” yang menyatakan bahwa Pengalaman Kerja dan Kompensasi secara
simultan berpengaruh terhadap produktivitas kerja karyawan yang dibuktikan
dengan uji statistik Path Analysis menunjukan bahwa besar pengaruh pengalaman
kerja dan kompensasi adalah sebesar 55,80%, sedangkan besar pengaruh pengalaman
kerja dan produktivitas kerja sebesar 52,10%. Untuk memaksimalkan produktivitas kerja karyawan yang telah didapatkan
pada pengalaman kerja dan kompensasi tersebut bisa diimplementasikan melalui
produktivitas kerja yang meningkat. Menurut Sedarmayanti (2001) menyebutkan
produktivitas kerja menunjukkan bahwa individu merupakan perbandingan dari
efektivitas keluaran (pencapaian unjuk kerja maksimal) dengan efisiensi salah satu
masukan (tenaga kerja) yang mencangkup kuantitas, kualitas dalam waktu tertentu. Peneliti melakukan penelitian mengenai pengaruh variabel kompensasi (X1)
terhadap produktivitas kerja (Y) secara parsial dengan hasil tidak terdapat pengaruh
yang signifikan antara variabel kompensasi dan variabel produktivitas kerja. Hal ini
dapat dilihat dari uji-t. Uji-t ini digunakan untuk mengetahui seberapa kuat pengaruh
yang diberikan oleh variabel kompensi nilai Thitung yang didapat sebesar 0,978 dan
signifikansinya di atas 0,05 yaitu 0,342. Hal tersebut berarti Thitung lebih kecil dari
Ttabel dengan α = 5% : 2 = 2,5% (uji 2 sisi) yaitu 1,734 sehingga dapat disimpulkan
bahwa Ho diterima, dengan kata lain tidak ada pengaruh signifikan antara variabel
kompensasi (X1) terhadap variabel produktivitas kerja (Y) secara simultan. Hasil dari
penelitian ini berbanding balik dengan “kompensasi memiliki pengaruh dengan
produktivitas kerja” maka dapat disimpulkan bahwa tidak terbukti kebenarannya
atau artinya H0 diterima. https://paris.ipb-intl.ac.id/
Except where otherwise noted, content on this site is licensed under a Creative Commons
Attribution 4.0 International license. (CC BY 4.0) Pembahasan Hasil dari penelitian ini bertolak belakang dengan penelitian
yang dilakukan oleh Nelli (2019) yang berjudul “Pengaruh Kompensasi Terhadap
Produktifitas Kerja Karyawan Pada Perusahaan Daerah Air Minum (PDAM) Tirta
Rangga Kabupaten Subang” menyatakan bahwa hasil analisis menunjukkan adanya
pengaruh positif Kompensasi terhadap produktivitas kerja karyawan. Artinya
semakin tinggi Kompensasi yang diberikan kepada karyawan, maka akan semakin
tinggi produktivitas kerja karyawan tersebut. Sehingga produktivitas kerja karyawan
yang tinggi dapat dibentuk dengan adanya kompensasi yang tinggi, sementara itu
dalam hal kompesasi sangatlah berpengaruh pada produktivitas kerja karyawan
terutama pada hasil kerja yang dilakukan karyawan Hasil dari penelitian ini didukung oleh penelitian oleh Syahrul (2019) yang
berjudul “Pengaruh Kompensasi dan Pelatihan Kerja Terhadap Produktivitas
Karyawan CV. Rodajati Karanganyar” menyatakan bahwa hasil analisis menunjukan
tidak ada pengaruh yang signifikan terhadap produktifitas karyawan. Kompensasi
terdiri dari indikator upah, gaji, insentif, fasilitas, tunjangan. Penelitian lain yang
dilakukan oleh Dwi (2014) dengan judul “Pengaruh Kompensasi Terhadap
Produktivitas Kerja Pada Karyawan Tetap dan Karyawan Kontrak PT. Dan Liris
Indonesia” menyatakan bahwa gaji/upah tidak berpengaruh dan tidak signifikan 1110 Jurnal Ilmiah Pariwisata dan Bisnis
Vol 02 No 5, 2023: 1101 - 1113
https://dx.doi.org/10.22334/paris.v2i5 EISSN: 2828-3325 EISSN: 2828-3325 terhadap produktivitas kerja karyawan. Hal ini terjadi karena karyawan kontrak
dalam bentuk finansial yang mereka terima belum sesuai dengan keinginannya. terhadap produktivitas kerja karyawan. Hal ini terjadi karena karyawan kontrak
dalam bentuk finansial yang mereka terima belum sesuai dengan keinginannya. Setelah melakukan penelitian di departemen Food and Beverage Service di Hotel
Swiss-Belinn Singkawang dengan hasil yang menjelaskan bahwa kompensasi tidak
berpengaruh secara signifikan dengan produktivitas kerja karyawan. Hal ini terjadi
dikarenakan para karyawan merasa imbalan atau balas jasa yang diberikan kepada
mereka yang paling berarti adalah balas jasa finansial atau dalam bentuk uang. Selain
itu, hal ini juga terjadi karena pemberian kompensasi yang dirasakan kurang begitu
baik bagi karyawan yang masih dalam status kontrak atau outsourcing. Oleh karena
itu dapat disimpulkan bahwa semakin naik nilai kompensasi maka semakin
meningkat nilai produtivitas kerja, maka pihak hotel diharapkan mampu untuk
memberikan
kompensasi
yang
layak
kepada
karyawannya
untuk
dapat
mempertahankan produktivitas kerja karyawan. Penelitian mengenai pengaruh variabel pengalaman kerja (X2) terhadap
produktivitas kerja (Y) karyawan secara parsial. Terdapat pengaruh yang signifikan
antara variabel pengalaman kerja terhadap produtivitas kerja karyawan, hal ini dapat
dilihat dari hasil uji-t. Uji-t ini digunakan untuk mengetahui seberapa kuat pengaruh
yang diberikan oleh variabel bebas terhadap variabel terikat. Pembahasan Hasil uji-t menunjukkan
bahwa thitung sebesar 2,351 lebih daripada ttabel dengan α = 5% : 2 = 2,5% (uji 2 sisi)
yaitu 1,723 dengan signifikansi 0,031. Oleh karena itu semakin naik nilai pengalaman kerja maka semakin meningkat
nilai produtivitas kerja. Maka pihak hotel diharapkan mampu untuk melakukan tahap
seleksi yang sesuai dengan pengalaman kerja dan departemen yang dibutuhkan,
karena data yang peneliti dapatkan adalah pengalaman kerja berpengaruh terhadap
produktivitas kerja. Semakin tinggi pengalaman kerja yang dimiliki, semakin tinggi
pula tingkat produktivitas karyawan. https://paris.ipb-intl.ac.id/
Except where otherwise noted, content on this site is licensed under a Creative Commons
Attribution 4.0 International license. (CC BY 4.0) 4. SIMPULAN Berdasarkan rumusan masalah, tujuan, hasil analisis, dan pembahasan pada bab
sebelumnya tentang pengaruh kompensasi dan pengalaman kerja terhadap
produktivitas kerja karyawan Food and Beverage Sercie di Hotel Swiss-Belinn
Singkawang secara simultan dan parsial, maka kesimpulan dari penelitian bahwa
tidak terdapat pengaruh secara parsial kompensasi terhadap Produktivitas kerja
karyawan departemen Food and Beverage Service di Hotel Swiss-Belinn Singkawang
secara parsial dengan nilai signifikansi 0,342 > 0,05. Hal ini terjadi dikarenakan para karyawan merasa imbalan atau balas jasa yang
diberikan kepada mereka yang paling berarti adalah balas jasa finansial atau dalam
bentuk uang. Selain itu, hal ini juga terjadi karena pemberian kompensasi yang
dirasakan kurang begitu baik bagi karyawan yang masih dalam status kontrak. Dapat
disimpulkan bahwa semakin naik nilai kompensasi maka semakin meningkat nilai
produtivitas kerja, maka pihak hotel diharapkan mampu untuk memberikan 1111 Jurnal Ilmiah Pariwisata dan Bisnis
Vol 02 No 5, 2023: 1101 - 1113
https://dx.doi.org/10.22334/paris.v2i5 EISSN: 2828-3325 EISSN: 2828-3325 EISSN: 2828-3325 kompensasi yang layak kepada karyawannya untuk dapat mempertahankan
produktivitas kerja karyawan. Terdapat pengaruh positif dan signifikan antara Pengalaman Kerja terhadap
Produktivitas karyawan departemen Food and Beverage Service dengan nilai
signifikasi 0,05 < 0,00. Sehingga dapat disimpulkan bahwa jika kedua variabel
ditingkatkan secara bersama–sama, maka produktivitas karyawan juga akan
meningkat. Kompensasi dan pengalaman kerja memiliki pengaruh positif terhadap
produktivitas karyawan departemen Food and Beverage Service Di Hotel Swiss-
Belinn Singkawang secara parsial, dalam peningkatan kompensasi dan pengalaman
kerja memiliki kontribusi sebesar 80,8% dalam peningkatan produktivitas kerja. Sedangkan variabel lain yang tidak diteliti dalam penelitian ini sebesar 19,2% adalah
penelitian yang tidak dilakukan dalam penelitian ini. Dapat disimpulkan bahwa
semakin tinggi kompensasi dan pengalaman kerja, semakin tinggi pula produktivitas
kerja karyawan, dan demikian pula sebaliknya semakin kurangnya/rendah
kompensasi dan pengalaman kerja, maka produktivitas karyawan juga semakin
rendah. Dari pemaparan di atas menandakan bahwa kompensasi dan pengalaman kerja
memiliki pengaruh baik secara simultan dan berpengaruh secara parsial terhadap
produktivitas kerja karyawan departemen Food and Beverage Service di Hotel Swiss-
Belinn Singkawang. Artinya, seorang karyawan dapat meningkatkan produktivitas
dengan memiliki kompensasi yang bagus sekaligus memiliki banyak pengalaman
dalam bekerja. https://paris.ipb-intl.ac.id/
Except where otherwise noted, content on this site is licensed under a Creative Commons
Attribution 4.0 International license. (CC BY 4.0) Saran Berdasarkan simpulan tentang pengaruh kompensasi dan pengalaman kerja
terhadap produktivitas kerja karyawan Food and Beverage Service di Hotel Swiss-
Belinn Singkawang yang dilakukan pada penelitian ini, dapat disarankan beberapa
hal sebagai berikut. 1. Dalam peningkatan produktivitas kerja karyawan departemen Food and
Beverage Serive di Hotel Swiss-Belinn Singkawang terkait dengan kompensasi
dan pengalaman kerja, dinilai masih kurang dan perlu ditingkatkan, dalam hal
ini pihak hotel perlu mempertimbangkan karyawan yang memiliki pengalaman
kerja yang sesuai sesuai dapat ditempatkan pada posisi jabatan yang sesuai
dengan kualifikasi pengalaman kerja tersebut. Dengan demikian karyawan
dapat memberikan produktivitas kerja yang baik bagi perusahaan. 2. Dalam peningkatan produktivitas kerja, dapat dilihat bahwa kompensasi saja
tidak cukup, tetapi harus ditunjang dengan faktor lainnya seperti pengalaman
kerja. Maka dari itu Swiss-Belinn Hotel Singkawang sebaiknya lebih
mengutamakan untuk melihat pengalamankerja yang di miliki seorang calon
karyawan karena pengalaman kerja dalam penelitian ini terbukti berpengaruh 1112 Jurnal Ilmiah Pariwisata dan Bisnis
Vol 02 No 5, 2023: 1101 - 1113
https://dx.doi.org/10.22334/paris.v2i5 EISSN: 2828-3325 terhadap produktivitas kerja departemen Food and Beverage Service di Hotel
Swiss-Belinn Singkawang. terhadap produktivitas kerja departemen Food and Beverage Service di Hotel
Swiss-Belinn Singkawang. 5. DAFTAR PUSTAKA Cahyani. 2005. Dasar – Dasar Organisasi dan Manajemen, Jakarta: PT. Grasindo. Foster, Bill. 2001. Pembinaan UntukPeningkatan Kinerja Karyawan. Jakarta: PPM
Mondy, Wayne. 2008 Manajemen Sumber Daya Manusia Jilid 1. Jakarta: Erlangga
Putri, Emita Devi Hari. 2018. Pengantar Akomodasi dan Restoran. Yogyakarta:
D
bli h Cahyani. 2005. Dasar – Dasar Organisasi dan Manajemen, Jakarta: PT. Grasindo. Foster, Bill. 2001. Pembinaan UntukPeningkatan Kinerja Karyawan. Jakarta: PPM
Mondy, Wayne. 2008 Manajemen Sumber Daya Manusia Jilid 1. Jakarta: Erlangga Putri, Emita Devi Hari. 2018. Pengantar Akomodasi dan Restoran. Yogyakarta:
Deepublish. Sedarmayanti, M. 2001, Sumber Daya Manusia dan Produktivitas Kerja, Bandung: CV. Mandar Maju. Sutrisno, Edi. 2009. Manajemen Sumber Daya Manusia, Kencana, Jakarta Fajar, S. 2019. Pengaruh Kompensasi Dan Pelatihan Kerja Terhadap Produktivitas
Karyawan CV. Rodajati Karanganyar (Doctoral dissertation, Universitas
Muhammadiyah Surakarta). Faris, R., Bagia, I. W., & Suwendra, I. W. 2016. Pengaruh Pengalaman Kerja dan
Kompensasi Terhadap Produktivitas Kerja Karyawan. Jurnal Manajemen
Indonesia, 4 (1). Muslimah, D. A. 2014. Pengaruh Kompensasi Terhadap Produktivitas Kerja Pada
Karyawan Tetap dan Karyawan Kontrak PT. Dan Liris Indonesia Sulistiana, N. 2019. “Pengaruh Kompensasi Terhadap Produktivitas Kerja Karyawan
Pada Perusahaan Daerah Air Minum (PDAM) Tirta Rangga Kabupaten
Subang.”The
World
of
Business
Administration
Journal,
70-92. https://doi.org/10.37950/wbaj.vi.747 1113 https://paris.ipb-intl.ac.id/
Except where otherwise noted, content on this site is licensed under a Creative Commons
Attribution 4.0 International license. (CC BY 4.0)
|
https://openalex.org/W2980594759
|
https://www.frontiersin.org/articles/10.3389/fonc.2019.01094/pdf
|
English
| null |
A Qualitative Transcriptional Signature for Predicting Recurrence Risk for High-Grade Serous Ovarian Cancer Patients Treated With Platinum-Taxane Adjuvant Chemotherapy
|
Frontiers in oncology
| 2,019
|
cc-by
| 7,931
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Yixin Liu 1†, Zheyang Zhang 2†, Tianhao Li 2, Xin Li 2, Sainan Zhang 2, Ying Li 2,
Wenyuan Zhao 2, Yunyan Gu 2, Zheng Guo 2,3,4* and Lishuang Qi 2* Yixin Liu 1†, Zheyang Zhang 2†, Tianhao Li 2, Xin Li 2, Sainan Zhang 2, Ying Li 2,
Wenyuan Zhao 2, Yunyan Gu 2, Zheng Guo 2,3,4* and Lishuang Qi 2* Edited by:
Connie Irene Diakos,
Royal North Shore Hospital, Australia Edited by:
Connie Irene Diakos,
Royal North Shore Hospital, Australia 1 Basic Medicine College, Harbin Medical University, Harbin, China, 2 College of Bioinformatics Science and Technology,
Harbin Medical University, Harbin, China, 3 Key Laboratory of Ministry of Education for Gastrointestinal Cancer, Department of
Bioinformatics, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, China, 4 Key Laboratory of Medical
Bioinformatics, Fuzhou, China ,
Royal North Shore Hospital, Australia
Reviewed by:
Hitoshi Tsuda,
National Defense Medical
College, Japan
Sophia H. L. George,
University of Miami, United States
*Correspondence:
Lishuang Qi
qilishuang7@ems.hrbmu.edu.cn
Zheng Guo
guoz@ems.hrbmu.edu.cn
†These authors have contributed
equally to this work Reviewed by:
Hitoshi Tsuda,
National Defense Medical
College, Japan
Sophia H. L. George,
University of Miami, United States Resistance to platinum and taxane adjuvant chemotherapy (ACT) is the main cause of
the recurrence and poor prognosis of high-grade serous ovarian cancer (HGS-OvCa)
patients receiving platinum-taxane ACT after surgery. However, currently reported
quantitative transcriptional signatures, which are commonly based on risk scores
summarized from gene expression, are unsuitable for clinical application because of
their high sensitivity to experimental batch effects and quality uncertainties of clinical
samples. Using 226 samples of HGS-OvCa patients receiving platinum-taxane ACT
in TCGA, we developed a qualitative transcriptional signature, consisting of four
gene pairs whose within-samples relative expression orderings could robustly predict
patient recurrence-free survival (RFS). In two independent test datasets, the predicted
non-responders had significantly shorter RFS than the predicted responders (log-rank
p < 0.05). In a test dataset containing data for patient pathological response state,
the signature reclassified 12 out of 22 pathological complete response patients as
non-responders and two out of 16 pathological non-complete response patients as
responders. Notably, the 12 predicted non-responders in the pathological complete
response group had significantly shorter RFS than the predicted responders (log-
rank p = 0.0122). ORIGINAL RESEARCH
published: 18 October 2019
doi: 10.3389/fonc.2019.01094 ORIGINAL RESEARCH
published: 18 October 2019
doi: 10.3389/fonc.2019.01094 This qualitative transcriptional signature, which is insensitive to
experimental batch effects and quality uncertainties of clinical samples, can individually
identify HGS-OvCa patients who are more likely to benefit from platinum-taxane
adjuvant chemotherapy. *Correspondence:
Lishuang Qi
qilishuang7@ems.hrbmu.edu.cn
Zheng Guo
guoz@ems.hrbmu.edu.cn †These authors have contributed
equally to this work †These authors have contributed
equally to this work †These authors have contributed
equally to this work Specialty section:
This article was submitted to
Women’s Cancer,
a section of the journal
Frontiers in Oncology Received: 19 April 2019
Accepted: 04 October 2019
Published: 18 October 2019 Keywords: ovarian cancer, platinum chemotherapy, taxane chemotherapy, predictive signature, relative
expression orderings Data Sources and Data Pre-processing Data Sources and Data Pre-processing
In this study, one gene expression dataset of HGS-OvCa
was downloaded from The Cancer Genome Atlas (TCGA,
https://portal.gdc.cancer.gov/, 2017) and two gene expression
datasets of HGS-OvCa were downloaded from the Gene
Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/). TCGA expression profiles of 226 stage II-IV HGS-OvCa patients
receiving platinum-taxane ACT after surgery was used to
train a qualitative predictive signature. In the training dataset
(TCGA), 163 patients were diagnosed as having a pathological
complete response (CR), while the other 63 patients were
diagnosed as having a pathological non-complete response
(non-CR), including 32 partial responses, 17 stable disease,
and 14 progressive disease patients (13). One independent
dataset [GSE30161 (14)], denoted as test 1, recorded the RFS
information and the pathological response states of patients
receiving platinum-taxane ACT after surgery, including 22 CR
patients and 16 non-CR patients (15 partial response and 1
progressive disease). The dataset was used to test the performance
of the signature in predicting recurrence risk and pathological
response state of patients receiving platinum-taxane ACT. Another independent dataset [GSE9891 (15)], denoted as test 2,
which only provide the RFS information of 132 patients, was used
to test the performance of the qualitative signature in predicting
recurrence risk for patients receiving platinum-taxane ACT after
surgery. Notably, all the primary tumor samples used in this study
were extracted from the HGS-OvCa patients before receiving
ACT. The clinical information of patients in the three datasets
is described in Table 1. Recently, some studies have been devoted to developing
predictive signatures for platinum-taxane ACT, based on gene
expression profiles of primary tumor samples (4–6). However,
most of the reported transcriptional signatures, such as 40-
gene signature (5) and 23-gene signature (6), are based on risk
scores to distinguish patients with shorter and longer survival
rates after receiving platinum-based ACT, and defined them
as non-responders and responders to platinum-based ACT,
respectively. While, responders (or non-responders) predicted by
these signatures may be resistant (or sensitive) to platinum-based
ACT because of a low (or high) degree of malignancy of their own
cancer cells (7), the signature is not directly applied to predict the
sensitivity of platinum or taxane. Therefore, information about a
patient’s pathological response should be utilized to increase the
relevance of signatures to platinum-taxane ACT. INTRODUCTION In this study, we combined the pathological response
states and the recurrence-free survival (RFS) information to
extract a qualitative transcriptional signature for predicting
the RFS of patients receiving platinum-taxane ACT after
surgery. The performance of the signature was validated in two
independent datasets. Epithelial ovarian cancer has the highest mortality rate of all
gynecologic cancers. The majority of patients with ovarian cancer
are diagnosed as high grade (i.e., grade 2–3) (1). The standard
treatment for high-grade serous ovarian cancer (HGS-OvCa)
is surgery and platinum-based adjuvant chemotherapy (ACT)
which is typically administered concurrently with taxane (2),
denoted as platinum-taxane ACT. However, more than 70% of
HGS-OvCa patients will develop recurrent disease within a few
years after receiving platinum-taxane ACT, resulting in a 5-year
survival rate of <40% (1, 3). Therefore, it is necessary to develop a
predictive signature to distinguish responders who should receive
platinum-taxane ACT from non-responders who should receive
alternative therapies. Citation: Liu Y, Zhang Z, Li T, Li X, Zhang S,
Li Y, Zhao W, Gu Y, Guo Z and Qi L
(2019) A Qualitative Transcriptional
Signature for Predicting Recurrence
Risk for High-Grade Serous Ovarian
Cancer Patients Treated With
Platinum-Taxane Adjuvant
Chemotherapy. Front. Oncol. 9:1094. doi: 10.3389/fonc.2019.01094 October 2019 | Volume 9 | Article 1094 Frontiers in Oncology | www.frontiersin.org Qualitative Signature for Ovarian Cancer Liu et al. Data Sources and Data Pre-processing More importantly, our recent work (8) has shown that this
kind of signature, based on risk scores summarized from gene
expression measurements of the signature genes, tends to be
impractical in clinical settings because its application requires the
pre-collection of samples for data normalization to overcome the
large measurement batch effects between different datasets (9). Therefore, sample risk classification will be influenced by the risk
composition of other samples adopted for data normalization. Additionally, the signature based on the quantitative expression
measurements lacks robustness for quality uncertainties of
clinical samples, including varied proportions of tumor epithelial
cells in tumor tissues from different tumor locations in the
body of the same patient (10), partial RNA degradation during
specimen storage and preparation (11), and RNA amplification
bias for minimum specimens (12). For TCGA data derived from Illumina HiSeq 2000 RNA
Sequencing Version 2 (Illumina, San Diego, CA, USA), the
normalized count values determined by fragments per kilobase
of exon per million fragments mapped (FPKM) method were
obtained and log2-transformed was used for the gene expression. For data generated by Affymetrix platforms, the robust multi-
array average algorithm (RMA) was used for pre-processing the
raw data. Probe IDs were matched with Gene IDs using the
corresponding platform files. For each sample, the expression
measurements of all probe IDs corresponding to the same Gene
ID were averaged to obtain a single measurement. Probes that did
not match any Gene ID or that matched multiple Gene IDs were
deleted (16). In contrast, the within-sample relative expression orderings
(REOs) of genes are the qualitative transcriptional characteristics
of samples, which are robust against the experimental batch
effects, and disease signatures based on REOs can be directly
applied to individual level samples (8). Most importantly, we have
reported that the within-sample REOs of genes are highly robust
against the above-mentioned quality uncertainties of clinical
samples, which are common factors that can lead to the failure of
the quantitative transcriptional signature in clinical applications. Therefore, it is worthwhile to apply the within-sample REOs to
find a robust qualitative transcriptional signature to predict the
response states of platinum-taxane ACT for HGS-OvCa patients. Frontiers in Oncology | www.frontiersin.org Survival Analyses The RFS of patients were truncated at 5 years (60 months)
such that patients with more than 5 years of follow-up were
censored at 5 years. Survival curves were estimated using the
Kaplan–Meier method and compared using the log-rank test. October 2019 | Volume 9 | Article 1094 2 Liu et al. Qualitative Signature for Ovarian Cancer Liu et al. TABLE 1 | Clinical information of datasets analyzed in this study. Variable
Training
Test 1
Test 2
Data source
TCGA
GSE30161
GSE9891
Sample size
Total
226
38
132
Treatment
Drug
Platinum-
taxane
Platinum-
taxane
Platinum-
taxane
Survival data
–
RFS
RFS
RFS
Age
–
59 (34-87)
62 (44–78)
59 (23-79)
Grade
G2
30
14
56
G3
196
24
76
Tumor stage
II
14
–
3
III
181
34
118
IV
31
4
11
Residual tumor
0–10 nm
109
14
76
≥11 nm
55
23
43
Un
62
1
13
Response state
CR
163
22
–
Non-CR
63
16
–
PR
32
15
–
SD
17
0
–
PD
14
1
-
Platform
–
Illu.HiSeqV2
Affy.U133
Plus2.0
Affy.U133
Plus2.0
RFS, Recurrence-free survival; Un, unknown; CR, Complete response; Non-CR, Non-
Complete response including Partial response (PR), Stable disease (SD), and Progressive
disease (PD); Affy., Affymetrix; Illu., Illumina. prognosis-associated genes, a and b, respectively. From all the
possible gene pairs, we selected prognosis-associated gene pairs
whose specific REO patterns (e.g., Ea > Eb) were significantly
associated with longer RFS of patients using the univariate Cox
model with 5% FDR control. For each prognosis-associated gene
pair, the patients with the specific REO pattern (e.g., Ea > Eb)
were voted as responders, otherwise (e.g., Ea < Eb) voted as
non-responders. The C-index was used to evaluate predictive
performance of each prognosis-associated gene pair. Based on the prognosis-associated gene pairs, we applied
a forward selection procedure to search for a signature that
achieved the largest C-index value for predicting the RFS of
patient receiving platinum-taxane ACT. Here, we chose each
prognosis-associated gene pair as a seed and added to the optimal
gene pair one at a time until the C-index did not increase. During the procedure, we adopted a simple majority-voting rule
as follows: a sample was predicted as the responder, if more than
half of the REOs (Ea > Eb) of gene pairs in the set voted as
a responder; otherwise, it will be considered a non-responder. Statistics Analyses RFS, Recurrence-free survival; Un, unknown; CR, Complete response; Non-CR, Non-
Complete response including Partial response (PR), Stable disease (SD), and Progressive
disease (PD); Affy., Affymetrix; Illu., Illumina. The Chi-square test was used to examine the association of two
response groups predicted by the GPS with the four known
molecular subtypes of HGS-OvCa patients. Fisher’s exact test
was used to examine the association of two response groups
predicted by the signature with the pathological response states. Student’s t-test was used to examine the difference in levels of
gene expression between the two response groups predicted by
the signature. For exploring the biological function of DE genes,
we conducted the gene functional enrichment analysis using
the R package clusterProfiler (18), where a hypergeometric test,
based on the current Kyoto Encyclopedia of Genes and Genomes
(KEGG) databases, was employed. Significance was defined as p
< 0.05 or FDR < 0.05 for multiple testing. All statistical analyses
were performed using the R 3.4.3 (http://www.r-project.org/). The univariate Cox proportional-hazards regression model was
used to identify risk factors for recurrence of HGS-OvCa patients,
including known prognostic clinical factors: age (≥60 vs. <60
years), stage (IV vs. III vs. II), histological grade (3 vs. 2)
and residual tumor (≥11 vs. 0–10 mm). The multivariate Cox
proportional-hazards regression model was used to evaluate the
independent performance of the signature after adjusting for
the clinical factors with p-value <0.2 in the univariate Cox
model. Hazard ratios (HRs) and 95% confidence intervals (CIs)
were generated using the Cox proportional hazards model. The
concordance index (C-index) (17) was used to estimate the
predictive performance of a signature for patient survival. Survival Analyses We selected the set of gene pairs that had the largest C-index
in the procedure as a predictive gene pair signature (GPS) for
platinum-taxane ACT. Frontiers in Oncology | www.frontiersin.org Developing a Qualitative Predictive
Signature for Platinum-Taxane ACT g
Step 1: Finding prognosis-associated genes: Figure 1 describes the flowchart of this study. Here, we focused
on analyzing the 9,819 genes commonly measured by the two
platforms, IlluminaHiSeq_RNASeqV2 and Affymetrix U133 Plus
2.0, used in this study. First, we extracted 555 potential DE
genes between 163 pathological CR and 63 pathological non-
CR patients from the TCGA dataset (Student’s t-test, p < 0.05). Among the potential DE genes, we pre-selected 70 potential
prognosis-associated genes whose expressions were significantly
associated with patients’ RFS (univariate Cox model, p < 0.05). Next, from all the gene pairs constructed by the prognosis-
associated genes, we identified 30 prognosis-associated gene
pairs whose REOs were significantly associated with patients’ In the training data, Student’s t-test with 5% p control was
used to select the potentially differentially expressed genes (DE
genes) between pathological CR and non-CR groups. From
the potential DE genes, we selected the potential prognosis-
associated genes whose gene expressions were significantly
associated with patients’ RFS using the univariate Cox model with
5% p control. p
Step 2: Finding prognosis-associated gene pairs: All possible gene pairs were constructed from both prognosis-
associated genes. The samples could be classified into two groups
according to the within sample REO (Ea > Eb or Ea < Eb) of each
gene pair. Here, Ea and Eb represent the expression levels of two October 2019 | Volume 9 | Article 1094 Frontiers in Oncology | www.frontiersin.org 3 Liu et al. Qualitative Signature for Ovarian Cancer RFS (univariate Cox model, FDR < 0.05). With a forward
selection procedure using each of the 30 prognosis-associated
gene pairs as a seed separately (see Materials and Methods),
we obtained 30 sets of gene pairs, among which a set of four
gene pairs reached the largest C-index of 0.63. Thus, the four
gene pairs were selected as the predictive gene pair signature
for platinum-taxane ACT, denoted as 4-GPS (Table 2). The
classification rule of 4-GPS is that a sample was predicted as
a responder, if more than two of the four gene pairs have the
specific REOs (Ea > Eb); otherwise, it will be predicted to be a
non-responder (Figure 2A). The R function for the classification
of 4-GPS for a cohort or an individual is available in the
Supplementary R function: HGS-OvCa response prediction for
platinum-taxane ACT (see Supplementary Material). Developing a Qualitative Predictive
Signature for Platinum-Taxane ACT According
to the majority voting rule, 83 patients were predicted as non-
responders, and had significantly shorter RFS than 143 patients
predicted as responders (log-rank p = 2.11E-09, HR = 2.57,
95% CIs: 1.87–3.53, C-index = 0.63, Figure 2B). A univariate
Cox analysis showed that only 4-GPS (non-response vs. response,
p = 6.80E-09, HR = 2.57, 95% CIs: 1.87–3.53, Figure 3C)
was statistically significantly associated with patients’ RFS. The
univariate Cox result of the clinical factors are also displayed
in Figure 3C. FIGURE 1 | The flowchart for developing and validating the qualitative
predictive GPS for platinum-taxane ACT. Next, we analyzed the association of two response groups
predicted by 4-GPS with the four molecular subtypes of the
HGS-OvCa patients in the TCGA dataset, which were stratified
by the TCGA original article (26). Here, 216 of 226 patients
used in this study had molecular subtype information, which
were classified as immunoreactive (n = 51), differentiated (n
= 60), proliferative (n = 58), and mesenchymal (n = 47)
subtypes (27). We found that 42 (89.36%) of 47 patients in the
mesenchymal subtype were predicted to be non-responders by
4-GPS, with its proportion significantly higher than that in the
other three subtypes (Chi-square test, p = 2.20E-16, Figure 2D). The result was in accordance with a previous report that shows
that the mesenchymal subtype is associated with a poor prognosis
of HGS-OvCa patients receiving platinum-taxane ACT after
surgery (28), providing the biological evidence that 4-GPS has the
ability to identify non-responders of platinum-taxane ACT. FIGURE 1 | The flowchart for developing and validating the qualitative
predictive GPS for platinum-taxane ACT. 1.18–7.75, Figure 3C), after adjusting for histological grade (p =
0.1594, HR = 1.75, 95% CIs: 0.80–3.80, Figure 3C) and residual
tumor (p = 0.1915, HR = 1.73, 95% CIs: 0.76–3.95, Figure 3C). Notably, test 1 also provides patients’ pathological response
states for platinum-taxane ACT, including 22 pathological CR
patients and 16 pathological non-CR patients. The result showed
that the non-responders predicted by 4-GPS were significantly
enriched in the pathological non-CR group (Fisher’s exact
test, p = 0.0403, Figure 3D). It is worth noting that 4-GPS
reclassified 12 out of 22 pathological CR patients as non-
responders and two out of 16 pathological non-CR patients as
responders. Developing a Qualitative Predictive
Signature for Platinum-Taxane ACT In the pathological CR group, we found that 12 non-
responders reclassified by 4-GPS had significantly shorter RFS
than 10 responders consistently predicated by 4-GPS (log-rank
p = 0.0122, HR = 4.06, 95% CIs: 1.26–13.08, C-index =0.62,
Figure 3E). The above result indicates a better classification of 4-
GPS for platinum-taxane response states of HGS-OvCa patients. In addition, the accuracy of the two pathological non-CR patients
reclassified as responders by 4-GPS needs further validation, as
the small sample size is unfit for survival analysis. Independent Validation of 4-GPS
The performance of 4-GPS was tested in two independent
datasets, which were detected in different laboratories with
different microarray platforms. Independent Validation of 4-GPS The performance of 4-GPS was tested in two independent
datasets, which were detected in different laboratories with
different microarray platforms. y
In test 1, 26 non-responders predicted by 4-GPS had
significantly shorter RFS than 12 predicted responders (log-rank
p = 0.0022, HR = 3.86, 95% CIs: 1.54–9.70, C-index= 0.61,
Figure 3A) after receiving platinum-taxane ACT. A univariate
Cox analysis showed that 4-GPS (non-response vs. response,
p = 0.0040, HR = 3.86, 95% CIs: 1.54–9.70, Figure 3B),
histological grade (3 vs. 2, p = 0.1740, HR = 1.70, 95% CIs:
0.79–3.64, Figure 3B) and residual tumor (≥11 vs. 0–10 mm,
p = 0.0114, HR = 2.78, 95% CIs: 1.26–6.12, Figure 3B) were
significantly or marginally significantly associated with patients’
RFS. Multivariate Cox analysis for 37 patients with complete
clinical information showed that 4-GPS remained significantly
associated with patients’ RFS (p = 0.0209, HR = 3.03, 95% CIs: October 2019 | Volume 9 | Article 1094 Frontiers in Oncology | www.frontiersin.org 4 Qualitative Signature for Ovarian Cancer Liu et al. TABLE 2 | Composition and Biologic functional characterizations of 4-GPS. Signature REO
(Ea > Eb)
HR
P-value
C-index
Biologic functional characterizations
Gene pair 1 (FUS >
THBS2)
0.54
4.12E-4
0.57
FUS: A DNA/RNA-binding protein that plays a role in DNA repair and damage response
(19). THBS2: This gene encodes a disulfide-linked homotrimeric glycoprotein that mediates
cell-to-cell and cell-to-matrix interactions (20). Gene pair 2 (GUCY2C >
RCVRN)
0.47
5.61E-5
0.58
GUCY2C: This gene encodes a transmembrane protein that functions as a receptor for
endogenous peptides guanylin and uroguanylin (21). RCVRN: This gene encodes a member of the recoverin family of neuronal calcium
sensors. Recoverin may be the antigen responsible for cancer-associated (22). Gene pair 3 (PCSK6 >
ZNF365)
0.57
7.96E-4
0.58
PCSK6: A pro-protein convertase that plays an important role in cancer cell proliferation
(23). ZNF365: A DNA repair pathway gene in the homologous recombination pathway that is
important in the repair of complex double-stranded lesions (24). Gene pair 4 (PASK >
DNAJB14)
0.55
2.66E-4
0.58
PASK: Alternatively spliced transcript variants encoding multiple isoforms have been
observed for this gene (25). DNAJB14: Required to promote protein folding and trafficking, prevent aggregation of
client proteins, and promote unfolded proteins to endoplasmic reticulum-associated
degradation (ERAD) pathway. REO represents the relative expression ordering of gene pair (Ea > Eb); HR and P-value are the statistic calculated from the univariate Cox regression model. Independent Validation of 4-GPS HR represents the risk
coefficient of the REO for gene pair (a, b), where HR < 1 indicates that Ea > Eb is a protective factor, otherwise a risk factor; P-value represents the significance of the REO for gene
pair (a, b). TABLE 2 | Composition and Biologic functional characterizations of 4-GPS. REO represents the relative expression ordering of gene pair (Ea > Eb); HR and P-value are the statistic calculated from the univariate Cox regression model. HR represents the risk
coefficient of the REO for gene pair (a, b), where HR < 1 indicates that Ea > Eb is a protective factor, otherwise a risk factor; P-value represents the significance of the REO for gene
pair (a, b). Similarly, in test 2, 115 non-responders predicted by 4-
GPS also had significantly shorter RFS than 17 predicted
responders (log-rank p = 0.0123, HR = 2.35, 95% CIs:
1.18–4.69, C-index = 0.55, Figure 4A). A univariate Cox
analysis showed that 4-GPS (non-response vs. response, p
=0.0150, HR = 2.35, 95% CIs: 1.18–4.69, Figure 4B), stage
(IV vs. III vs. II, p =0.0669, HR = 1.61, 95% CIs: 0.97–
2.68, Figure 4B) and residual tumor (≥11 vs. 0–10 mm,
p = 0.0591, HR = 1.50, 95% CIs: 0.98–2.29, Figure 4B)
were significantly or marginally significantly associated with
patients’ RFS. And, a multivariate Cox analysis for 119 patients
with complete clinical information also showed that 4-GPS
remained significantly associated with patients’ RFS (p =
0.0112, HR = 2.58, 95% CIs: 1.24–5.35, Figure 4C), after
adjusting for the significant clinical factors including stage
(p = 0.0990, HR = 1.66, 95% CIs: 0.91–3.02, Figure 4C)
and residual tumor (p = 0.1350, HR = 1.39, 95% CIs:
0.90–2.15, Figure 4C). implications in tumor progression. For example, PCSK6, a
pro-protein convertase, plays an important role in cancer cell
proliferation (23) and ZNF365, a DNA repair pathway gene in the
homologous recombination (HR) pathway (24). In the training
dataset, the expression level of PCSK6 in the predicted response
group was significantly higher than that in the predicted non-
response group (Student’s t-test, p = 2.45E-11, Figure 5A), while
the expression level of ZNF365 in the predicted response group
was significantly lower than that in predicted non-response group
(Student’s t-test, p = 3.77E-09, Figure 5B). Independent Validation of 4-GPS Therefore, the relative
order of PCSK6 expression level was higher than that of ZNF365
in the predicted responders and reversed in the predicted non-
responders. This indicates that the responders predicted by 4-
GPS might have higher cell proliferative capacity and lower DNA
repair capacity than the predicted non-responders. Therefore, the
cancer cells in the predicted responders were more easily attacked
by platinum agents and could not repair the lesions induced by
platinum agents. The functional enrichment analyses also supported the
ability of 4-GPS in distinguishing the response and non-
response to platinum-taxane ACT. In the training dataset,
we identified 2,410 DE genes between 83 non-responders
and 143 responders predicted by 4-GPS (Student’s t-test,
FDR < 0.05). The DE genes were significantly enriched
in 18 KEGG functional terms (hypergeometric test, FDR
< 0.05, Figure 5C), including several functions related with
platinum resistance for HGS-OvCa patients, such as PI3K-
Akt signaling pathway (31), and cell adhesion molecules
(32). In addition, some other functions have been reported
to be related to taxane sensitivity for HGS-OvCa patients,
such as focal adhesion (33), and cytokine-cytokine receptor
interaction (34). Functional Characterizations of 4-GPS A sample is predicted to be responder (R)
if more than two of the four gene pairs have the specific REOs (Ea > Eb); otherwise, it will be predicted to be non-responder (NR). The sample exemplar (pink) is
predicted to be responder because the count of the specific REOs (Ea > Eb) in the sample is 3. (B) The Kaplan–Meier curves of RFS for the 226 HGS-OvCa patients
receiving platinum-taxane ACT in the training dataset. Hazard ratio (HR) and 95% confidence intervals (CIs) were determined using univariate Cox regression models. (C) Univariate Cox analyses of 4-GPS, age, stage, grade, and residual tumor. Solid circles represent the HRs for risk of recurrence, and the open-ended horizontal
lines represent the 95% CIs. (D) The Confusion Matrix for the response prediction of 4-GPS and the HGS-OvCa molecular subtypes in TCGA. Chi-square test was
used to compare the association. Functional Characterizations of 4-GPS The detailed information of 4-GPS is described in Table 2. Functional annotation showed that six signature genes involved
in all four gene pairs were included in cell adhesion and
regulation of angiogenesis [THBS2 (20)], cell proliferation
[GUCY2C (21)], and the regulation of cellular and metabolic
processes [FUS (19); PASK (25); PCSK6 (29); RCVRN (22)],
which have been reported to be related with platinum and/or
taxane sensitivity. Several genes including ZNF365 (30), THBS2
(20), GUCY2C (21), PCSK6 (29), and RCVRN (22), have been
reported to be associated with a poor prognosis of HGS-OvCa
patients or other cancer patients treated with ACT after surgery. The REO of two genes in a gene pair has intuitive biological October 2019 | Volume 9 | Article 1094 Frontiers in Oncology | www.frontiersin.org 5 Liu et al. Qualitative Signature for Ovarian Cancer
FIGURE 2 | The predictive 4-GPS for risk classification and recurrence analyses for HGS-OvCa patients in TCGA. (A) Risk classification of 4-GPS for HGS-OvCa
patients based on the within-sample relative expression orderings (REOs) of the four gene pairs with the majority-voting rule. A sample is predicted to be responder (R)
if more than two of the four gene pairs have the specific REOs (Ea > Eb); otherwise, it will be predicted to be non-responder (NR). The sample exemplar (pink) is
predicted to be responder because the count of the specific REOs (Ea > Eb) in the sample is 3. (B) The Kaplan–Meier curves of RFS for the 226 HGS-OvCa patients
receiving platinum-taxane ACT in the training dataset. Hazard ratio (HR) and 95% confidence intervals (CIs) were determined using univariate Cox regression models. (C) Univariate Cox analyses of 4-GPS, age, stage, grade, and residual tumor. Solid circles represent the HRs for risk of recurrence, and the open-ended horizontal
lines represent the 95% CIs. (D) The Confusion Matrix for the response prediction of 4-GPS and the HGS-OvCa molecular subtypes in TCGA. Chi-square test was
used to compare the association. Comparison of 4-GPS With Other
Signatures
which were not the training datasets for the two signatures. The
40-gene signature (6) cited in the introduction was not analyzed Liu et al. Qualitative Signature for Ovarian Cancer FIGURE 2 | The predictive 4-GPS for risk classification and recurrence analyses for HGS-OvCa patients in TCGA. (A) Risk classification of 4-GPS for HGS-OvCa
patients based on the within-sample relative expression orderings (REOs) of the four gene pairs with the majority-voting rule. Comparison of 4-GPS With Other
Signatures which were not the training datasets for the two signatures. The
40-gene signature (6) cited in the introduction was not analyzed
because its application to independent data needs resetting risk
thresholds, which makes it a non-independent validation. The We also compared the performance of 4-GPS with the published
23-gene signature in test 1 and test 2 of this study, respectively, October 2019 | Volume 9 | Article 1094 Frontiers in Oncology | www.frontiersin.org 6 Liu et al. Liu et al. Qualitative Signature for Ovarian Cancer FIGURE 3 | The validation of 4-GPS in test 1. (A) The Kaplan–Meier curves of RFS for 38 HGS-OvCa patients treated with platinum-taxane ACT. (B) Univariate Cox
analyses of 4-GPS, age, stage, grade, and residual tumor in test 1. (C) Multivariate Cox analyses of 4-GPS after adjusting for significantly prognostic clinical factors in
test 1. (D) The Confusion Matrix for the response prediction of 4-GPS with pathological response states. Fisher’s exact test was used to compare the association. (E) The Kaplan–Meier curves of RFS for 22 HGS-OvCa patients receiving platinum-taxane ACT in the pathological complete response group. FIGURE 3 | The validation of 4-GPS in test 1. (A) The Kaplan–Meier curves of RFS for 38 HGS-OvCa patients treated with platinum-taxane ACT. (B) Univariate Cox
analyses of 4-GPS, age, stage, grade, and residual tumor in test 1. (C) Multivariate Cox analyses of 4-GPS after adjusting for significantly prognostic clinical factors in
test 1. (D) The Confusion Matrix for the response prediction of 4-GPS with pathological response states. Fisher’s exact test was used to compare the association. (E) The Kaplan–Meier curves of RFS for 22 HGS-OvCa patients receiving platinum-taxane ACT in the pathological complete response group. were analyzed together for comparison. The requirement of a
comparison with the other samples needs pre-collection of a
set of samples, and the risk prediction of an individual sample
will rely on the risk composition of other samples adopted for
comparison. This provided further evidence that the type of
quantitative signatures would be unfit to direct clinical settings,
as reported in our previous study (8). other published signatures, such as the 422-gene signature (4),
were not analyzed because the author did not provide the
predictive model. Comparison of 4-GPS With Other
Signatures Briefly, for 23-gene signature (5), a point of
each sample was given for each gene if its high expression was
associated with longer (or shorter) survival in its training dataset
and if its expression in the sample was higher (or lower) than the
median expression of all samples. The risk score was the sum of
these points calculated by 23 genes. The samples were categorized
as a high-risk (non-response) group when their scores were lower
than 11 (training cut-off), and vice versa. The survival results
showed that 23-gene signatures failed to predict the RFS of
patients receiving platinum-taxane ACT in the two test datasets
(Figures 6A,B). Moreover, 23-gene signature could not predict
the response states of individual samples when no other samples Frontiers in Oncology | www.frontiersin.org DISCUSSION In this study, we constructed a qualitative transcriptional
signature consisting of four gene pairs (4-GPS) by combining the
pathological response state and RFS information of HGS-OvCa October 2019 | Volume 9 | Article 1094 Frontiers in Oncology | www.frontiersin.org Qualitative Signature for Ovarian Cancer Liu et al. FIGURE 4 | The validation of 4-GPS in test 2. (A) The Kaplan–Meier curves of RFS for 132 HGS-OvCa patients receiving platinum-taxane ACT in test 2. (B) Univariate
Cox analyses of 4-GPS, age, stage, grade, and residual tumor in test 2. (C) Multivariate Cox analyses of 4-GPS after adjusting for significantly prognostic clinical
factors in test 2. FIGURE 4 | The validation of 4-GPS in test 2. (A) The Kaplan–Meier curves of RFS for 132 HGS-OvCa patients receiving platinum-taxane ACT in test 2. (B) Univariate
Cox analyses of 4-GPS, age, stage, grade, and residual tumor in test 2. (C) Multivariate Cox analyses of 4-GPS after adjusting for significantly prognostic clinical
factors in test 2. ACT. While, the predicted non-responders could be resistant to
both platinum and taxane, require further testing (such as BRCA
mutation), and receive alternate therapies [e.g., bevacizumab
(37)] after surgery. patients. The signature could identify the individual platinum-
taxane responders with longer RFS after receiving platinum-
taxane ACT, and its performance was effectively validated in two
independent datasets. According to response evaluation criteria in solid tumors
(RECIST), a certain percentage of pathological response states
of HGS-OvCA patients may be misclassified by the conventional
iconographies, especially near the cut-offpoints for the short-
term reduction of tumor size after platinum-taxane ACT
(35). Therefore, the National Comprehensive Cancer Network
(NCCN) clinical guidelines for HGS-OvCa patients recommend
that patients with a pathological complete response but relapse
within 6 months after receiving platinum-taxane ACT should
be deemed resistant. In this study, we found that 4-GPS could
reclassify 12 clinical diagnosed CR patients as non-responders
in test 1 and their RFS was significantly shorter than the other
CR patients (log-rank p = 0.0122; Figure 3E), suggesting a better
performance of 4-GPS in identifying patients who are resistant
to platinum-taxane. Notably, there were large differences in the predicted
response/non-response ratios among cohorts. A previous study
reported
that
different
cohorts
collected
in
the
datasets
had different risk compositions (8), such as the ratios of
patients with potential metastases or resistance to drugs
in the cohort. Frontiers in Oncology | www.frontiersin.org DISCUSSION In order to support the accuracy of the
signature in each dataset, we additionally performed the cross
comparison of RFS between the predicted responders and
non-responders derived from different cohorts. The results
showed that the responders predicted by 4-GPS in the
training data had significantly longer RFS than the non-
responders predicted in test 1 (log-rank p = 4.04E−09,
Figure 7A) and test 2 (log-rank p = 0.0009, Figure 7B), and
the predicted non-responders also had significantly shorter
RFS than the responders predicted in test 1 (log-rank p
= 0.0059, Figure 7C) and test 2 (log-rank p = 0.0002,
Figure 7D). Similar results for the cross comparisons were
observed in test 1 and test 2 (Figure S1). The results also
provided the indirect evidence for the rationality of the
large difference in predicted responders/non-responders among
different cohorts. The standard treatment for HGS-OvCa is surgery and
platinum-based
ACT,
which
is
typically
administered
concurrently with a taxane (2). Recently, our study has proved
that genes related to single drug sensitivity could be identified in
clinical samples of patients who received a combination of ACT. This is because the drugs used in combination had no or limited
pharmacological antagonism (36). In this study, we developed
a predicted signature based on the patients receiving platinum
combined with taxane. We considered that the responders
predicted by the signature could be sensitive to either platinum
or taxane, and should receive platinum combined with taxane In conclusion, the qualitative predictive signature could be
applied to the gene expression profile of the postoperative sample,
obtained from an individual, to determine the response state
for the platinum-taxane ACT. For the predicted responders,
they should be advised to receive platinum combined with October 2019 | Volume 9 | Article 1094 8 Liu et al. Qualitative Signature for Ovarian Cancer GURE 5 | Functional characterizations of 4-GPS. (A) The boxplot of expression level of PCSK6 in predicted response and non-response groups in the training
aset. (B) The boxplot of expression level of ZNF365 in predicted response and non-response groups in the training dataset. (C) KEGG functional enrichment
alyses of DE genes between non-responders and responders predicted by 4-GPS in the training dataset. FIGURE 5 | Functional characterizations of 4-GPS. (A) The boxplot of expression level of PCSK6 in predicted response and non-response groups in the training
dataset. DISCUSSION (B) The boxplot of expression level of ZNF365 in predicted response and non-response groups in the training dataset. (C) KEGG functional enrichment
analyses of DE genes between non-responders and responders predicted by 4-GPS in the training dataset. Frontiers in Oncology | www.frontiersin.org DATA AVAILABILITY STATEMENT taxane ACT after surgery, while for the predicted non-
responders, they should be evaluated for the other therapies,
which requires for further exploration. The signature is highly
robust against experimental batch effects and uncertainties of
quality of clinical samples, which is convenient in clinical
settings and requires further validation in a prospective
clinical trial. Publicly
available
datasets
were
analyzed
in
this
study. This
data
can
be
found
here:
https://portal.gdc.cancer. gov/,
https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=
GSE30161,
https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? acc=GSE9891. October 2019 | Volume 9 | Article 1094 Frontiers in Oncology | www.frontiersin.org 9 Liu et al. Qualitative Signature for Ovarian Cancer FIGURE 6 | Prognostic performance of 23-gene signature in 2 test datasets. (A) The Kaplan–Meier curves of RFS for 38 HGS-OvCa patients treating with
platinum-taxane ACT in test 1. (B) The Kaplan–Meier curves of RFS for 132 HGS-OvCa patients treating with platinum-taxane ACT in test 2. FIGURE 6 | Prognostic performance of 23-gene signature in 2 test datasets. (A) The Kaplan–Meier curves of RFS for 38 HGS-OvCa patients treating with
platinum-taxane ACT in test 1. (B) The Kaplan–Meier curves of RFS for 132 HGS-OvCa patients treating with platinum-taxane ACT in test 2. FIGURE 7 | The cross comparisons of RFS between the predicted responders and non-responders derived from different cohorts. (A) The Kaplan–Meier curves of
RFS for 143 responders predicted by 4-GPS in the training dataset and 26 non-responders predicted in test 1. (B) The Kaplan–Meier curves of RFS for 143
responders predicted by 4-GPS in training dataset and 115 non-responders predicted in test 2. (C) The Kaplan–Meier curves of RFS for 83 non-responders predicted
by 4-GPS in the training dataset and 12 responders predicted in test 1. (D) The Kaplan–Meier curves of RFS for 83 non-responders predicted by 4-GPS in the training
dataset and 17 responders predicted in test 2. FIGURE 7 | The cross comparisons of RFS between the predicted responders and non-responders derived from different cohorts. (A) The Kaplan–Meier curves of
RFS for 143 responders predicted by 4-GPS in the training dataset and 26 non-responders predicted in test 1. (B) The Kaplan–Meier curves of RFS for 143
responders predicted by 4-GPS in training dataset and 115 non-responders predicted in test 2. (C) The Kaplan–Meier curves of RFS for 83 non-responders predicted
by 4-GPS in the training dataset and 12 responders predicted in test 1. REFERENCES (2016) 6:33366. doi: 10.1038/srep33366 7. Earl CS, Keong TW, An SQ, Murdoch S, McCarthy Y, Garmendia J, et al. Haemophilus influenzae responds to glucocorticoids used in asthma therapy
by modulation of biofilm formation and antibiotic resistance. EMBO Mol
Med. (2015) 7:1018–33. doi: 10.15252/emmm.201505088 21. Liu Y, Cheng G, Qian J, Ju H, Zhu Y, Stefano M, et al. Expression
of guanylyl cyclase C in tissue samples and the circulation of rectal
cancer patients. Oncotarget. (2017) 8:38841–9. doi: 10.18632/oncotarget. 16406 8. Qi L, Chen L, Li Y, Qin Y, Pan R, Zhao W, et al. Critical limitations of
prognostic signatures based on risk scores summarized from gene expression
levels: a case study for resected stage I non-small-cell lung cancer. Brief
Bioinform. (2016) 17:233–42. doi: 10.1093/bib/bbv064 22. Golovastova MO, Tsoy LV, Bocharnikova AV, Korolev DO, Gancharova
OS, Alekseeva EA, et al. The cancer-retina antigen recoverin as a
potential biomarker for renal tumors. Tumour Biol. (2016) 37:9899–907. doi: 10.1007/s13277-016-4885-5 9. Guan Q, Yan H, Chen Y, Zheng B, Cai H, He J, et al. Quantitative or qualitative
transcriptional diagnostic signatures? A case study for colorectal cancer. BMC
Genomics. (2018) 19:99. doi: 10.1186/s12864-018-4446-y 23. Longuespee R, Couture F, Levesque C, Kwiatkowska A, Desjardins R, Gagnon
S, et al. Implications of proprotein convertases in ovarian cancer cell
proliferation and tumor progression: insights for PACE4 as a therapeutic
target. Transl Oncol. (2014) 7:410–9. doi: 10.1016/j.tranon.2014.04.008 10. Cheng J, Guo Y, Gao Q, Li H, Yan H, Li M, et al. Circumvent the
uncertainty in the applications of transcriptional signatures to tumor
tissues sampled from different tumor sites. Oncotarget. (2017) 8:30265–75. doi: 10.18632/oncotarget.15754 get. Transl Oncol. (2014) 7:410–9. doi: 10.1016/j.tranon.2014.04.0 24. Zhang Y, Park E, Kim CS, Paik JH. ZNF365 promotes stalled replication
forks recovery to maintain genome stability. Cell Cycle. (2013) 12:2817–28. doi: 10.4161/cc.25882 11. Chen R, Guan Q, Cheng J, He J, Liu H, Cai H, et al. Robust
transcriptional
tumor
signatures
applicable
to
both
formalin-fixed
paraffin-embedded and fresh-frozen samples. Oncotarget. (2017) 8:6652–62. doi: 10.18632/oncotarget.14257 25. MacDonald PE, Rorsman P. Per-arnt-sim (PAS) domain kinase (PASK)
as a regulator of glucagon secretion. Diabetologia. (2011) 54:719–21. doi: 10.1007/s00125-011-2072-1 26. Cancer Genome Atlas Research N. Integrated genomic analyses of ovarian
carcinoma. Nature. (2011) 474:609–15. doi: 10.1038/nature10166 12. Liu H, Li Y, He J, Guan Q, Chen R, Yan H, et al. Robust transcriptional
signatures for low-input RNA samples based on relative expression orderings. BMC Genomics. (2017) 18:913. REFERENCES in ovarian carcinoma: predicting platinum resistance. PLoS ONE. (2012)
7:e30550. doi: 10.1371/journal.pone.0030550 in ovarian carcinoma: predicting platinum resistance. PLoS ONE. (2012)
7:e30550. doi: 10.1371/journal.pone.0030550 1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2018. CA Cancer J Clin. (2018)
68:7–30. doi: 10.3322/caac.21442 1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2018. CA Cancer J Clin. (2018)
68:7–30. doi: 10.3322/caac.21442 15. Tothill RW, Tinker AV, George J, Brown R, Fox SB, Lade S, et al. Novel molecular subtypes of serous and endometrioid ovarian cancer
linked
to
clinical
outcome. Clin
Cancer
Res. (2008)
14:5198–208. doi: 10.1158/1078-0432.CCR-08-0196 2. Markman M, Liu PY, Wilczynski S, Monk B, Copeland LJ, Alvarez RD,
et al. Phase III randomized trial of 12 versus 3 months of maintenance
paclitaxel in patients with advanced ovarian cancer after complete response to
platinum and paclitaxel-based chemotherapy: a Southwest Oncology Group
and Gynecologic Oncology Group trial. J Clin Oncol. (2003) 21:2460–5. doi: 10.1200/JCO.2003.07.013 16. Dai M, Wang P, Boyd AD, Kostov G, Athey B, Jones EG, et al. Evolving gene/transcript definitions significantly alter the interpretation
of GeneChip data. Nucleic Acids Res. (2005) 33:e175. doi: 10.1093/nar/
gni179 3. Matulonis
UA,
Sood
AK,
Fallowfield
L,
Howitt
BE,
Sehouli
J,
Karlan BY. Ovarian cancer. Nat
Rev
Dis
Primers. (2016) 2:16061. doi: 10.1038/nrdp.2016.61 17. Harrell FE Jr, Lee KL, Mark DB. Multivariable prognostic models: issues in
developing models, evaluating assumptions and adequacy, and measuring
and reducing errors. Stat Med. (1996) 15:361–87. doi: 10.1002/(SICI)1097-
0258(19960229)15:4<361::AID-SIM168>3.0.CO;2-4 4. Gonzalez Bosquet J, Newtson AM, Chung RK, Thiel KW, Ginader T,
Goodheart MJ, et al. Prediction of chemo-response in serous ovarian cancer. Mol Cancer. (2016) 15:66. doi: 10.1186/s12943-016-0548-9 18. Yu G, Wang LG, Han Y, He QY. clusterProfiler: an R package for
comparing biological themes among gene clusters. OMICS. (2012) 16:284–7. doi: 10.1089/omi.2011.0118 5. Kang J, D’Andrea AD, Kozono D. A DNA repair pathway-focused score
for prediction of outcomes in ovarian cancer treated with platinum-based
chemotherapy. J Natl Cancer Inst. (2012) 104:670–81. doi: 10.1093/jnci/djs177 19. Ward CL, Boggio KJ, Johnson BN, Boyd JB, Douthwright S, Shaffer SA, et al. A loss of FUS/TLS function leads to impaired cellular proliferation. Cell Death
Dis. (2014) 5:e1572. doi: 10.1038/cddis.2014.508 6. Wang JY, Chen LL, Zhou XH. Identifying prognostic signature in
ovarian
cancer
using
DirGenerank. Oncotarget. (2017)
8:46398–413. doi: 10.18632/oncotarget.18189 20. Wang X, Zhang L, Li H, Sun W, Zhang H, Lai M. THBS2 is a
potential prognostic biomarker in colorectal cancer. Sci Rep. DATA AVAILABILITY STATEMENT (D) The Kaplan–Meier curves of RFS for 83 non-responders predicted by 4-GPS in the training
dataset and 17 responders predicted in test 2. October 2019 | Volume 9 | Article 1094 Frontiers in Oncology | www.frontiersin.org 10 Qualitative Signature for Ovarian Cancer Liu et al. FUNDING The Supplementary Material for this article can be found
online
at:
https://www.frontiersin.org/articles/10.3389/fonc. 2019.01094/full#supplementary-material This work was supported by the National Natural Science
Foundation
of
China
(Grant
Nos. 61701143,
81572935, This work was supported by the National Natural Science
Foundation
of
China
(Grant
Nos. 61701143,
81572935, AUTHOR CONTRIBUTIONS 81872396,
61673143,
and
61601151),
the
Joint
Scientific
and
Technology
Innovation
Fund
of
Fujian
Province
(Grant No. 2016Y9044), the Health and Family Planning
Commission Scientific Research of Heilongjiang Province
(Grant No. 2016-175), and the Fundamental Research Funds
for
the
Provincial
Universities
(Grant
Nos. 2017JCZX48
and 31041180016). ZG and LQ conceived the idea. LQ and YLiu conceived and
designed the experiments and wrote the manuscript. YLiu and
ZZ designed the experiments. YLi, ZZ, TL, XL, and SZ performed
the experiments and analyzed the data. YG and WZ helped in
interpreting the results and writing the manuscript. All authors
approved the final version. REFERENCES doi: 10.1186/s12864-017-4280-7 27. Yang D, Sun Y, Hu L, Zheng H, Ji P, Pecot CV, et al. Integrated analyses
identify a master microRNA regulatory network for the mesenchymal
subtype
in
serous
ovarian
cancer. Cancer
Cell. (2013)
23:186–99. doi: 10.1016/j.ccr.2012.12.020 13. Eisenhauer EA, Therasse P, Bogaerts J, Schwartz LH, Sargent D, Ford R, et al. New response evaluation criteria in solid tumours: revised RECIST guideline
(version 1.1). Eur J Cancer. (2009) 45:228–47. doi: 10.1016/j.ejca.2008.10.026 28. Gupta
N,
Xu
Z,
El-Sehemy
A,
Steed
H,
Fu
Y. Notch3
induces
epithelial-mesenchymal
transition
and
attenuates
carboplatin-induced 14. Ferriss JS, Kim Y, Duska L, Birrer M, Levine DA, Moskaluk C, et al. Multi-
gene expression predictors of single drug responses to adjuvant chemotherapy October 2019 | Volume 9 | Article 1094 Frontiers in Oncology | www.frontiersin.org 11 Liu et al. Liu et al. Qualitative Signature for Ovarian Cancer apoptosis in ovarian cancer cells. Gynecol Oncol. (2013) 130:200–6. doi: 10.1016/j.ygyno.2013.03.019 35. Gruber
IV,
Rueckert
M,
Kagan
KO,
Staebler
A,
Siegmann
KC,
Hartkopf A, et al. Measurement of tumour size with mammography,
sonography and magnetic resonance imaging as compared to histological
tumour size in primary breast cancer. BMC Cancer. (2013) 13:328. doi: 10.1186/1471-2407-13-328 29. Couture F, Sabbagh R, Kwiatkowska A, Desjardins R, Guay SP, Bouchard
L, et al. PACE4 undergoes an oncogenic alternative splicing switch
in cancer. Cancer Res. (2017) 77:6863–79. doi: 10.1158/0008-5472.CAN-
17-1397 36. Tong M, Zheng W, Lu X, Ao L, Li X, Guan Q, et al. Identifying
clinically relevant drug resistance genes in drug-induced resistant cancer
cell lines and post-chemotherapy tissues. Oncotarget. (2015) 6:41216–27. doi: 10.18632/oncotarget.5649 30. Couch FJ, Gaudet MM, Antoniou AC, Ramus SJ, Kuchenbaecker KB, Soucy
P, et al. Common variants at the 19p13.1 and ZNF365 loci are associated
with ER subtypes of breast cancer and ovarian cancer risk in BRCA1 and
BRCA2 mutation carriers. Cancer Epidemiol Biomarkers Prev. (2012) 21:645–
57. doi: 10.1158/1055-9965.EPI-11-0888 37. Aghajanian C, Blank SV, GoffBA, Judson PL, Teneriello MG, Husain A, et
al. OCEANS: a randomized, double-blind, placebo-controlled phase III trial
of chemotherapy with or without bevacizumab in patients with platinum-
sensitive recurrent epithelial ovarian, primary peritoneal, or fallopian tube
cancer. J Clin Oncol. (2012) 30:2039–45. doi: 10.1200/JCO.2012.42.0505 31. Gasparri ML, Besharat ZM, Farooqi AA, Khalid S, Taghavi K, Besharat RA,
et al. MiRNAs and their interplay with PI3K/AKT/mTOR pathway in ovarian
cancer cells: a potential role in platinum resistance. J Cancer Res Clin Oncol. (2018) 144:2313–8. Frontiers in Oncology | www.frontiersin.org October 2019 | Volume 9 | Article 1094 REFERENCES doi: 10.1007/s00432-018-2737-y Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest. 32. Scalici JM, Arapovic S, Saks EJ, Atkins KA, Petroni G, Duska LR, et al. Mesothelium expression of vascular cell adhesion molecule-1 (VCAM-1) is
associated with an unfavorable prognosis in epithelial ovarian cancer (EOC). Cancer. (2017) 123:977–84. doi: 10.1002/cncr.30415 Copyright © 2019 Liu, Zhang, Li, Li, Zhang, Li, Zhao, Gu, Guo and Qi. This is an
open-access article distributed under the terms of the Creative Commons Attribution
License (CC BY). The use, distribution or reproduction in other forums is permitted,
provided the original author(s) and the copyright owner(s) are credited and that the
original publication in this journal is cited, in accordance with accepted academic
practice. No use, distribution or reproduction is permitted which does not comply
with these terms. 33. Levy A, Alhazzani K, Dondapati P, Alaseem A, Cheema K, Thallapureddy K,
et al. Focal adhesion kinase in ovarian cancer: a potential therapeutic target
for platinum and taxane-resistant tumors. Curr Cancer Drug Targets. (2019)
19:179–88. doi: 10.2174/1568009618666180706165222 34. Che CL, Zhang YM, Zhang HH, Sang YL, Lu B, Dong FS, et al. DNA
microarray reveals different pathways responding to paclitaxel and docetaxel
in non-small cell lung cancer cell line. Int J Clin Exp Pathol. (2013) 6:1538–48. October 2019 | Volume 9 | Article 1094 Frontiers in Oncology | www.frontiersin.org 12
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https://openalex.org/W2414586725
|
http://www.journalijar.com/uploads/406_IJAR-9689.pdf
|
English
| null |
RELATIONSHIP OF DEPRESSION AND LONELINESS INDEPRESSED AND NORMAL POPULATION.
|
International journal of advanced research
| 2,016
|
cc-by
| 4,142
|
Key words: *Corresponding Author
Fatima khurram Bukhari. Manuscript Info Manuscript Info
Manuscript History:
Received: 19 March 2016
Final Accepted: 22 April 2016
Published Online: May 2016
Key words:
Depression, loneliness, population,
Social Skills,
*Corresponding Author
Fatima khurram Bukhari. RELATIONSHIP OF DEPRESSION AND LONELINESS INDEPRESSED AND NORMA Fatima khurram Bukhari1, IramAwan2,Samar Fahd3. 1. Assistant Professor, Department of Applied Psychology, The Islamia University of Bahawalpur. 2. Assistant Professor, Department of Applied Psychology, Bahaudin Zakariya University, Multan. 3. Lecturer, Department of Applied Psychology, The Islamia University of Bahawalpur. Fatima khurram Bukhari1, IramAwan2,Samar Fahd3. 1. Assistant Professor, Department of Applied Psychology, The Islamia University of Bahawalpur. 2. Assistant Professor, Department of Applied Psychology, Bahaudin Zakariya University, Multan. 3. Lecturer, Department of Applied Psychology, The Islamia University of Bahawalpur. 1. Assistant Professor, Department of Applied Psychology, The Islamia University of Bahawalpur
2. Assistant Professor, Department of Applied Psychology, Bahaudin Zakariya University, Multan
3. Lecturer, Department of Applied Psychology, The Islamia University of Bahawalpur. Abstract The present study was designed to explore the relationship among depression
and Loneliness. The sample was consisted on 120 individuals, 60 from
normal population and 60 from clinically diagnosed depressed patients. The
normal sample i.e.60 individuals were further divided into 30 males’ and30
females. Similarly in the sample of clinically diagnosed depressed patients
30 were males and 30 were females. Depressed patients were selected from
Bahawal Victoria Hospital, Bahawalpur and normal populations were
selected from the different areas of Bahawalpur. Beck’s depression inventory
was used to measure the level of depression and UCLA loneliness was used
to measure loneliness. Result of the study reveals that there is a strong
relationship among depression and loneliness. People who are depressed
have strong feelings of loneliness. Which shows that people who are less
assertive and lack social skills are more prone to depression which further
leads to have loneliness feelings? Received: 19 March 2016
Final Accepted: 22 April 2016
Published Online: May 2016 ISSN 2320-5407 ISSN 2320-5407 International Journal of Advanced Research (2016), Volume 4, Issue 5, 85-89 INTERNATIONAL JOURNAL
OF ADVANCED RESEARCH Introduction:- It is often accompanied by some physical ailments such as, indigestion,
constipation, dizzy spells, unpleasant sensation in the chest and generalized pain. Psychological manifestation of
depression people describes themselves as feeling apathetic, discouraged and hopeless in emotional aspects. Their
expectancy for a happy and cheerful event in the future in very low and them always describe themselves in negative
terms (Gara, 1995). Many depressed people feel intensely sad and dejected. They describe themselves as feeling
miserable, empty and humiliated. Some of them also experience anxiety, anger or agitation. Many seem to loss their
feeling of affection for friends and relatives (Comer, 1995). Womenfolk are supplementary susceptible to have depression for the reason that the effects of hormones. Tennov
found that all forms of depression are more frequently found in women than man. Women are at high risk of
depression because of biological difference. Social reason may also contribute to depression among women because
of larger pressures of work and family responsibility. The roles and expectations of women and increased rates of
sexual abuse and poverty are the major cause of high ratio of depression in females. (Kessler,2003). If depression is common cold of psychotherapy than loneliness is the headache (Myers, 2000).The state of
loneliness, unlike solitude is a chronic distressful mental condition whereby an individual feels estranged from peers
and starved from the emotional intimacy found in relationship. Aloneness is simply being away from others. Loneliness is an enthusiastic state in which an individual encounters an effective sentiment void and disconnection
loneliness is more than the sentiment needing organization or needing to accomplish something with someone else. Loneliness constitutes a ruinous type of self-recognition. The lonely get a handle on left, overlooked, unneeded and
disregarded. It appears to be likely that considerations concerning the loss of past and those in it and the high see for
others as appeared by counter-intuitive convictions force irrational requests on the person. Such requests ordinarily
go unfulfilled making people feel more secluded in view of them and to decipher their current condition as
calamitous. (Jackson2000). Loneliness is a state of psychological discomfort which occurs when one’s social relationship are smaller or less
satisfactory than a person desires. The psychological discomfort are felt when our social relations lack some
essential feature. This deficit may be qualitative so (too few relationships) or quantitative (unsatisfying
relationships) (Sarson & Sarson, 2000). Loneliness is neither depression nor grief. Introduction:- Depression is an enthusiastic state set apart by incredible pity feelings of uselessness and Guilt, withdrawal from
other and loss of rest, voracity sexual longing and hobby and joy in regular exercises. Just for instance most of us
experience unhappiness throughout the way of our lives though may be not to a degree or with a frequency that
warrants the diagnosis of depression is frequently associated with other psychological problems. For instance pain
attacks substance abuse, sexual dysfunction and personality disorders. (Davison & Neale, 2001). Depression in our day by day life incorporates negative sentiments, for example, shortcoming dissatisfaction
disappointment, despair helplessness and sadness. It is a little depressed state in which existence appears miserable
and its contests irresistible. Clinical depression is a grave mental disorder with no positive characterization. The
mental discomfort it passes is unadorned and long lasting and way intensify as the month and year go by. It is so
devastating that clinically depressed individual might reach a point where they are incapable to do the simplest of
life’s activities and some even try to end their lives. (Davison & Neale, 2001). Depression belongs to the class of affective disorder in DSM-V (American Psychiatric Association, 2013). Depressed individuals face different mood changes thus it is also included in the class of mood disorders (American
Psychiatric Association, 2013). At the point when depressed people are stood up to with an issue they don't appear
to discover any answer for that issue. They discover each minute substantial and their heads fill and resonate with
self-recrimination. Depressed individuals might likewise disregard individual cleanliness and appearance and make
various hypochondriacally grievances of a throbbing painfulness that have no physical premise. They feel
completely sad and lose trust and no ability to start any work. They might feel misgiving uneasiness and melancholy
a significant part of the time when depression gets to be interminable, the patient does not generally snap back a
prior level of working. (Davison and Neale, 2001). 85 ISSN 2320-5407 International Journal of Advanced Research (2016), Volume 4, Issue 5, 85-89 Depressed people report physical as well as psychological problems. In physical symptoms people who feel
depressed might lead to weight gain or weight loss. They have broken sleep by wakeful restless spell, headaches,
backaches and pain in neck and face. Introduction:- Though depression may result
from loneliness. The two are not identical lonely persons are compelled to create new relationship to eliminate the
stressful state they endure. Depressed individuals nevertheless are reluctant to execute their grief on others and
consequently remain in separation (Schultz & Moore, 1990). Loneliness seems to become predominantly dominant in current periods. Toward the start of the most recent century
families were commonly bigger more steady, separation was uncommon and generally few individuals lived alone
in 1995, 24 million America lived in single individual family units by 2010, it is evaluated that number will have
expanded to around 31 million. Physical division regularly debilitates families’ securities, and these days it is not
under any condition bizarre for relatives to be isolated by hundred or even a great many miles. Loneliness is often
associated with negative states such as depression, anxiety and self-destructive behaviors (Rokach, 1996). Females might be more adept to recognize their loneliness than man in light of the fact that the negative outcomes of
conceding loneliness are less for females. Loneliness appears to bring down our self-esteem and low self-esteem
appears to add to depression. It can be an endless loop and both add to depression. Loneliness has been observed to
be identified with segregation structure kinfolk and non-family, low training low salary, passing of huge others, and
unemployment, being unmarried, it is identified with negative state of mind, wrong attributions and social abilities
insufficiency. Rationale of the study:- y
The present research is done to explore what relationship depression and loneliness. Loneliness has been found to
relate with locus of control relational style, social network relations, interpersonal skills, social support, risk taking
anxiety, depression and self-critics as well as to demographic variables. The main reason to conduct this study was
to see the individual opinion about locus of control in their lives. How people relate their behavior to locus of
control and how it affects their outcomes and their psychological mindedness. 86 ISSN 2320-5407 International Journal of Advanced Research (2016), Volume 4, Issue 5, 85-89 Another reason was that in Pakistani culture people know very little about loneliness. Pakistani society is based on
extended family systems so there is a collective culture it may be possible that people might feel less lonely and
have internal locus of control. The third and most important reason was that such type of research was not conducted
in the context of Pakistani culture. The many of the researches were grounded on western culture so it is very
necessary that these types of researches might be done in the Pakistani context. Although one study was carried out in India by Dewan and Jaswal(1997) to find the relationship of depression
&locus of control in this research loneliness is not found while it is proved by many previous researches that it has
an important role in depression, but Indian culture is also different from Pakistani culture and the study was on
depression and locus of control but in this research another relating factor is added which might have different
results. The results of research in India showed positive relation between external locus of control and depression. One of the studies was also conducted by Ayesha (2003) in order to find out the relation between depression and
locus of control but loneliness is missing which is also an important factor. Her research also shows the relationship
among these factors. One of the most important purposes of this study was in this locality in the lower Punjab this topic was new and no
research was conducted in this area. So the purpose was to know that the relationship either exist or not, it is a small
effort to explore these dimensions which surely will help clinical psychologists, psychiatrists, and mental health
professionals. It will help them to understand the cognitive styles and emotional states of the patients. Procedure:- The sample was consisted on 120 individuals. First of all permission from administration of hospitals was granted to
conduct the study. Before starting the study, informed consent of all the participants was obtained. They were
assured that their information would be kept confidential and the data will be used only for research purpose. The
first scale was Beck depression inventory BDI- Urdu version and the second scale was UCLA (University of
California Los Angelis) Loneliness scale. When the Performa’s were filled by participants, each Performa was then
checked individually and rated according to the scoring manual. Method:- The sample for this research was taken from Bahawalpur City one hundred and twenty individuals were taken, 60
from normal population and 60 from clinically depressed patients. These groups were further divided on the basis of
gender differences and marital status. The age range was from 20-50 years having at least proper comprehension
and understanding of Urdu language. Clinically depressed were taken from Bahawal Victoria Hospital Bahawalpur. The demographic variables used were age, marital status, education, occupation, & socioeconomic class. Instruments:- The present study is done by the use of BDI- Urdu version (BDI-U) (Khan, 1996) to measure depression. The Beck
depression inventory is a 21 item self-report questionnaire that assesses the presence and severity of cognitive,
affective, motivational and physical symptoms of depression. Score range is form 0-63 higher score indicates greater
symptoms severity. The second scale was loneliness scale it was used to measure loneliness score of the individual. Every individual
were rated accordingly on the basis of scoring given. Russell and Colleagues (1980) developed a revised version of
UCLA (University of California Los Angelis) Loneliness scale that included positively worded or non-lonely items
and negatively worded or lonely items. Result:- BDI
EDU
BDI
__
-0.375
Sig
__
.000**
(** P < .05) (
P < .05)
Results presented in the table indicate that there is a significant negative correlation between the two measures. The
table suggest that the correlation of BDI score and Education table (edu) is r = .000 (** P < .05). So regarding the
results depression is negatively correlated with education as education increases depression decreases. Table No 5: Model summary of the relationship between BDI scores and Education. Model
R
R2
Adjusted 2
SEE
1
.375
.140
.133
15.82
The table shows the model summary of the relationship between BDI and education. Result in the table indicates
that the value of R2 shows the variation in BDI explained by the variation in the independent variable i-e education. Table No 6: Co-efficient of multiple regressions of BDI and education
Un standardized Co-efficient
Standardized Co efficient
Model
B
S. E
Beta
t
Sig
Constant
44.300
4.721
--
9.384
.000
EDU
-5.818
1.325
-.375
-4.391
.000
Results in the table show the regression coefficient of education (edu) which is -5.232. It means that one degree
increase in the education tends then it would lead to -5.818 degree decrease in the BDI score of that independent
variable. So, the negative correlation exists between these two variables. Table No 6: Co-efficient of multiple regressions of BDI and education
d
di
d C
ffi i
S
d
di
d C
ffi i Result:- Table No 5: Model summary of the relationship between BDI scores and Education. Model
R
R2
Adjusted 2
SEE
1
.375
.140
.133
15.82
The table shows the model summary of the relationship between BDI and education. Result in the table indicates
that the value of R2 shows the variation in BDI explained by the variation in the independent variable i-e education. Table No 6: Co-efficient of multiple regressions of BDI and education
Un standardized Co-efficient
Standardized Co efficient
Model
B
S. E
Beta
t
Sig Table No 2: Model summary of the relationship between BDI Score and LONE Score. Model
R
R2
Adjusted 2
SEE
1
.405
.164
.157
15.60
The table indicates the model summary of the relationship between BDI and LONE. The value of R2 shows the
variation in the BDI explained 16.4% variation in the BDI score is explained by the variation in the loneliness score. Table No 2: Model summary of the relationship between BDI Score and LONE Score. Table No 2: Model summary of the relationship between BDI Score and LONE Score. Model
R
R2
Adjusted 2
SEE
1
.405
.164
.157
15.60
The table indicates the model summary of the relationship between BDI and LONE. The value of R2 shows the
variation in the BDI explained 16.4% variation in the BDI score is explained by the variation in the loneliness score. Table No 3: Co-efficient of multiple regressions of BDI and Lone. Table No 3: Co-efficient of multiple regressions of BDI and Lone. Un standardized Co-efficient
Standardized Co efficient
Model
B
S. E
Beta
t
Sig
Constant
-11.13
7.562
--
-1.473
.143
Lone
0.677
0.141
.405
4.808
.00
The regression co-efficient of loneliness is 0.677. It means that one degree increase in the loneliness will lead to
6.77 degree increase in BDI score of that independent variable. So, the relationship between these two variables is The regression co-efficient of loneliness is 0.677. It means that one degree increase in the loneliness will lead to
6.77 degree increase in BDI score of that independent variable. So, the relationship between these two variables is
highly strong and they are positively correlated to each other. Table No 4:Correlation matrix of education (edu) and BDI score. Result:- 1. Multiple regressions were calculated to see the relationship between depression and loneliness. 2. Multiple regressions were used to see the relationship between depression and education. Table No1: Correlation matrix between BDI Score and Loneliness Score LONE . BDI
LONE
DBI
__
0.405
Sig
___
.000**
** p<0.05 Table No1: Correlation matrix between BDI Score and Loneliness Score LONE . BDI
LONE
DBI
__
0.405
Sig
___
.000**
** p<0.05 87 International Journal of Advanced Research (2016), Volume 4, Issue 5, 85-89 ISSN 2320-5407 Results presented in the table indicate that there is a significant correlation between the two measures. The table
suggests that the correlation of BDI and LONE is r = .000 (** P < .05). So, regarding the results depression is highly
positively correlated with loneliness. Results presented in the table indicate that there is a significant correlation between the two measures. The table
suggests that the correlation of BDI and LONE is r = .000 (** P < .05). So, regarding the results depression is highly
positively correlated with loneliness. p
y
Table No 2: Model summary of the relationship between BDI Score and LONE Score. Model
R
R2
Adjusted 2
SEE
1
.405
.164
.157
15.60
The table indicates the model summary of the relationship between BDI and LONE. The value of R2 shows the
variation in the BDI explained 16.4% variation in the BDI score is explained by the variation in the loneliness score. Table No 3: Co-efficient of multiple regressions of BDI and Lone. Un standardized Co-efficient
Standardized Co efficient
Model
B
S. E
Beta
t
Sig
Constant
-11.13
7.562
--
-1.473
.143
Lone
0.677
0.141
.405
4.808
.00
The regression co-efficient of loneliness is 0.677. It means that one degree increase in the loneliness will lead to
6.77 degree increase in BDI score of that independent variable. So, the relationship between these two variables is
highly strong and they are positively correlated to each other. Table No 4:Correlation matrix of education (edu) and BDI score. BDI
EDU
BDI
__
-0.375
Sig
__
.000**
(** P < .05)
Results presented in the table indicate that there is a significant negative correlation between the two measures. The
table suggest that the correlation of BDI score and Education table (edu) is r = .000 (** P < .05). So regarding the
results depression is negatively correlated with education as education increases depression decreases. Discussion:- Every person has a wish to live a life in which he gets all the luxuries of life. It is a mechanical world today. Everyone has to work as a machine to get the luxuries of life. Everyone is running towards comfort. So there is a
competition between people. Everyone is sacrificing his/her peace of mind to get all comfort. So, this type of
environment causes many problems which arises because of the failure is depression. People who do not cope with
this fast world may become depressed.People sometimes feeling depressed are also left the societies they lost their
confidence in themselves and then they remain apart from all social gatherings, so, a sense of loneliness developed. Those people preferred to live a lonely life. They don’t have friends. They make their own fantasies and live in
those fantasies. Their psychological awareness is very poor. In our society people don’t have any concept about the
psychological disorders. So, the unawareness of the psychological disorder plays an important role in the
development if these disorders. 88 International Journal of Advanced Research (2016), Volume 4, Issue 5, 85-89 ISSN 2320-5407 Present study was assumed to investigate relationship of depression and loneliness among normal and depressed
individuals. The research was conducted on the basis of two different scales. For the measurement of depression,
Beck depression inventory was used. In contrast for the measurement of loneliness UCLA loneliness scale was used
to measure loneliness. The main aim of the study was to see the relationship between depression and loneliness. The results show that a very strong relationship exist between these two variables. So, depressed people also feel
lonely and they developed loneliness. According to Rubinstein and Shaver loneliness seem to lower self-esteem and
contribute to depression. It can be a vicious circle and both contribute to loneliness. According to researches it is
found that person who exhibited cognitive strategy called pessimistic avoidance (Characterized by failure
expectations negative effects, avoidance and self-handicapping) was related to loneliness that persisted over a period
of one year. This finding has support from other researches, which show that lonely person has a general pessimistic
attitude towards social situations (Parkhust&Hopmeyer, 1999). This pessimistic attitude in parts prevent lonely
individuals from forming new attachments with other for fear of rejection perceived as inevitable because of a
possible combination between expectations of failure and feelings of social inadequacy. Conclusion:- This research reveals that the relationship exists between depression and loneliness. It is concluded that because of
the stress in life people became disappointed and they feel lonely and separate themselves from their environment. It is concluded form the research that these variables are interrelated and occurrence of one of the variable leads to
the cause of other. Discussion:- Related to the general concept of social skills deficits is the somewhat more specific idea that constant seeking a
reassurance is the critical variable in depression. Depressed people seek reassurance that other truly care but even
when reassured they are only temporarily satisfied. Their negative self- concept causes them to double the truth of
the feedback they have received. So, these people leave the society and they developed feelings of loneliness
(Davison & Neale, 2001). Another objective was to see relation of education and depression. Result suggests that
education is negative correlated to depression. Education plays important role as education increases then depression
decreases, because it gives awareness of the disturbance and a can better cope with the depression. All these play important role in the etiology of depression. Some social factors such as family environment and
people attitude. The behavior of the people stress others to become pessimistic they lack self-confidence because of
the negative attitude of people. References:- 1. American Psychiatric Association. (2013). Diagnostic and Statistical Manual of Mental Disorders (DSM-5). American Psychiatric Pub. d y
2. Comer R.J. (1995).Abnormal Psychology (2nd Ed.) USA: W.H. Freeman &Company. th 3. Davison G.C & Neale, J.M. (2001).Abnormal Psychology (8th Ed.). New York:John Wiley & Sons. 4. Jackson, K. (2000). Family Homelessness: More than Simply a Lack of Housing. Taylor & Francis. 5. Jaswal, S., &Dewan, A. (1997).The Relationship between Locus of Control and Depression. Journal of Personality
and Clinical Studies, 13, 25-28. 5. Jaswal, S., &Dewan, A. (1997).The Relationship between Locus of Control and Depression. Journal of Personality
and Clinical Studies, 13, 25-28. (
)
p
gy
p
( )
7. Khan, A. A., Marwat, S. K., Noor, M. M., & Fatima, S. (2015). Reliability and Validity of Beck Depression Inventory
among General Population in Khyber Pakhtunkhwa, Pakistan. Journal of Ayub Medical College Abbottabad, 27(3),
573-575. 7. Khan, A. A., Marwat, S. K., Noor, M. M., & Fatima, S. (2015). Reliability and Validity of Beck Depression Inventory
among General Population in Khyber Pakhtunkhwa, Pakistan. Journal of Ayub Medical College Abbottabad, 27(3),
573-575. 8. Myers, D. G. (2000). The Funds, Friends, and Faith of Happy People. American Psychologist, 55(1), 56. 8. Myers, D. G. (2000). The Funds, Friends, and Faith of Happy People. American Psychologist, 55(1), 56. y
ppy
p
y
g
9. Parkhurst, J. T., &Hopmeyer, A. (1999). Developmental Change in the Sources of Loneliness in Childhood
Adolescence: Constructing a Theoretical Model. Loneliness in Childhood and Adolescence, 56-79. 9. Parkhurst, J. T., &Hopmeyer, A. (1999). Developmental Change in the Sources of Loneliness in Childhood an
Adolescence: Constructing a Theoretical Model. Loneliness in Childhood and Adolescence, 56-79. 10. Rokach, A., & Brock, H. (1996). The Causes of Loneliness. Psychology: A Journal of Human Behav & Brock, H. (1996). The Causes of Loneliness. Psychology: A Journal of Human Behavior. 11. Samreen, H., &Zubair, A. (2013).Locus of Control and Death Anxiety among Police Personnel. Pakistan Journal of
Psychological Research, 28(2), 261. 11. Samreen, H., &Zubair, A. (2013).Locus of Control and Death Anxiety among Police Personnel. Pakistan Journal of
Psychological Research, 28(2), 261. y
g
( )
12. Sarson, GJ. &Sarson, R.B. (2000).Abnormal Psychology (6th Ed). Prentice Hall, New Jersey. &Sarson, R.B. (2000).Abnormal Psychology (6th Ed). Prentice Hall, New Jersey. 13. Schultz, N. R., & Moore, D. (1986). The Loneliness Experience of College Students Sex Differences. Personality and
Social Psychology Bulletin,12(1), 111-119. 13. References:- Schultz, N. R., & Moore, D. (1986). The Loneliness Experience of College Students Sex Differences. Personality and
Social Psychology Bulletin,12(1), 111-119. 14. Woolfolk, R. L., Novalany, J., Gara, M. A., Allen, L. A., &Polino, M. (1995). Self-Complexity, Self-Evaluation, and
Depression: An Examination of Form and Content within the Self-Schema. Journal of Personality and Social
Psychology, 68(6), 1108. 89
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An IoT based Automatic Room Temperature and Health Monitoring System
|
International journal of engineering and advanced technology
| 2,022
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cc-by
| 2,809
|
International Journal of Engineering and Advanced Technology (IJEAT)
ISSN: 2249-8958 (Online), Volume-11 Issue-6, August 2022 International Journal of Engineering and Advanced Technology (IJEAT)
ISSN: 2249-8958 (Online), Volume-11 Issue-6, August 2022 C. Industrial Tool These IOT devices ranges from normal house hold objects
to highly developed industrial tools. Keywords: IoT, Heart monitoring sensor, Pulse sensor,
Temperature and humidity sensor. B. Business Intelligent To exchange the data’s with other devices that are
connected to the internet IoT is used. Once all the relevant
things are connected effective business intelligence is built
through the data’s collected. [7] I. INTRODUCTION The temperature and Pulse monitoring system is a real
time and simple project that can be used in day-to-day life. [3] The system is mainly more useful in hospitals, at old age
homes, etc. [5] A temperature that is configured by the user
is constantly maintained as the room temperature through
triggering the cooling appliances if necessary. Th The room temperature and health monitoring system is
used to monitor the heart beat of the patient it also adjusts
the temperature of the room suitable to the need of the
patient by continuous monitoring of heart rate and room
temperature. [4] The system aims to prevent attack for heart
patients. The system also provides suitable environmental
conditions according to the preferences in hotels, Old age
homes, hospitals, etc. [5] The system is mostly used for
people with lack of ability like old age people and disabled
people to open/close the windows and on/off of the
appliances like fan, air conditioner automatically to maintain
the room temperature according to their need. This device
can also be used manually by triggering the window
operations through a mobile application. Thus, this project
make less severe the stress and workload of older and
disabled people. The purpose of this project is to maintain
the body temperature and reduce the risks of heart attack. [6] The hardware’s that are used in this project are as
follows: A. ESP32 Microcontroller: B. Relay B. Relay C. Max30100 Pulse sensor D. Temperature Sensor (LM35, DHT11) E. Stepper motor E. Stepper motor F. Sim 900A GSM module G. Neo-6m GPS module A. Power of an Internet The Internet of Things are about extending the power of
the internet. It also describes the network of physical objects
that are fixed with sensors and other technologies. [7] An IoT based Automatic Room Temperature
and Health Monitoring System Hemanth Kumar V, Padma Priya D Abstract: There are so many tasks being automated nowadays. Likewise, the automatic room temperature and health monitoring
system is a process to monitor heart beats using pulse sensor and
the body temperature of patient. [3] There is also an automated
operation to control the room temperature by closing and
opening of windows in old age homes, hospitals, factories, etc. [5]
Using IoT the heart beat of patent is monitored, room
temperature and patient's body temperature is continuously
monitored. [6] The temperature can also be adjusted accordingly. These features can also be manually controlled through a mobile
application from anywhere. As the data's are gathered and
uploaded to a server the temperature of a closed environment can
be easily retrieved. The appliances that are connected like fan
and air conditioner will automatically adjust to the temperature
[1] in and out of the room according to the patient's body. Thus,
this project helps us to control the room (closed environment)
temperature without manpower. [1] A. ESP32 Microcontroller: ESP32 chip microcontroller is a low power system that
are with dual-mode Bluetooth and integrated wi-fi. The
ESP32 microcontroller is well suited for the IOT projects. As ESP32 is a game changer, there will be no need to
connect a separate device for cloud communication. Published By:
Blue Eyes Intelligence Engineering
and Sciences Publication (BEIESP)
© Copyright: All rights reserved. p
Fig 1. ESP32 Fig 1. ESP32 Manuscript received on 09 June 2022 | Revised Manuscript
received on 13 June 2022 | Manuscript Accepted on 15 August
2022 | Manuscript published on 30 August 2022. * Correspondence Author
Hemanth Kumar V, Assistant Professor, Department of Physics,
Bharath Institute of Higher Education and Research (BIHER), Selaiyur,
Chennai (Tamil Nadu), India. Email: hemmatsci@gmail.com
Padma Priya D*, Masters, Department of Physics, Bharath Institute of
Higher Education and Research (BIHER), Selaiyur, Chennai (Tamil Nadu),
India. Email: padmadurai.07@gmail.com Manuscript received on 09 June 2022 | Revised Manuscript
received on 13 June 2022 | Manuscript Accepted on 15 August
2022 | Manuscript published on 30 August 2022. * Correspondence Author Hemanth Kumar V, Assistant Professor, Department of Physics,
Bharath Institute of Higher Education and Research (BIHER), Selaiyur,
Chennai (Tamil Nadu), India. Email: hemmatsci@gmail.com Fig 1. ESP32 g
Padma Priya D*, Masters, Department of Physics, Bharath Institute of
Higher Education and Research (BIHER), Selaiyur, Chennai (Tamil Nadu),
India. Email: padmadurai.07@gmail.com Published By:
Blue Eyes Intelligence Engineering
and Sciences Publication (BEIESP)
© Copyright: All rights reserved. © The Authors. Published by Blue Eyes Intelligence Engineering and
Sciences Publication (BEIESP). This is an open access article under the
CC-BY-NC-ND license http://creativecommons.org/licenses/by-nc-nd/4.0/ Published By:
Blue Eyes Intelligence Engineering
and Sciences Publication (BEIESP)
© Copyright: All rights reserved. Published By:
Blue Eyes Intelligence Engineering
and Sciences Publication (BEIESP)
© Copyright: All rights reserved. Published By:
Blue Eyes Intelligence Engineering
and Sciences Publication (BEIESP)
© Copyright: All rights reserved. Retrieval Number: 100.1/ijeat.F36490811622
DOI: 10.35940/ijeat.F3649.0811622
Journal Website: www.ijeat.org E. Stepper Motor: Relay modules are the electronic components that are
found
everywhere. Wherever,
the
low
voltage
microcontrollers are used the relay modules are required. The device detects the intolerable conditions in the assigned
area. It also gives commands to disconnect the affected area
to the circuit breaker which protects the system from
damage. A motor which converts electrical to mechanical power is
called stepper motor also known as DC motor. It has a
capacity to rotate in 360 degree and other motors like servo
motors has capability only to rotate at 180 degree and the
stepper motor is used to move the window to any angle. Fig 5. Stepper Motor Fig 2. Relay Fig 5. Stepper Motor Retrieval Number: 100.1/ijeat.F36490811622
DOI: 10.35940/ijeat.F3649.0811622
Journal Website: www.ijeat.org 1 F. SIM900A Gsm Module: Fig 2. Relay The module SIM900a is a readily available GPRS/GSM
module that are used in PDA and many mobile phones. This
module is also used to develop IOT and Embedded
Applications. The SIM900 module looks like a single chip
but in total there are 68 pins that helps to build the
applications. G. NEO-6M Gps Module: This module is a GPS receiver that is able to locate all
the locations on Earth. Also, it is able to roughly track about
22 satellites. The NEO-6M GPS module works with low
power consumption. The module has EEPROM for storing
the configuration settings and also has a battery for power
backup. Fig 3. MAX30100 D. Temperature Sensors (LM35, DHT11): A sensor called temperature sensor is used to monitor an
object and its surroundings. Here to measure body
temperature LM35 sensor is used the capacity to measure is
up to 150 degree Celsius. Another sensor called DHT11 is
used to monitor moisture and temperature of surroundings. The temperature by measuring the change in resistance can
be successfully integrated with Node MCU. p
Fig 7. Neo-6m GPS Fig 4. Temperature sensors (DHT11 and LM35) Fig 7. Neo-6m GPS Fig 7. Neo-6m GPS 2
Published By:
Blue Eyes Intelligence Engineering
and Sciences Publication (BEIESP)
© Copyright: All rights reserved. Fig 4. Temperature sensors (DHT11 and LM35) 2
Retrieval Number: 100.1/ijeat.F36490811622
DOI: 10.35940/ijeat.F3649.0811622
Journal Website: www.ijeat.org
Published By:
Blue Eyes Intelligence Engineeri
and Sciences Publication (BEIES
© Copyright: All rights reserved. Published By:
Blue Eyes Intelligence Engineering
and Sciences Publication (BEIESP)
© Copyright: All rights reserved. Retrieval Number: 100.1/ijeat.F36490811622
DOI: 10.35940/ijeat.F3649.0811622
Journal Website: www.ijeat.org C. MAX30100 Pulse sensor: C. MAX30100 Pulse sensor: MAX30100 sensor is used to monitor the heart rate with
pulse oximeter. To measure the heart rate by calculating
light intensity changes in various organs and blood flow
changes through those organs is known as pulse sensor and
it is also called as photoplethysmography. The pulse sensor
contains LED and photodiode where the photodiode absorbs
the light which is reflected when it passes through blood. This is measured as an electric signal that is processed by a
microcontroller. Fig 6. SIM900A GSM Fig 3. MAX30100 Fig 6. SIM900A GSM V. RESULT V. RESULT Retrieval Number: 100.1/ijeat.F36490811622
DOI: 10.35940/ijeat.F3649.0811622
Journal Website: www.ijeat.org
Fig 1. Monitored Data REFERENCES 3
Retrieval Number: 100.1/ijeat.F36490811622
DOI: 10.35940/ijeat.F3649.0811622
Journal Website: www.ijeat.org
Published By:
Blue Eyes Intelligence Engineering
and Sciences Publication (BEIESP)
© Copyright: All rights reserved. Fig 1. Monitored Data
REFERENCES
1. Gaurav Waradkar, Vinay Maitry,Pa
TejasviAnsurkar, Asha Rawat(2016), "A
with visitor counter", Imperial Journa
(IJIR). 2. Katia Obraczka, Sam Mansfield, Er
System for Autonomous, Continuous,
and Its Application to Pressure Injury M
3. Soh Zhi Ping, Seng Kheau Chung, Hoe
Chekima, Jamal A.Dargham(2020) “I
Patient Monitoring System”
4. M Ramesha., P. Saleem Akram, Vali
Sohail, K.Teja Samba Siva Rao (2021
Health Monitoring system”
5. Sharnil Pandya, Warish Patel, Vira
Developing an IoT Based Intelligent M
Remote Health Monitoring 3
Retrieval Number: 100.1/ijeat.F36490811622
DOI: 10.35940/ijeat.F3649.0811622
Journal Website: www.ijeat.org
Published By:
Blue Eyes Intelligence Engineering
and Sciences Publication (BEIESP)
© Copyright: All rights reserved. Fig 1. Monitored Data
REFERENCES
1. Gaurav Waradkar, Vinay Maitry,Parth Das , Hitesh Ramina,
TejasviAnsurkar, Asha Rawat(2016), "Automated room light controller
with visitor counter", Imperial Journal of Interdisciplinary Research
(IJIR). 2. Katia Obraczka, Sam Mansfield, Eric Vin(2021) “An IoT-Based
System for Autonomous, Continuous, Real-Time Patient Monitoring
and Its Application to Pressure Injury Management
3. Soh Zhi Ping, Seng Kheau Chung, Hoe Tung Yew, Ming Fung Ng, Ali
Chekima, Jamal A.Dargham(2020) “IoT Based Real-Time Remote
Patient Monitoring System”
4. M Ramesha., P. Saleem Akram, Valiveti;Shaik Sai Aamani Sindhu
Sohail, K.Teja Samba Siva Rao (2021) ” IoT based Remote Patient
Health Monitoring system”
5. Sharnil Pandya, Warish Patel, Viral Mistry (2016) i-MsRTRM:
Developing an IoT Based Intelligent Medicare System for Real-Time
Remote Health Monitoring 1. Gaurav Waradkar, Vinay Maitry,Parth Das , Hitesh Ramina,
TejasviAnsurkar, Asha Rawat(2016), "Automated room light controller
with visitor counter", Imperial Journal of Interdisciplinary Research
(IJIR). 1. Gaurav Waradkar, Vinay Maitry,Parth Das , Hitesh Ramina,
TejasviAnsurkar, Asha Rawat(2016), "Automated room light controller
with visitor counter", Imperial Journal of Interdisciplinary Research
(IJIR). (
)
2. Katia Obraczka, Sam Mansfield, Eric Vin(2021) “An IoT-Based
System for Autonomous, Continuous, Real-Time Patient Monitoring
and Its Application to Pressure Injury Management (
)
2. Katia Obraczka, Sam Mansfield, Eric Vin(2021) “An IoT-Based
System for Autonomous, Continuous, Real-Time Patient Monitoring
and Its Application to Pressure Injury Management 3. Soh Zhi Ping, Seng Kheau Chung, Hoe Tung Yew, Ming Fung Ng, Ali
Chekima, Jamal A.Dargham(2020) “IoT Based Real-Time Remote
Patient Monitoring System” 3. IV. WORKING Fig 2. Mobile Application
This project is based on the ESP32 Micro-controller,
which has an inbuilt Wi-Fi module for sending data
over TCP/IP.
The role of this microcontroller is to gather data from
all the sensors, aggregate them, and perform assigned
tasks efficiently.
The first scope of this project is Automatic/Manual
room temperature controlling and monitoring. [2] For
gathering the temperature of the room and the
environment, we'll be using DHT11 sensors.
The user will set his/her suitable temperature for the
room using the cloud platform.
Once the ESP32 micro-controller gets the user's
temperature, it will analyze the user's temperature with
room and environment temperatures.
If the room temperature is not suitable with the user's
room
temperature,
but
the
outside(environment)
temperature is suitable, we'll open the window using a
stepper motor, so that the room temperature will be
adjusted with the user's temperature.
If the temperature is not suitable even after opening the
window, we'll turn on the fan to adjust the temperature
and AC even further to adjust the temperature. On this
basis,
the
room
temperature
will
be
adjusted
automatically with the user's temperature.
The user can also be able to control them manually. The
second scope of application will be patient monitoring
[2], where we monitor the user's body temperature,
heart rate, and blood oxygen level using Lm35 and
Max30100 sensors. [6] Retrieval Number: 100.1/ijeat.F36490811622
DOI: 10.35940/ijeat.F3649.0811622
Journal Website: www.ijeat.org 2 International Journal of Engineering and Advanced Technology (IJEAT)
ISSN: 2249-8958 (Online), Volume-11 Issue-6, August 2022 International Journal of Engineering and Advanced Technology (IJEAT)
ISSN: 2249-8958 (Online), Volume-11 Issue-6, August 2022 VI. CONCLUSION
If the user's health is critical, it will send an automatic
alert message to the closed ones, using the cloud
platform. [4] Here we've added GSM and GPS services
so that we'll get the location of the user and using GSM,
we can send the status of the user to neighbors and the
local hospital/police station when the user has critical
health status. [5] [8]. The temperature controlling and patient monitoring system
helps in serving people at risk by reducing cases of heart
attacks. [2] The system also reduces other medical
emergencies as it maintains and constantly monitors the
temperatures in and around of the patient. [5] All these tasks
that the system does are automated and it also has an option
to control it manually if needed. [8] Thus, this project
perform duties for Patients, Old-aged people and especially
for the people with disability, etc. [6] REFERENCES Soh Zhi Ping, Seng Kheau Chung, Hoe Tung Yew, Ming Fung Ng, Ali
Chekima, Jamal A.Dargham(2020) “IoT Based Real-Time Remote
Patient Monitoring System” 4. M Ramesha., P. Saleem Akram, Valiveti;Shaik Sai Aamani Sindhu
Sohail, K.Teja Samba Siva Rao (2021) ” IoT based Remote Patient
Health Monitoring system” 4. M Ramesha., P. Saleem Akram, Valiveti;Shaik Sai Aamani Sindhu
Sohail, K.Teja Samba Siva Rao (2021) ” IoT based Remote Patient
Health Monitoring system” 5. Sharnil Pandya, Warish Patel, Viral Mistry (2016) i-MsRTRM:
Developing an IoT Based Intelligent Medicare System for Real-Time
Remote Health Monitoring 3
Published By:
Blue Eyes Intelligence Engineering
and Sciences Publication (BEIESP)
© Copyright: All rights reserved. International Journal of Engineering and Advanced Technology (IJEAT)
ISSN: 2249-8958 (Online), Volume-11 Issue-6, August 2022 6. Rani G, Utekar;Jayant Rani G, Umale (2018) “Automated IoT Based
Healthcare System for Monitoring of Remotely Located Patients”
[CrossRef] 7. Alok Kumar Gupta, Rahul Johari (2019). “IOT based Electrical Device
Surveillance and Control System of Internet of Things”: Smart
Innovation and Usages, International Conference On IEEE. 8. Mohammad Salah Uddin, Suraiya Banu, Jannat Binta Alam (2017),
“Real time patient monitoring system based on Internet of Things” ,
2017 4th International Conference on Advances in Electrical
Engineering (ICAEE) Retrieval Number: 100.1/ijeat.F36490811622
DOI: 10.35940/ijeat.F3649.0811622
Journal Website: www.ijeat.org 3 International Journal of Engineering and Advanced Technology (IJEAT)
ISSN: 2249-8958 (Online), Volume-11 Issue-6, August 2022 International Journal of Engineering and Advanced Technology (IJEAT)
ISSN: 2249-8958 (Online), Volume-11 Issue-6, August 2022 Retrieval Number: 100.1/ijeat.F36490811622
DOI: 10.35940/ijeat.F3649.0811622
Journal Website: www.ijeat.org AUTHORS PROFILE Hemanth Kumar V, is working as an assistant
professor in the department of Physics at Bharath
Institute of Higher Education and Research (BIHER),
Selaiyur, Chennai, India. He has completed M.sc college
of Engineering from National Institute of Technology,
Karnataka, Surathkal. He has completed his Ph.D in
Metallurgical and Materials Engineering from National Institute of
Technology, Karnataka, Surathkal. He has 5 years of teaching experience in
the field of Science and Physics. Also, he is a Talented Sociological
professor driven to inspire students to pursue academic and personal
excellence. He consistently strive to create a challenging and engaging
learning
in
which
students
become
life-long
scholar. Email:
hemmatsci@gmail.com Hemanth Kumar V, is working as an assistant
professor in the department of Physics at Bharath
Institute of Higher Education and Research (BIHER),
Selaiyur, Chennai, India. He has completed M.sc college
of Engineering from National Institute of Technology,
Karnataka, Surathkal. He has completed his Ph.D in
Metallurgical and Materials Engineering from National Institute of
Technology, Karnataka, Surathkal. He has 5 years of teaching experience in
the field of Science and Physics. Also, he is a Talented Sociological
professor driven to inspire students to pursue academic and personal
excellence. He consistently strive to create a challenging and engaging
learning
in
which
students
become
life-long
scholar. Email:
hemmatsci@gmail.com Padma Priya D, is currently doing her Masters in the
department of Physics in Bharath Institute of Higher
Education and Research (BIHER), Selaiyur, Chennai,
India. She has completed her Bachelor’s Degree in the
department of Physics in Bharath Institute of Higher
Education and Research (BIHER), Selaiyur, Chennai,
India. She did her schoolings in Jeeva Velu Residential School (JVRS) at
Tiruvannamalai. She is most particularly interested in Internet of Things
(IoT) automation. Email: padmadurai.07@gmail.com. 4
Retrieval Number: 100.1/ijeat.F36490811622
DOI: 10.35940/ijeat.F3649.0811622
Journal Website: www.ijeat.org
Published By:
Blue Eyes Intelligence Engineering
and Sciences Publication (BEIESP)
© Copyright: All rights reserved. 4
Published By:
Blue Eyes Intelligence Engineering
and Sciences Publication (BEIESP)
© Copyright: All rights reserved. Published By:
Blue Eyes Intelligence Engineering
and Sciences Publication (BEIESP)
© Copyright: All rights reserved. Retrieval Number: 100.1/ijeat.F36490811622
DOI: 10.35940/ijeat.F3649.0811622
Journal Website: www.ijeat.org Retrieval Number: 100.1/ijeat.F36490811622
DOI: 10.35940/ijeat.F3649.0811622
Journal Website: www.ijeat.org 4
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https://openalex.org/W3000484773
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https://gmd.copernicus.org/articles/13/139/2020/gmd-13-139-2020.pdf
|
English
| null |
Machine dependence and reproducibility for coupled climate simulations: the HadGEM3-GC3.1 CMIP Preindustrial simulation
|
Geoscientific model development
| 2,020
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cc-by
| 9,040
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1
Introduction The UK CMIP6 (Coupled Model Intercomparison Project
Phase 6) community runs individual MIP (Model Intercom-
parison Project) experiments on differing computing plat-
forms but will generally compare results against the refer-
ence simulations run on the UK Met Office platform. For
this reason, within the UK CMIP community, the possible
influence of machine dependence on simulation results is of-
ten informally discussed among scientists, but surprisingly
an analysis to quantify its impact has not been attempted. Here we investigate the behaviour of the Preindustrial (PI)
simulation prepared by the UK Met Office for the forth-
coming CMIP6 (Coupled Model Intercomparison Project
Phase 6) under different computing environments. The issue of being able to reproduce identical simula-
tion results across different supercomputers, or following
a system upgrade on the same supercomputer, has long
been known by numerical modellers and computer scientists. However, the impact that a different computing environment
can have on otherwise identical numerical simulations ap-
pears to be little known by climate model users and model
data analysts. In fact, the subject is rarely ever addressed in
a way that helps the community understand the magnitude
of the problem or to develop practical guidelines that take
account of the issue. Discrepancies between the means of key climate variables
were analysed at different timescales, from decadal to cen-
tennial. We found that for the two simulations to be statis-
tically indistinguishable, a 200-year averaging period must
be used for the analysis of the results. Thus, constant-forcing
climate simulations using the HadGEM3-GC3.1 model are
reproducible on different HPC platforms provided that a suf-
ficiently long duration of simulation is used. In regions where El Niño–Southern Oscillation (ENSO)
teleconnection patterns were detected, we found large
sea surface temperature and sea ice concentration differ-
ences on centennial timescales. This indicates that a 100-
year constant-forcing climate simulation may not be long
enough to adequately capture the internal variability of the
HadGEM3-GC3.1 model, despite this being the minimum
simulation length recommended by CMIP6 protocols for
many MIP (Model Intercomparison Project) experiments. To the extent of our knowledge, only a few authors have
discussed the existence of machine dependence uncertainty
and highlighted the importance of bit-for-bit numerical re-
producibility in the context of climate model simulations. Song et al. (2012) and Hong et al. (2013) investigated the
uncertainty due to the round-off error in climate simulations. Liu et al. Machine dependence and reproducibility for coupled climate
simulations: the HadGEM3-GC3.1 CMIP Preindustrial simulation
Maria-Vittoria Guarino1, Louise C. Sime1, David Schroeder2, Grenville M. S. Lister3, and Rosalyn Hatcher3
1British Antarctic Survey, Cambridge, UK
2Department of Meteorology, University of Reading, Reading, UK
3National Centre for Atmospheric Science, University of Reading, Reading, UK Correspondence: Maria-Vittoria Guarino (m.v.guarino@bas.ac.uk) Received: 28 March 2019 – Discussion started: 16 May 2019
Revised: 29 November 2019 – Accepted: 4 December 2019 – Published: 16 January 2020 Abstract. When the same weather or climate simulation is
run on different high-performance computing (HPC) plat-
forms, model outputs may not be identical for a given ini-
tial condition. While the role of HPC platforms in delivering
better climate projections is to some extent discussed in the
literature, attention is mainly focused on scalability and per-
formance rather than on the impact of machine-dependent
processes on the numerical solution. 1
Introduction (2015a, b) discussed the importance of bitwise iden-
tical reproducibility in climate models. On the basis of our findings, we recommend a minimum
simulation length of 200 years whenever possible. In this paper, we investigate the behaviour of the
UK CMIP6 Preindustrial (PI) control simulation with the Geosci. Model Dev., 13, 139–154, 2020
https://doi.org/10.5194/gmd-13-139-2020
© Author(s) 2020. This work is distributed under
the Creative Commons Attribution 4.0 License. Geosci. Model Dev., 13, 139–154, 2020
https://doi.org/10.5194/gmd-13-139-2020
© Author(s) 2020. This work is distributed under
the Creative Commons Attribution 4.0 License. M.-V. Guarino et al.: Machine dependence and reproducibility for coupled climate simulations 140 HadGEM3-GC3.1 model on two different high-performance
computing (HPC) platforms. We first study whether the two
versions of the PI simulation show significant differences in
their long-term statistics. This answers our first question of
whether the HadGEM3-GC3.1 model gives different results
on different HPC platforms. tialized with (x0,y0,z0) ≡(1, 1, 1) and numerically inte-
grated with a 4th-order Runge–Kutta scheme using a time
step of 0.01. The Lorenz model was run on two HPC plat-
forms, namely the UK Met Office Supercomputer (here-
inafter simply “MO”) and ARCHER. To first demonstrate the implications of switching between
different computing environments, the Lorenz model was run
on the ARCHER platform using the following: Machine-dependent processes can influence the model in-
ternal variability by causing it to be sampled differently on
the two platforms (i.e. similarly to what happens to en-
semble members initiated from different initial conditions). Therefore, our second objective is to quantify discrepancies
between the two simulations at different timescales (from
decadal to centennial) in order to identify an averaging pe-
riod and/or simulation length for which the two simulations
return the same internal variability. – two different FORTRAN compilers (cce8.5.8 and in-
tel17.0; see Fig. 1a and b); – same FORTRAN compiler (cce8.5.8) but different
degrees of floating-point optimization (-hfp0 and
-hfp3; see Fig. 1c and d); and – the same FORTRAN (cce8.5.8) compiler and compil-
ing options, but the x component in Eqs. (1)–(3) was
perturbed by adding a noise term obtained using the
random_number and random_seed intrinsic FOR-
TRAN functions. In particular, the seed of the random
number generator was set to 1 and 3 in two separate ex-
periments; see Fig. 1e and f. Note that the PI control simulation is a constant-forcing
simulation. Therefore, no ensemble members are required for
such an experiment because, provided that the simulation is
long enough, it will return a picture of the natural climate
variability. The remainder of the paper is organized as follows. In
Sect. 2, mechanisms by which the computing environment
can influence the numerical solution of chaotic dynamical
systems are reviewed and discussed. In Sect. 3, the numerical
simulations are presented, and the methodology used for the
data analysis is described. In Sect. 4, the simulation results
are presented and discussed. In Sect. 5, the main conclusions
of the present study are summarized. M.-V. Guarino et al.: Machine dependence and reproducibility for coupled climate simulations Finally, to illustrate the role of using different HPC plat-
forms, the Lorenz model was run on the ARCHER and MO
platforms using the same compiler (intel17.0) and identical
compiling options (i.e. level of code optimization, floating-
point precision, vectorization) (Fig. 1g and h). The divergence of the solutions in Fig. 1a and b can likely
be explained by the different “computation order” of the two
compilers (i.e. the order in which the same arithmetic expres-
sion is computed). In Fig. 1c and d, solutions differ because
of the round-off error introduced by the different precision
of floating-point computation. In Fig. 1e and f, the different
seed used to generate random numbers caused the system to
be perturbed differently in the two cases. While this conclu-
sion is straightforward, it is worth mentioning that the use
of random numbers is widespread in weather and climate
modelling. Random number generators are largely used in
physics parameterizations for initialization and perturbation
purposes (e.g. clouds, radiation, and turbulence parameteri-
zations) as well as in stochastic parameterizations. The pro-
cesses by which initial seeds are selected within the model
code are thus crucial in order to ensure numerical repro-
ducibility. Furthermore, different compilers may have differ-
ent default seeds. Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. Geosci. Model Dev., 13, 139–154, 2020 www.geosci-model-dev.net/13/139/2020/ 2
The impact of machine dependence on the numerical
solution In this section, possible known ways in which machine-
dependent processes can influence the numerical solution of
chaotic dynamical systems are reviewed and discussed. In this section, possible known ways in which machine-
dependent processes can influence the numerical solution of
chaotic dynamical systems are reviewed and discussed. Different compiling options, degrees of code optimization,
and basic library functions all have the potential to affect the
reproducibility of model results across different HPC plat-
forms and on the same platform under different computing
environments. Here we provide a few examples of machine-
dependent numerical solutions using the 3-D Lorenz model
(Lorenz, 1963), which is a simplified model for convection
in deterministic flows. The Lorenz model consists of the fol-
lowing three differential equations: As for Fig. 1g and h, this is probably the most relevant
result for the present paper. It highlights the influence of the
HPC platform (and of its hardware specifications) on the final
numerical solution. In Fig. 1g and h the two solutions diverge
in time similarly to Fig. 1a–d; however, identifying reasons
for the observed differences is not straightforward. While we
speculate that reasons may be down to the machine archi-
tecture and/or chip set, further investigations on the subject
were not pursued as this would be beyond the scope of this
study. dx
dt = α(y −x),
(1)
dy
dt = γ x −y −zx,
(2)
dz
dt = xy −βz,
(3) (1) (2) (3) where the parameters α = 10, γ = 28, and β = 8/3 were
chosen to allow for the generation of flow instabilities and
obtain chaotic solutions (Lorenz, 1963). The model was ini- Geosci. Model Dev., 13, 139–154, 2020 Geosci. Model Dev., 13, 139–154, 2020 www.geosci-model-dev.net/13/139/2020/ M.-V. Guarino et al.: Machine dependence and reproducibility for coupled climate simulations M.-V. 2
The impact of machine dependence on the numerical
solution Guarino et al.: Machine dependence and reproducibility for coupled climate simulations 141 uarino et al.: Machine dependence and reproducibility for coupled climate simulations
Attractor (left-hand side) and time series of the x component (right-hand side) of the 3-D Lorenz model for sim
R using the cce8.5.8 and intel17.0 compilers (a, b), the same compiler (cce8.5.8) but a different level of floating-po
p3) (c, d), and the same compiler (cce8.5.8) and compiling options but a different seed for the random number gene
els (g) and (h) are the Lorenz attractor and the x component time series for the Lorenz model run on MO and ARC
mpiler (intel17.0) and compiling options. osci model dev net/13/139/2020/
Geosci Model Dev 13 1 Figure 1. Attractor (left-hand side) and time series of the x component (right-hand side) of the 3-D Lorenz model for simulations run on
ARCHER using the cce8.5.8 and intel17.0 compilers (a, b), the same compiler (cce8.5.8) but a different level of floating-point optimization
(hfp0, hfp3) (c, d), and the same compiler (cce8.5.8) and compiling options but a different seed for the random number generator (seed 1, 3)
(e, f). Panels (g) and (h) are the Lorenz attractor and the x component time series for the Lorenz model run on MO and ARCHER using the
same compiler (intel17.0) and compiling options. Figure 1. Attractor (left-hand side) and time series of the x component (right-hand side) of the 3-D Lorenz model for simulations run on
ARCHER using the cce8.5.8 and intel17.0 compilers (a, b), the same compiler (cce8.5.8) but a different level of floating-point optimization
(hfp0, hfp3) (c, d), and the same compiler (cce8.5.8) and compiling options but a different seed for the random number generator (seed 1, 3)
(e, f). Panels (g) and (h) are the Lorenz attractor and the x component time series for the Lorenz model run on MO and ARCHER using the
same compiler (intel17.0) and compiling options. Geosci. Model Dev., 13, 139–154, 2020 3.1
Numerical simulations In this study, we consider two versions of the Preindustrial
(PI) control simulation prepared by the UK Met Office for
the Sixth Coupled Model Intercomparison Project, CMIP6
(Eyring et al., 2016). This PI control experiment is used to
study the (natural) unforced variability of the climate system,
and it is one of the reference simulations against which many
of the other CMIP6 experiments will be analysed. Table 1 provides an overview of the hardware and software
specifications of the two HPC platforms on which the model
was run. p
y
The PI simulation considered in this paper uses the
N96 resolution version of the HadGEM3-GC3.1 climate
model (N96ORCA1). The model set-up, initialization, per-
formance, and physical basis are documented in Menary
et al. (2018) and Williams et al. (2018), to which the
reader is referred for a detailed description. In summary,
HadGEM3-GC3.1 is a global coupled atmosphere–land–
ocean–ice model that comprises the Unified Model (UM) at-
mosphere model (Walters et al., 2017), the JULES land sur-
face model (Walters et al., 2017), the NEMO ocean model
(Madec and the NEMO Team, 2015), and the CICE sea ice
model (Ridley et al., 2018b). The UM vertical grid contains
85 pressure levels (terrain-following hybrid height coordi-
nates), while the NEMO vertical grid contains 75 depth lev-
els (rescaled height coordinates). In the N96 resolution ver-
sion, the atmospheric model utilizes a horizontal grid spac-
ing of approximately 135 km on a regular latitude–longitude
grid. The grid spacing of the ocean model, which employs an
orthogonal curvilinear grid, is 1◦everywhere but decreases
down to 0.33◦between 15◦N and 15◦S of the Equator, as
described by Kuhlbrodt et al. (2018). Of the possible mechanisms discussed in Sect. 2, the
ARCHER and MO simulations were likely affected by differ-
ences in compiler, processor type, number of processors, and
processor decomposition (alongside the different machine). p
p
(
g
)
Note that the porting of the HadGEM3-GC3.1 model from
the Met Office computing platform to the ARCHER plat-
form was tested by running 50 ensemble members (each 24 h
long) on both platforms (this was done by the UK Met Office
and NCAS-CMS teams). Each ensemble member was cre-
ated by adding a random bit-level perturbation to a set of se-
lected variables (x and y components of the wind, air poten-
tial temperature, specific humidity, longwave radiation, etc.). M.-V. Guarino et al.: Machine dependence and reproducibility for coupled climate simulations M.-V. Guarino et al.: Machine dependence and reproducibility for coupled climate simulations M.-V. Guarino et al.: Machine dependence and reproducibility for coupled climate simulation Table 1. Hardware and software specifications of the ARCHER and
MO HPC platforms as used to run the HadGEM3-GC3.1 model. The three mechanisms discussed above were selected be-
cause they are illustrative of the problem and easily testable
via a simple model such as the Lorenz model. However, there
are a number of additional software and hardware specifica-
tions that can influence numerical reproducibility and that
only emerge when more complex codes, like weather and
climate models, are run. These are the number of processors
and processor decomposition, communications software (i.e. MPI libraries), and threading (i.e. OpenMP libraries). on the MO HPC platform on 2500 cores. The model was
at first run for 700 model years to allow the atmospheric
and oceanic masses to attain a steady state (model spin-
up) and then run for a further 500 model years (actual run
length) (see Menary et al., 2018 for details). A copy of the
PI control simulation was ported to the ARCHER HPC plat-
form (hereinafter referred to as PIAR), initialized using the
atmospheric and oceanic fields from the end of the spin-
up, and run for 200 model years using 1500 cores. The
source codes of the atmosphere and ocean models were
compiled on the two platforms using the same levels of
code optimization (-O option), vectorization (-Ovector
option), and floating-point precision (-hfp option) and, for
numerical reproducibility purposes, selecting the least tol-
erant behaviour in terms of code optimization when the
number of ranks or threads varies (-hflex_mp option). For the atmosphere component the following options were
used: -O2 -Ovector1 -hfp0 -hflex_mp=strict. For the ocean component the following options were used:
-O3 -Ovector1 -hfp0 -hflex_mp=strict. We conclude this section by stressing that the four case
studies presented in Fig. 1 (and the additional mechanisms
discussed in this section) are all essentially a consequence of
the chaotic nature of the system. When machine-dependent
processes introduce a small perturbation or error into the sys-
tem (no matter by which means), they cause it to evolve dif-
ferently after a few time steps. www.geosci-model-dev.net/13/139/2020/ ndence and reproducibility for coupled climate simulations
Table 1. Hardware and software specifications of the ARCHER and
MO HPC platforms as used to run the HadGEM3-GC3.1 model. HPC platform
Machine
Compiler
Processor
MO
Cray XC40
cce 8.3.4
Broadwell
ARCHER
Cray XC30
cce 8.5.8
Ivy Bridge 142 4.1
Multiple timescales The long-term means of the selected variables and the associ-
ated SNR are shown in Figs. 2 and 3. All the variables exhibit
SNR < 1, indicating that on multi-centennial timescales the
differences observed between the two simulations fall into
the expected range of variability of the PI control run. Note that, when calculating (PIMO −PIAR) differences,
PIMO and PIAR segments are subtracted in chronological or-
der. Thus, for example, the first 10 years of PIAR are sub-
tracted from the first 10 years of PIMO and so on. In fact, be-
cause the PI control simulation is run with a constant climate
forcing, using a “chronological order” in the strictest sense is
meaningless, as every 10-year segment is equally represen-
tative of the pre-industrial decadal variability. We acknowl-
edge that an equally valid alternative approach would be to
subtract the PIAR and PIMO segments without a prescribed
order. When maps like the ones in Figs. 2 and 3 are computed
using 10-, 30-, 50-, and 100-year averaging periods (not
shown), the magnitude of the anomalies increases and (PIMO
−PIAR) differences become significant (SNR ≫1). This be-
haviour is discussed below. Figures 4 to 9 show annual mean time series of spatially
averaged SST, SIA, SAT, SW TOA, LW TOA, and P, respec-
tively. Figures 4d to 9d show (PIMO −PIAR) differences as
a function of the averaging timescale for each variable (see
Sect. 3.2 for details on the computation of the means). The
200-year global mean and standard deviation of each variable
are shown in Table 2. Discrepancies in the results between the two runs were
quantified by computing the signal-to-noise ratio (SNR) for
each considered variable at each timescale. The signal is
represented by the mean of the differences between PIMO
and PIAR (µMO−AR), and the noise is represented by the
standard deviation of PIMO (σMO), our “reference” simula-
tion. Because of the basic properties of variance, for which
VarX−Y = VarX +VarY −2Cov(X,Y) (Loeve, 1977), we can
more conveniently express the noise as σMO = σMO−AR
√
2
un-
der the assumption that PIMO and PIAR are uncorrelated
(Cov(MO,AR) = 0) and have the same variance (VarMO =
VarAR). 3.2
Data post-processing and analysis When SNR < 1, (PIMO −PIAR) differences can be inter-
preted as fluctuations within the estimated range of internal
variability. When SNR > 1, (PIMO −PIAR) differences in the
mean are outside the expected range of internal variability. This eventuality indicates either a true difference in the mean
or that the expected range of variability is underestimated. During the analysis of the results, the following climate vari-
ables were considered: sea surface temperature (SST), sea ice
area and sea ice concentration (SIA, SIC), 1.5 m air temper-
ature (SAT), the outgoing longwave and shortwave radiation
fluxes at top of the atmosphere (LW TOA and SW TOA),
and the precipitation flux (P ). These variables were selected
as representative of the ocean and atmosphere domains and
because they are commonly used to evaluate the status of the
climate system. For the final step of the analysis, the El Niño–Southern
Oscillation (ENSO) signal was computed for the ARCHER
and MO simulations. We used the Niño 3.4 index, with a 3-
month running mean, defined as follows: NINO3.4 = SSTmnth −SST30 yr
if 5 ◦N ≤latitude ≤5 ◦S and
120 ◦W ≤longitude ≤170 ◦W,
(5) NINO3.4 = SSTmnth −SST30 yr
if 5 ◦N ≤latitude ≤5 ◦S and
120 ◦W ≤longitude ≤170 ◦W,
(5) Discrepancies between the means of the selected vari-
ables were analysed at different timescales, from decadal to
centennial. To compute 10-, 30-, 50-, and 100-year means,
(PIMO −PIAR) 200-year time series were divided into 20,
6, 4, and 2 segments, respectively. Spatial maps were simply
created by averaging each segment over time. Additionally,
to create the scatter plots presented in Sect. 4.1, the time av-
erage was combined with an area-weighted spatial average. Except for SIC, all the variables were averaged globally. Ad-
ditionally, SIC, SST, and SAT were regionally averaged over
the Northern and Southern Hemisphere, while SW TOA, LW
TOA, and P were regionally averaged over the tropics, north-
ern extratropics, and southern extratropics according to the
underlying physical processes. (5) where SSTmnth is the monthly sea surface temperature and
SST30 yr is the climatological mean of the first 30 years of
simulation used to compute the anomalies. 3.1
Numerical simulations Variables from each set of ensembles were then tested for
significance using a Kolmogorov–Smirnov test to determine
whether they can be assumed to be drawn from the same
distribution. These tests did not reveal any significant prob-
lem with the porting of the HadGEM3-GC3.1 model (David
Case, National Centre for Atmospheric Science, University
of Reading, Reading, UK, personal communications, 2019). However, this method is restricted to timescales shorter than
1 d. The centennial simulations presented in this paper will
help us understand whether or not differences can arise on
longer timescales in the HadGEM3-GC3.1 model. Following the CMIP6 guidelines, the model was initial-
ized using constant 1850 greenhouse gas (GHG), ozone,
solar, tropospheric aerosol, stratospheric volcanic aerosol,
and land use forcings. The UK CMIP6 PI control simu-
lation (hereinafter referred to as PIMO) was originally run Geosci. Model Dev., 13, 139–154, 2020 www.geosci-model-dev.net/13/139/2020/ 143 4.1
Multiple timescales This allowed us to compute SNR on one grid and
avoid divisions by (nearly) zero when the sea ice field be-
tween PIMO and PIAR evolved differently, resulting in unre-
alistically high SNR values along the sea ice edges. Finally,
SNR is defined as For all the considered variables, PIMO and PIAR start di-
verging quickly after the first few time steps once the system
has lost memory of the initial conditions (Figs. 4 to 9, pan-
els a, b, c). See Sect. 2 (Fig. 1) for a further discussion on
how machine-dependent processes can influence the tempo-
ral evolution of the system. SST, SAT, SW TOA, and LW TOA differ the most
in the Northern Hemisphere (and particularly on decadal
timescales) (yellow diamonds in Figs. 4d, 6d, 7d, 8d), while
SIA anomalies are particularly high in the Southern Hemi-
sphere (red crosses in Fig. 5d) and P anomalies in the trop-
ics (green circles in Fig. 9d). Overall, discrepancies are the
largest at decadal timescales at which the spread between the
two simulations can reach |0.2| ◦C in global mean air tem-
perature (Fig. 6d), |1.2| million km2 in Southern Hemisphere SNR = |µMO−AR|
σMO
= |µMO−AR|
σMO−AR
√
2
. (4) (4) www.geosci-model-dev.net/13/139/2020/ Geosci. Model Dev., 13, 139–154, 2020 -V. Guarino et al.: Machine dependence and reproducibility for coupled climate simulations 144 year means and corresponding SNR of (PIMO −PIAR) differences for NH SST (a, b), SH SST (c, d), NH SIC Figure 2. The 200-year means and corresponding SNR of (PIMO −PIAR) differences for NH SST (a, b), SH SST (c, d), NH SIC (e, f), and
SH SIC (g, h). Geosci. Model Dev., 13, 139–154, 2020 www.geosci-model-dev.net/13/139/2020/ M.-V. Guarino et al.: Machine dependence and reproducibility for coupled climate simulations M.-V. Guarino et al.: Machine dependence and reproducibility for coupled climate simulations 145 gure 3. The 200-year means and corresponding SNR of (PIMO −PIAR) differences for SAT (a, b), SW TOA (c, d), LW
(g, h). Figure 3. The 200-year means and corresponding SNR of (PIMO −PIAR) differences for SAT (a, b), SW TOA (c, d), LW TOA (e, f), and
P (g, h). ans and corresponding SNR of (PIMO −PIAR) differences for SAT (a, b), SW TOA (c, d), LW TOA (e, f), an Large differences in the mean and SNR ≫1 are thus not sur-
prising when analysing decadal periods. sea ice area (Fig. 5d), or |1| W m−2 in global TOA outgoing
LW flux (Fig. 8d). On decadal timescales, the averaging period is too short to
adequately sample the model interannual variability; there-
fore, the estimated mean is not stable, and the estimated stan-
dard deviation is likely to be underestimated compared with
the true standard deviation of the model internal variability. On longer timescales, the estimates of the mean and stan-
dard deviation converge toward their “true” values. Accord-
ingly, we see that the differences in the mean between PIMO
and PIAR become smaller and approach zero as the timescale
increases (Figs. 4d to 9d). When we consider the 200-year
timescale, we find no SNR value greater than 1 (Figs. 2 and www.geosci-model-dev.net/13/139/2020/ www.geosci-model-dev.net/13/139/2020/ Geosci. Model Dev., 13, 139–154, 2020 M.-V. Guarino et al.: Machine dependence and reproducibility for coupled climate simulations M.-V. Guarino et al.: Machine dependence and reproducibility for coupled climate simulations 146 Figure 4. Annual mean time series of global SST (a), Northern Hemisphere SST (b), and Southern Hemisphere SST (c) for PIMO (grey line)
and PIAR (dashed line). Panel (d) shows how SST differences vary as a function of the timescale. Figure 4. Annual mean time series of global SST (a), Northern Hemisphere SST (b), and Southern Hemisphere SST (c) for PIMO (grey line)
and PIAR (dashed line). Panel (d) shows how SST differences vary as a function of the timescale. Figure 5. Annual mean time series of Northern Hemisphere SIA (a) and Southern Hemisphere SIA (b) for PIMO (grey line) and PIAR
(dashed line). The 200-year mean of the NH and SH SIA seasonal cycle is shown in (c). Panel (d) shows how SIA differences vary as a
function of the timescale. Figure 5. Annual mean time series of Northern Hemisphere SIA (a) and Southern Hemisphere SIA (b) for PIMO (grey line) and PIAR
(dashed line). The 200-year mean of the NH and SH SIA seasonal cycle is shown in (c). Panel (d) shows how SIA differences vary as a
function of the timescale. Geosci. Model Dev., 13, 139–154, 2020 Geosci. Model Dev., 13, 139–154, 2020 www.geosci-model-dev.net/13/139/2020/ M.-V. Guarino et al.: Machine dependence and reproducibility for coupled climate simulations 4.2
The 100-year timescale The large differences observed on timescales shorter than
200 years are a direct consequence of the (potentially under-
estimated) internal variability of the model and triggered (at
least initially) by machine-dependent processes (compiler,
machine architecture, etc.; see Sects. 2 and 3.1 for details). The two simulations behave similarly to ensemble members
initiated from different initial conditions. Therefore, they ex-
hibit different phases of the same internal variability, but over
longer timescales differences converge to zero (Figs. 4–9). In Figs. 4d to 9d, the variation of (PIMO −PIAR) differ-
ences with the timescale suggests the existence of a power-
law relationship.1 To investigate this behaviour, a base-10
logarithmic transformation was applied to the x and y axes
in Figs. 4d to 9d, and linear regression was used to find the
straight lines that best fit the data. Figure 10 shows log–log plots for SST, SAT, SW TOA,
LW TOA, and P for the maximum (PIMO −PIAR) values
at each timescale. To ease the comparison, all the variables
were averaged globally and over the Southern Hemisphere
(SH) and Northern Hemisphere (NH). Global, NH, and SH
mean data all align along a straight line, supporting the ex-
istence of a power law. However, the most interesting result
emerges at the global scale on which (PIMO −PIAR) dif-
ferences vary following the same power-law relationship, re-
gardless of the physical quantity considered. More precisely,
the actual slope values for SST, SAT, SW TOA, LW TOA,
and P are −0.65, −0.65, −0.64, −0.66, and −0.67, respec-
tively. Thus, the straight lines that best fit the global mean
data in Fig. 10 all have a slope of ≈2/3. The existence of
a ≈2/3 power law, which does not depend on the single
quantity, shows a consistent scaling of (PIMO −PIAR) dif-
ferences with the timescale that approaches a plateau near
the 200-year timescale (note that an actual plateau can only
be reached for longer simulations, as differences computed
over all timescales longer than 200 years would be ≈0). While in Sect. 4.1 we showed that PIMO and PIAR neces-
sitate 200 years to become statistically indistinguishable, an
interesting case to look at is the 100-year timescale. g
y
For instance, the minimum simulation length required by
CMIP6 protocols for a few of the MIP experiments (ex-
cluding the DECK and Historical simulations) is 100 years
or less, and ensembles are not always requested (e.g. 1Note that, for readability, the ticks of the x axes in Figs. 4d
to 9d were equally spaced. This partially masks the power-law be-
haviour discussed in the paper, which can be better detected when
the natural x axes are used. M.-V. Guarino et al.: Machine dependence and reproducibility for coupled climate simulations M.-V. Guarino et al.: Machine dependence and reproducibility for coupled climate simulations
Table 2 The 200-year global mean and standard deviation for SST
variables considered here) and thus M.-V. Guarino et al.: Machine dependence and reproducibility for coupled climate simulations 149 variables considered here), and thus represents a signal of a
different nature. In summary, although large differences can be observed
at smaller timescales (see the next section for a further dis-
cussion), the climate of PIMO and PIAR is indistinguishable
on the 200-year timescale. We thus conclude that the mean
climate properties simulated by the HadGEM3-GC3.1 model
are reproducible on different HPC platforms, provided that a
sufficiently long simulation length is used. MO
ARCHER
Mean, SD
Mean, SD
SST (◦C)
17.93, 0.07
17.95, 0.08
SIA (106 km2)
21.44, 0.65
21.30, 0.68
SAT (°C)
13.71, 0.10
13.75, 0.12
SW TOA (W m−2)
98.83, 0.24
98.76, 0.27
LW TOA (W m−2)
241.29, 0.27
241.36, 0.33
P (10−6 kg m−2 s−1)
36.22, 0.12
36.25, 0.14 Our results also show that HadGEM3-GC3.1 does not suf-
fer from compiler bugs that would make the model behave
differently on different machines for integration times longer
than 24 h (for which the model was previously tested; see
Sect. 3.1). 3). Following this diagnostic and for the variables we as-
sessed, our results show that there is no significant differ-
ence in the simulated mean between the two PIMO and PIAR
HadGEM3-GC3.1 simulations when a 200-year-long period
is considered. M.-V. Guarino et al.: Machine dependence and reproducibility for coupled climate simulations MO
ARCHER
Mean, SD
Mean, SD
SST (◦C)
17.93, 0.07
17.95, 0.08
SIA (106 km2)
21.44, 0.65
21.30, 0.68
SAT (°C)
13.71, 0.10
13.75, 0.12
SW TOA (W m−2)
98.83, 0.24
98.76, 0.27
LW TOA (W m−2)
241.29, 0.27
241.36, 0.33
P (10−6 kg m−2 s−1)
36.22, 0.12
36.25, 0.14
variables considered here), and th
different nature. In summary, although large di
at smaller timescales (see the nex
cussion), the climate of PIMO an
on the 200-year timescale. We th
climate properties simulated by th
are reproducible on different HPC
sufficiently long simulation length
Our results also show that HadG
fer from compiler bugs that wou
differently on different machines
than 24 h (for which the model M.-V. Guarino et al.: Machine dependence and reproducibility for coupled climate simulations 147 M.-V. Guarino et al.: Machine dependence and reproducibility for coupled climate simulations
147
Figure 6. As in Fig. 4 but for SAT. Figure 7. Annual mean time series of SW TOA in the tropics (a), SW TOA in the northern extratropics (b), and SW TOA in the southern
extratropics (c) for PIMO (grey line) and PIAR (dashed line). Panel (d) shows how SW TOA differences vary as a function of the timescale. www.geosci-model-dev.net/13/139/2020/
Geosci. Model Dev., 13, 139–154, 2020 Figure 6. As in Fig. 4 but for SAT. Figure 6. As in Fig. 4 but for SAT. Figure 6. As in Fig. 4 but for SAT. Figure 6. As in Fig. 4 but for SAT. Figure 7. Annual mean time series of SW TOA in the tropics (a), SW TOA in the northern extratropics (b), and SW TOA in the southern
extratropics (c) for PIMO (grey line) and PIAR (dashed line). Panel (d) shows how SW TOA differences vary as a function of the timescale. Figure 7. Annual mean time series of SW TOA in the tropics (a), SW TOA in the northern extratropics (b), and SW TOA in the southern
extratropics (c) for PIMO (grey line) and PIAR (dashed line). Panel (d) shows how SW TOA differences vary as a function of the timescale. Geosci. Model Dev., 13, 139–154, 2020 www.geosci-model-dev.net/13/139/2020/ www.geosci-model-dev.net/13/139/2020/ 148
M.-V. Guarino et al.: Machine dependence and reproducibility for coupled climate simulations
Figure 8. As in Fig. 4 but for LW TOA. Figure 9. As in Fig. 4 but for P . Geosci. Model Dev., 13, 139–154, 2020
www.geosci-model-dev.net/13/139/2020/ M.-V. Guarino et al.: Machine dependence and reproducibility for coupled climate simulations 148 Figure 8. As in Fig. 4 but for LW TOA. Figure 8. As in Fig. 4 but for LW TOA. Figure 8. As in Fig. 4 but for LW TOA. Figure 9. As in Fig. 4 but for P . Geosci. Model Dev., 13, 139–154, 2020
www.geosci-model-dev.net/13/139/2020/ Figure 9. As in Fig. 4 but for P . Geosci. Model Dev., 13, 139–154, 2020 Geosci. Model Dev., 13, 139–154, 2020 www.geosci-model-dev.net/13/139/2020/ www.geosci-model-dev.net/13/139/2020/ M.-V. Guarino et al.: Machine dependence and reproducibility for coupled climate simulation
Table 2. The 200-year global mean and standard deviation for SST,
SIA, SAT, SW TOA, LW TOA, and P . 4.2
The 100-year timescale some
of the Tier 1, 2, and 3 experiments of PMIP, Otto-Bleisner
et al., 2017; nonlinMIP, Good et al., 2016; GeoMIP, Kravitz
et al., 2015; HighResMIP, Haarsma et al., 2016; FAFMIP,
Gregory et al., 2016). This is likely because longer fully
coupled climate simulations are not always possible. They
demand significant computational resources or impractically
long running times (for example, simulating 200 years with
the HadGEM3-GC3.1 model on ARCHER in its CMIP6 con-
figuration takes about 4 months). Our results show that 100 years may not be long enough to
sample the same climate variability when HadGEM3-GC3.1
is run on different HPC platforms. This is particularity evi-
dent when we look at the spatial patterns of (PIMO −PIAR)
differences and at the associated SNR (see below). SIA (not shown) was the only variable that did not show
a ≈2/3 power-law relationship. This, however, should not
invalidate the analysis presented above. The sea ice area is
an integral computed over a limited area, and not a mean
computed on a globally uniform surface (like all the other In Fig. 11, (PIMO −PIAR) differences materialize into spa-
tial patterns that are signatures of physical processes. SST
(Fig. 11a, b) and SIC (Fig. 11c, d) anomalies are the largest
in West Antarctica where ENSO teleconnection patterns are
expected; they correspond to regions where SNR becomes
equal to or larger than 1. This suggests that (PIMO −PIAR)
differences are driven by two different ENSO regimes (the
connection between SIC (and SST) anomalies in the South- www.geosci-model-dev.net/13/139/2020/ Geosci. Model Dev., 13, 139–154, 2020 M.-V. Guarino et al.: Machine dependence and reproducibility for coupled climate simulation M.-V. Guarino et al.: Machine dependence and reproducibility for coupled climate simulations 150 Figure 10. Log–log plots of SST (a), SAT (b), SW TOA (c), LW TOA (d), and P (e) representing maximum (PIMO −PIAR) differences as a
function of the timescale. All the variables were averaged globally (green circles) and over the SH (red crosses) and NH (yellow diamonds). The straight lines represent the best-fit lines for the data obtained by linear regression. Figure 10. Log–log plots of SST (a), SAT (b), SW TOA (c), LW TOA (d), and P (e) representing maximum (PIMO −PIAR) differences as a
function of the timescale. All the variables were averaged globally (green circles) and over the SH (red crosses) and NH (yellow diamonds). 4.2
The 100-year timescale The straight lines represent the best-fit lines for the data obtained by linear regression. ern Hemisphere and ENSO has been widely documented in
the literature; e.g. Kwok and Comiso, 2002; Liu et al., 2002;
Turner, 2004; Welhouse et al., 2016; Pope et al., 2017). sembles the one shown by (PIMO −PIAR) differences
in Fig. 11b. Thus, we conclude that differences between
ARCHER and MO are comparable to differences between
ensemble members run on a single machine. This hypothesis is confirmed by the ENSO signal in
Fig. 12. A few times, a strong El Niño (La Niña) event in
PIMO corresponds to a strong La Niña (El Niño) event in
PIAR. This opposite behaviour enlarges SIC (and SST) dif-
ferences between the two runs and strengthens the µMO−AR
signal, resulting in a strong SNR. As for PIMO, in Fig. 11f large differences (and SNR >
1) between the two ensemble members are found in East
Antarctica. While this suggests that in this case a climate
process other than ENSO is in action, the large SNR con-
firms that 100 years is too short a length for constant-forcing
HadGEM3-GC3.1 simulations even on the same machine. As ENSO provides a medium-frequency modulation of the
climate system, it is not surprising that it takes longer than
100 years for its variability to be fully represented (see e.g. Wittenberg, 2009). In summary, the analysis above confirms that (PIMO −
PIAR) differences, while triggered by the computing environ-
ment, are largely dominated by the internal variability as they
persist among ensemble members on the same machine (in
Fig. 11 SNR > 1). Finally, we want to know whether the two ENSO regimes
in PIMO and PIAR are a reflection of the different computing
environment or solely the result of natural variability (i.e. if
a similar behaviour can be detected for simulations run on
the same machine). This can be done by splitting the 200-
year simulations in two segments and assuming that each
100-year period of PIMO and PIAR is a member of an ensem-
ble of size two. Therefore, the ARCHER ensemble is made
of PIAR1st and PIAR2nd, and the MO ensemble comprises
PIMO1st and PIMO2nd. M.-V. Guarino et al.: Machine dependence and reproducibility for coupled climate simulations 151 ure 11. The 100-year means and corresponding SNR of (PIMO −PIAR) differences for SH SST (a, b) and SH SIC (c, d). Panels
how SNR of (PIAR1st −PIAR2nd) and (PIMO1st −PIMO2nd) differences for SH SST, respectively. Figure 11. The 100-year means and corresponding SNR of (PIMO −PIAR) differences for SH SST (a, b) and SH SIC (c, d). Panels (e) and
(f) show SNR of (PIAR1st −PIAR2nd) and (PIMO1st −PIMO2nd) differences for SH SST, respectively. Discrepancies between the means of key climate variables
(SST, SIA / SIC, SAT, SW TOA, LW TOA, and P ) were
analysed at different timescales, from decadal to centennial
(see Sect. 3.2 for details on methodology). Although the two versions of the same PI control simula-
tion do not bit-compare, we found that the long-term statis-
tics of the two runs are similar and that, on multi-centennial
timescales, the considered variables show a signal-to-noise
ratio (SNR) less than 1. We conclude that in order for PIMO www.geosci-model-dev.net/13/139/2020/ 5
Discussion and conclusions In this paper, the effects of different computing environments
on the reproducibility of coupled climate model simulations
are discussed. Two versions of the UK CMIP6 PI control
simulation, one run on the UK Met Office supercomputer
(MO) (PIMO) and the other run on the ARCHER (PIAR) HPC
platform, were used to investigate the impact of machine-
dependent processes of the N96ORCA1 HadGEM3-GC3.1
model. Figure 11e and f show the signal-to-noise ratio corre-
sponding to SST differences between PIAR1st and PIAR2nd
and between PIMO1st and PIMO2nd. In Fig. 11e, the SNR
pattern exhibited by the ARCHER ensemble members re- Geosci. Model Dev., 13, 139–154, 2020 www.geosci-model-dev.net/13/139/2020/ www.geosci-model-dev.net/13/139/2020/ .-V. Guarino et al.: Machine dependence and reproducibility for coupled climate simulations www.geosci-model-dev.net/13/139/2020/ Geosci. Model Dev., 13, 139–154, 2020 152
M.-V. Guarino et al.: Machine dependence and reproducibility for coupled climate simulations
Figure 12. The Niño 3.4 index for PIMO and PIAR. A 3-month running mean was applied to the ENSO signal, and values greater and smaller
than or equal to ±0.5 are shaded in orange and green. M.-V. Guarino et al.: Machine dependence and reproducibility for coupled climate simulation 152 Figure 12. The Niño 3.4 index for PIMO and PIAR. A 3-month running mean was applied to the ENSO signal, and values greater and smaller
than or equal to ±0.5 are shaded in orange and green. Figure 12. The Niño 3.4 index for PIMO and PIAR. A 3-month running mean was applied to the ENSO signal
than or equal to ±0.5 are shaded in orange and green. and PIAR to be statistically indistinguishable, a 200-year av-
eraging period must be used for the analysis of the results. This indicates that simulations using the HadGEM3-GC3.1
model are reproducible on different HPC platforms (in their
mean climate properties), provided that a sufficiently long
simulation length is used. the HadGEM3-GC3.1 model (on the same or on a different
machine). While this result is not unexpected per se, it is rele-
vant to CMIP6 experiments as CMIP6 protocols recommend
a minimum simulation length of 100 years (or less) for many
of the MIP experiments. This result has immediate implications for members of the
UK CMIP6 community who will run individual MIP exper-
iments on the ARCHER HPC platform and will compare
results against the reference PI simulation run on the MO
platform by the UK Met Office. The magnitude of (PIMO
−PIAR) differences presented in this paper should be re-
garded as threshold values below which differences between
ARCHER and MO simulations must be interpreted with cau-
tion (as they might be the consequence of a wrong sampling
of the model internal variability rather than the climate re-
sponse to a different forcing). Additionally, the relationship between global mean differ-
ences and timescale exhibits a ≈2/3 power-law behaviour,
regardless the physical quantity considered, that approaches
a plateau near the 200-year timescale. Thus, there is a con-
sistent time-dependent scaling of (PIMO −PIAR) differences
across the whole climate simulation so that variables con-
verge toward their true values at the same rate, independently
of the physical processes that they represent. M.-V. Guarino et al.: Machine dependence and reproducibility for coupled climate simulations tal design and organization, Geosci. Model Dev., 9, 1937–1958,
https://doi.org/10.5194/gmd-9-1937-2016, 2016. https://jules-lsm.github.io/ (Joint UK Land Environment Simula-
tor, 2020). The NEMO model code is available from http://www. nemo-ocean.eu (NEMO Consortium, 2020). The model code for
CICE can be downloaded from https://code.metoffice.gov.uk/trac/
cice/browser (CICE Consortium, 2020). Good, P., Andrews, T., Chadwick, R., Dufresne, J.-L., Gregory,
J. M., Lowe, J. A., Schaller, N., and Shiogama, H.: nonlin-
MIP contribution to CMIP6: model intercomparison project for
non-linear mechanisms: physical basis, experimental design and
analysis principles (v1.0), Geosci. Model Dev., 9, 4019–4028,
https://doi.org/10.5194/gmd-9-4019-2016, 2016. Data availability. Access to the data used in the paper has been
granted to the editor. The CMIP6 PI simulation run by the UK
Met Office will be made available on the Earth System Grid Fed-
eration (ESGF) (https://cera-www.dkrz.de/WDCC/ui/cerasearch/
cmip6?input=CMIP6.CMIP.MOHC.HadGEM3-GC31-LL,
https://doi.org/10.22033/ESGF/CMIP6.419; Ridley et al., 2018a),
the data repository for all CMIP6 output. CMIP6 outputs are
expected to be public by 2020. The dataset used for the analysis
of the PI simulation ported to ARCHER can be shared, under
request, via the CEDA platform (https://help.ceda.ac.uk, last
access: 13 January 2020). Please contact the authors. Data availability. Access to the data used in the paper has been
granted to the editor. The CMIP6 PI simulation run by the UK
Met Office will be made available on the Earth System Grid Fed-
eration (ESGF) (https://cera-www.dkrz.de/WDCC/ui/cerasearch/
cmip6?input=CMIP6.CMIP.MOHC.HadGEM3-GC31-LL, Gregory, J. M., Bouttes, N., Griffies, S. M., Haak, H., Hurlin, W. J.,
Jungclaus, J., Kelley, M., Lee, W. G., Marshall, J., Romanou,
A., Saenko, O. A., Stammer, D., and Winton, M.: The Flux-
Anomaly-Forced Model Intercomparison Project (FAFMIP) con-
tribution to CMIP6: investigation of sea-level and ocean climate
change in response to CO2 forcing, Geosci. Model Dev., 9, 3993–
4017, https://doi.org/10.5194/gmd-9-3993-2016, 2016. p
p
https://doi.org/10.22033/ESGF/CMIP6.419; Ridley et al., 2018a),
the data repository for all CMIP6 output. CMIP6 outputs are
expected to be public by 2020. The dataset used for the analysis
of the PI simulation ported to ARCHER can be shared, under
request, via the CEDA platform (https://help.ceda.ac.uk, last
access: 13 January 2020). Please contact the authors. Haarsma, R. J., Roberts, M. J., Vidale, P. L., Senior, C. A., Bellucci,
A., Bao, Q., Chang, P., Corti, S., Fuˇckar, N. S., Guemas, V., von
Hardenberg, J., Hazeleger, W., Kodama, C., Koenigk, T., Leung,
L. R., Lu, J., Luo, J.-J., Mao, J., Mizielinski, M. Competing interests. The authors declare that they have no conflict
of interest. Competing interests. The authors declare that they have no conflict
of interest. Kravitz, B., Robock, A., Tilmes, S., Boucher, O., English, J. M.,
Irvine, P. J., Jones, A., Lawrence, M. G., MacCracken, M.,
Muri, H., Moore, J. C., Niemeier, U., Phipps, S. J., Sillmann, J.,
Storelvmo, T., Wang, H., and Watanabe, S.: The Geoengineering
Model Intercomparison Project Phase 6 (GeoMIP6): simulation
design and preliminary results, Geosci. Model Dev., 8, 3379–
3392, https://doi.org/10.5194/gmd-8-3379-2015, 2015. Acknowledgements. We would like to thank the two anonymous
referees and the topical editor, Sophie Valcke, for their time and
their valuable comments. Maria-Vittoria Guarino and Louise C. Sime acknowledge the
financial support of NERC research grants NE/P013279/1 and
NE/P009271/1. This work used the ARCHER UK National Su-
percomputing Service (http://www.archer.ac.uk, last access: 13 Jan-
uary 2020). The authors acknowledge the use of the UK Met Office
supercomputing facility in providing data for model comparisons. Kuhlbrodt, T., Jones, C. G., Sellar, A., Storkey, D., Blockley, E.,
Stringer, M., Hill, R., Graham, T., Ridley, J., Blaker, A., Calvert,
D., Copsey, D., Ellis, R., Hewitt, H., Hyder, P., Ineson, S., Mulc-
ahy, J., Siahaan, A., and Walton, J.: The Low-Resolution Version
of HadGEM3 GC3. 1: Development and Evaluation for Global
Climate, J. Adv. Model. Earth Sy., 10, 2865–2888, 2018. Kwok, R. and Comiso, J. C.: Spatial patterns of variability
in Antarctic surface temperature: Connections to the South-
ern Hemisphere Annular Mode and the Southern Oscillation,
Geophys. Res. Lett., 29, https://doi.org/10.1029/2002GL015415,
2002. Financial support. This research has been supported by NERC
(grant nos. NE/P013279/1 and NE/P009271/1). Liu, J., Yuan, X., Rind, D., and Martinson, D. G.: Mechanism study
of the ENSO and southern high latitude climate teleconnections,
Geophys. Res. Lett., 29, https://doi.org/10.1029/2002GL015143,
2002. Review statement. This paper was edited by Sophie Valcke and re-
viewed by two anonymous referees. Liu, L., Li, R., Zhang, C., Yang, G., Wang, B., and Dong, L.: En-
hancement for bitwise identical reproducibility of Earth system
modeling on the C-Coupler platform, Geosci. Model Dev. Dis-
cuss., 8, 2403–2435, https://doi.org/10.5194/gmdd-8-2403-2015,
2015a. M.-V. Guarino et al.: Machine dependence and reproducibility for coupled climate simulations S., Mizuta, R.,
Nobre, P., Satoh, M., Scoccimarro, E., Semmler, T., Small, J.,
and von Storch, J.-S.: High Resolution Model Intercomparison
Project (HighResMIP v1.0) for CMIP6, Geosci. Model Dev., 9,
4185–4208, https://doi.org/10.5194/gmd-9-4185-2016, 2016. Author contributions. MVG ran the simulation on the ARCHER
supercomputer, designed and carried out the tests in Sect. 2, and
analysed all simulation results with the contribution of LCS and DS. GL and RH ported the HadGEM3 PI simulation to the ARCHER su-
percomputer, provided technical support, and advised on the nature
of machine-dependent processes. All authors revised the paper. Hong, S.-Y., Koo, M.-S., Jang, J., Esther Kim, J.-E., Park, H., Joh,
M.-S., Kang, J.-H., and Oh, T.-J.: An evaluation of the soft-
ware system dependency of a global atmospheric model, Mon. Weather Rev., 141, 4165–4172, 2013. Joint UK Land Environment Simulator: JULES, available at: https:
//jules-lsm.github.io/, last access: 13 January 2020. References CICE
Consortium:
CICE,
available
at:
https://github.com/
CICE-Consortium last access: 13 January 2020. Eyring, V., Bony, S., Meehl, G. A., Senior, C. A., Stevens, B.,
Stouffer, R. J., and Taylor, K. E.: Overview of the Coupled
Model Intercomparison Project Phase 6 (CMIP6) experimen- Liu, L., Peng, S., Zhang, C., Li, R., Wang, B., Sun, C., Liu, Q.,
Dong, L., Li, L., Shi, Y., He, Y., Zhao, W., and Yang, G.: Impor-
tance of bitwise identical reproducibility in earth system mod- www.geosci-model-dev.net/13/139/2020/ Larger inconsistencies between the two runs were found
for shorter timescales (at which SNR ≥1), the largest be-
ing at decadal timescales. For example, when a 10-year av-
eraging period is used, discrepancies between the runs can
be up to |0.2| ◦C global mean air temperature anomalies, or
|1.2| million km2 Southern Hemisphere sea ice area anoma-
lies. The observed differences are a direct consequence of
the different sampling of the internal variability when the
same climate simulation is run on different machines. They
become approximately zero when a 200-year averaging pe-
riod is used, confirming that the overall physical behaviour
of the model was not affected by the different computing en-
vironments. In light of our results, our recommendation to the UK
MIPs studying the climate response to different forcings is
to run HadGEM3-GC3.1 for at least 200 years, even when
CMIP6 minimum requirements are 100 years (see, for exam-
ple, the PMIP protocols; Otto-Bleisner et al., 2017). Finally, although the quantitative analysis presented in this
paper applies strictly to HadGEM3-GC3.1 constant-forcing
climate simulations only, this study has the broader purpose
of increasing awareness in the climate modelling community
of the subject of the machine dependence of climate simula-
tions. On a 100-year timescale, large SST and SIC differences
(with SNR ≥1) were found where ENSO teleconnection pat-
terns are expected. Medium-frequency climate processes like
ENSO need longer than 100 years to be fully represented. Thus, a 100-year constant-forcing simulation may not be
long enough to correctly capture the internal variability of Code availability. Access to the model code used in the paper has
been granted to the editor. The source code of the UM is available
under licence. To apply for a licence, go to http://www.metoffice. gov.uk/research/modelling-systems/unified-model (UK Met Office,
2020). JULES is available under licence free of charge; see Geosci. Model Dev., 13, 139–154, 2020 www.geosci-model-dev.net/13/139/2020/ 153 M.-V. Guarino et al.: Machine dependence and reproducibility for coupled climate simulations 154 eling and status report, Geosci. Model Dev. Discuss., 8, 4375–
4400, https://doi.org/10.5194/gmdd-8-4375-2015, 2015b. eling and status report, Geosci. Model Dev. Discuss., 8, 4375–
4400, https://doi.org/10.5194/gmdd-8-4375-2015, 2015b. Song, Z., Qiao, F., Lei, X., and Wang, C.: Influence of par-
allel computational uncertainty on simulations of the Cou-
pled General Climate Model, Geosci. Model Dev., 5, 313–319,
https://doi.org/10.5194/gmd-5-313-2012, 2012. Loeve, M.: Elementary probability theory, in: Probability Theory I,
Springer, 1–52, 1977. Lorenz, E. N.: Deterministic nonperiodic flow, J. Atmos. Sci., 20,
130–141, 1963. Turner, J.: The El Niño–Southern Oscillation and Antarctica, Int. J. Climatol., 24, 1–31, 2004. UK Met Office: Unified Model, available at: http://www.metoffice. gov.uk/research/modelling-systems/unified-model, last access:
13 January 2020. Madec,
G. and
the
NEMO
Team:
NEMO
ocean
engine,
available at: https://epic.awi.de/id/eprint/39698/1/NEMO_book_
v6039.pdf (last access: 13 January 2020), 2015. Walters, D., Boutle, I., Brooks, M., Melvin, T., Stratton, R., Vosper,
S., Wells, H., Williams, K., Wood, N., Allen, T., Bushell, A.,
Copsey, D., Earnshaw, P., Edwards, J., Gross, M., Hardiman,
S., Harris, C., Heming, J., Klingaman, N., Levine, R., Man-
ners, J., Martin, G., Milton, S., Mittermaier, M., Morcrette, C.,
Riddick, T., Roberts, M., Sanchez, C., Selwood, P., Stirling,
A., Smith, C., Suri, D., Tennant, W., Vidale, P. L., Wilkinson,
J., Willett, M., Woolnough, S., and Xavier, P.: The Met Office
Unified Model Global Atmosphere 6.0/6.1 and JULES Global
Land 6.0/6.1 configurations, Geosci. Model Dev., 10, 1487–
1520, https://doi.org/10.5194/gmd-10-1487-2017, 2017. Menary, M. B., Kuhlbrodt, T., Ridley, J., Andrews, M. B., Dimdore-
Miles, O. B., Deshayes, J., Eade, R., Gray, L., Ineson, S.,
Mignot, J., Roberts, C. and Robson, J., Wood, R., and Xavier, P.:
Preindustrial Control Simulations With HadGEM3-GC3. 1 for
CMIP6, J. Adv. Model. Earth Sy., 10, 3049–3075, 2018. NEMO
Consortium:
NEMO,
available
at:
https://www. nemo-ocean.eu/, last access: 13 January 2020. Otto-Bliesner, B. L., Braconnot, P., Harrison, S. P., Lunt, D. J.,
Abe-Ouchi, A., Albani, S., Bartlein, P. J., Capron, E., Carlson,
A. E., Dutton, A., Fischer, H., Goelzer, H., Govin, A., Hay-
wood, A., Joos, F., LeGrande, A. N., Lipscomb, W. H., Lohmann,
G., Mahowald, N., Nehrbass-Ahles, C., Pausata, F. S. R., Peter-
schmitt, J.-Y., Phipps, S. J., Renssen, H., and Zhang, Q.: The
PMIP4 contribution to CMIP6 – Part 2: Two interglacials, scien-
tific objective and experimental design for Holocene and Last
Interglacial simulations, Geosci. Model Dev., 10, 3979–4003,
https://doi.org/10.5194/gmd-10-3979-2017, 2017. www.geosci-model-dev.net/13/139/2020/ Geosci. Model Dev., 13, 139–154, 2020 M.-V. Guarino et al.: Machine dependence and reproducibility for coupled climate simulations J., Willett, M., Woolnough, S., and Xavier, P.: The Met Office
Unified Model Global Atmosphere 6.0/6.1 and JULES Global
Land 6.0/6.1 configurations, Geosci. Model Dev., 10, 1487–
1520, https://doi.org/10.5194/gmd-10-1487-2017, 2017. Welhouse, L. J., Lazzara, M. A., Keller, L. M., Tripoli, G. J., and
Hitchman, M. H.: Composite analysis of the effects of ENSO
events on Antarctica, J. Climate, 29, 1797–1808, 2016. Williams, K., Copsey, D., Blockley, E., Bodas-Salcedo, A., Calvert,
D., Comer, R., Davis, P., Graham, T., Hewitt, H., Hill, R., Hyder,
P., Ineson, S., Johns, T. C., Keen, A. B., Lee, R. W., Megann,
A., Milton, S. F., Rae, J. G. L., Roberts, M. J., Scaife, A. A.,
Schiemann, R., Storkey, D., Thorpe, L., Watterson, I. G., Walters,
D. N., West, A., Wood, R. A., Woollings, T., and Xavier, P. K.:
The Met Office global coupled model 3.0 and 3.1 (GC3.0 and
GC3.1) configurations, J. Adv. Model. Earth Sy., 10, 357–380,
2018. Pope, J. O., Holland, P. R., Orr, A., Marshall, G. J., and Phillips, T.:
The impacts of El Niño on the observed sea ice budget of West
Antarctica, Geophys. Res. Lett., 44, 6200–6208, 2017. Ridley, J., Menary, M., Kuhlbrodt, T., Andrews, M., and An-
drews, T.: MOHC HadGEM3-GC31-LL model output pre-
pared for CMIP6 CMIP, Earth System Grid Federation,
https://doi.org/10.22033/ESGF/CMIP6.419, 2018a. Wittenberg, A. T.: Are historical records sufficient to con-
strain ENSO simulations?, Geophys. Res Lett., 36, L12702,
https://doi.org/10.1029/2009GL038710, 2009. Ridley, J. K., Blockley, E. W., Keen, A. B., Rae, J. G. L.,
West, A. E., and Schroeder, D.: The sea ice model compo-
nent of HadGEM3-GC3.1, Geosci. Model Dev., 11, 713–723,
https://doi.org/10.5194/gmd-11-713-2018, 2018b. Geosci. Model Dev., 13, 139–154, 2020 Geosci. Model Dev., 13, 139–154, 2020 www.geosci-model-dev.net/13/139/2020/
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Interplay between spin polarization and color superconductivity in high density quark matter
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Downloaded from orbit.dtu.dk on: Oct 24, 2024 Downloaded from orbit.dtu.dk on: Oct 24, 2024 Downloaded from orbit.dtu.dk on: Oct 24, 2024 Interplay between spin polarization and color superconductivity in high density quark
matter Tsue, Yasuhiko; da Providência, João; Providência, Constança; Yamamura, Masatoshi; Bohr, Henrik Document Version
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and investigate your claim. Citation (APA):
Tsue, Y., da Providência, J., Providência, C., Yamamura, M., & Bohr, H. (2013). Interplay between spin
polarization and color superconductivity in high density quark matter. Progress of Theoretical and Experimental
Physics, 2013(10), 103D01. https://doi.org/10.1093/ptep/ptt076 General rights
Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright
owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. General rights
Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright
owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.
U
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h †These authors contributed equally to this work Interplay between spin polarization and color
superconductivity in high density quark matter It follows that a transition from one to the other phase occurs, passing through true
minima with both a spin polarization and a color superconducting gap. It is shown that the quark
spin polarized phase is realized at rather high density, while the two-flavor color superconducting
phase is realized in a lower density region. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Subject Index
D30 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Here, it is suggested that a four-point interaction of the tensor type may lead to spin polarization
in quark matter at high density. It is found that the two-flavor superconducting phase and the spin
polarized phase correspond to distinct local minima of a certain generalized thermodynamical
potential. It follows that a transition from one to the other phase occurs, passing through true
minima with both a spin polarization and a color superconducting gap. It is shown that the quark
spin polarized phase is realized at rather high density, while the two-flavor color superconducting
phase is realized in a lower density region. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Subject Index
D30 ( )
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This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © The Author(s) 2013. Published by Oxford University Press on behalf of the Physical Society of Japan. © The Author(s) 2013. Published by Oxford University Press on behalf of the Physical Society of Japan.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Prog. Theor. Exp. Phys. 2013, 103D01 (20 pages)
DOI: 10.1093/ptep/ptt076 Interplay between spin polarization and color
superconductivity in high density quark matter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Here, it is suggested that a four-point interaction of the tensor type may lead to spin polarization
in quark matter at high density. It is found that the two-flavor superconducting phase and the spin
polarized phase correspond to distinct local minima of a certain generalized thermodynamical
potential. It follows that a transition from one to the other phase occurs, passing through true
minima with both a spin polarization and a color superconducting gap. It is shown that the quark
spin polarized phase is realized at rather high density, while the two-flavor color superconducting
phase is realized in a lower density region. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Subject Index
D30 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Here, it is suggested that a four-point interaction of the tensor type may lead to spin polarization
in quark matter at high density. It is found that the two-flavor superconducting phase and the spin
polarized phase correspond to distinct local minima of a certain generalized thermodynamical
potential. © The Author(s) 2013. Published by Oxford University Press on behalf of the Physical Society of Japan.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creat Link back to DTU Orbit Link back to DTU Orbit Citation (APA):
Tsue, Y., da Providência, J., Providência, C., Yamamura, M., & Bohr, H. (2013). Interplay between spin
polarization and color superconductivity in high density quark matter. Progress of Theoretical and Experimental
Physics, 2013(10), 103D01. https://doi.org/10.1093/ptep/ptt076 1.
Introduction Under extreme conditions, such as high temperature and/or high baryon density, it is interesting
to study the behavior of quark and gluonic matter and/or hadronic matter governed by quantum
chromodynamics (QCD) in the context of the physics of relativistic heavy ion collisions and of the
interior of compact stars. In low temperature and high density regions [1] such as the interior of a
neutron star, it has been widely investigated and pointed out that various phases may be realized, e.g.,
various meson condensed phases in hadronic matter [2], the two-flavor color superconducting (2SC)
and color-flavor locked (CFL) phases [3–5], the quarkyonic phase [6], the quark ferromagnetic phase
[7–9] in quark matter, and so forth. In particular, we may conjecture that the quark ferromagnetic
phase exists at high density quark matter, since the existence of the magnetar was reported [10]. In
preceding work [7–9], the pseudovector-type interaction between quarks was considered and it was
pointed out that the quark spin polarization was realized. However, it was shown that the quark spin
alignment disappears if the quark mass is zero, e.g., in the chiral symmetric phase [11]. Thus, it is
interesting to investigate whether there is a possibility of spontaneous spin polarization under another
interaction between quarks. Recently, the present authors have indicated that the quark spin polarization may occur at high
density quark matter even in the chiral symmetric phase due to the tensor-type four-point interaction PTEP 2013, 103D01 Y. Tsue et al. between quarks, which leads to the quark spin polarization [12,13]. In these papers, it was shown
that a second-order phase transition occurs from normal quark phase to quark spin polarized phase. However, in quark matter at high baryon density, it is believed that the two-flavor color supercon-
ducting phase appears. Thus, it is interesting to investigate the stability of the quark spin polarized
phase against the 2SC phase. In this paper, we investigate the color-symmetric superconducting phase [14] and the quark spin
polarized phase and clarify which phase is stable by using the Nambu–Jona-Lasinio (NJL) model
[15,16], including quark-pair interaction [17] and the tensor-type four-point interaction [12,13]. The
quark-pair interaction is derived by a Fierz transformation from the original NJL model Lagrangian. As for the color superconducting phase, many possibilities, such as the gapless superconducting
phase, which is still controversial for realistic situations, have been considered. 1.
Introduction However, in this
paper, the simple 2SC phase derived by the NJL model is treated. We thus consider the quark-pairing
gap and the quark spin polarization F such that ̸= 0 and/or F ̸= 0 leads to the 2SC phase
and/or quark spin polarized phase, respectively. In this paper, the system is treated by the mean field
approximation and the BCS state is introduced [18]. This paper is organized as follows: In the next section, the basic Hamiltonian is introduced and
expressions of the Hamiltonian under some basis sets are given. In Appendix A, one of the expres-
sions of the Hamiltonian is given, in which good helicity states are used. In Sect. 3, the BCS state
is defined, where a detailed derivation is given in Appendix B, and the thermodynamic potential is
derived. In Sect. 4, numerical results and discussions are given, together with Appendix C, and the
last section is devoted to a summary and concluding remarks. 2.1.
Basic Hamiltonian Let us start with the following Lagrangian density: L = L0 + LS + LT + Lc,
L0 = ¯ψiγ μ∂μψ,
LS = GS(( ¯ψψ)2 + ( ¯ψiγ5⃗τψ)2),
LT = −G
4
( ¯ψγ μγ ν ⃗τψ)( ¯ψγμγν ⃗τψ) + ( ¯ψiγ5γ μγ νψ)( ¯ψiγ5γμγνψ)
,
Lc = Gc
2
A=2,5,7
( ¯ψiγ5τ2λAψC)( ¯ψCiγ5τ2λAψ) + ( ¯ψτ2λAψC)( ¯ψCτ2λAψ)
. (1) (1) Here, ψC = C ¯ψT with C = iγ 2γ 0 being the charge conjugation operator. Also, τ2 is the second
component of the Pauli matrices representing the isospin su(2)-generator and λA are the antisymmet-
ric Gell–Mann matrices representing the color su(3)c-generator. As is well known, the Lagrangian
density L0 + LS corresponds to the original NJL model. We add Lc, which can be derived by the
Fierz transformation from LS, and which represents the quark-pair interaction. Also, we introduce
LT , which represents the tensor-type interaction between quarks. In this paper, we concentrate on quark matter at high baryon density where the chiral symmetry is
restored in the density region considered here. Thus, the chiral condensate, ⟨¯ψψ⟩, is assumed to be 2/20 PTEP 2013, 103D01 Y. Tsue et al. Y. Tsue et al. equal to zero in this high density region. Under the mean field approximation, the above Lagrangian
density is recast into equal to zero in this high density region. Under the mean field approximation, the above Lagrangian
density is recast into LMF = L0 + LMF
T
+ LMF
c
,
LMF
T
= −F( ¯ψ 3τ3ψ) −F2
2G ,
F = −G⟨¯ψ 3τ3ψ⟩,
3 = −iγ 1γ 2 =
σ3
0
0
σ3
,
LMF
c
= −1
2
A=2,5,7
( ¯ψCiγ5τ2λA + h.c.) −32
2Gc
,
A = ∗
A = −Gc⟨¯ψiγ5τ2λAψ⟩,
= 2 = 5 = 7,
(2) LMF = L0 + LMF
T
+ LMF
c
,
LMF
T
= −F( ¯ψ 3τ3ψ) −F2
2G , F = −G⟨¯ψ 3τ3ψ⟩,
3 = −iγ 1γ 2 =
σ3
0
0
σ3
,
LMF
c
= −1
2
A=2,5,7
( ¯ψCiγ5τ2λA + h.c.) −32
2Gc
, A = ∗
A = −Gc⟨¯ψiγ5τ2λAψ⟩,
= 2 = 5 = 7,
(2) A = ∗
A = −Gc⟨¯ψiγ5τ2λAψ⟩,
= 2 = 5 = 7,
(2) (2) where h.c. represents the Hermitian conjugate term of the preceding one. 2.1.
Basic Hamiltonian Here, we have used a Dirac
representation for the Dirac gamma matrices and σ3 represents the third component of the 2 × 2 Pauli
spin matrices. The symbol ⟨· · · ⟩represents the expectation value with respect to a vacuum state. The
expectation value F corresponds to the order parameter of the spin alignment that leads to quark
ferromagnetization. The expectation value corresponds to the quark-pair condensate, which means
the existence of the color superconducting phase if ̸= 0. Here, in order to ensure color symmetry,
we assume that all quark-pair condensates have the same expectation values, 2 = 5 = 7. The mean field Hamiltonian density with quark chemical potential μ is easily obtained as The mean field Hamiltonian density with quark chemical potential μ is easily obtaine HMF −μN = K0 + HMF
T
+ HMF
c
,
K0 = ¯ψ(−γ · ∇−μγ0)ψ,
HMF
T
= −LMF
T ,
HMF
c
= −LMF
c
(3) (3) with N = ψ†ψ. In the Dirac representation for the Dirac gamma matrices, the Hamiltonian matrix
of the spin polarization part, HSP
MF =
d3x (K0 + HMF
T ), is written as hSP
MF = p · α + Fτ3β 3
=
⎛
⎜⎜⎜⎝
Fτ3
0
p3
p1 −ip2
0
−Fτ3
p1 + ip2
p3
p3
p1 −ip2
−Fτ3
0
p1 + ip2
−p3
0
Fτ3
⎞
⎟⎟⎟⎠,
(4) (4) where αi = γ 0γ i and β = γ 0. For good helicity states, this Hamiltonian matrix is diagonalized with
F = 0. For simplicity, we rotate around the p3 axis and we set p2 = 0 without loss of generality. In
this case, we derive κ = U−1hSP
MFU as follows: where αi = γ 0γ i and β = γ 0. For good helicity states, this Hamiltonian matrix is diagonalized with
F = 0. For simplicity, we rotate around the p3 axis and we set p2 = 0 without loss of generality. 2.1.
Basic Hamiltonian In
this case, we derive κ = U−1hSP
MFU as follows: U =
1
2√p
⎛
⎜⎜⎜⎜⎜⎝
√p + p3
√p −p3
−√p + p3
−√p −p3
p1
|p1|
√p −p3
−p1
|p1|
√p + p3
−p1
|p1|
√p −p3
p1
|p1|
√p + p3
√p + p3
−√p −p3
√p + p3
−√p −p3
p1
|p1|
√p −p3
p1
|p1|
√p + p3
p1
|p1|
√p −p3
p1
|p1|
√p + p3
⎞
⎟⎟⎟⎟⎟⎠
, U =
1
2√p
⎛
⎜⎜⎜⎜⎜⎝
√p + p3
√p −p3
p1
|p1|
√p −p3
−p1
|p1|
√p + p3
√p + p3
−√p −p3
p1
|p1|
√p −p3
p1
|p1|
√p + p3 −√p + p3
−√p −p3
−p1
|p1|
√p −p3
p1
|p1|
√p + p3
√p + p3
−√p −p3
p1
|p1|
√p −p3
p1
|p1|
√p + p3
⎞
⎟⎟⎟⎟⎟⎠
, PTEP 2013, 103D01 Y. Tsue et al. κ = U−1hSP
MFU
=
⎛
⎜⎜⎜⎝
p
0
0
0
0
p
0
0
0
0
−p
0
0
0
0
−p
⎞
⎟⎟⎟⎠+ Fτ3
p
⎛
⎜⎜⎜⎝
0
|p1|
−p3
0
|p1|
0
0
p3
−p3
0
0
|p1|
0
p3
|p1|
0
⎞
⎟⎟⎟⎠. (5) (5) Finally, in the original basis rotated around the p3-axis, |p1| is replaced with
p2
1 + p2
2. As for the
Hamiltonian matrix of the color superconducting part, HMF
c
=
d3xHMF
c
, the result has already
been obtained in Ref. [17], based on Ref. [14]. 2.1.
Basic Hamiltonian As a result, on the basis of good helicity states,
the relevant combination of the mean field Hamiltonian HMF =
d3xHMF and the quark number
N =
d3xN is given by HMF −μN =
pητα
(p −μ)c†
pηταcpητα −(p + μ)˜c†
pητα ˜cpητα
+ F
pητα
φτ
p2
1 + p2
2
p
c†
pηταcp−ητα + ˜c†
pητα ˜cp−ητα
−η p3
p
c†
pητα ˜cpητα + ˜c†
pηταcpητα
+
2
pηαα′α′′ττ ′
(c†
pηταc†
−pητ ′α′ + ˜c†
pητα ˜c†
−pητ ′α′ + F
pητα
φτ
p2
1 + p2
2
p
c†
pηταcp−ητα + ˜c†
pητα ˜cp−ητα
−η p3
p
c†
pητα ˜cpητα + ˜c†
pηταcpητα
+
2
pηαα′α′′ττ ′
(c†
pηταc†
−pητ ′α′ + ˜c†
pητα ˜c†
−pητ ′α′ + c−pητ ′α′cpητα + ˜c−pητ ′α′ ˜cpητα)φτϵαα′α′′ϵττ ′
+ V · F2
2G + V · 32
2Gc
,
(6) + c−pητ ′α′cpητα + ˜c−pητ ′α′ ˜cpητα)φτϵαα′α′′ϵττ ′ (6) where V represents the volume in the box normalization.1 Here, c†
pητα and ˜c†
pητα represent the quark
and antiquark creation operators with momentum p, helicity η = ±, isospin index τ = ±, and color
α. Further, φτ = 1 for τ = 1 (up quark) and φτ = −1 for τ = −1 (down quark). Also, ϵττ ′ and
ϵαα′α′′ represent the complete antisymmetric tensor for the isospin and color indices. We define p =
p2
1 + p2
2 + p2
3, i.e., the magnitude of momentum. nce, we rewrite the above Hamiltonian as
3
For later convenience, we rewrite the above Hamiltonian as
For later convenience, we rewrite the above Hamiltonian as HMF −μN = HSP
MF −μN + Vcs + V · F2
2G + V · 32
2Gc
(7) (7) c
with with HSP
MF −μN =
pττ ′αα′α′′
⎛
⎜⎜⎜⎜⎝
c†
p+τα
c†
p−τα
˜c†
p+τα
˜c†
p−τα
⎞
⎟⎟⎟⎟⎠
T
(κ −μ · 1)δττ ′δαα′
⎛
⎜⎜⎜⎜⎝
cp+τ ′α′
cp−τ ′α′
˜cp+τ ′α′
˜cp−τ ′α′
⎞
⎟⎟⎟⎟⎠
, 1 We hope that no confusion will occur, although notations Vcs and V (p) appear later, which represent the
quark-pair interaction in (7) and a matrix in (A1) diagonalizing a certain part of the Hamiltonian matrix,
respectively. 2.1.
Basic Hamiltonian 1 We hope that no confusion will occur, although notations Vcs and V (p) appear later, which represent the
quark-pair interaction in (7) and a matrix in (A1) diagonalizing a certain part of the Hamiltonian matrix,
respectively. 4/20 PTEP 2013, 103D01 Y. Tsue et al. κ =
⎛
⎜⎜⎜⎝
p
0
0
0
0
p
0
0
0
0
−p
0
0
0
0
−p
⎞
⎟⎟⎟⎠+ Fτ3
p
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
0
p2
1 + p2
2
−p3
0
p2
1 + p2
2
0
0
p3
−p3
0
0
p2
1 + p2
2
0
p3
p2
1 + p2
2
0
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
,
Vcs =
2
pηαα′α′′ττ ′
(c†
pηταc†
−pητ ′α′ + ˜c†
pητα ˜c†
−pητ ′α′ + c−pητ ′α′cpητα + ˜c−pητ ′α′ ˜cpητα) Vcs =
2
pηαα′α′′ττ ′
(c†
pηταc†
−pητ ′α′ + ˜c†
pητα ˜c†
−pητ ′α′ + c−pητ ′α′cpητα + ˜c−pητ ′α′ ˜cpητα) × φτϵαα′α′′ϵττ ′. (8) (8) As for Vcs, we can easily sum up with respect to τ and τ ′, which leads to As for Vcs, we can easily sum up with respect to τ and τ ′, which leads to As for Vcs, we can easily sum up with respect to τ and τ ′, which leads to Vcs =
pηαα′α′′
ϵαα′α′′(c†
pη+αc†
−pη−α′ + ˜c†
pη+α ˜c†
−pη−α′ + c−pη−α′cpη+α + ˜c−pη−α′ ˜cpη+α). (9) Vcs =
pηαα′α′′
ϵαα′α′′(c†
pη+αc†
−pη−α′ + ˜c†
pη+α ˜c†
−pη−α′ + c−pη−α′cpη+α + ˜c−pη−α′ ˜cpη+α). (9) (9) 2.2. Expressions of the Hamiltonian under other basis sets 2.2. Expressions of the Hamiltonian under other basis sets If the quark-pair condensate is equal to zero, the operator given by Eq. (7) becomes HSP
MF −μN +
V F2/(2G). Since the quark number N is already diagonal, let us diagonalize the Hamiltonian matrix
κ. First, we set up τ3 = 1 because the contribution of τ3 = −1 is the same as the contribution of
τ3 = 1, as was seen in (9), which results in a factor 2. Then, the Hamiltonian matrix κ can be simply
expressed as κ =
⎛
⎜⎜⎜⎝
q
e
−g
0
e
q
0
g
−g
0
−q
e
0
g
e
−q
⎞
⎟⎟⎟⎠,
(10) (10) where q = p, e = F
p2
1 + p2
2/p and g = Fp3/p. 2.1.
Basic Hamiltonian The eigenvalues of κ are easily obtained as where q = p, e = F
p2
1 + p2
2/p and g = Fp3/p. The eigenvalues of κ are easily obtained as ± ε(±)
p
= ±
g2 + (e ± q)2 = ±
p2
3 +
F ±
p2
1 + p2
2
2
. (11) (11) By introducing new fermion operators (apητα, a†
pητα, ˜apητα, ˜a†
pητα) by ⎛
⎜⎜⎜⎜⎝
ap+τα
ap−τα
˜ap+τα
˜ap−τα
⎞
⎟⎟⎟⎟⎠
= W †V †(p)
⎛
⎜⎜⎜⎜⎝
cp+τα
cp−τα
˜cp+τα
˜cp−τα
⎞
⎟⎟⎟⎟⎠
,
(12) (12) where the operators V (p) and W are given in Appendix A, the mean field Hamiltonian in which
both quark spin polarization and the quark-pair condensate are simultaneously considered can be
expressed as −μN =
pητα
(ε(η)
p
−μ)a†
pηταapητα −(ε(η)
p
+ μ)˜a†
pητα ˜apητα
HMF −μN =
pητα
(ε(η)
p
−μ)a†
pηταapητα −(ε(η)
p
+ μ)˜a†
pητα ˜apητα
+
2
pηττ ′αα′α′′
a†
pητα′a†
−pητ ′α′′ −˜a†
pητα′ ˜a†
−pητα′′ + h.c.
ϵαα′α′′ϵττ ′φτ
+ V · F2
2G + V · 32
2Gc
. (1 (13) 5/20 PTEP 2013, 103D01 Y. Tsue et al. Y. Tsue et al. Here, the summation with respect to isospin indices τ and τ ′ is explicitly rewritten. The above
mean field Hamiltonian is the starting point for discussing the quark spin polarized phase and color
superconducting phase. 3. BCS state and thermodynamic potential
We introduce the BCS state following Ref. [18]: 3.
BCS state and thermodynamic potential We introduce the BCS state following Ref. [18]: We introduce the BCS state following Ref. [18]: |⟩= eS|0⟩,
|0⟩=
pητα(ε(η)
p <μ)
a†
pητα|0⟩, |⟩= eS|0⟩,
|0⟩=
pητα(ε(η)
p <μ)
a†
pητα|0⟩,
S =
pη(ε(η)
p >μ)
Kpη
2
αα′α′′ττ ′
a†
pηταa†
−pητ ′α′ϵαα′α′′ϵττ ′φτ S =
pη(ε(η)
p >μ)
Kpη
2
αα′α′′ττ ′
a†
pηταa†
−pητ ′α′ϵαα′α′′ϵττ ′φτ +
pη(ε(η)
p ≤μ)
Kpη
2
αα′α′′ττ ′
apηταa−pητ ′α′ϵαα′α′′ϵττ ′φτ,
(14) (14) where |0⟩is the vacuum state with respect to apητα, and Kpη = K−pη and
Kpη =
K−pη are satisfied. Here, the contribution of quark-pairing with negative energy is not considered in quark matter. Then,
the state |⟩is a vacuum state with respect to new operators dpητα: dpητα =
apητα −Kpη(a†
−pητ ′β −a†
−pητ ′γ )
for
ε(η)
p
> μ
a†
pητα +
Kpη(a−pητ ′β −a−pητ ′γ )
for
ε(η)
p
≤μ
,
(15) (15) where (α, β, γ ) is a cyclic permutation for color indices and τ ′ = −τ. For the above operators, the
state |⟩satisfies the following relation: where (α, β, γ ) is a cyclic permutation for color indices and τ ′ = −τ. For the above operators, the
state |⟩satisfies the following relation: dpητα|⟩= 0. (16) (16) The state |⟩is identical with the BCS state. In order to calculate the expectation value of the mean field Hamiltonian (13), it is necessary to
obtain the expectation values of a†
pηταapητα, a†
pηταa†
−pητ ′α′, and so on with respect to the BCS state
|⟩. The detailed calculations are given in Appendix B. Thus, by summing up with respect to color
and isospin indices, we obtain the expectation value of the mean field Hamiltonian for the BCS
state as ⟨|HMF −μN|⟩= 3
pητ(ε(η)
p >μ)
(ε(η)
p
−μ)
2K 2
pη
1 + 3K 2pη
+ 2
Kpη
1 + 3K 2pη
+ 3
pητ(ε(η)
p ≤μ)
(ε(η)
p
−μ)
1 −
2
K 2
pη
1 + 3
K 2pη
+ 2
Kpη
1 + 3
K 2pη
+ V · F2
2G + V · 32
2Gc
. 3.
BCS state and thermodynamic potential (1 + 3
pητ(ε(η)
p ≤μ)
(ε(η)
p
−μ)
1 −
2
K 2
pη
1 + 3
K 2pη
+ 2
Kpη
1 + 3
K 2pη
+ V · F2
2G + V · 32
2Gc
. (17) 6/20 PTEP 2013, 103D01 Y. Tsue et al. In order to determine the BCS state, namely, to determine the variational variables Kpη and
Kpη, we
introduce new variational variables θpη and ˜θpη instead of Kpη and
Kpη: sin θpη =
√
3Kpη
1 + 3K 2pη
,
cos θpη =
1
1 + 3K 2pη
,
sin ˜θpη =
√
3
Kpη
1 + 3
K 2pη
,
cos ˜θpη =
1
1 + 3
K 2pη
. (18) (18) Thus, the Hamiltonian and color superconducting gap can be expressed in terms of θpη and ˜θpη as |HMF −μN|⟩= 2
pη(ε(η)
p >μ)
2(ε(η)
p
−μ) sin2 θpη + 2
√
3 sin θpη cos θpη
+ 2
pη(ε(η)
p ≤μ)
(ε(η)
p
−μ)(3 −2 sin2 ˜θpη) + 2
√
3 sin ˜θpη cos ˜θpη
+ V · F2
2G + V · 32
2Gc
,
(19)
= 2 = 5 = 7 = −Gc · 1
V
pηβγ τ
⟨|a†
pητβa†
−pη−τγ |⟩
= −2Gc
⎛
⎜⎝1
V
pητ(ε(η)
p >μ)
Kpη
1 + 3K 2pη
+ 1
V
pητ(ε(η)
p ≤μ)
Kpη
1 + 3
K 2pη
⎞
⎟⎠
= −4Gc
√
3
⎛
⎜⎝1
V
pη(ε(η)
p >μ)
sin θpη cos θpη + 1
V
pη(ε(η)
p ≤μ)
sin ˜θpη cos ˜θpη
⎞
⎟⎠. (20) ⟨|HMF −μN|⟩= 2
pη(ε(η)
p >μ)
2(ε(η)
p
−μ) sin2 θpη + 2
√
3 sin θpη cos θpη
+ 2
pη(ε(η)
p ≤μ)
(ε(η)
p
−μ)(3 −2 sin2 ˜θpη) + 2
√
3 sin ˜θpη cos ˜θpη
+ V · F2
2G + V · 32
2Gc
,
(19) (19) = 2 = 5 = 7 = −Gc · 1
V
pηβγ τ
⟨|a†
pητβa†
−pη−τγ |⟩ (20) Here, in the first line in (19) and the third line in (20), the isospin indices are summed up and an extra
factor 2 appears with respect to the corresponding expression in Ref. 3.
BCS state and thermodynamic potential [14]. Also, in the second line
in (20), the color indices β and γ are summed up and an extra factor 2 also appears. Here, in the first line in (19) and the third line in (20), the isospin indices are summed up and an extra
factor 2 appears with respect to the corresponding expression in Ref. [14]. Also, in the second line
in (20), the color indices β and γ are summed up and an extra factor 2 also appears. Next, we impose the minimization condition for ⟨|HMF −μN|⟩with respect to , θpη, and
˜θpη. First, we impose the minimization condition with respect to : ∂
∂⟨|HMF −μN|⟩= 0. (21) (21) This minimization condition leads to the gap equation in (20) exactly. Secondly, we minimize
⟨|HMF −μN|⟩with respect to θpη and ˜θpη: This minimization condition leads to the gap equation in (20) exactly. Secondly, we minimize
⟨|HMF −μN|⟩with respect to θpη and ˜θpη: ∂
∂θpη
⟨|HMF −μN|⟩= 0,
∂
∂˜θpη
⟨|HMF −μN|⟩= 0,
(22) (22) 7/20 PTEP 2013, 103D01
Y. Tsue et al. PTEP 2013, 103D01 Y. Tsue et al. which lead to the following equations: which lead to the following equations: owing equations: (ε(η)
p
−μ) sin 2θpη +
√
3 cos 2θpη = 0,
−(ε(η)
p
−μ) sin 2 ˜θpη +
√
3 cos 2 ˜θpη = 0. (23) (23) Thus, we obtain tan 2θpη = −
√
3/(ε(η)
p
−μ) and tan 2 ˜θpη =
√
3/(ε(η)
p
−μ), which gives Thus, we obtain tan 2θpη = −
√
3/(ε(η)
p
−μ) and tan 2 ˜θpη =
√
3/(ε(η)
p
−μ), which gives sin θ2
pη = 1
2
⎡
⎣1 −
ε(η)
p
−μ
(ε(η)
p
−μ)2 + 32
⎤
⎦,
sin ˜θ2
pη = 1
2
⎡
⎣1 +
ε(η)
p
−μ
(ε(η)
p
−μ)2 + 32
⎤
⎦. (24) (24) Since the variational parameters θpη and ˜θpη are determined completely, namely, Kpη and
Kpη are
determined, the BCS state is obtained. 4.
Numerical results and discussions In this section, first, we investigate the possible phases with F = 0 and ̸= 0 or with F ̸= 0 and
= 0 at high density, namely, two-flavor color superconducting phase or spin polarized phase,
separately. First, as an extreme situation in the quark spin polarized phase with = 0, the
thermodynamic potential reduces to ( = 0, F, μ) = 6 · 1
V
p (ε(+)
p
≤μ)
(ε(+)
p
−μ) + 6 · 1
V
p (ε(−)
p
≤μ)
(ε(−)
p
−μ) + F2
2G ,
(27) (27) where it should be noted that
(ε(η)
p
−μ)2 = ε(η)
p
−μ for ε(η)
p
> μ or −(ε(η)
p
−μ) for ε(η)
p
< μ,
respectively, when (27) is derived from (25). Of course, the sum over momentum is replaced by the
momentum-integration as 1
V
p
−→
! d3p
(2π)3 . (28) (28) Then, the thermodynamic potential derived here is identical with the one that has been previously
obtained by the present authors, i.e., Eq. (3.1) in Ref. [1].2 Thus, the thermodynamic potential with
= 0 has already been given in Ref. [12] in the analytical form as ⎧
⎪⎪⎪⎪⎪⎪⎪⎪
F2
2G −1
π2
&
μ2 −F2
4
(3F2μ + 2μ3)+Fμ3 arctan
F
&
μ2 −F2 ( = 0, F, μ) =
⎧
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎨
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎩
F2
2G −1
π2
&
μ2 −F2
4
(3F2μ + 2μ3)+Fμ3 arctan
F
&
μ2 −F2
−F4
4 ln μ +
&
μ2 −F2
F
for
F < μ
F2
2G −1
2π μ3F
for
F > μ
. (29) ( = 0, F, μ) =
⎪⎨
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎩
−F4
4 ln μ +
&
μ2 −F2
F
for
F < μ
F2
2G −1
2π μ3F
for
F > μ
. (29) (29) If the minimum of the thermodynamical potential exists in the range of F < μ, the gap equation for
F is derived from ∂( = 0, F, μ)/∂F = 0, which leads to F = G
π2
2Fμ
&
μ2 −F2 + μ3 arctan
F
&
μ2 −F2 −F3 ln μ +
&
μ2 −F2
F
. (30) (30) In the other case, namely, the F = 0 case, the two-flavor color superconducting phase may be
realized with ̸= 0. 3.
BCS state and thermodynamic potential Thus, we can derive the thermodynamic potential (, F, μ) at zero temperature from (19) with
(24) as (, F, μ) = 1
V ⟨|HMF −μN|⟩
= 2 · 1
V
pη(ε(η)
p ≤μ)
2(ε(η)
p
−μ) −
(ε(η)
p
−μ)2 + 32
+ 2 · 1
V
pη(ε(η)
p >μ)
(ε(η)
p
−μ) −
(ε(η)
p
−μ)2 + 32
+ F2
2G + 32
2Gc
,
(25) (25) where ε(±)
p
=
p2
3 +
F ±
p2
1 + p2
2
2
. Here, we explicitly introduce and write a three-
momentum cutoff parameter . The above derivation is nothing but that of the usual method in
the BCS theory. Therefore, it is possible to derive the same result using the equations of motion
by eliminating so-called dangerous terms in the algebraic method [18]. It may be interesting to
observe that Eq. (25) means, physically, that only two “effective colors” participate in the pairing
process, the third “color” remaining inert. The contribution of the active “colors” to the energy is
2(ε(η)
p
−μ) −2
(ε(η)
p
−μ)2 + 32
/2, both for ε(η)
p
< μ and for ε(η)
p
> μ, while the contri-
bution of the inert “color"is simply ε(η)
p
−μ, but only for ε(η)
p
< μ. The gap equation in (20), i.e. ∂(, F, μ)/∂ = 0, is obtained as
⎡
⎣2 · 1
V
pη=±
1
(ε(η)
p
−μ)2 + 32
−1
Gc
⎤
⎦= 0. (26) (26) 8/20 PTEP 2013, 103D01 Y. Tsue et al. 2 In Eq. (3.1) in Ref. [12], F-integration has to be carried out. 2 In Eq. (3.1) in Ref. [12], F-integration has to be carried out. 4.
Numerical results and discussions When F = 0, the quasiparticle energy ε(η)
p
= p is obtained for η = ±. The
thermodynamic potential (25) is then evaluated as In the other case, namely, the F = 0 case, the two-flavor color superconducting phase may be
realized with ̸= 0. When F = 0, the quasiparticle energy ε(η)
p
= p is obtained for η = ±. The
thermodynamic potential (25) is then evaluated as (, F = 0, μ) = 4 · 1
V
p<μ
2(p −μ) −
(p −μ)2 + 32
+ 4 · 1
V
p>μ
(p −μ) −
(p −μ)2 + 32
+ 32
2Gc
. (31) (31) 2 In Eq. (3.1) in Ref. [12], F-integration has to be carried out. 2 In Eq. (3.1) in Ref. [12], F-integration has to be carried out. 9/20 PTEP 2013, 103D01 Y. Tsue et al. Table 1. Parameter set. / GeV
G / GeV−2
Gc / GeV−2
0.631
20.0
6.6 After replacement of the sum over momentum into momentum-integration in (28), the analytical
form of the thermodynamic potential is obtained with a three-momentum cutoff parameter : After replacement of the sum over momentum into momentum-integration in (28), the analytical
form of the thermodynamic potential is obtained with a three-momentum cutoff parameter : (, F = 0, μ) = 32
2Gc
−μ4
6π2 + 4
2π2 −2μ3
3π2
−
1
12π2
(2μ3 −392μ)
&
μ2 + 32 −
&
(μ −)2 + 32
+ (63 + 92 −22μ −2μ2)
&
(μ −)2 + 32
+ 3(122μ2 −94) ln −μ +
&
(μ −)2 + 32
−μ +
&
μ2 + 32
. (32) (32) The gap equation becomes The gap equation becomes The gap equation becomes 1
π2
−3μ
&
μ2 + 32 + (3μ + )
&
(μ −)2 + 32
+ (2μ2 −32) ln −μ +
&
(μ −)2 + 32
−μ +
&
μ2 + 32
= 1
Gc
. (33) (33) The quark number density ρ is calculated from the thermodynamical relation as The quark number density ρ is calculated from the thermodynamical relation as ρ = −∂(, F, μ)
∂μ
. (34) (34) In our model Hamiltonian, the model parameters are G, Gc, and . 3 In Ref. [17], GC/GS = 0.6 is adopted, where GC in Ref. [17] corresponds to Gc/2 here. 4.
Numerical results and discussions In the original NJL model, the
coupling strength GS appears where GS = 5.5 GeV−2 is adopted [19], although this parameter does
not appear explicitly in the model considered here. Then, following Ref. [17], the strength of the
quark-pair interaction, Gc, is taken3 as (Gc/2)/GS = 0.6. Thus, we adopt Gc = 6.6 GeV−2. As for
the three-momentum cutoff , a standard value [19] is adopted here, namely, = 0.631 GeV. If the
tail stretching beyond the Fermi momentum of occupation number should be fully taken into account
in the BCS theory, a larger value of three-momentum cutoff should be adopted, such as = 0.8
GeV [18]. However, it will be later seen that the tail of the occupation number is already sufficiently
taken into account in the case = 0.631 GeV. As for G in the strength of the tensor-type interaction
between quarks, we put G = 20 GeV−2, which was used in our previous paper [12]. As was discussed
in Ref. [12], if the effect of the vacuum polarization is taken into account, the coupling constant
G should be replaced with the renormalized coupling Gr, in which 1/Gr = 1/G −2/π2. Then,
Gr = 20 GeV−2 corresponds to the bare coupling G = 11.1 GeV−2 for = 0.631 GeV or G = 8.7
GeV−2 for = 0.8 GeV. Thus, the strengths of the quark-pairing and tensor-type interactions are
comparable. The parameter set used here is summarized in Table 1. 10/20 PTEP 2013, 103D01 Y. Tsue et al. Fig. 1. (a) The pressures for normal (thin curve), two-flavor color superconducting (2SC) (dash-dotted curve),
and quark spin polarized (SP) (solid curve) phases are shown as functions with respect to quark chemical
potential μ in the case of = 0.631 GeV. (b) The details are depicted around μ ≈0.442 GeV. Fig. 1. (a) The pressures for normal (thin curve), two-flavor color superconducting (2SC) (dash-dotted curve),
and quark spin polarized (SP) (solid curve) phases are shown as functions with respect to quark chemical
potential μ in the case of = 0.631 GeV. (b) The details are depicted around μ ≈0.442 GeV. Fig. 2. The occupation number is depicted as a function of |p| in the cases μ = 0.442 GeV and μ = 0.30 GeV. Fig. 2. 4.
Numerical results and discussions The occupation number is depicted as a function of |p| in the cases μ = 0.442 GeV and μ = 0.30 GeV. First, let us estimate the thermodynamic potential numerically in two phases, namely, color super-
conducting phase with F = 0 and ̸= 0 and quark spin polarized phase with F ̸= 0 and = 0,
separately. The state with (F = 0, = 0) gives a local minimum of the thermodynamic potential,
where 0 is the solution of the gap equation (26) with F = 0 or Eq. (33). However, the state with
(F = F0, = 0) gives a local minimum or a saddle point of the thermodynamic potential corre-
sponding to the value of μ, where F0 is the solution of the gap equation ∂( = 0, F, μ)/∂F = 0
or Eq. (30). This is shown in Appendix C. We compare the pressure in the color superconducting
phase (F = 0, = 0) with that in the quark spin polarized phase (F = F0, = 0). The pressure
p is given by (35) p = −(, F, μ). (35) In Fig. 1(a), the pressures for normal (thin curve), two-flavor color superconducting (dash-dotted
curve), and quark spin polarized (solid curve) phases are shown in the case of = 0.631 GeV as
functions with respect to quark chemical potential μ. Up to μ = μc = 0.442 GeV, where ( =
0, F = 0, μc) = ( = 0, F = F0, μc), the two-flavor color superconducting (2SC) phase is
realized. However, above μ = μc, the quark spin polarized phase is favored. In Fig. 1(b), details are
depicted around μ ≈0.442(= μc) GeV. As for the baryon number density, up to ρB = 4.73ρ0, where
ρ0 = 0.17 fm−3 is the normal nuclear matter density, the color superconducting phase is realized. Thus, it is enough to include the effects of the tail of the occupation number with the three-momentum
cutoff = 0.631 GeV. In Fig. 2, the occupation number is depicted as a function of the magnitude In Fig. 1(a), the pressures for normal (thin curve), two-flavor color superconducting (dash-dotted
curve), and quark spin polarized (solid curve) phases are shown in the case of = 0.631 GeV as
functions with respect to quark chemical potential μ. 4.
Numerical results and discussions Up to μ = μc = 0.442 GeV, where ( =
0, F = 0, μc) = ( = 0, F = F0, μc), the two-flavor color superconducting (2SC) phase is
realized. However, above μ = μc, the quark spin polarized phase is favored. In Fig. 1(b), details are
depicted around μ ≈0.442(= μc) GeV. As for the baryon number density, up to ρB = 4.73ρ0, where
ρ0 = 0.17 fm−3 is the normal nuclear matter density, the color superconducting phase is realized. Thus, it is enough to include the effects of the tail of the occupation number with the three-momentum
cutoff = 0.631 GeV. In Fig. 2, the occupation number is depicted as a function of the magnitude 11/20 PTEP 2013, 103D01 Y. Tsue et al. PTEP 2013, 103D01
Y. Tsue et al. 0
0.00
0.05
0.10
0.15
0.20
0.00
0.01
0.02
0.03
0.04
thermodynamic potential Φ (Δ,F,µ=0.40 GeV)
= 0.0309665
F0 = 0
F
0.01340
0.01332
0.01320
0.01324
0.01328
0.01336
true minimum
(a)
0
0.00
0.05
0.10
0.15
0.20
0.00
0.01
0.02
0.03
0.04
thermodynamic potential Φ (Δ,F,µ=0.42 GeV)
F0 = 0.112113
= 0.0344522
F
0.0016400
0.0016300
0.0016200
0.0015900
true minimum
0.0016425
(b)
0
0.00
0.05
0.10
0.15
0.20
0.00
0.01
0.02
0.03
0.04
F
0.00203
0.00201
0.00195
0.00201582
0.00201582
F0 = 0.200391
= 0.0377968
thermodynamic potential Φ (Δ,F,µc=0.442 GeV)
true minimum
0.00205
(c)
0.00
0.05
0.10
0.15
0.20
0.25
0.00
0.01
0.02
0.03
0.04
0.05
thermodynamic potential Φ (Δ,F,µ=0.45 GeV)
F0 = 0.228493
F
0 = 0.0388851
0.00222
0.00220
0.00218
0.00216
0.00216
true minimum
(d)
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.00
0.01
0.02
0.03
0.04
0.05
thermodynamic potential Φ (Δ,F,µ=0.46 GeV)
F0 = 0.261598
0= 0.0401157
F
0.00230
0.00245
0.00240
0.00235
true minimum
(e)
0.0
0.1
0.2
0.3
0.00
0.01
0.02
0.03
0.04
0.05
0.06
thermodynamic potential Φ (Δ,F,µ=0.48 GeV)
F0 = 0.324172
F
0 = 0.0421576
0.0029
0.0028
0.0030
true minimum
(f)
Fig. 3. The contour map of the thermodynamic potential (, F, μ) is depicted in the cases (a) μ = 0.40
GeV, (b) μ = 0.42 GeV, (c) μ = 0.442 GeV, (d) μ = 0.45 GeV, (e) μ = 0.46 GeV, and (f) μ = 0.48 GeV. The
horizontal and vertical axes represent F and , respectively. 4.
Numerical results and discussions 0
0.00
0.05
0.10
0.15
0.20
0.00
0.01
0.02
0.03
0.04
thermodynamic potential Φ (Δ,F,µ=0.42 GeV)
F0 = 0.112113
= 0.0344522
F
0.0016400
0.0016300
0.0016200
0.0015900
true minimum
0.0016425
(b) 0
0.00
0.05
0.10
0.15
0.20
0.00
0.01
0.02
0.03
0.04
thermodynamic potential Φ (Δ,F,µ=0.40 GeV)
= 0.0309665
F0 = 0
F
0.01340
0.01332
0.01320
0.01324
0.01328
0.01336
rue minimum
(a) (a) (b) thermodynamic potential Φ (Δ,F,µ=0.42 GeV) thermodynamic potential Φ (Δ,F,µ=0.40 GeV) F0 = 0.112113 0
0.00
0.05
0.10
0.15
0.20
0.00
0.01
0.02
0.03
0.04
F
0.00203
0.00201
0.00195
0.00201582
0.00201582
F0 = 0.200391
= 0.0377968
thermodynamic potential Φ (Δ,F,µc=0.442 GeV)
true minimum
0.00205
(c)
t 0.00
0.05
0.10
0.15
0.20
0.25
0.00
0.01
0.02
0.03
0.04
0.05
thermodynamic potential Φ (Δ,F,µ=0.45 GeV)
F0 = 0.228493
F
0 = 0.0388851
0.00222
0.00220
0.00218
0.00216
0.00216
true minimum
(d) (c) (d) thermodynamic potential Φ (Δ,F,µ=0.45 GeV) true minimum (f) (e) 0.0
0.1
0.2
0.3
0.00
0.01
0.02
0.03
0.04
0.05
0.06
thermodynamic potential Φ (Δ,F,µ=0.48 GeV)
F0 = 0.324172
F
0 = 0.0421576
0.0029
0.0028
0.0030
true minimum
(f) 0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.00
0.01
0.02
0.03
0.04
0.05
thermodynamic potential Φ (Δ,F,µ=0.46 GeV)
F0 = 0.261598
0= 0.0401157
F
0.00230
0.00245
0.00240
0.00235
true minimum
(e) thermodynamic potential Φ (Δ,F,µ=0.48 GeV) true minimum Fig. 3. The contour map of the thermodynamic potential (, F, μ) is depicted in the cases (a) μ = 0.40
GeV, (b) μ = 0.42 GeV, (c) μ = 0.442 GeV, (d) μ = 0.45 GeV, (e) μ = 0.46 GeV, and (f) μ = 0.48 GeV. The
horizontal and vertical axes represent F and , respectively. 12/20 PTEP 2013, 103D01 Y. Tsue et al. of momentum p(= |p|). It seems from Fig. 2 that the effects of the tail of the occupation number are
fully taken into account. Secondly, let us consider the gap and F simultaneously. Figure 3 shows contour maps of the
thermodynamic potential (, F, μ), where the horizontal and vertical axis represent F and ,
respectively, in the cases (a) μ = 0.40 GeV, (b) μ = 0.42 GeV, (c) μ = μc = 0.442 GeV, (d) μ =
0.45 GeV, (e) μ = 0.46 GeV, and (f) μ = 0.48 GeV. In case (a), the gap equations give F = 0 and
= 0. 4.
Numerical results and discussions The point (F = 0, = 0) is a true minimum of the thermodynamic potential. Then, up to
μ ≈0.40 GeV, the color superconducting phase is realized. In the region with μ>∼0.407 GeV, which
corresponds to the baryon number density ρ divided by the normal nuclear matter density ρ0, being
ρ/ρ0 = 3.71, the gap equation ∂( = 0, F, μ)/∂F has a nontrivial solution with a nonzero F
value. Then, the true minimum of the thermodynamic potential moves to the point (F ̸= 0, ≈0)
from the point (F = 0, = 0), as is seen in Fig. 3(b). In Fig. 3(c) with μ = μc = 0.442 GeV, in
which ( = 0, F = 0, μ = μc) = ( = 0, F = F0, μ = μc), the true minimum is located at
(F ≈0.8F0, ≈0.80). This state may be interpreted as a state in a mixed phase, in the sense that
this phase is intermediate between a pure spin polarized phase and a pure 2SC one. Further, in case
(d), μ = 0.45 GeV, the true minimum is located at (F ≈0.8F0, ≈0.70). In cases (e) and (f),
namely μ>∼0.46 GeV, the point (F = F0, = 0) becomes a true minimum and the spin polarized
phase is realized. However, in case (e), two minima appear. As seen in case (e), it is most likely that
a jump occurs from (F = F0, = 0) to (F = F0, = 0). Thus, starting from the 2SC phase, the
second-order phase transition starts at the onset of the spin polarization F = F0, and finally it seems
that the transition occurs from intermediate phase to spin polarized phase. 5.
Summary and concluding remarks In this paper, it was shown within the mean field approximation that the quark spin polarized phase
may be realized after the two-flavor color superconducting phase and the mixed phase as the baryon
density increases. We first calculated the pressure of the two phases separately, namely, in the 2SC
( ̸= 0 and F = 0) and the quark spin polarized ( = 0 and F ̸= 0) phases. As a result, in a certain
lower density region, the 2SC phase is realized. However, in rather high density regions, the quark
spin polarized phase is realized. If the large F expansion is carried out in Eq. (26), then the gap
equation for is expressed approximately as
⎡
⎣
! d3p
(2π)3
⎛
⎝1 +
2μ
p2
1 + p2
2 −32
2F2
⎞
⎠−
F
4Gc
⎤
⎦≈0. (36) (36) For large F, the above equation has only the trivial solution = 0. Thus, it is expected that in
fact the quark spin polarized phase with = 0 and F ̸= 0 is realized in the high baryon density
region with large F. The situation is the same even if another parameter is reasonably adopted. For
example, when we adopt G = 10 GeV−2, which is one-half of the value used in this paper, the
behavior of the phase transition is similar in the case of G = 20 GeV−2 except for the value of
chemical potential μ = μc = 0.605 GeV, where ( = 0, F = 0, μc) = ( = 0, F = F0, μc)
is satisfied. But since μc = 0.605 GeV is close to the three-momentum cutoff , it may be impossible
to draw definite conclusions about the phase transition. It might be thought that, as a result, the spin polarized (SP) phase may be more favorable than
the two-flavor color superconducting (2SC) phase at high density. It should be noted that the
thermodynamic potential can be re-expressed as = −
μ
0 dμ N, which is proportional to −μ× 13/20 PTEP 2013, 103D01 Y. Tsue et al. (volume surrounded by the Fermi surface). 5.
Summary and concluding remarks In the quark matter with F = 0 at low baryon density
or small quark chemical potential, the thermodynamic potential depends on μ4 (= μ × μ3), namely
∝−μ4, in which μ3 is nothing but the volume surrounded by the Fermi surface because the shape
of the Fermi surface is a sphere. However, at very high baryon density or large quark chemical poten-
tial, in which the relation F > μ is satisfied, the shape of the Fermi surface becomes a torus, namely,
(
p2
1 + p2
2 −F)2 + p2
3 = μ2. Here, the volume of the torus is 2π2μ2 × F, where μ is the smaller
radius and F is the other larger radius of the torus. In the case F > μ, namely the full polarization
case, from Eq. (29), F = (G/(2π)) × μ3 gives the minimum of the thermodynamic potential. As a
result, the thermodynamic potential is proportional to −μ× (volume surrounded by the Fermi sur-
face) ∝−μ × μ2 × F ∝−μ6, as shown in our previous paper [12]. Thus, at high baryon density
or large quark chemical potential, the spin polarized phase is favored. It might be concluded that the
mechanism that causes the SP phase to be more favored than the 2SC phase at high densities is the
distortion effect of the Fermi surface. For the three-momentum cutoff = 0.631 GeV, it is sufficient to take into account the tail of the
occupation number since the values of the chemical potential on the phase transition point from
the 2SC phase to the mixed phase and from the mixed phase to the spin polarized phase are about
0.407 GeV and 0.46 GeV, respectively. In general, in the system with finite chemical potential, a
chemical-potential dependent cutoff may be used [20–22]. However, for the sake of comparison, if
a rather large fixed value of the three-momentum cutoff, = 0.8 GeV, is adopted where the tail of
the occupation number is more fully taken into account, the critical chemical potential from the 2SC
phase to the mixed phase is unchanged. Above μ = μc = 0.491 GeV, the state with (F = F0, = 0)
is favored against the state with (F = 0, = 0). With this larger fixed three-momentum cutoff, it
is concluded that, up to ρB = 7.42ρ0, the color superconducting phase is realized. Acknowledgements One of the authors (Y.T.) would like to express his sincere thanks to Professor J. da Providência and Professor
C. Providência, two of the co-authors of this paper, for their warm hospitality during his visit to Coimbra in
summer of 2012. One of the authors (Y.T.) is partially supported by Grants-in-Aid for Scientific Research (No. 23540311) from the Ministry of Education, Culture, Sports, Science and Technology in Japan. C. Providência, two of the co-authors of this paper, for their warm hospitality during his visit to Coimbra in
summer of 2012. One of the authors (Y.T.) is partially supported by Grants-in-Aid for Scientific Research (No. 23540311) from the Ministry of Education, Culture, Sports, Science and Technology in Japan. (
)
p
y
pp
y
23540311) from the Ministry of Education, Culture, Sports, Science and Technology in Japan. 5.
Summary and concluding remarks The use of a large
three-momentum cutoff leads to the shift of the phase transition density, i.e., the phase transition
density is higher with a larger three-momentum cutoff. However, if a larger is used, it is necessary
to re-adjust the coupling constants. Secondly, the quark-pairing gap and the spin polarization F were considered simultaneously. Under the present treatment, the phase transition behavior from the 2SC phase with ̸= 0 and F = 0
to the quark spin polarized phase with = 0 and F ̸= 0 may be clear and the transition occurs
passing through the state with (F ̸= 0, ̸= 0), which can be interpreted as the mixed phase. In future work, it would be interesting and important to investigate the phase equilibrium at finite
temperature and in the presence of an external magnetic field. Also, matter in beta equilibrium
could be analyzed, while symmetric matter is considered in the present paper. Additionally, it is
important to investigate the interplay between the color-flavor locked (CFL) phase and the spin
polarized phase in higher density regions in quark matter. Further, it may be interesting to inves-
tigate the interplay between two quark spin polarized phases, namely, the quark spin polarized phase
originating from the tensor-type four-point interaction and that originating from the pseudovector-
type four-point interaction between quarks. In this paper, the mean field approximation for the
established BCS theory is adopted, under which the thermodynamic potential is considered in the
standard manner. Of course, there are other approaches to investigating the phase transition, such as
with the use of the CJT potential [23], the Landau potential [24], and so on. It would be interest-
ing to clarify the difference between the standard BCS mean field approach and the CJT potential
approach, including the effects of two-loop order as a general problem of the phase transition phe-
nomena. However, that is beyond the scope of the present paper. These topics are left for future
investigations. 14/20 PTEP 2013, 103D01 Y. Tsue et al. Appendix A. Mean field Hamiltonian in the basis of good helicity states The following matrix is introduced in order to diagonalize the Hamiltonian matrix in Eq. (8) with
Eq. (10): V (p) =
⎛
⎜⎜⎜⎜⎜⎜⎜⎜⎜⎜⎜⎜⎝
ε(+)
p
+e+q
2
ε(+)
p
−
ε(−)
p
−e+q
2
ε(−)
p
−
ε(+)
p
−e−q
2
ε(+)
p
ε(−)
p
+e−q
2
ε(−)
p
ε(+)
p
+e+q
2
ε(+)
p
ε(−)
p
−e+q
2
ε(−)
p
−
ε(+)
p
−e−q
2
ε(+)
p
−
ε(−)
p
+e−q
2
ε(−)
p
−
g
2
ε(+)
p
(ε(+)
p
+e+q)
g
2
ε(−)
p
(ε(−)
p
−e+q)
−
g
2
ε(+)
p
(ε(+)
p
−e−q)
g
2
ε(−)
p
(ε(−)
p
+e−q)
g
2
ε(+)
p
(ε(+)
p
+e+q)
g
2
ε(−)
p
(ε(−)
p
−e+q)
g
2
ε(+)
p
(ε(+)
p
−e−q)
g
2
ε(−)
p
(ε(−)
p
+e−q)
⎞
⎟⎟⎟⎟⎟⎟⎟⎟⎟⎟⎟⎟⎠
. (A1)
Then, we can diagonalize the Hamiltonian matrix κ as V †κV . Namely, −
ε(+)
p
−e−q
2
ε(+)
p
−
ε(+)
p
−e−q
2
ε(+)
p
−
g
2
ε(+)
p
(ε(+)
p
−e−q)
g
2
ε(+)
p
(ε(+)
p
−e−q) V (p) =
⎛
⎜⎜⎜⎜⎜⎜⎜⎜⎜⎜⎜⎜⎝
ε(+)
p
+e+q
2
ε(+)
p
ε(+)
p
+e+q
2
ε(+)
p
−
g
2
ε(+)
p
(ε(+)
p
+e+q)
g
2
ε(+)
p
(ε(+)
p
+e+q) −
ε(−)
p
−e+q
2
ε(−)
p
ε(−)
p
−e+q
2
ε(−)
p
g
2
ε(−)
p
(ε(−)
p
−e+q)
g
2
ε(−)
p
(ε(−)
p
−e+q) (A1) Then, we can diagonalize the Hamiltonian matrix κ as V †κV . Namely, Then, we can diagonalize the Hamiltonian matrix κ as V †κV . Appendix A. Mean field Hamiltonian in the basis of good helicity states Namely, HSP
MF =
pτα
⎛
⎜⎜⎜⎜⎜⎝
b†
p+τα
b†
p−τα
˜b†
p+τα
˜b†
p−τα
⎞
⎟⎟⎟⎟⎟⎠
T
diag κ
⎛
⎜⎜⎜⎜⎝
bp+τα
bp−τα
˜bp+τα
˜bp−τα
⎞
⎟⎟⎟⎟⎠
,
(A2)
h
d fi HSP
MF =
pτα
⎛
⎜⎜⎜⎜⎜⎝
b†
p+τα
b†
p−τα
˜b†
p+τα
˜b†
p−τα
⎞
⎟⎟⎟⎟⎟⎠
T
diag κ
⎛
⎜⎜⎜⎜⎝
bp+τα
bp−τα
˜bp+τα
˜bp−τα
⎞
⎟⎟⎟⎟⎠
,
(A2) HSP
MF =
pτα
⎛
⎜⎜⎜⎜⎜⎝
b†
p+τα
b†
p−τα
˜b†
p+τα
˜b†
p−τα
⎞
⎟⎟⎟⎟⎟⎠
T
diag κ
⎛
⎜⎜⎜⎜⎝
bp+τα
bp−τα
˜bp+τα
˜bp−τα
⎞
⎟⎟⎟⎟⎠
,
(A2)
where we define
⎛
ε(+)
p
0
0
0
0
(−)
0
0
⎞ HSP
MF =
pτα
⎛
⎜⎜⎜⎜⎜⎝
b†
p+τα
b†
p−τα
˜b†
p+τα
˜b†
p−τα
⎞
⎟⎟⎟⎟⎟⎠
T
diag κ
⎛
⎜⎜⎜⎜⎝
bp+τα
bp−τα
˜bp+τα
˜bp−τα
⎞
⎟⎟⎟⎟⎠
,
(A2)
where we define
diag κ =
⎛
⎜⎜⎜⎝
ε(+)
p
0
0
0
0
ε(−)
p
0
0
0
0
−ε(+)
p
0
0
0
0
−ε(−)
p
⎞
⎟⎟⎟⎠,
⎛
⎜⎜⎜⎝
bp+τα
bp−τα
˜bp+τα
˜bp−τα
⎞
⎟⎟⎟⎠= V †(p)
⎛
⎜⎜⎜⎝
cp+τα
cp−τα
˜cp+τα
˜cp−τα
⎞
⎟⎟⎟⎠. (A3) (A2) diag κ =
⎛
⎜⎜⎜⎝
ε(+)
p
0
0
0
0
ε(−)
p
0
0
0
0
−ε(+)
p
0
0
0
0
−ε(−)
p
⎞
⎟⎟⎟⎠,
⎛
⎜⎜⎜⎝
bp+τα
bp−τα
˜bp+τα
˜bp−τα
⎞
⎟⎟⎟⎠= V †(p)
⎛
⎜⎜⎜⎝
cp+τα
cp−τα
˜cp+τα
˜cp−τα
⎞
⎟⎟⎟⎠. (A3) (A3) Secondly, let us consider the total Hamiltonian matrix with the quark-pair interaction term in the
basis derived above. In this basis, Vcs in (9) is written as Secondly, let us consider the total Hamiltonian matrix with the quark-pair interaction term in the
basis derived above. In this basis, Vcs in (9) is written as Vcs =
pαα′α′′
⎛
⎜⎜⎜⎜⎜⎝
b†
p++α
b†
p−+α
˜b†
p++α
˜b†
p−+α
⎞
⎟⎟⎟⎟⎟⎠
T
V †(p)V (−p)
⎛
⎜⎜⎜⎜⎜⎝
b†
−p+−α′
b†
−p−−α′
˜b†
−p+−α′
˜b†
−p−−α′
⎞
⎟⎟⎟⎟⎟⎠
ϵαα′α′′ . (A4) (A4) 15/20 PTEP 2013, 103D01 Y. Tsue et al. Here, if we replace p with −p, then q, e, g, and ε(±)
p
in (10) are changed or unchanged to q, e, −g,
and ε(±)
p
. Appendix A. Mean field Hamiltonian in the basis of good helicity states Thus, we obtain V †(p)V (−p) =
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
q+e
ε(+)
p
0
−|g|
ε(+)
p
0
0
q−e
ε(−)
p
0
−|g|
ε(−)
p
−|g|
ε(+)
p
0
−q+e
ε(+)
p
0
0
−|g|
ε(−)
p
0
−q−e
ε(−)
p
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
. (A5) (A5) Finally, let us diagonalize the above matrix V †(p)V (−p). We introduce the following matrix: Finally, let us diagonalize the above matrix V †(p)V (−p). We introduce the following matrix: W =
⎛
⎜⎜⎜⎜⎜⎜⎜⎜⎜⎝
−
'
ε(+)
p
+e+q
2ε(+)
p
0
'
ε(+)
p
−e−q
2ε(+)
p
0
0
−
'
ε(−)
p
−e+q
2ε(−)
p
0
'
ε(−)
p
+e−q
2ε(−)
p
|g|
2ε(+)
p
(ε(+)
p
+e+q)
0
|g|
2ε(+)
p
(ε(+)
p
−e−q)
0
0
|g|
2ε(−)
p
(ε(−)
p
−e+q)
0
|g|
2ε(−)
p
(ε(−)
p
+e−q)
⎞
⎟⎟⎟⎟⎟⎟⎟⎟⎟⎠
. (A6) (A6) Then, by using the matrix W, the matrix V †(p)V (−p) that appears in the quark-pair interaction part
can be diagonalized as Then, by using the matrix W, the matrix V †(p)V (−p) that appears in the quark-pair interaction part
can be diagonalized as W †V †(p)V (−p)W =
⎛
⎜⎜⎜⎝
1
0
0
0
0
1
0
0
0
0
−1
0
0
0
0
−1
⎞
⎟⎟⎟⎠. (A7) (A7) By introducing new fermion operators (apητα, a†
pητα, ˜apητα, ˜a†
pητα) by By introducing new fermion operators (apητα, a†
pητα, ˜apητα, ˜a†
pητα) by ⎛
⎜⎜⎜⎜⎝
ap+τα
ap−τα
˜ap+τα
˜ap−τα
⎞
⎟⎟⎟⎟⎠
= W †
⎛
⎜⎜⎜⎜⎝
bp+τα
bp−τα
˜bp+τα
˜bp−τα
⎞
⎟⎟⎟⎟⎠
,
(A8) (A8) the mean field Hamiltonian in which both quark spin polarization and the quark-pair condensate are
simultaneously considered can be expressed in Eq. (13). Appendix B. Expectation values of bilinear operators with respect to the BCS state The expectation values for the BCS state are summarized as follows: Xpη = ⟨|a†
pηταapητα′|⟩,
(for
α ̸= α′)
Npη = ⟨|a†
pηταapητα|⟩,
Dpη = ⟨|a−pη−τα′apητα|⟩,
(for
α ̸= α′)
Ppη = ⟨|a−pη−ταapητα|⟩,
(B (B1) Ppη = ⟨|a−pη−ταapητα|⟩,
(B1) Ppη = ⟨|a−pη−ταapητα|⟩, 16/20 PTEP 2013, 103D01 Y. Tsue et al. where we take α = 1 and α′ = 2 for the following calculations because color symmetry is retained. We can easily calculate the above expectation values by using the relation (16) with (15). For ε(η)
p
> μ, the following relations are obtained: ) > μ, the following relations are obtained: Xpη = −K 2
pη + K 2
pη(Npη −Xpη),
Npη = 2K 2
pη −2K 2
pη(Npη −Xpη),
Dpη = Kpη −3K 2
pηDpη + K 2
pηPpη,
Ppη = −2K 2
pηPpη. (B2) Ppη = −2K 2
pηPpη. (B2) (B2) In the same way, for ε(η)
p
≤μ, the following relations are also obtained: In the same way, for ε(η)
p
≤μ, the following relations are also obtained: In the same way, for ε(η)
p
≤μ, the following relations are also obtained: Xpη = −
K 2
pη(Xpη −Npη),
Npη = 1 + 2
K 2
pη(Xpη −Npη),
Dpη =
Kpη −3
K 2
pηDpη −
K 2
pηPpη,
Ppη = 2
K 2
pηPpη. (B3) (B3) pectation values are calculated as As a result, the expectation values are calculated as For ε(η)
p
> μ
Xpη = −
K 2
pη
1 + 3K 2pη
,
Npη =
2K 2
pη
1 + 3K 2pη
,
Dpη =
Kpη
1 + 3K 2pη
,
Ppη = 0,
(B4)
For ε(η)
p
≤μ
Xpη =
K 2
pη
1 + 3
K 2pη
,
Npη =
1 +
K 2
pη
1 + 3K 2pη
= 1 −
2
K 2
pη
1 + 3
K 2pη
,
Dpη =
Kpη
1 + 3
K 2pη
,
Ppη = 0. (B5) (B4) (B5) Appendix C. The states with ( ̸= 0, F = 0) and ( = 0, F ̸= 0) Appendix C. The states with ( ̸= 0, F = 0) and ( = 0, F ̸= 0) Appendix C. Appendix B. Expectation values of bilinear operators with respect to the BCS state The states with ( ̸= 0, F = 0) and ( = 0, F ̸= 0)
It may be shown that (0, F = 0) is a local minimum of the thermodynamic potential (, F, μ),
where 0 is the solution of the gap equation ∂(, F = 0)/∂ = 0. Here, the states with (0, F =
0) and ( = 0, F0) give extrema of the thermodynamic potential, where F0 is the solution of the gap
equation ∂( = 0, F)/∂F = 0: ∂(, F, μ)
∂
= 3
⎡
⎣−2 · 1
V
p,η
1
(ε(η)
p
−μ)2 + 32
+ 1
Gc
⎤
⎦,
(C1) (C1) PTEP 2013, 103D01 Y. Tsue et al. ⎧
⎪⎨
⎪⎩
∂(0, F = 0, μ)
∂
= 0,
(gap equation)
∂( = 0, F, μ)
∂
= 0,
∂(, F, μ)
∂F
= 2 · 1
V
p,η,εp≤μ
⎡
⎣2 −
ε(η)
p
−μ
(ε(η)
p
−μ)2 + 32
⎤
⎦∂ε(η)
p
∂F
+ 2 · 1
V
p,η,εp≥μ
⎡
⎣1 −
ε(η)
p
−μ
(ε(η)
p
−μ)2 + 32
⎤
⎦∂ε(η)
p
∂F + F
G ,
(C2)
⎧
⎪⎨
⎪⎩
∂( = 0, F, μ)
∂F
= 0,
(gap equation)
∂(, F = 0, μ)
∂F
= 0, ⎧
⎪⎨
⎪⎩
∂(0, F = 0, μ)
∂
= 0,
(gap equation)
∂( = 0, F, μ)
∂
= 0,
∂(, F, μ)
∂F
= 2 · 1
V
p,η,εp≤μ
⎡
⎣2 −
ε(η)
p
−μ
(ε(η)
p
−μ)2 + 32
⎤
⎦∂ε(η)
p
∂F
+ 2 · 1
V
p,η,εp≥μ
⎡
⎣1 −
ε(η)
p
−μ
(ε(η)
p
−μ)2 + 32
⎤
⎦∂ε(η)
p
∂F + F
G ,
(C2)
⎧
⎪∂( = 0, F, μ) = 0,
(gap equation) (gap equation) (C2) ⎧
⎪⎨
⎪⎩
∂( = 0, F, μ)
∂F
= 0,
(gap equation)
∂(, F = 0, μ)
∂F
= 0, ∂F where the last equality is satisfied due to ∂ε(η)
p /∂F|F=0 = η
p2
1 + p2
2/p, ε(η)
p
= |p|, and η = ±1. Appendix B. Expectation values of bilinear operators with respect to the BCS state Further, by using the gap equation
for (̸= 0), we obtain ∂2(0, F = 0, μ)/∂F∂ = 0 due to ε(η)
p
= |p| and η = ±1. Further, by using the gap equation
for (̸= 0), we obtain ∂2(0, F = 0, μ)/∂F∂ = 0 due to ε(η)
p
= |p| and η = ±1. Further, by using the gap equation
for (̸= 0), we obtain ∂2(0, F = 0, μ)
∂2
= 6 · 1
V
p,η
32
0
[(p −μ)2 + 32
0]3/2 > 0 . (C5) (C5) As for the other diagonal matrix element, ∂2(0, F = 0, μ)/∂F2, the positiveness is not shown
analytically. Instead of analytical calculation, numerical estimation is useful just before and after
the chemical potential μ = μc with model parameters G = 20 GeV−2, Gc = 6.6 GeV−2, and =
0.631 GeV used in our paper, where μc gives (0, F = 0, μc) = ( = 0, F0, μc). From Table 2,
it is seen that the eigenvalues of the stability matrix M(0, F = 0) are positive together with (C5). Thus, the state with ( = 0, F = 0) is identified with a local minimum state of the thermodynamic
potential. Next, let us consider the state with ( = 0, F0). The stability matrix M( = 0, F0) is also a
diagonal matrix. We easily obtain the inequality ∂2( = 0, F0, μ)
∂F2
= 6 · 1
V
p,η,ε(η)
p ≤μ
p2
3
ε(η)3
p
+ 1
G > 0. (C6) (C6) As for the other matrix element, ∂2( = 0, F0, μ)/∂2 , numerical estimation, just before and
after the point μ = μc, may also be useful. However, the integral diverges at ε(η)
p
= μ. This is not
surprising, in view of a well known “anomaly” of the BCS theory, according to which a perturbation
expansion in powers of the coupling constant G is not valid, even if this parameter is infinitesimally
small. As a result, the thermodynamical potential cannot be expanded in powers of , even if this
parameter is infinitesimally small. We find that ∂( = 0, F0, μ)/∂ = 0, but the second derivative
diverges. We wish to see if, in the direction, the extremum point ( = 0, F0) is a maximum or
a minimum. Appendix B. Expectation values of bilinear operators with respect to the BCS state Figure 3 shows that the point with ( = 0, F0) is a minimum point in the F direction,
but is a maximum point in the direction in the region μ < 0.46 GeV. Thus, in this region, a point
( = 0, F0) is a saddle point. However, the case μ>∼0.46 GeV, the extremum point with ( = 0, F0)
is a minimum point in the direction. Thus, it is concluded that the point ( = 0, F0) gives a true
minimum of the thermodynamic potential , which results in the quark spin polarized phase. [ ]
,
p
g
y
,
(
)
[2] T. Kunihiro, T. Muto, T. Takatsuka, R. Tamagaki, and T. Tatsumi, Prog. Theor. Phys. Suppl.
112, 1 (1993). ,
(
)
[3] M. Alford, K. Rajagopal, and F. Wilczek, Nucl. Phys. B 357, 443 (1999). [ ]
j g p
y
[4] K. Iida and G. Baym, Phys. Rev. D 63, 074018 (2001). [1] K. Fukushima and T. Hatsuda, Rep. Prog. Phys. 74, 014001 (2011). [ ]
,
p
g
y
,
(
)
[2] T. Kunihiro, T. Muto, T. Takatsuka, R. Tamagaki, and T. Tatsumi, Prog. Theor. Phys. Suppl.
112, 1 (1993).
[3] M. Alford, K. Rajagopal, and F. Wilczek, Nucl. Phys. B 357, 443 (1999).
[4] K. Iida and G. Baym, Phys. Rev. D 63, 074018 (2001). ]
,
j g p ,
,
y
,
(
)
] K. Iida and G. Baym, Phys. Rev. D 63, 074018 (2001). Appendix B. Expectation values of bilinear operators with respect to the BCS state The condition that the states corresponding to (0, F = 0) or ( = 0, F0) are a local minima is
that the eigenvalues are positive for the following stability matrix M(, F), which consists of the
second derivatives: M(, F) =
⎛
⎜⎜⎝
∂2(, F, μ)
∂2
∂2(, F, μ)
∂F∂
∂2(, F, μ)
∂∂F
∂2(, F, μ)
∂F2
⎞
⎟⎟⎠,
(C3) (C3) where e
∂2(, F, μ)
∂2
= 3
⎡
⎣−2 · 1
V
p,η
(ε(η)
p
−μ)2
[(ε(η)
p
−μ)2 + 32]3/2 + 1
Gc
⎤
⎦,
(C4a) (C4a) ∂2(, F, μ)
∂F2
= 2 · 1
V
p,η,εp≤μ
−
32
[(ε(η)
p
−μ)2 + 32]3/2
F + η
p2
1 + p2
2
2
ε(η)2
p
+ 2 · 1
V
p,η,εp≤μ
⎡
⎣2 −
ε(η)
p
−μ
(ε(η)
p
−μ)2 + 32
⎤
⎦p2
3
ε(η)3
p
+ 2 · 1
V
p,η,εp≥μ
−
32
[(ε(η)
p
−μ)2 + 32]3/2
(F + η
p2
1 + p2
2)2
ε(η)2
p
+ 2 · 1
V
p,η,εp≥μ
⎡
⎣1 −
ε(η)
p
−μ
(ε(η)
p
−μ)2 + 32
⎤
⎦p2
3
ε(η)3
p
+ 1
G ,
(C4b)
∂2(, F, μ)
∂F∂
= 6 · 1
V
p,η
ε(η)
p
−μ
[(ε(η)
p
−μ)2 + 32]3/2 ·
F + η
p2
1 + p2
2
ε(η)
p
. (C4c) (C4b) (C4c) Then, if ( = 0, F = 0) is a local minimum, the eigenvalues of the stability matrix M(0, F = 0)
have to be positive. Here, it should be noted that M(0, 0) is a diagonal matrix because Then, if ( = 0, F = 0) is a local minimum, the eigenvalues of the stability matrix M(0, F = 0)
have to be positive. Here, it should be noted that M(0, 0) is a diagonal matrix because 18/20 PTEP 2013, 103D01 Y. Tsue et al. Table 2. Numerical estimations. μ / GeV
0 / GeV
∂2(0, F = 0, μ)
∂F2
/ GeV2
0.44
0.037 5255
0.007 743 56
0.441 90
0.037 7933
0.007 492 14
μc
0.441 95
0.037 8003
0.007 485 53
0.45
0.038 8851
0.006 447 47 Table 2. Numerical estimations. ∂2(0, F = 0, μ)/∂F∂ = 0 due to ε(η)
p
= |p| and η = ±1. References 19/20 PTEP 2013, 103D01 Y. Tsue et al. [5] M. G. Alford, A. Schmitt, K. Rajagopal, and T. Schafer, Rev. Mod. Phys. 80, 1455 (2008), and
references therein. [5] M. G. Alford, A. Schmitt, K. Rajagopal, and T. Schafer, Rev. Mod. Phys. 80, 1455 (2008), and
references therein. [6] L. McLerran and R. D. Pisarski, Nucl. Phys. A 796, 83 (2007). [6] L. McLerran and R. D. Pisarski, Nucl. Phys. A 796, 83 (2007). L. McLerran and R. D. Pisarski, Nucl. Phys. A 79 [7] T. Tatsumi, Phys. Lett. B 489, 280 (2000). [ ]
y
(
)
[8] T. Maruyama and T. Tatsumi, Nucl. Phys. A 693, 710 (2001). y
(
)
[8] T. Maruyama and T. Tatsumi, Nucl. Phys. A 693, 710 (2001). [8] T. Maruyama and T. Tatsumi, Nucl. Phys. A [9] E. Nakano, T. Maruyama, and T. Tatsumi, Phys. Rev. D 68, 105001 (2003). [9] E. Nakano, T. Maruyama, and T. Tatsumi, Phys. Rev. D 68, 105001 (2003). [10] C. Kouveliotou et al., Nature 393, 235 (1998). [11] S. Maedan, Prog. Theor. Phys. 118, 729 (2007). 2] Y. Tsue, J. da Providência, C. Providência, and M. Yamamura, Prog. Theor. Phys. 128, 507 (2012 [12] Y. Tsue, J. da Providência, C. Providência, and M. Yamamura, Prog. Theor. Phys. 128, ] Y. Tsue, J. da Providência, C. Providência, and M [12] Y. Tsue, J. da Providência, C. Providência, and M. Yamamura, Prog. Theor. Phys. 128, 507 (2012). [13] H. Bohr, P. K. Panda, C. Providência, and J. da Providência, Int. J. Mod. Phys. E 22, 1350019 [13] H. Bohr, P. K. Panda, C. Providência, and J. da Providência, Int. J. Mod. Phys. E 22 [14] H. Bohr, C. Providência, and J. da Providência, Eur. Phys. J. A 41, 355 (2009). [14] H. Bohr, C. Providência, and J. da Providência, Eur. Phys. J. A 41, 355 (2009). [15] Y. Nambu and G. Jona-Lasinio, Phys. Rev. 122, 345 (1961). [15] Y. Nambu and G. Jona-Lasinio, Phys. Rev. 122, 345 (1961). [16] Y. Nambu and G. Jona-Lasinio, Phys. Rev 124, 246 (1961). [16] Y. Nambu and G. Jona-Lasinio, Phys. Rev 124, 246 (1961). [17] M. Kitazawa, T. Koide, T. Kunihiro, and Y. Nemoto, Prog. Theor. Phys. 108, 929 (2002). [17] M. Kitazawa, T. Koide, T. Kunihiro, and Y. Nemoto, Prog. Theor. Phys. 108, 929 (2002). ] H. Bohr, P. K. Panda, C. Providência, and J. da P [19] T. References Hatsuda and T. Kunihiro, Phys. Rep. 247, 221 (1994). [19] T. Hatsuda and T. Kunihiro, Phys. Rep. 247, C. H. Lenzi, A. S. Schneider, C. Providência, and R. M. Marinho Jr., Phys. Rev. C 82, 015809 (2010 [20] C. H. Lenzi, A. S. Schneider, C. Providência, and R. M. Marinho Jr., Phys. Rev. C 8 [20] C. H. Lenzi, A. S. Schneider, C. Providência, and R. M. Marinho Jr., Phys. Rev. C 82, 015809 (2010). [21] R. Casalbuoni, R. Gatto, G. Nardulli, and M. Ruggoeri, Phys. Rev. D 68, 034024 (2003). [
]
,
,
,
,
y
,
(
)
[21] R. Casalbuoni, R. Gatto, G. Nardulli, and M. Ruggoeri, Phys. Rev. D 68, 034024 (2003). [21] R. Casalbuoni, R. Gatto, G. Nardulli, and M. Ruggoeri, Phys. Rev. D 68, 034024 (2003). Baldo, G. F. Burgio, P. Castorina, S. Plumari, and Z. Zappalà, Phys. Rev. C 75, 035804 (2007). [22] M. Baldo, G. F. Burgio, P. Castorina, S. Plumari, and Z. Zappalà, Phys. Rev. C 75, 0 J. M. Cornwall, R. Jackiw, and E. Tombolis, Phys. Rev. D 10, 2428 (1974). [23] J. M. Cornwall, R. Jackiw, and E. Tombolis, Phys. Rev. D 10, 2428 (1974). [24] Y. Hashimoto, Y. Tsue, and H. Fujii, Prog. Theor. Phys. 114, 595 (2005). 20/20
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https://openalex.org/W4396668515
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https://www.nature.com/articles/s41598-024-61084-5.pdf
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English
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Angulation and curvature of aortic landing zone affect implantation depth in transcatheter aortic valve implantation
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Scientific reports
| 2,024
|
cc-by
| 9,308
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www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Angulation and curvature of aortic
landing zone affect implantation
depth in transcatheter aortic valve
implantation
OPEN Riccardo Gorla 1*, Omar A. Oliva 1, Luca Arzuffi 1, Valentina Milani 2, Simone Saitta 3,
Mattia Squillace 1, Enrico Poletti 1, Maurizio Tusa 1, Emiliano Votta 3,4, Nedy Brambilla 1,
Luca Testa 1, Francesco Bedogni 1,5 & Francesco Sturla 3,4,5 In transcatheter aortic valve implantation (TAVI), final device position may be affected by device
interaction with the whole aortic landing zone (LZ) extending to ascending aorta. We investigated
the impact of aortic LZ curvature and angulation on TAVI implantation depth, comparing short-frame
balloon-expanding (BE) and long-frame self-expanding (SE) devices. Patients (n = 202) treated with
BE or SE devices were matched based on one-to-one propensity score. Primary endpoint was the
mismatch between the intended (HPre) and the final (HPost) implantation depth. LZ curvature and
angulation were calculated based on the aortic centerline trajectory available from pre-TAVI computed
tomography. Total LZ curvature ( kLZ,tot ) and LZ angulation distal to aortic annulus ( αLZ,Distal ) were
greater in the SE compared to the BE group (P < 0.001 for both). In the BE group, HPost was significantly
higher than HPre at both cusps (P < 0.001). In the SE group, HPost was significantly deeper than HPre
only at the left coronary cusp (P = 0.013). At multivariate analysis, αLZ,Distal was the only independent
predictor (OR = 1.11, P = 0.002) of deeper final implantation depth with a cut-off value of 17.8°. Aortic
LZ curvature and angulation significantly affected final TAVI implantation depth, especially in high
stent-frame SE devices reporting, upon complete release, deeper implantation depth with respect to
the intended one. Keywords Aortic stenosis, TAVI, Innovation, Risk stratification ords Aortic stenosis, TAVI, Innovation, Risk stratificat Abbreviations
αSTJ
Angulation of the STJ plane with respect to the aortic annulus plane
αLZ,Proximal
Angulation of the proximal landing zone plane with respect to the aortic annulus plane
αLZ,Distal
Angulation of the distal landing zone plane with respect to the aortic annulus plane
AA
Aortic angulation
AR
Aortic root
AUC
Area under the curve
BE
Balloon-expandable
BSA
Body surface area
CT
Computed tomography
H
Implantation depth
HPre
Intended implantation depth
HPost
Final implantation depth
kAR,tot
Total curvature of the aortic root centerline
kLZ,tot
Total curvature of the landing zone centerline
LAR
Aortic root length
1Department of Clinical and Interventional Cardiology, IRCCS Policlinico San Donato, P.Zza Edmondo Malan
2, 20097 San Donato Milanese, Milan, Italy. 2Scientific Directorate, IRCCS Policlinico San Donato, San
Donato Milanese, Italy. Angulation and curvature of aortic
landing zone affect implantation
depth in transcatheter aortic valve
implantation
OPEN 3Department of Electronics, Information and Bioengineering, Politecnico di Milano,
Milano, Italy. 43D and Computer Simulation Laboratory, IRCCS Policlinico San Donato, San Donato Milanese,
Italy. 5These authors contributed equally: Francesco Bedogni and Francesco Sturla. *email: riccardo.gorla@
grupposandonato.it Abbreviations
αSTJ
Angulation of the STJ plane with respect to the aortic annulus plane
αLZ,Proximal
Angulation of the proximal landing zone plane with respect to the aortic annulus plane
αLZ,Distal
Angulation of the distal landing zone plane with respect to the aortic annulus plane
AA
Aortic angulation
AR
Aortic root
AUC
Area under the curve
BE
Balloon-expandable
BSA
Body surface area
CT
Computed tomography
H
Implantation depth
HPre
Intended implantation depth
HPost
Final implantation depth 1Department of Clinical and Interventional Cardiology, IRCCS Policlinico San Donato, P.Zza Edmondo Malan
2, 20097 San Donato Milanese, Milan, Italy. 2Scientific Directorate, IRCCS Policlinico San Donato, San
Donato Milanese, Italy. 3Department of Electronics, Information and Bioengineering, Politecnico di Milano,
Milano, Italy. 43D and Computer Simulation Laboratory, IRCCS Policlinico San Donato, San Donato Milanese,
Italy. 5These authors contributed equally: Francesco Bedogni and Francesco Sturla. *email: riccardo.gorla@
grupposandonato.it | https://doi.org/10.1038/s41598-024-61084-5 Scientific Reports | (2024) 14:10409 www.nature.com/scientificreports/ LCC
Left coronary cusp
LZ
Landing zone
MPR
Multiplanar reconstruction
NCC
Non-coronary cusp
PCI
Percutaneous coronary intervention
PPI
Permanent pacemaker implantation
PVL
Paravalvular leakage
ROC
Receiver-operating characteristic
SE
Self-expandable
STJ
Sinotubular junction
STS
Society of Thoracic Surgeons
TAVI
Transcatheter aortic valve implantation
THV
Transcatheter heart valve
∆H
Implantation depth mismatch Transcatheter aortic valve implantation (TAVI) has emerged as the treatment of choice for patients with severe
aortic stenosis at high or intermediate surgical risk1,2. An increased aortic angulation, as evident in the so-called
horizontal aorta, has already been included among the anatomical factors increasing the complexity of TAVI
procedures3 and, in some reports, also affecting procedural success4–6. Based on computed tomography (CT),
aortic angulation (AA) is generally defined on a coronal projection at the level of the aortic annulus as the angle
between the horizontal (i.e., axial) plane and the plane of the aortic annulus6,7. Abramovitz et al.6 reported that an
increased AA adversely influences acute procedural success of TAVIs performed by implanting self-expandable
(SE) transcatheter heart valves (THVs) but not balloon-expandable (BE) THVs. Conversely, in a recent study,
Medranda et al. Study design and data collectionh This is an observational, retrospective and single-center registry enrolling patients with severe aortic stenosis
treated from December 2016 to September 2021 with one of the following THVs: Evolut R/Pro (Medtronic,
Minneapolis, MN, USA), Portico (Abbott, Minneapolis, MN, USA), Myval (Meril Life Sciences Pvt Ltd, Vapi,
Gujarat, India), and Sapien 3 (Edwards Lifesciences, Irvine, CA, USA). j
p
Valve-in-valve TAVI, and bicuspid aortic valve were exclusion criteria. Patients were divided in two groups:
(i) patients implanted with a BE short stent frame THV (Myval and Sapien 3) and (ii) patients receiving a SE
long stent frame THV (Evolut R/Pro and Portico). g
All the collected records on the enrolled patients were retrieved from the institutional TAVI database, includ-
ing data regarding baseline conditions, preprocedural CT angiography, echocardiography, TAVI procedure and
outcome. For each patient, consensus to proceed with TAVI was reached following Heart Team discussion, as
per protocol of our Institute. One-to-one propensity score matching was employed to balance the comparison between the two groups
and remove the potential bias due to baseline characteristics, namely age, Society of Thoracic Surgeons (STS)
score, body surface area (BSA), calcium volume 800 HU and aortic angulation. The propensity score was created
using a multivariable logistic regression model. Matching was performed with a fixed ratio 1:1 optimal match-
ing protocol without replacement and using a caliper width equal to 0.28 of the logit of the standard deviation.h The study was conducted in accordance with the Declaration of Helsinki and approved by the local Ethics
Committee of IRCCS Ospedale San Raffaele (protocol code “AI4TAVI”, No. 33/INT/2023, accepted on March
15th, 2023); informed consent was waived because of the retrospective nature of the study and the anonymized
data analysis. Angulation and curvature of aortic
landing zone affect implantation
depth in transcatheter aortic valve
implantation
OPEN suggested that AA does not affect TAVI outcomes using the new-generation BE (SAPIEN 3) or
SE (CoreValve Evolut PRO) THVs7.l Based on this conflicting evidence, we hypothesized that the “whole” aortic landing zone, which may extend
up to the proximal tract of the ascending aorta in case of TAVI with SE THVs, may be relevant in addition to
other anatomical features (e.g., bicuspid aortic valve, elliptic aortic annulus and ascending aorta diameter) in
predicting final THV position and outcome.i i
To test this hypothesis, we retrospectively quantified the anatomy of the aortic landing zone, namely in terms
of curvature and angulation, in patients receiving SE and BE THVs and evaluated the impact of these features
on the final THV implantation depth in the two groups. Analysis of the aortic landing zoneh y
g
The aortic landing zone was assessed in terms of the geometric features of its centerline, which were analyzed
using a dedicated script written in Matlab (The MathWorks Inc., Natick, MA, USA)11. To this purpose, each
centerline profile was described in the parametric form r(s) =
x(s), y(s), z(s)
as a function of its arc length
( s ) and interpolated through non-uniform rational basis splines (NURBS). For each dataset, the nominal LZ
for TAVI was defined in accordance with the nominal height of each THV, which varies according to the label
size. Specifically, within the SE long stent frame group it ranges between 50 and 53 mm for Portico, and between
45 and 46 mm for Evolut R/Pro. Conversely, in the BE short stent frame group, the nominal height ranges
between 17 and 21 mm for Myval and between 15.5 and 22.5 mm for Sapien 3. Further details are available in
Supplementary Table S1. The initial proximal point of the nominal LZ, i.e., PLZ,Proximal (Fig. 1.A) was automati-
cally positioned 4 mm below the annular point (PAnn) for all the analyzed centerlines, as representative of target
implantation depth in TAVR (9,10). The distal LZ extremity, i.e., PLZ,Distal, was defined on the centerline taking
the THV-specific nominal height into account.i pi
g
AR length (LAR) was measured as the length of the portion of the centerline profile between the annulus plane
and the STJ plane (Fig. 1.B).h The following angular characteristics were extracted from the centerline trajectory (Fig. 1): the STJ angulation
αSTJ with respect to the aortic annulus, expressed as the angle between the tangent unit vector to the centerline
on the annulus plane and the corresponding tangent unit vector on the STJ plane (Fig. 1.C); the angulation of
both the distal and proximal LZ extremities with respect to the aortic annulus, namely αLZ,Distal and αLZ,Proximal
respectively, each one defined as the angle between the tangent unit vector to the centerline on the distal/proximal
LZ plane and the corresponding tangent unit vector on the annulus plane (Fig. 1.D). www.nature.com/scientificreports/ For BE valves, the intended implantation depth was measured with the valve fully closed, before deployment
under rapid pacing. For SE valves, the intended implantation depth was measured with the valve opened up to
the non-recapture point, prior to complete release. For BE valves, the intended implantation depth was measured with the valve fully closed, before deployment
under rapid pacing. For SE valves, the intended implantation depth was measured with the valve opened up to
the non-recapture point, prior to complete release.tiii p
p
p
p
Device success after TAVI was defined according to VARC-3 definition upon fulfilling all the following
criteria9: technical success, freedom from mortality, freedom from surgery or intervention related to the THV
device or to a major vascular or access-related or cardiac structural complication, intended performance of the
THV (mean gradient < 20 mmHg, peak velocity < 3 m/s, Doppler velocity index ≥ 0.25, and less than moderate
aortic regurgitation). All these endpoints were evaluated during the index hospitalization. CT acquisitions and image processing CT angiography was acquired on a 64-row multidetector scanner (SOMATOM Definition, Siemens Healthineers,
Erlangen, Germany). Image sequential acquisition was performed with retrospective ECG-gating. The optimal
systolic reconstruction (BestSyst) was considered for the subsequent analysis. Due to the retrospective nature
of the study, patients with CT imaging not available were excluded from the analysis. Pixel spacing ranged from
0.26 × 0.26 mm2 to 0.87 × 0.87 mm2, while slice thickness ranged between 0.25 and 1 mm. g
Each dataset was imported in 3mensio Structural Heart (version 8.2, Pie Medical Imaging BV, Maastricht,
The Netherlands) and post-processed by a qualified operator. Aortic centerline was automatically detected and
verified by the user through multiplanar reconstruction views, with the possibility to adjust the position of the
control points lying on the centerline (Fig. 1.A). The annulus plane, which is the reference for all the measure-
ments along the centerline, was defined as the plane passing through the user-specified position of the three
nadirs of the aortic leaflets (PAnn on the centerline). The sinotubular junction (STJ) was identified annotating its
position along the centerline with respect to the annular plane (PSTJ). Several measurements (e.g., area, perimeter,
and diameters) were extracted as part of the standard preprocedural evaluation of the aortic root (AR) anatomy10. p
p p
y
Aortic angulation was calculated on CT angiography from the implantation projection in which the three
coronary cusps were aligned and was defined as the angle between the horizontal plane and the plane of the
aortic annulus5.i Finally, the profile of the centerline was exported from 3 mensio as a set of points, whose 3D coordinates were
stored in a file with extensible markup language (xml) format. Analysis of the aortic landing zoneh −→
−→
−→ g
g
g
Also, on each point of the centerline, the tangent ( →
T (s) ), normal ( →
N (s) ), and binormal ( →
B (s) ) unit vectors
were calculated according to the Frenet–Serret frame12 (Supplementary Material) to compute the pointwise
absolute value of curvature k , which quantifies the local bending of the centerline, i.e., the deviation of the curve
from a straight line, expressed in mm−1 (Fig. 1.E). Accordingly, the cumulative k was calculated along the AR
and LZ regions, namely as kAR,tot and kLZ,tot , respectively. Study endpoint and definitionsh The primary endpoint of the study was the mismatch (∆H) between the intended (HPre) and the final (HPost)
implantation depth of each TAVI THV, defined for each dataset as: H = HPost −HPre Implantation depth was defined as the maximal distance between the intraventricular edge of the biopros-
thesis and the aortic annulus at the level of both the non-coronary cusp (NCC) and left coronary cusp (LCC)8
calculated from the implantation projection where the inflow edges are aligned. H was computed at both cusps,
yielding HNCC and HLCC , respectively, and their mean value Hmean . The choice of implantation projection
was left to the operator discretion; for SE-devices, both three-cusps view and cusp overlap view were employed,
with the latter increasingly used after 2019; for BE-devices, three cusps view was employed in all cases. https://doi.org/10.1038/s41598-024-61084-5 Scientific Reports | (2024) 14:10409 | www.nature.com/scientificreports/ Statistical analysis Normal distribution was checked by Kolgomorov-Smirgov and Shapiro–Wilk tests. Continuous variables fol-
lowing a normal distribution are reported as mean and standard deviation; otherwise, median and interquartile
range are presented. Covariates following a normal distribution were compared using unpaired Student’s t-test,
while Mann–Whitney U tests were used to compare continuous variables with skewed distribution. Categorical
and dichotomous variables are presented as counts and percentages and were compared by Pearson chi-square
or Fisher exact tests, as appropriate. pp
p
Receiver-operating characteristic (ROC) curve analysis was employed to investigate the predictive value of
the analyzed LZ features on the mismatch Hmean in implantation depth. To this purpose, the study population
was dichotomized in terms of Hmean classifying each dataset as below or above the pre-defined threshold of
Hmean , i.e., the average value of Hmean over the study population. For each feature, the area under the curve
(AUC) was calculated and the best cutoff was evaluated using the maximal Youden Index. Scientific Reports | (2024) 14:10409 | https://doi.org/10.1038/s41598-024-61084-5 www.nature.com/scientificreports/ Univariate and multivariate logistic regression analysis were carried out to sort out the analyzed variables
di
h i
i l i
h bi
d
i
f TAVI i
l
i
d
h V i bl
i h P
Figure 1. Landing zone analysis. Identification of the landing zone (LZ) on the aortic centerline for a SE valve
(A), definition of the AR length (LAR, B) and extraction of STJ plane angulation (αSTJ, C), proximal LZ plane
angulation (αLZ,Proximal) and distal LZ plane angulation (αLZ,Distal) with respect to the aortic annulus plane (D). Quantification of the cumulative absolute curvature of the aortic centerline (E) in the AR ( kAR,tot ) and LZ
( kLZ,tot ) regions, respectively. Ann, aortic annulus; AR, aortic root; LZ, landing zone; STJ, sinotubular junction. Figure 1. Landing zone analysis. Identification of the landing zone (LZ) on the aortic centerline for a SE valve
(A), definition of the AR length (LAR, B) and extraction of STJ plane angulation (αSTJ, C), proximal LZ plane
angulation (αLZ,Proximal) and distal LZ plane angulation (αLZ,Distal) with respect to the aortic annulus plane (D). Quantification of the cumulative absolute curvature of the aortic centerline (E) in the AR ( kAR,tot ) and LZ
( kLZ,tot ) regions, respectively. Ann, aortic annulus; AR, aortic root; LZ, landing zone; STJ, sinotubular junction. Results
f h Of the 784 TAVI patients eligible for the study, 143 received a BE THV while 641 received a SE THV (Figure S1). Based on the propensity score matching, 266 patients were selected and matched obtaining 133 patients for BE
and SE group, respectively. The results of propensity score matching are detailed in Table S2 while baseline main
characteristics before (unmatched) and after (matched) propensity score matching are summarized in Table S3.i t
p
p
y
g
Due to unavailability of CT imaging for 34 patients, the final matched study cohort included 202 TAVI
patients, i.e., 101 for each group; baseline characteristics are summarized in Table 1. Differences between the
two groups were not statistically significant in terms of age, cardiovascular risk factors, STS score, creatinine
clearance, calcium score, index of eccentricity and aortic angulation. Patients in the BE group showed lower left
ventricle ejection fraction (P < 0.001), lower mean AV gradient (P = 0.002) and, on CT angiography, a larger and
higher aortic root as compared to the patients in the SE group. Also, in the BE group both αSTJ and kAR,tot were
higher (P = 0.024 and P = 0.035, respectively) than in the SE group.i g
(
,
p
y)
g
p
Focusing on the THV-specific landing zone, in the SE group mean kLZ,tot was three times greater than in the
BE group (1.23 10–1·mm−1 vs. 0.41 10–1·mm-1, P < 0.001), mean αLZ,Distal was almost four times wider (28.5° vs. 7.4°, P < 0.001), but αLZ,Proximal remained comparable (P = 0.16). Baseline characteristics clustered according to
Hmean are available in Table S4.h The THVs employed for TAVI are detailed in Table 2; transfemoral access was used in the majority of patients
(88.6%) while subclavian access route was more frequent in the SE group (P = 0.03), which also reported larger
contrast volume (P = 0.02), longer radiation time (P < 0.001) and higher rates of predilatation (P = 0.001) and post-
dilatation (P < 0.001) with respect to the BE group. Negligible differences were noted between BE and SE in terms
of vascular complications, stenting of the access site and concomitant percutaneous coronary intervention (PCI).i In terms of intended and final implantation depth (i.e., HPre and HPost, respectively), BE and SE THVs show a
different behavior (Fig. 2). Results
f h In the BE group, HPost was significantly higher than HPre at both LCC and NCC cusps
(P < 0.001), so that HNCC and HLCC were both negative. Instead, in the SE group, HPost was significantly deeper
than HPre at the LCC cusp (P = 0.013) but not at the NCC (P = 0.64) one. Hmean , i.e., the average of Hmean
over the entire study cohort, was equal to − 1.5 mm; when computed separately for each group, it was equal to
-4.2 mm and 1.1 mm in the BE and SE group, respectively (P < 0.001).i g
p
p
y
Hence, on average, the final implantation depth was higher than the desired one (∆Hmean < 0) with BE THVs
and deeper (∆Hmean > 0) with SE THVs (Fig. 3A). More in detail, we observed Hmean < Hmean in 84.2%
(85/101) of BE THV recipients, but Hmean ≥ Hmean in 91.1% (92/101) of SE THV recipients. Procedural data
and clinical in-hospital outcome clustered according to Hmean are reported in Table S5. At ROC analysis (Table 2), both kLZ,tot (Fig. 3B) and αLZ,Distal (Fig. 3C) reported the highest predictive value
(P < 0.001) on Hmean , reporting the same AUC equal to 0.85 and cut-off values of 0.76 10–1·mm−1 and 17.8°,
respectively. Both variables were found to have the strongest association (P < 0.001) with Hmean at univariate
logistic regression analysis (Table 3). Correct and incorrect classifications are detailed in the Supplementary
Table S6. At multivariate logistic regression analysis, αLZ,Distal arose as the only independent predictor (OR: 1.11; 95%
CI 1.04 to 1.19, P = 0.002) of positive Hmean , i.e., of a final implantation depth deeper than the initially intended
one.if Overall device success was satisfying and equal to 92.6% without significant differences between BE and SE
groups (P = 0.28, Table 2), also in terms of permanent pacemaker implantation (PPI, P = 0.85), stroke (P = 0.62)
and in-hospital mortality (P = 0.28). Ejection fraction remained higher in SE group (P = 0.012), as at baseline. Rate of absent/trivial PVL was higher in BE group (51.5 vs. 36.6%, P = 0.034), though the rate of PVL ≥ moderate
remained comparable (5.0% in BE vs. 10.8% in SE, P = 0.13). Statistical analysis Univariate and multivariate logistic regression analysis were carried out to sort out the analyzed variables
according to their potential impact on the binary endpoint of TAVI implantation depth. Variables with P https://doi.org/10.1038/s41598-024-61084-5 www.nature.com/scientificreports/ values < 0.05 at univariate analysis were entered simultaneously in the multivariate logistic regression analysis. All P-values were two-sided with values < 0.05 considered statistically significant. Analyses were performed
using SPSS 28.0 statistical analysis software (IBM Italia, Milano, Italy). values < 0.05 at univariate analysis were entered simultaneously in the multivariate logistic regression analysis. All P-values were two-sided with values < 0.05 considered statistically significant. Analyses were performed
using SPSS 28.0 statistical analysis software (IBM Italia, Milano, Italy). Discussionhi The main findings of the present study can be summarized as follows: (i) curvature and angulation of the aortic
landing zone may significantly affect the final implantation depth of the THV in TAVI; (ii) high-frame SE valves,
due to their nominal height, proved to be more sensitive than BE valves to the landing zone anatomy, with the
interaction of the upper part of the device with the proximal ascending aorta potentially explaining SE deeper
and asymmetrical implantation depth upon complete release.hf y
p
p
p
p
The so-called horizontal aorta has been considered a debated factor in the recent years negatively affecting
procedural success with SE valves according to some authors3–6, whereas no significant differences in outcomes
between BE and SE devices were reported by others7,13.h p
y
The technical challenge of TAVI with SE devices (i.e., Portico and Evolut-R) in the horizontal aorta is repre-
sented by the difficulty to perform valve release maintaining the device coaxial to the aortic root (i.e., perpendicu-
lar to the aortic annulus) over the entire procedure. This may lead to an increased or asymmetrical implantation
depth between NCC and LCC, though the issue is not systematically observed in all the horizontal anatomies
as confirmed by the discrepancy of the data reported in the clinical literature. In this scenario, considering only
the angulation of the aortic annulus may be too simplistic.hf g
y
Therefore, to provide additional insight into the factors potentially affecting implantation depth in TAVI,
we deepened the characterization of the aortic landing zone in terms of both curvature and angulation. Discussionhi Also,
to balance confounding factors and reduce selection bias, we performed a propensity score matching between Scientific Reports | (2024) 14:10409 | https://doi.org/10.1038/s41598-024-61084-5 www.nature.com/scientificreports/ Variables
Overall (n = 202)
BE (n = 101)
SE (n = 101)
P value§
Age (years)
81 (77, 85)
81 (78, 86)
81 (77, 85)
0.56
Female sex
64 (31.7)
26 (25.7)
38 (37.6)
0.07
BSA (m2)
1.86 ± 0.19
1.85 ± 0.19
1.86 ± 0.20
0.74
Hypertension
151 (74.8)
78 (77.2)
73 (72.3)
0.42
Diabetes
57 (28.2)
30 (29.7)
27 (26.7)
0.64
Dyslipidemia
81 (40.1)
38 (37.6)
43 (42.6)
0.47
COPD
27 (13.4)
12 (11.9)
15 (14.9)
0.54
CAD
48 (23.8)
20 (19.8)
28 (27.7)
0.19
Prior CABG
23 (11.4)
8 (7.9)
15 (14.9)
0.12
Prior AMI
15 (7.4)
9 (8.9)
6 (5.9)
0.42
Prior AF
58 (28.7)
31 (30.7)
27 (26.7)
0.53
STS score (%)
3.2 (2.1, 5.6)
3.2 (2.4, 5.8)
3.2 (2.0, 5.3)
0.38
Creatinine clearance (mL/min/1.73 m2)
58.5 (43.0, 74.0)
60.5 (43.8, 72.3)
57.0 (43.0, 73.3)
0.35
Haemoglobin (g/dL)
12.5 ± 1.9
12.6 ± 2.0
12.4 ± 1.8
0.62
Ejection fraction (%)
55.0 (44.0, 63.0)
51.0 (40.0, 60.0)
58.5 (50.0, 65.0)
< 0.001
Mean AV gradient (mmHg)
42.6 ± 15.0
38.9 ± 14.4
45.7 ± 14.8
0.002
Aortic regurgitation ≥ moderate
35 (17.3)
18 (17.8)
17 (16.8)
0.85
LM height (mm)
15.8 ± 3.8
16.1 ± 3.8
15.5 ± 3.8
0.21
RCA height (mm)
19.5 ± 3.6
19.7 ± 3.4
19.2 ± 3.9
0.27
Annulus minimal diameter (mm)
22.5 ± 3.0
23.4 ± 2.7
21.5 ± 2.9
< 0.001
Annulus maximal diameter (mm)
28.4 ± 2.9
29.3 ± 2.8
27.4 ± 2.6
< 0.001
Annulus mean diameter (mm)
25.4 ± 2.6
26.3 ± 2.5
24.5 ± 2.4
< 0.001
Annulus perimeter (mm)
80.0 ± 8.2
82.7 ± 8.1
77.2 ± 7.4
< 0.001
Annulus area (mm2)
496.5 ± 103.3
531.2 ± 101.7
461.7 ± 93.1
< 0.001
LVOT diameter (mm)
24.9 ± 3.2
26.0 ± 3.1
23.9 ± 3.1
< 0.001
Valsalva diameter (mm)
34.0 ± 3.8
34.7 ± 3.8
33.4 ± 3.7
0.004
Calcium volume 800 HU (mm3)
249 (119, 474)
268 (126, 499)
184 (112, 470)
0.39
Aortic angulation (°)
48.1 ± 9.5
48.4 ± 9.8
47.8 ± 9.3
0.63
Index of eccentricity
0.20 (0.17, 0.25)
0.20 (0.16, 0.24)
0.21 (0.17, 0.26)
0.17
LAR (mm)
22.4 ± 3.6
23.1 ± 3.5
21.7 ± 3.6
0.009
kAR,tot (10–1·mm-1)
0.37 (0.26, 0.51)
0.41 (0.27, 0.53)
0.35 (0.25, 0.45)
0.035
kLZ,tot (10–1·mm-1)
0.83 (0.41, 1.27)
0.41 (0.26, 0.56)
1.23 (1.00, 1.48)
< 0.001
αSTJ (°)
9.1 (5.7, 13.0)
10.7 (6.2, 14.2)
8.3 (5.1, 12.1)
0.024
αLZ,Proximal (°)
2.6 (1.4, 4.3)
2.9 (1.7, 4.8)
2.5 (1.3, 3.9)
0.16
αLZ,Distal (°)
15.8 (6.9, 28.6)
7.4 (4.7, 11.0)
28.5 (21.5, 37.1)
< 0.001 Table 1. Discussionhi Baseline patient characteristics. Values are mean ± SD, median (IQR) or n (% of column total). AF,
atrial fibrillation; AMI, acute myocardial infarction; AR, aortic root; AV, aortic valve; BE, balloon-expandable;
BSA, body surface area; CABG, coronary artery bypass grafting; CAD, coronary artery disease; COPD, chronic
obstructive pulmonary disease; HU, Hounsfield Units; kAR,tot, total (cumulative) curvature of the aortic root
centerline; kLZ,tot, total (cumulative) curvature of the landing zone centerline; LZ, landing zone; LAR, aortic root
length; LM, left main; LVOT, left ventricular outflow tract; RCA, right coronary artery; SE, self-expandable;
STJ, sinotubular junction; STS, Society of Thoracic Surgeons; αSTJ, angulation of the STJ plane with respect to
the aortic annulus plane; αLZ,Proximal, angulation of the proximal LZ plane with respect to the aortic annulus
plane; αLZ,Distal, angulation of the distal LZ plane with respect to the aortic annulus plane. § BE vs. SE. Significant
values (P<0.05) are in bold. patients treated with BE and SE THVs according to the relevant anatomical features with an already known effect
on TAVI outcome, such as aortic angulation and calcium volume 800 HU5,14.i patients treated with BE and SE THVs according to the relevant anatomical features with an already known effect
on TAVI outcome, such as aortic angulation and calcium volume 800 HU5,14.i To the best of our knowledge, this is the first attempt to elucidate the potential factors impacting on the
final device implantation depth through a quantitative assessment of specific geometrical features of the device
landing zone. Of note, the evaluation of both angulation and curvature of the aortic landing zone can be easily
embedded in the planning of TAVI procedure; input data (aortic centerline and anatomical landmarks) can be
directly extracted from software tools already available in clinical practice and the calculation process is not
time-consuming.i g
Both angulation and curvature of the aortic landing zone led to a significant variation of implantation depth
between the intended (i.e., measured at the non-recapture point for SE device) and the final one, upon com-
plete release. Additionally, angulation of the distal landing zone arose as an independent predictor of increased
implantation depth, also reporting at ROC analysis a cut off value equal to 17.8°. Table 2. Procedural data and clinical in-hospital outcome. Values are mean ± SD, median (IQR) or n (% Discussionhi Scientific Reports | (2024) 14:10409 | https://doi.org/10.1038/s41598-024-61084-5 www.nature.com/scientificreports/ Table 2
Procedural data and clinical in-hospital outcome Values are mean±SD med
Variables
Overall (n = 202)
BE (n = 101)
SE (n = 101)
P value§
Implanted THV type
Myval
81 (40.1)
81 (80.2)
–
–
Sapien 3
20 (9.9)
20 (19.8)
–
–
Evolut Pro
22 (10.9)
–
22 (21.8)
–
Evolut R
53 (26.2)
–
53 (52.5)
–
Portico
26 (12.9)
–
26 (27.7)
–
Femoral route
179 (88.6)
91 (90.1)
88 (87.1)
0.51
Subclavian route
11 (5.4)
2 (2.0)
9 (8.9)
0.03
EPS
5 (2.5)
3 (3.0)
2 (2.0)
0.67
Any vascular complications
11 (5.4)
7 (6.9)
4 (4.0)
0.35
PTA with stenting of access site
10 (5.0)
7 (6.9)
3 (3.0)
0.19
PCI with stenting
17 (8.4)
10 (9.9)
7 (6.9)
0.45
Predilatation
82 (40.6)
32 (31.7)
50 (49.5)
0.001
Implantation depth
NCC HPre (mm)
7.0 (5.0, 9.0)
8.0 (7.0, 10.0)
5.0 (4.0, 8.0)
< 0.001
LCC HPre (mm)
8.0 (6.0, 10.0)
9.0 (8.0, 11.0)
7.0 (5.0, 9.0)
< 0.001
NCC HPost (mm)
5.0 (4.0, 7.0)
5.0 (4.0, 6.0)
7.0 (4.6, 8.0)
< 0.001
LCC HPost (mm)
6.0 (4.0, 8.0)
5.0 (4.0, 6.0)
8.0 (6.0, 10.0)
< 0.001
NCC ∆H (mm)
− 1.0 (− 4.0, 1.0)
− 4.0 (− 6.0, − 2.0)
0.0 (− 1.0, 2.5)
< 0.001
LCC ∆H (mm)
− 1.0 (− 5.0, 1.0)
− 5.0 (− 7.0, − 2.0)
1.0 (0.0, 3.0)
< 0.001
Hmean(mm)
− 1.5 ± 3.7
–4.2 ± 2.5
1.1 ± 2.6
< 0.001
Hmean < Hmean
94 (46.5)
85 (84.2)
9 (8.9)
< 0.001
Hmean ≥ Hmean
108 (53.5)
16 (15.8)
92 (91.1)
< 0.001
Postdilatation
58 (28.7)
6 (5.9)
52 (51.5)
< 0.001
Emergent cardiac surgery
0 (0.0)
0 (0.0)
0 (0.0)
–
Need for second valve
0 (0.0)
0 (0.0)
0 (0.0)
–
Contrast volume (mL)
150 (120, 180)
140 (111, 170)
150 (125, 184)
0.02
Radiation time (min)
19.4 (14.6, 26.4)
18.1 (12.7, 23.5)
22.2 (16.1, 28.5)
< 0.001
In-hospital outcome
Ejection fraction (%)
56.0 (50.0, 63.0)
55.0 (45.0, 61.0)
58.5 (52.3, 65.0)
0.012
Mean gradient (mmHg)
7.0 (5.0, 9.0)
7.0 (6.0, 9.0)
7.0 (5.0, 10.0)
0.19
PVL absent/trivial
89 (44.1)
52 (51.5)
37 (36.6)
0.034
PVL mild
89 (44.1)
38 (37.6)
51 (50.5)
0.09
PVL > moderate
17 (8.4)
5 (5.0)
12 (10.8)
0.13
Device success
187 (92.6)
91 (90.1)
96 (95.1)
0.28
PPI
31 (15.3)
15 (14.9)
16 (15.8)
0.85
Stroke*
4 (2.0)
1 (1.0)
3 (3.0)
0.62
In-hospital mortality
1 (0.5)
1 (1.0)
0 (0.0)
0.28 Table 2. Table 2. Procedural data and clinical in-hospital outcome. Values are mean ± SD, median (IQR) or n (%
of column total). Mismatch in implantation depth (∆H) calculated as HPost—HPre; * Including not disabling
stroke. EPS, embolic protection system; HPre, final implantation depth; HPre, pre-implantation intended depth;
LCC, left coronary cusp; NCC, non-coronary cusp; PCI, percutaneous coronary intervention; PPI, permanent
pacemaker implantation; PTA, percutaneous transluminal angioplasty; PVL, paravalvular leakage; ∆H,
variation of implantation depth. § BE vs. SE. Significant values (P<0.05) are in bold. Discussionhi Procedural data and clinical in-hospital outcome. Values are mean ± SD, median (IQR) or n (%
of column total). Mismatch in implantation depth (∆H) calculated as HPost—HPre; * Including not disabling
stroke. EPS, embolic protection system; HPre, final implantation depth; HPre, pre-implantation intended depth;
LCC, left coronary cusp; NCC, non-coronary cusp; PCI, percutaneous coronary intervention; PPI, permanent
pacemaker implantation; PTA, percutaneous transluminal angioplasty; PVL, paravalvular leakage; ∆H,
variation of implantation depth. § BE vs. SE. Significant values (P<0.05) are in bold. Table 2. Procedural data and clinical in-hospital outcome. Values are mean ± SD, median (IQR) or n (%
of column total). Mismatch in implantation depth (∆H) calculated as HPost—HPre; * Including not disabling
stroke. EPS, embolic protection system; HPre, final implantation depth; HPre, pre-implantation intended depth;
LCC, left coronary cusp; NCC, non-coronary cusp; PCI, percutaneous coronary intervention; PPI, permanent
pacemaker implantation; PTA, percutaneous transluminal angioplasty; PVL, paravalvular leakage; ∆H,
variation of implantation depth. § BE vs. SE. Significant values (P<0.05) are in bold. Furthermore, a different behavior was evident when comparing short-frame BE and high-frame SE THVs. On
the one hand, BE devices systematically shortened during valve opening resulting in a final implantation depth
slightly higher than the intended one, irrespectively of the degree of curvature and angulation of the landing
zone. On the other hand, high-frame SE devices resulted in a final implantation depth deeper than the intended
one, in particular at the LCC (Fig. 2). This may be due to the interaction of the upper part of the device with the
proximal ascending aorta (Fig. 4), which locally exhibits higher values of curvature and angulation if compared
to the AR region. Indeed, the initial, i.e., intended, positioning of the SE device (Fig. 4, panel A) is generally
characterized on both NCC and LCC by a symmetrical implantation depth, which is preserved during the first
phase of the device release (Fig. 4, panel B). However, in the final phase of the device release, the terminal por-
tion of SE frame may directly interact with the aortic wall while realigning with the already deployed part of the
device (Fig. 4, panel C). Accordingly, as also evident in Supplementary Video S1, a significant variation can be
noticed for LCC implantation depth while this variation remains negligible for NCC side. Discussionhi Depending on the local https://doi.org/10.1038/s41598-024-61084-5 Scientific Reports | (2024) 14:10409 | www.nature.com/scientificreports/ l
d
f h l
d
h
l
b
h
(
) f h
Figure 2. Box and whiskers plots of the intended (Pre) and final (Post) implantation depth (H) reported within
BE (A) and SE (B) groups as mean (Hmean), NCC (HNCC) and LCC (HLCC) values. BE, balloon-expandable; LCC,
left coronary cusp; NCC, non-coronary cusp; SE, self-expandable; THV, transcatheter heart valve. Figure 2. Box and whiskers plots of the intended (Pre) and final (Post) implantation depth (H) reported within
BE (A) and SE (B) groups as mean (Hmean), NCC (HNCC) and LCC (HLCC) values. BE, balloon-expandable; LCC, Figure 2. Box and whiskers plots of the intended (Pre) and final (Post) implantation depth (H) reported within
BE (A) and SE (B) groups as mean (Hmean), NCC (HNCC) and LCC (HLCC) values. BE, balloon-expandable; LCC,
left coronary cusp; NCC, non-coronary cusp; SE, self-expandable; THV, transcatheter heart valve. Figure 2. Box and whiskers plots of the intended (Pre) and final (Post) implantation depth (H) reported within
BE (A) and SE (B) groups as mean (Hmean), NCC (HNCC) and LCC (HLCC) values. BE, balloon-expandable; LCC,
left coronary cusp; NCC, non-coronary cusp; SE, self-expandable; THV, transcatheter heart valve. angulation and curvature of the landing zone, mechanical interaction between the outer curve (i.e., NCC) of the
ascending aorta and the prosthetic valve may occur, inducing a partial rotation of the device and increasing its
axial motion along the contralateral (i.e., LCC) side of the landing zone. For instance, in a patient with remarkable angulation and curvature of the proximal ascending aorta (Fig. 5,
panel A), TAVI with BE valve generally reveals a symmetrical reduction of implantation depth on both LCC and
NCC cusps since no mechanical interaction is expected between the device and the proximal ascending aorta
(Fig. 5, panel B). Conversely, in patients referred to TAVI with SE devices, a pronounced angulation and curvature
of the distal aortic landing zone can induce an axial motion and partial tilting of the device, in particular on LCC
(Fig. 5, panels C and D) while this alteration in the final implantation depth is not evident with a less angulated
distal aortic landing zone (Fig. Discussionhi 5, panels E and F).hi g
g
p
The clinical implications of our findings may be relevant since the implantation depth is associated with the
need of postprocedural PPI15 and PVL5, with both the conditions increasing the risk of all-cause mortality and
cardiovascular mortality7,16. y
Furthermore, there will be an increasing number of low-risk and younger patients undergoing TAVI17,
the future, in whom the need for postprocedural PPI will become less and less acceptable.hi Thus, it will be of utmost importance to further improve procedural outcome. In this regard, the finding
on CT scan of an angulated aortic landing zone may be relevant during preprocedural planning and should be
interpreted as an anatomical feature supporting the choice of a short-frame device. It is worth noting that Portico and Evolut-R THVs were grouped together within the high-frame SE group:
despite a similar stent height, the Evolut-R stent frame is more rigid due the smaller cell design and has higher
radial force as compared to the Portico19. Also, the EnVeo-R (Medtronic, Minneapolis, MN, USA) delivery sys-
tem used for Evolut-R/Pro THV is made of a double spine technology, allowing steering only in two directions, https://doi.org/10.1038/s41598-024-61084-5 Scientific Reports | (2024) 14:10409 | www.nature.com/scientificreports/ Figure 3. Bar plot of the patient-specific mismatch in the mean implantation depth Hmean (A) within BE and
SE groups, reporting data as paired values according to the results of one-to-one propensity score matching. Scatter plots of the association of (B) kLZ,tot curvature and (C) αLZ,Distal angulation with Hmean and ROC curves
evaluating the predictive value of each landing zone characteristic on the final variation of THV implantation
depth. Other abbreviations as in Figs. 1 and 2. Figure 3. Bar plot of the patient-specific mismatch in the mean implantation depth Hmean (A) within BE and
SE groups, reporting data as paired values according to the results of one-to-one propensity score matching. Scatter plots of the association of (B) kLZ,tot curvature and (C) αLZ,Distal angulation with Hmean and ROC curves
evaluating the predictive value of each landing zone characteristic on the final variation of THV implantation
depth. Other abbreviations as in Figs. 1 and 2. while the FlexNav (Abbott, Minneapolis, MN, USA) delivery system used for Portico THV is made of a single
spine technology and it is provided with a stabilization layer, which improves stability during valve release. https://doi.org/10.1038/s41598-024-61084-5 Scientific Reports | (2024) 14:10409 | www.nature.com/scientificreports/ Table 3. Discussionhi AR, aortic root; HU, Hounsfield Units; LAR,
aortic root length; LVOT, left ventricular outflow tract; LZ, landing zone; kAR,tot, total (cumulative) curvature
of the aortic root centerline; kLZ,tot, total (cumulative) curvature of the landing zone centerline; αSTJ, angulation
of the STJ plane with respect to the aortic annulus plane; αLZ,Proximal, angulation of the proximal LZ plane with
respect to the aortic annulus plane; αLZ,Distal, angulation of the distal LZ plane with respect to the aortic annulus
plane; STJ, sinotubular junction. Significant values (P<0.05) are in bold. Table 3. Univariate and multivariate logistic regression analysis of the aortic root dimensions and landing
zone features associated with implant height variation after TAVI. AR, aortic root; HU, Hounsfield Units; LAR,
aortic root length; LVOT, left ventricular outflow tract; LZ, landing zone; kAR,tot, total (cumulative) curvature
of the aortic root centerline; kLZ,tot, total (cumulative) curvature of the landing zone centerline; αSTJ, angulation
of the STJ plane with respect to the aortic annulus plane; αLZ,Proximal, angulation of the proximal LZ plane with
respect to the aortic annulus plane; αLZ,Distal, angulation of the distal LZ plane with respect to the aortic annulus
plane; STJ, sinotubular junction. Significant values (P<0.05) are in bold. Figure 4. Mechanism of aortic landing zone interaction with SE valve. Initial positioning of the SE device with
a symmetrical implantation depth on both NCC and LCC cusps (green bars, A); the intended implantation
depth is maintained in the initial phase of SE release (B) while the final implantation depth of the device may
change depending on the interaction of the device with the aortic wall anatomy (C). Abbreviations as in Fig. 2. Figure 4. Mechanism of aortic landing zone interaction with SE valve. Initial positioning of the SE device with
a symmetrical implantation depth on both NCC and LCC cusps (green bars, A); the intended implantation
depth is maintained in the initial phase of SE release (B) while the final implantation depth of the device may
change depending on the interaction of the device with the aortic wall anatomy (C). Abbreviations as in Fig. 2. Nonetheless, mismatch in mean implantation depth Hmean proved to be comparable between Portico and
Evolut sub-groups (P = 0.44, Figure S2), with statistically negligible differences on both LCC and NCC sides
(P = 0.46 and P = 0.33, respectively).h (
p
y)
There are main limitations that should be taken into consideration when interpreting the results of
analysis. Discussionhi Univariate and multivariate logistic regression analysis of the aortic root dimensions and landing
zone features associated with implant height variation after TAVI. AR, aortic root; HU, Hounsfield Units; LAR,
aortic root length; LVOT, left ventricular outflow tract; LZ, landing zone; kAR,tot, total (cumulative) curvature
of the aortic root centerline; kLZ,tot, total (cumulative) curvature of the landing zone centerline; αSTJ, angulation
of the STJ plane with respect to the aortic annulus plane; αLZ,Proximal, angulation of the proximal LZ plane with
respect to the aortic annulus plane; αLZ,Distal, angulation of the distal LZ plane with respect to the aortic annulus
plane; STJ, sinotubular junction. Significant values (P<0.05) are in bold. Variables
ROC
Univariate (n = 202)
Multivariate (n = 202)
AUC
P value
OR (95% CI)
P value
OR (95% CI)
P value
Calcium Score 800 HU
0.57
0.11
0.99 (0.99 ÷ 1.00)
0.13
Aortic angulation
0.50
0.99
1.00 (0.97 ÷ 1.03)
0.89
Annulus minimal diameter
0.62
0.003
0.86 (0.77 ÷ 0.95)
0.003
1.29 (0.96 ÷ 1.72)
0.09
Annulus mean diameter
0.65
< 0.001
0.82 (0.73 ÷ 0.92)
0.001
0.66 (0.37 ÷ 1.19)
0.17
Annulus maximal diameter
0.65
< 0.001
0.84 (0.76 ÷ 0.93)
0.001
0.96 (0.69 ÷ 1.35)
0.82
Annulus perimeter
0.64
< 0.001
0.94 (0.91 ÷ 0.98)
0.001
1.23 (0.87 ÷ 1.73)
0.25
Annulus area
0.64
< 0.001
0.61 (0.45 ÷ 0.81)
0.001
0.61 (0.51 ÷ 7.43)
0.70
LVOT diameter
0.65
< 0.001
0.85 (0.77 ÷ 0.93)
0.001
0.77 (0.57 ÷ 1.03)
0.08
Valsalva diameter
0.59
0.04
0.94 (0.87 ÷ 1.02)
0.15
LAR
0.60
0.01
0.89 (0.82 ÷ 0.96)
0.004
0.92 (0.81 ÷ 1.05)
0.21
kAR,tot
0.58
0.07
0.0 (0.0 ÷ 23.1)
0.14
kLZ,tot
0.85
< 0.001
E14 (E10 ÷ E17)
< 0.001
E+5 (0.2 ÷ E+11)
0.15
αSTJ
0.58
0.06
0.97 (0.92 ÷ 1.01)
0.16
αLZ,Proximal
0.54
0.31
1.07 (0.97 ÷ 1.18)
0.21
αLZ,Distal
0.85
< 0.001
1.15 (1.11 ÷ 1.20)
< 0.001
1.11 (1.04 ÷ 1.19)
0.002 Table 3. Univariate and multivariate logistic regression analysis of the aortic root dimensions and landing
zone features associated with implant height variation after TAVI. Discussionhi First, this is a proof-of-concept retrospective study investigating a potential relation between novel anatomical
aortic features and implantation depth in TAVI based on a relatively small sample size, though we performed a
propensity score match to account for possible bias in the selected population. Nonetheless, due to the available
sample size, only 5 parameters (age, STS score, body surface area, calcium volume 800 HU and aortic angula-
tion) were selected for propensity score matching; otherwise, the study population would have been too small. Nonetheless, several parameters not included in the one-to-one matching (e.g., prior atrial fibrillation, chronic
obstructive pulmonary disease, coronary artery disease, prior coronary artery bypass grafting and prior acute
myocardial infarction) were comparable between BE and SE datasets before matching and remained comparable
also after one-to-one propensity matching. Scientific Reports | (2024) 14:10409 | https://doi.org/10.1038/s41598-024-61084-5 www.nature.com/scientificreports/ Figure 5. BE valve deployment in a patient with angulated proximal ascending aorta (A, B): (A) initial device
positioning and (B) almost symmetrical higher final implantation depth (H) on both NCC and LCC cusps. SE
valve implantation in a patient with high angulation of the proximal ascending aorta (C, D): (C) symmetrical
initial implantation depth and (D) final tilted valve configuration with a deeper than expected implantation
depth on LCC. SE valve implantation in a patient with restrained angulation of the proximal ascending aorta
(E, F): (E) initial symmetrical THV positioning and (F) final THV implantation depth with symmetrical
implantation depth on both LCC and NCC. Abbreviations as in Figs. 1 and 2. Figure 5. BE valve deployment in a patient with angulated proximal ascending aorta (A, B): (A) initial device
positioning and (B) almost symmetrical higher final implantation depth (H) on both NCC and LCC cusps. SE
valve implantation in a patient with high angulation of the proximal ascending aorta (C, D): (C) symmetrical
initial implantation depth and (D) final tilted valve configuration with a deeper than expected implantation
depth on LCC. SE valve implantation in a patient with restrained angulation of the proximal ascending aorta
(E, F): (E) initial symmetrical THV positioning and (F) final THV implantation depth with symmetrical
implantation depth on both LCC and NCC. Abbreviations as in Figs. 1 and 2. Conclusions During TAVI procedure, an increased angulation of the distal portion of THV landing zone proved to signifi-
cantly impact on the final release of the device in terms of mismatch between the final and the intended implan-
tation depth. Specifically, due to their remarkable frame height extending the surface of interaction with the
proximal ascending aorta, the final implantation depth of SE devices may be deeper with respect to the intended
one, in particular on LCC, as a consequence of the mechanical interplay between the device and the aortic wall
in the final phase of device release. Discussionhi Second, clinical validation of our findings with respect to current TAVI outcomes (i.e., PVL, PPI) shoul
investigated on a larger and prospective population study.h Third, calculation of the angulation and curvature of the aortic landing zone was performed through the
combined use of different commercial tools. Nonetheless, the proposed metrics as well as the way they are
calculated can be effectively automated and made accessible to clinicians in routine TAVI planning, directly
embedding these measurements in commercial software already in use or further leveraging deep learning-based
dedicated workflows20. References et al. Permanent pacemaker insertion reduction and optimized temporary pacemaker management after contempo-
rary transcatheter aortic valve implantation with self-expanding valves (from the pristine TAVI study). Am. J. Cardiol. 189, 1–10. https://doi.org/10.1016/j.amjcard.2022.11.026 (2023).t 15. Yoon, S. H. et al. Permanent pacemaker insertion reduction and optimized temporary pacemaker management after contempo-
rary transcatheter aortic valve implantation with self-expanding valves (from the pristine TAVI study). Am. J. Cardiol. 189, 1–10. https://doi.org/10.1016/j.amjcard.2022.11.026 (2023).t p
g
j
j
16. Pagnesi, M. et al. Incidence, predictors, and prognostic impact of new permanent pacemaker implantation after TAVR with self-
expanding valves. JACC Cardiovasc. Interv. https://doi.org/10.1016/j.jcin.2023.05.020 (2023). p
g
j
j
16. Pagnesi, M. et al. Incidence, predictors, and prognostic impact of new permanent pacemaker implantation after TAVR with self-
expanding valves. JACC Cardiovasc. Interv. https://doi.org/10.1016/j.jcin.2023.05.020 (2023). p
g
p
g
j j
17. Mack, M. J. et al. Transcatheter aortic-valve replacement with a balloon-expandable valve in low-risk patients. N. Engl. J. Med. 380, 1695–1705. https://doi.org/10.1056/NEJMoa1814052 (2019). p
g
p
g
j j
17. Mack, M. J. et al. Transcatheter aortic-valve replacement with a balloon-expandable valve in low-risk patients. N. Engl. J. Med. 380, 1695–1705. https://doi.org/10.1056/NEJMoa1814052 (2019). p
g
J
(
)
8. Popma, J. J. et al. Transcatheter aortic-valve replacement with a self-expanding valve in low-risk patients. N. Engl. J. Med. 380
1706–1715. https://doi.org/10.1056/NEJMoa1816885 (2019). 1706–1715. https://doi.org/10.1056/NEJMoa1816885 (2019). p
g
19. Finotello, A. et al. Finite element analysis of transcatheter aortic valve implantation: Insights on the modelling of self-expandable
devices. J. Mech. Behav. Biomed. Mater. 123, 104772. https://doi.org/10.1016/j.jmbbm.2021.104772 (2021). p
g
19. Finotello, A. et al. Finite element analysis of transcatheter aortic valve implantation: Insights on the modelling of self-expandable
devices. J. Mech. Behav. Biomed. Mater. 123, 104772. https://doi.org/10.1016/j.jmbbm.2021.104772 (2021). p
g
j j
20. Saitta, S. et al. A CT-based deep learning system for automatic assessment of aortic root morphology for TAVI planning. Com
Biol. Med. 163, 107147. https://doi.org/10.1016/j.compbiomed.2023.107147 (2023). Acknowledgementsh g
This work was supported by IRCCS Policlinico San Donato, a clinical research hospital partially funded by the
Italian Ministry of Health. Data availabilityh The data that support the findings of this study will be available from the corresponding author upon reason-
able request. Received: 4 December 2023; Accepted: 30 April 2024 Received: 4 December 2023; Accepted: 30 April 2024 Scientific Reports | (2024) 14:10409 | https://doi.org/10.1038/s41598-024-61084-5 www.nature.com/scientificreports/ www.nature.com/scientificreports/ References References
1. Mack, M. J. et al. 5-year outcomes of transcatheter aortic valve replacement or surgical aortic valve replacement for high surgical
risk patients with aortic stenosis (PARTNER 1): A randomised controlled trial. Lancet 385, 2477–2484. https://doi.org/10.1016/
s0140-6736(15)60308-7 (2015). 1. Mack, M. J. et al. 5-year outcomes of transcatheter aortic valve replacement or surgical aortic valve replacement for high surgical
risk patients with aortic stenosis (PARTNER 1): A randomised controlled trial. Lancet 385, 2477–2484. https://doi.org/10.1016/
s0140-6736(15)60308-7 (2015). (
)
(
)
2. Leon, M. B. et al. Transcatheter or surgical aortic-valve replacement in intermediate-risk patients. N. Engl. J. Med. 374, 1609–1620. https://doi.org/10.1056/NEJMoa1514616 (2016). (
)
(
)
2. Leon, M. B. et al. Transcatheter or surgical aortic-valve replacement in intermediate-risk patients. N. Engl. J. Med. 374, 1609–1620
https://doi.org/10.1056/NEJMoa1514616 (2016). p
g
J
(
)
3. Veulemans, V. et al. Novel insights on outcome in horizontal aorta with self-expandable new-generation transcatheter aortic valve
replacement devices. Catheter. Cardiovasc. Interv. 96, 1511–1519. https://doi.org/10.1002/ccd.28961 (2020). p
g
3. Veulemans, V. et al. Novel insights on outcome in horizontal aorta with self-expandable new-generation transcatheter aortic valve
replacement devices. Catheter. Cardiovasc. Interv. 96, 1511–1519. https://doi.org/10.1002/ccd.28961 (2020). p
,
p
g
(
4. Gallo, F. et al. Horizontal aorta in transcatheter self-expanding valves: Insights from the HORSE internation
Circ. Cardiovasc. Interv. 14, e010641. https://doi.org/10.1161/circinterventions.121.010641 (2021). p
p
g
(
)
4. Gallo, F. et al. Horizontal aorta in transcatheter self-expanding valves: Insights from the HORSE international multicentre registry
Circ. Cardiovasc. Interv. 14, e010641. https://doi.org/10.1161/circinterventions.121.010641 (2021). p
g
5. Gorla, R. et al. Impact of aortic angle on transcatheter aortic valve implantation outcome with Evolut-R, portico, and acurate-NEO. Catheter. Cardiovasc. Interv. 97, E135–E145. https://doi.org/10.1002/ccd.28957 (2021). g
5. Gorla, R. et al. Impact of aortic angle on transcatheter aortic valve implantation outcome with Evolut-R, portico, and acurate-NEO. Catheter. Cardiovasc. Interv. 97, E135–E145. https://doi.org/10.1002/ccd.28957 (2021). p
g
6. Abramowitz, Y. et al. Aortic angulation attenuates procedural success following self-expandable but not balloon-expandable TAVR
JACC Cardiovasc. Imaging 9, 964–972. https://doi.org/10.1016/j.jcmg.2016.02.030 (2016).h g
6. Abramowitz, Y. et al. Aortic angulation attenuates procedural success following self-expandable but not balloon-expandable TAVR. JACC Cardiovasc. Imaging 9, 964–972. https://doi.org/10.1016/j.jcmg.2016.02.030 (2016).h g g
p
g
j j
g
7. Medranda, G. A. et al. The impact of aortic angulation on contemporary transcatheter aortic valve replacement outcomes. JACC
Cardiovasc. Interv. 14, 1209–1215. https://doi.org/10.1016/j.jcin.2021.03.027 (2021). p
g
j j
(
)
8. Petronio, A. S. et al. Author contributions R.G. and F.S. conceived and designed the work. O.A.O. contributed to the study design and to collect data. L.A.,
E.P. and M.S. contributed to data collection and database integrity. S.S. and E.V. supported in house data process-
ing; F.S. and V.M. performed the statistical analysis. S.S., N.B., E.V. and L.T. contributed to the analysis of the
results. R.G., O.A.O. and F.S. wrote the first draft of the manuscript; E.V., L.A., M.S., V.M. and M.T. wrote sections
of the manuscript and contributed to critical revision of the intellectual content. L.T., N.B. and F.B. contributed
to the clinical interpretation of the results; F.B. contributed to funding acquisition. All the authors contributed
to the revision of the final version of the manuscript, read, and approved the submitted version. Competing interests p
g
Francesco Bedogni is proctor for Medtronic, Abbott, and Meryl Life Sciences; Nedy Brambilla and Luca Testa
are proctors for Abbott and and Meril Life Sciences. All the remaining authors declare no financial and non-
financial competing interests. References Optimal implantation depth and adherence to guidelines on permanent pacing to improve the results of
transcatheter aortic valve replacement with the medtronic corevalve system: The CoreValve prospective, international Post-Market
ADVANCE-II study. JACC Cardiovasc. Interv. 8, 837–846. https://doi.org/10.1016/j.jcin.2015.02.005 (2015).i p
g
j j
8. Petronio, A. S. et al. Optimal implantation depth and adherence to guidelines on permanent pacing to improve the results of
transcatheter aortic valve replacement with the medtronic corevalve system: The CoreValve prospective, international Post-Market
ADVANCE-II study. JACC Cardiovasc. Interv. 8, 837–846. https://doi.org/10.1016/j.jcin.2015.02.005 (2015). y J
,
p //
g/
/j j
(
)
9. Généreux, P. et al. Valve academic research consortium 3: Updated endpoint definitions for aortic valve clinical research. J. Am. Coll. Cardiol. 77, 2717–2746. https://doi.org/10.1016/j.jacc.2021.02.038 (2021).t y
p
g
j j
9. Généreux, P. et al. Valve academic research consortium 3: Updated endpoint definitions for aortic valve clinical research. J. Am. Coll. Cardiol. 77, 2717–2746. https://doi.org/10.1016/j.jacc.2021.02.038 (2021).t p
g
j j
10. Iacovelli, F. et al. Prosthesis depth and conduction disturbances after last generation balloon-expandable transcatheter aortic valve
implantation. Europace Eur. Pacing, Arrhythm. Card. Electrophysiol. J. Work. Groups Card. Pacing Arrhythm. Card. Cell. Electrophys. Eur. Soc. Cardiol. 20, 116–123. https://doi.org/10.1093/europace/euw310 (2018). ,
p
g
p
(
)
11. Marrocco-Trischitta, M. M. & Sturla, F. Blood flow helical pattern in type III arch configuration as a potential risk factor for
B aortic dissection. Eur. J. Cardiothorac. Surg. 61, 132–139. https://doi.org/10.1093/ejcts/ezab307 (2021). g
p
g
j
12. O’Flynn, P. M., O’Sullivan, G. & Pandit, A. S. Methods for three-dimensional geometric characterization of the arterial vasculature. Ann. Biomed. Eng. 35, 1368–1381. https://doi.org/10.1007/s10439-007-9307-9 (2007). g
,
p //
g/
/
(
)
13. Di Stefano, D. et al. Impact of horizontal aorta on procedural and clinical outcomes in second-generation transcatheter aortic
valve implantation. EuroIntervention 15, e749–e756. https://doi.org/10.4244/eij-d-19-00455 (2019). g
p
g
13. Di Stefano, D. et al. Impact of horizontal aorta on procedural and clinical outcomes in second-gene
valve implantation. EuroIntervention 15, e749–e756. https://doi.org/10.4244/eij-d-19-00455 (2019).i p
,
p
g
j
(
)
4. Jilaihawi, H. et al. A revised methodology for aortic-valvar complex calcium quantification for transcatheter aortic valve implanta
tion. Eur. Heart J. Cardiovasc. Imaging 15, 1324–1332. https://doi.org/10.1093/ehjci/jeu162 (2014).t g
j
14. Jilaihawi, H. et al. A revised methodology for aortic-valvar complex calcium quantification for tran
tion. Eur. Heart J. Cardiovasc. Imaging 15, 1324–1332. https://doi.org/10.1093/ehjci/jeu162 (2014 g g
p
g
j
j
15. Yoon, S. H. Open Access This article is licensed under a Creative Commons Attribution 4.0 International
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Lipid rafts promote liver cancer cell proliferation and migration by up-regulation of TLR7 expression
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Oncotarget
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Abstract Hepatocellular carcinoma (HCC) occurs predominantly in patients with underlying
chronic liver disease and cirrhosis. Toll-like receptors (TLRs) play an important role
in innate immune responses and TLR signaling has been associated with various
chronic liver diseases. Lipid rafts provide the necessary microenvironment for certain
specialized signaling events to take place, such as the innate immune recognition. The
purpose of this study was to determine the pattern of TLR7 expression in HCC, how to
recruit TLR7 into lipid rafts responded to ligands and whether targeting TLR7 might
have beneficial effects. The study group was comprised of 130 human liver tissues:
23 chronic hepatitis B (CHB), 18 liver cirrhosis (LC), 68 HCC and 21 normal livers. The
expression of TLR7 was evaluated using immunohistochemistry, western blotting,
and flow cytometry. Proliferation and migration of human HepG2 cells were studied
following stimulation of TLR7 using the agonist gardiquimod and inhibition with a
specific antagonist 20S-protopanaxadiol (aPPD). The activation of lipid raft-associated
TLR7 signaling was measured using western blotting, double immunohistochemistry
and immunoprecipitation in liver tissues and HepG2 cells. TLR7 expression was
up-regulated in human HCC tissues and hepatoma cell line. Proliferation and migration
of HepG2 cells in vitro increased significantly in response to stimulation of TLR7. TLR7
inhibition using aPPD significantly reduced HepG2 cell migration in vitro. The lipid raft
protein caveolin-1 and flotillin-1 were involved with enhanced TLR7 signaling in HCC. Conclusions: The data suggest that inhibiting TLR7 with antagonists, like aPPD, Conclusions: The data suggest that inhibiting TLR7 with antagonists, like
could potentially be used as a novel therapeutic approach for HCC. www.impactjournals.com/oncotarget/ www.impactjournals.com/oncotarget/ INTRODUCTION related to their immune status. The persistence of HBV
infection in human liver is believed to be associated to
impaired function in activation of type I interferons
(IFNs-α/β) [9, 10], a key anti-viral response. This may
involve inhibition of plasmacytoid dendritic cells (pDCs)
and alterations in natural killer (NK) cell activation. pDCs
play an essential role by recognizing foreign pathogens
and tumor cells through a panel of pattern recognition
receptors (PRR) [9–11], particularly toll-like receptor 7
(TLR7). Upon stimulation of TLR7, they produce and
release type I IFNs and other cytokines/chemokines,
and cause activation of NK cells and cross priming of
cytotoxic lymphocytes, thereby orchestrating both innate
and adaptive immune responses. Hepatocellular carcinoma (HCC) is the most
frequent primary malignancy of the liver and around 1%
of all deaths in the world were related to HCC. In light of
this, HCC is one of the cancers with the highest mortality
rates worldwide [1–4]. HCC is now the third leading
cause of cancer deaths in the Asia-Pacific region [5]. In
particular, 55% of the cases worldwide are estimated in
China [6]. Due to multiple etiologic factors affect HCC,
it makes HCC an extremely complex condition associated
with a poor prognosis and its underlying mechanisms
remain unknown. The risk of HCC development is mainly associated
with chronic hepatitis B virus (HBV) infection with
cirrhosis [7, 8]. The prognosis of HBV patients is closely A variety of chronic liver diseases activate the TLR7
signaling pathway. In turn, TLR7 signaling pathway plays www.impactjournals.com/oncotarget Oncotarget 63856 Although the roles of TLRs have been reported in
inflammation and cancer, TLR7 in HCC has not been much
explored. To gain a better understanding of the role of TLR7
in HCC proliferation and migration, this study focused
on TLR7 and HCC associated with the dysregulation of
intercellular signaling pathway caused by changes in raft-
associated TLR7 expression in the microenvironment of
the tumors. Does the increased TLR7 signaling root in
accumulated trafficking of TLR7 to lipid rafts? Alternatively,
is there a direct relationship among cholesterol content,
lipid raft content, and the hyper-responsiveness to TLR7
agonist? Finally, does TLR7-specific agonist also induce
increased inflammatory signaling in HCC? For this purpose,
we investigated the expression of TLR7 in vivo in the liver
tissues of HCC patients and in vitro in the hepatoma cell
line HepG2. Furthermore, we investigated that whether
lipid rafts associated with intensified TLR7 signaling in
HCC. TLR7 expression in human HCC tissues TLR7 was overexpressed in human HCC compared
with CHB and LC tissues. TLR7 was predominantly
membranous and perinuclear in malignant liver cells
but was expressed in the hepatocyte cytoplasm as the
scattered spots in normal, CHB and LC tissues (Figure
1A). The percentage of membranous TLR7 expression
in HCC was significantly higher than Normal, CHB
and LC patients (P = 0.001). Specifically, membranous
expression of TLR7 in HCC patients was 94.12% but
lacking in 95.24% of Normal livers, 86.96% of CHB and
72.22% of LC patients. Faint membranous TLR7 was
detected in 13.04% of CHB and 27.78% of LC (Table 1)
tissues. In the previous study, we found caveolin-1 and
flotillin-1 which were two typical lipid raft resident
proteins hyper-expressed in HCC compared to adjacent
noncancerous tissues (ANT), and confirmed that lipid
rafts played an essential role in HCC [22]. Cholesterol is
usually considered acting as a gasket between sphingolipid
hydrocarbon chains and functioning as dynamic glue
to make lipid rafts gather together [23]. Methyl-β-
cyclodextrin (MβCD) can deplete membrane cholesterol
and act as a lipid raft disruptor [24]. As a metabolite of
ginseng saponins, 20S-protopanaxadiol (aPPD), possessing
of cholesterol-like structure, can inhibit Akt activity and
lead to apoptosis in U87MG cells by interacting with lipid
rafts [24]. TLR7 is one of membrane-bound receptors
localized intracellularly on endosomal membranes which
act as foci for signaling complexes. Dysregulation of
endosomal signaling is causal to tumorigenesis [25],
and activation or inhibition of TLR7 is also familiar
to affect cell proliferation [26]. In addition, excessive
expression of TLR7 has been demonstrated in a variety of
malignant tumors [27, 28]. As a selective TLR7 agonist,
gardiquimod is a new small synthetic antiviral molecule of
imidazoquinoline, and its activity is 10 times stronger than
imiquimod [29]. Therefore, targeting TLR7 and modulating
TLR7 signaling have emerged as a novel therapeutic
approach for the prevention and treatment of cancer. Membranous TLR7 was observed in 94% of HCC
in score 2. Furthermore, membranous TLR7 was observed
in 41% of liver cancer with cirrhosis and in 28% of LC
patients in score 1 (Figure 1C). In general, the above
results suggested that the expression level of membranous
TLR7 in HCC patients was significantly higher than
that in patients with liver cirrhosis (P < 0.001), while
membranous TLR7 was rarely expressed in Normal liver. TLR7 expression in human HCC tissues The expression of TLR7 in the CHB, LC and HCC
groups significantly increased compared with the normal
group (Figure 1E). A rising trend of TLR7 expression was
found in the three patient groups. Correlation of TLR7 and Ki-67 in human HCC
tissues INTRODUCTION Finally, we investigated downstream NFκB pathway
which involved exaggerated raft-associated TLR7 signaling
in HCC. a crucial role in sustained chronic hepatic inflammation
[12]. Inappropriate activation of TLR7 can also lead to
inflammatory-related cancers. TLR7 initiates the myeloid
differentiation response protein 88 (MyD88)-dependent
signaling pathway [13]. The ligation of TLR7 and MyD88
results in the phosphorylation of IL1-receptor associated
kinase which in turn induces activation of the NFκB
pathway. Activation of downstream NFκB pathway
initiates the production of pro-inflammatory cytokines
such as TNF-α, IL-2, IL-6, IL-8, and especially IFNs-α/β. The activation of the MyD88-dependent TLR7 pathway
eventually results in the stimulation of NFκB, which in
turn leads to inflammation and carcinogenesis [14, 15]. l
g
[
]
When TLRs encounter their ligands, activation of
TLRs is largely governed by their subcellular localization
[13]. The binding of TLR7 and MyD88 results in the
assemblies gathering in the particular microdomains of the
cell surface, named as lipid rafts, enriching in cholesterol
and sphingolipid and functioning as activation platforms for
a variety of molecules and participate in many biological
processes, such as signal transduction and membrane
trafficking [16]. For instance, TLR4 transiently trafficks
to lipid rafts after stimulation of macrophages with
lipopolysaccharide (LPS), which results in subsequent
production of chemokines and inflammatory cytokines
[17, 18]. Besides, lipid rafts are also regarded as the
requirement for NFκB activation induced by IL-1, LPS,
CD40, or CD3/CD28 [19–21], and the destruction of lipid
rafts leads to the restraint of NFκB activation induced by
these stimuli. Enough these studies show that lipid rafts play
an increasingly vital role in the activation of TLR signaling. Flow cytometry analysis of TLR7 expression in
peripheral blood Furthermore, significantly higher MFI (P < 0.05) of TLR7
in HCC group was observed when compared to CHB
group. Immune cell phenotypes were identified and
evaluated by flow cytometry for the further observation
on TLR7-related immune cell infiltrates. We found that
NK cells and pDC were in association to TLR7 expression
levels. The MFI ± SEM values of CD56 for NK cells
were: Normal = 277.55 ± 212.21, CHB = 308.93 ± 250.96,
LC = 383.33 ± 23.57, and HCC = 653.74 ± 180.43. The expression level of CD56+ TLR7 in HCC group
was significantly higher (P < 0.05) than that in Normal To further understand pathogen-host interactions
and predict development stages, we characterized TLR7
expression in peripheral blood of patients with chronic
HBV infection and HBV-related LC and HCC. The
mean fluorescence intensity (MFI) values of TLR7 in
HCC group were significantly higher than Normal group
(Figure 2A). The MFI±SEM values were (Figure 2B):
Normal = 124.81 ± 21.07, CHB = 121.03 ± 25.26,
LC = 130.52 ± 15.48, and HCC = 191.77 ± 67.62. Oncota ge
63858
w impactjournals com/oncotarget
Figure 1: TLR7 expression in human HCC tissues. Immunohistochemistry showing (A) TLR7 was highly expressed on ce
membrane and perinuclei only in malignant hepatocytes but as the scattered spots in cytoplasm of hepatocytes of Normal, CHB an
LC. (B) Correlation of TLR7 and Ki-67 expression in HCC. Serial sections from the same paraffin block were stained with differe
ntibodies. Hepatic expression of the proliferative marker Ki-67 was high in nuclei. Membranous TLR7 expression was also high
malignant hepatocytes. (C) Percentage of cases from the validation set demonstrating no (0), weak (1) or high (2) expression of TLR7. (D
Correlation of TLR7 staining with Ki-67 index in HCC. (E) Western blotting analysis of TLR7 in liver tissue lysates. Bands were observe
t approximately 120 kDa and normalized by β-actin. There was a significant increase in the TLR7 expression of HCC group compare
with Normal control. *P < 0.05, results were representative of three independent experiments. Figure 1: TLR7 expression in human HCC tissues. Immunohistochemistry showing (A) TLR7 was highly expressed on cell
membrane and perinuclei only in malignant hepatocytes but as the scattered spots in cytoplasm of hepatocytes of Normal, CHB and
LC. (B) Correlation of TLR7 and Ki-67 expression in HCC. Serial sections from the same paraffin block were stained with different
antibodies. Correlation of TLR7 and Ki-67 in human HCC
tissues TLR7 expression was significantly correlated with
Ki-67 proliferation index (r = 0.3; P < 0.05) in the human
HCC tissues (Figure 1B and 1D). www.impactjournals.com/oncotarget Oncotarget 63857 Flow cytometry analysis of TLR7 expression in
peripheral blood Hepatic expression of the proliferative marker Ki-67 was high in nuclei. Membranous TLR7 expression was also high in
malignant hepatocytes. (C) Percentage of cases from the validation set demonstrating no (0), weak (1) or high (2) expression of TLR7. (D)
Correlation of TLR7 staining with Ki-67 index in HCC. (E) Western blotting analysis of TLR7 in liver tissue lysates. Bands were observed
at approximately 120 kDa and normalized by β-actin. There was a significant increase in the TLR7 expression of HCC group compared
with Normal control. *P < 0.05, results were representative of three independent experiments. www.impactjournals.com/oncotarget Oncotarget 63858 Table 1: Tissue clinical data and TLR7 staining Table 1: Tissue clinical data and TLR7 staining
Variables
% TLR7 negative in cytoplasm
(n = 57)
% TLR7 positive on cell
membrane
(n = 73)
P-value
Age (mean ± SD)
46.96 ± 10.49
49.51 ± 9.44
Sex
Female (n = 43)
60.47
39.53
0.000
Male (n = 87)
35.63
64.37
Histologic grade
G1 (n = 11)
0
100
0.000
G2 (n = 48)
6.25
93.75
G3 (n = 9)
11.11
88.89
HBV status
HBsAg + / HBeAg + (n = 50)
32
68
1.000
HBsAg + / HBeAg −(n = 59)
35.59
64.41
Tissue type
Normal (n = 21)
95.24
4.76
0.001
CHB (n = 23)
86.96
13.04
LC (n = 18)
72.22
27.78
HCC (n = 68)
5.88
94.12 Table 1: Tissue clinical data and TLR7 staining Table 1: Tissue clinical data and TLR7 staining HBV status HBsAg + / HBeAg + (n = 50)
HBsAg + / HBeAg −(n = 59) Tissue type Normal (n = 21)
CHB (n = 23)
LC (n = 18)
HCC (n = 68) group. The MFI ± SEM values of CD11c for pDC were:
Normal = 231.31 ± 70.30, CHB = 198.25 ± 75.73,
LC = 180.59 ± 52.41, and HCC = 127.33 ± 24.73. The
CD11c+ TLR7 expression in HCC was downregulated
compared with Normal group (P < 0.05) (Figure 2C). group. The MFI ± SEM values of CD11c for pDC were:
Normal = 231.31 ± 70.30, CHB = 198.25 ± 75.73,
LC = 180.59 ± 52.41, and HCC = 127.33 ± 24.73. The
CD11c+ TLR7 expression in HCC was downregulated
compared with Normal group (P < 0.05) (Figure 2C). Lipid rafts are engaged in TLR7 signaling
enhancement in human HCC tissues We isolated lipid rafts and nonrafts from normal
and HCC liver tissues using a simplified non-detergent
isolation method [22, 24]. Lipid raft and non-raft
fractions were respectively confirmed based on the
relative distribution of raft markers, caveolin-1 and
flotillin-1 and the non-raft marker, clathrin. Results of the
distribution of lipid rafts along gradient fractions were
shown in Figure 3A, in which caveolin-1 and flotillin-1
as the lipid raft resident proteins were mainly presented
in fraction 5 and 6 of both tumor and Normal tissues. Furthermore, levels of caveolin-1 and flotillin-1 proteins
in HCC samples elevated in fraction 5 and 6 while
declined in non-raft fractions compared with Normal
tissues. The expression of TLR7 was mainly located in
non-raft membrane fractions in Normal tissues. Upon
cancerization, a tendency toward increased translocation
of TLR7 into lipid rafts was observed in HCC tissues
(Figure 3A). The co-expression of TLR7 and lipid rafts
was analyzed by double-labeling immunohistochemistry. Flow cytometry analysis of TLR7 expression in
peripheral blood Lipid raft expression of TLR7 on the plasma membrane
was shown only in HCC tissues but as the scattered spots
in cytoplasm of hepatocytes in Normal, CHB and LC
tissues (Figure 3B). Collectively, these data suggest that
hepatocarcinogenesis results in a significant increase in
lipid raft contents, which concentrate and recruit more
TLR7 through augmented translocation or trafficking to
lipid rafts, leading to hyper-activation of downstream
signaling transduction and exaggerated proinflammatory
cytokine expression. The TLR7 agonist gardiquimod promotes
proliferation and migration of HepG2 cells non-raft fractions of normal and HCC groups (Figure 4A
and 4B). Taken together, our data suggested that an
increase in lipid raft content of HCC likely due to a
striking increase in cholesterol content of this fraction. To observe the effect of TLR7 agonist on hepatocyte
proliferation, HepG2 cells were treated with gardiquimod
for 4 h and 24 h respectively. We found that gardiquimod
promoted the proliferation of hepatocytes with a greater
effect at the dosage of 1.25 μg/ml, and 24 h seemed to be
more efficient than 4 h (Figure 5A). We thus chose a 1μg/ml
dose for subsequent experiments. To determine whether rapid alteration of cholesterol
content changes lipid raft content and TLR7 expression, we
incubated HepG2 cells with 10 mM MβCD or MβCD loaded
with 1 μg/ml of gardiquimod for 1h and then lipid raft and
non-raft fractions were pooled by centrifugation (Figure 4C). We found that the fraction 5 of HepG2 cells had the highest
level of cholesterol. Only MβCD depleted cholesterol,
but TLR7 agonist gardiquimod resulted in an increase of
cholesterol content in lipid rafts and TLR7 expression. To further observe whether gardiquimod could
enhance the hepatocyte migration, we measured the
cell migration rates of HepG2 cells treated with 1 μg/ml Figure 3: Increased TLR7 in lipid rafts of human HCC tissues. (A) Lipid raft and non-raft microdomains were isolated using
a non-detergent method and analyzed for caveolin-1 (lipid raft marker), flotillin-1 (lipid raft marker), clathrin (non-raft marker) and
TLR7 distribution by western blotting. The graph showed the expression of caveolin-1, flotillin-1, clathrin and TLR7 in HCC relative to
Normal tissue. *P < 0.05, lipid rafts compared with the non-rafts. (B) The co-expression of TLR7 and lipid rafts was analyzed by double
immunohistochemistry. The co-expression of TLR7 and lipid rafts was located in the cell membrane only in HCC group but as the scattered
spots in cytoplasm of live cells in Normal, CHB, LC groups. Interestingly, margination orientation of TLR7/Caveolin-1 or TLR7/Flotillin-1
from cytoplasm to membrane was observed in LC tissues. The microphotographs were magnified 1000 times. Figure 3: Increased TLR7 in lipid rafts of human HCC tissues. (A) Lipid raft and non-raft microdomains were isolated using
a non-detergent method and analyzed for caveolin-1 (lipid raft marker), flotillin-1 (lipid raft marker), clathrin (non-raft marker) and
TLR7 distribution by western blotting. Increased cholesterol in lipid rafts of human
HCC tissues and HepG2 cells To determine why lipid raft contents increased in
HCC, we isolated lipid rafts and nonrafts from Normal
and HCC liver tissues using a non-detergent isolation
method. We fractionated the gradient solution into 12
fractions of 1 ml per tube. Using raft markers (flotillin-1
and caveolin-1) and non-raft marker (clathrin), we
designated the fractions 4–6 as lipid rafts and the fractions
8–10 as nonrafts (Figure 3A). Interestingly, we found
that each lipid raft fraction contained more cholesterol in
HCC group than normal group (Figure 4A), resulting in
an average 21.27 ± 2.78% greater cholesterol content for
the whole lipid raft fraction (i.e., fractions 4–6) compared
with the same fractions from normal group (Figure 4B). On the other hand, cholesterol content was similar in www.impactjournals.com/oncotarget Oncotarget 63859 63860
urnals.com/oncotarget
ow cytometry analysis of TLR7 expression in peripheral blood. To evaluate the efficacy of the e
ed control was used. At least 3000 cells were assessed to calculate the mean fluorescence intensity values (MFI)
R7 MFI. Data were shown as mean ± SEM. *P < 0.05 compared with the Normal group. (B) Expression of TL
lysis. (C) The immune cells were identified and evaluated by flow cytometry for the further observation on
nfiltrates. NK cells and pDC were in association to TLR7 expression levels. CD3, for T cells; CD8, for cyt
ells; CD56, for NK cells or NKT cells; CD16, for granulocytes; CD14, for mononuclear phagocytes; CD11c, Figure 2: Flow cytometry analysis of TLR7 expression in peripheral blood. To evaluate the efficacy of the experiment, an
isotype-matched control was used. At least 3000 cells were assessed to calculate the mean fluorescence intensity values (MFI). (A) Statistical
analyses of TLR7 MFI. Data were shown as mean ± SEM. *P < 0.05 compared with the Normal group. (B) Expression of TLR7 using flow
cytometry analysis. (C) The immune cells were identified and evaluated by flow cytometry for the further observation on TLR7-related
immune cell infiltrates. NK cells and pDC were in association to TLR7 expression levels. CD3, for T cells; CD8, for cytotoxic T cells;
CD19, for B cells; CD56, for NK cells or NKT cells; CD16, for granulocytes; CD14, for mononuclear phagocytes; CD11c, for pDC. www.impactjournals.com/oncotarget Oncotarget 63860 The TLR7 agonist gardiquimod promotes
proliferation and migration of HepG2 cells The graph showed the expression of caveolin-1, flotillin-1, clathrin and TLR7 in HCC relative to
Normal tissue. *P < 0.05, lipid rafts compared with the non-rafts. (B) The co-expression of TLR7 and lipid rafts was analyzed by double
immunohistochemistry. The co-expression of TLR7 and lipid rafts was located in the cell membrane only in HCC group but as the scattered
spots in cytoplasm of live cells in Normal, CHB, LC groups. Interestingly, margination orientation of TLR7/Caveolin-1 or TLR7/Flotillin-1
from cytoplasm to membrane was observed in LC tissues. The microphotographs were magnified 1000 times. www.impactjournals.com/oncotarget Oncotarget 63861 eased cholesterol in lipid rafts of human HCC tissues and HepG2 cells. (A) Lipid raft an
ng a non-detergent method. Cholesterol concentration in each fraction of Normal and HCC tissues
cholesterol assay kit. Results were representative of at least three patients in each group. (B) Data
(fractions 4–6) and non-raft (fractions 8–10) cholesterol concentration. *P < 0.05, compared with Norm
h 10 mM MβCD or 1μg/ml gardiquimod for 1h and then lipid raft and non-raft fractions were poole
of cholesterol was detected in fraction 5 in both HepG2 cells and tissue samples. Only MβCD depl
ulted in an increase in lipid rafts. Results were representative of three independent experiments. Figure 4: Increased cholesterol in lipid rafts of human HCC tissues and HepG2 cells. (A) Lipid raft and non-raft fractions
were isolated using a non-detergent method. Cholesterol concentration in each fraction of Normal and HCC tissues was measured with
an Amplex Red cholesterol assay kit. Results were representative of at least three patients in each group. (B) Data from panel A were
presented as raft (fractions 4–6) and non-raft (fractions 8–10) cholesterol concentration. *P < 0.05, compared with Normal. (C) HepG2 cells
were treated with 10 mM MβCD or 1μg/ml gardiquimod for 1h and then lipid raft and non-raft fractions were pooled by centrifugation. The highest level of cholesterol was detected in fraction 5 in both HepG2 cells and tissue samples. Only MβCD depleted cholesterol, but
gardiquimod resulted in an increase in lipid rafts. Results were representative of three independent experiments. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 63862 gardiquimod or gardiquimod loaded with aPPD for
24 h using scratch assay in vitro. We also measured the
cell mortality of HepG2 cells treated with different
concentration of aPPD at different time points to determine
the toxic dose of aPPD to the cells. There was no significant
increase in the mortality of HepG2 cells with 10 μM aPPD
treatment both at 4 h (5.20 ± 2.45%, P = 0.196) and at 24 h
(12.63 ± 13.57%, P = 0.089). We observed a significant
cell death of HepG2 cells at 24 h loaded with 20 μM aPPD
(42.78 ± 14.78%, P = 0.002) (Figure 5B). Thus, we chose
a 10 μM dose for subsequent experiments. Our previous
results showed that aPPD inhibited tumor cell migration
[24]. Migration rate of HepG2 cells was significantly
upregulated after gardiquimod treatment in Figure 5C
and 5D (137.34 ± 12.46% of control, P < 0.05), but
effectively downregulated after aPPD treatment compared
with gardiquimod treatment (80.34 ± 11.19%, P < 0.001). HCC patients and in HepG2 cells. Importantly, patients with
viral hepatitis or cirrhosis rarely express TLR7; (2) TLR7
activation is involved with marked increase in tumor cell
proliferation and migration; (3) lipid rafts are positively
associated with enhanced TLR7 signaling in HCC; (4) an
increase in lipid raft content of HCC is likely attributable
to a striking increase in cholesterol accumulation; (5)
downstream NFκB pathway is involved with enhanced raft-
associated TLR7 signaling in HCC. These results indicate
that TLR7 may be a target for the treatment of HCC in
future. Inhibiting TLR7 with antagonists, like aPPD,
becomes a focus for their potential use as a novel therapeutic
approach for HCC based on the results of our study. pp
y
TLRs are usually involved in innate immune
response and TLR signaling is closely related to various
chronic liver diseases. Especially, TLR7 is excessive
expression in many malignancies [30–32]. However,
there is little research on the role of TLR7 in HCC. In the
present study, TLR7 expression was higher in human HCC
cases than that with viral hepatitis, cirrhosis and Normal
controls. Mohamed et al. had previously reported the high
expression of TLR7 in HCC patients [14]. In their study,
TLR7 tended to gather in the perinuclei of hepatoma cells. However, we found that TLR7 was mainly expressed in
the cell membrane and perinuclei. www.impactjournals.com/oncotarget The unusual localization
within HCC cells may be related to the mitogenic activity
of TLR7 contributing towards hepatocarcinogenesis. Then
the effect of activating TLR7 was further confirmed in
human HCC cell line using a TLR7 agonist gardiquimod,
which can substantially induce cell proliferation and
migration. On the one hand, overactive TLR signal
provides a microenvironment that is necessary for
malignant cell proliferation and migration. On the other
hand, TLR antagonists can also be used to inhibit cancer
cell growth. This leads to the further understanding that
the activation of TLRs acts as a “double-edged sword” in
the progression or treatment of cancer. Unlike results from
our studies, some papers showed that TLR7 expression
down-regulated in HCC [7, 33]. Lin et al. found the
down-regulated expression of TLR7 through cancerous
and non-cancerous liver tissues from HBV/HCV-related
HCC [7]. We studied the expression of TLR7 in normal
liver tissues by resecting parts of liver from hepatic cyst,
calculus of intrahepatic duct, gallbladder polyps et al. and
HBV-related HCC. Most of adjacent non-cancerous tissues
had cirrhosis background, so the expression of TLR7 may
differ in normal livers and non-cancerous liver tissues. i Enhanced lipid raft-associated TLR7 signaling
results in the activation of downstream NFκB
signaling pathway in HepG2 cells TLR7 was shown to translocate from the
endoplasmic reticulum to the endosome upon stimulation
by specific agonists, resulting in activation of NFκB
pathway [13]. To determine whether the downstream
NFκB pathway is upregulated in gardiquimod-treated
HepG2 cells, we stimulated HepG2 cells with 10 mM
MβCD or MβCD loaded with 1 μg/ml gardiquimod for
1h before isolating cell lysates for immunoblotting. As
shown in Figure 6A and 6B, we observed TLR7, MyD88
and NFκB content were significantly declined in MβCD-
treated cells (P < 0.05), but a trend of increased content in
gardiquimod-treated cells, especially the MyD88 content
(P < 0.05). Because gardiquimod stimulation was shown
not only to induce trafficking of TLR7 to the endosome
but also to induce translocation of TLR7 to lipid rafts,
we then tested the hypothesis that gardiquimod may
induce more TLR7 translocation to lipid rafts. Next,
we pooled lipid raft (fractions 4–6) fractions isolated
from MβCD ± gardiquimod treatment and performed
immunoprecipitation (Figure 6C and 6D) to visualize
and quantify the translocation of TLR7 to lipid rafts. We
found that gardiquimod stimulation significantly triggered
increased translocation of TLR7 to lipid raft fractions in
gardiquimod-treated cells vs. MβCD-treated cells. These
combined results suggest that the appreciably increased
lipid raft content increases MyD88-dependent activation
of plasma membrane and intracellular TLR7 through
augmented translocation or trafficking to lipid rafts,
resulting in activation of downstream NFκB pathway and
exaggerated pro-inflammatory cytokine expression. The main finding in this study was on TLR7
mediated mechanism of HCC development and
progression via lipid rafts on the cell membrane. Lipid
rafts are liquid-ordered membrane microdomains
enriched in highly ordered saturated sphingolipids and
cholesterol, which are conducive to lateral movement. Lipid rafts are not only considered as a means to explain
the spatial segregation of certain signaling pathways
deriving from the cell surface, but also provide the DISCUSSION Conclusions can be drawn from the above results
that (1) the expression of TLR7 is obviously increased in www.impactjournals.com/oncotarget Oncotarget 63863 e 5: Gardiquimod promotes proliferation and migration of HepG2 cells. (A) HepG2 cells were treated
ntration of gardiquimod for 4 h and 24 h respectively, and the proliferation rate was detected by MTT assay. *P < 0.0
< 0.001 compared to the untreated control (n = 3). (B) HepG2 cells were treated with different concentration of aPP
espectively, and the cell mortality was detected by MTT assay. *P < 0.05, **P < 0.01 compared to the untreated c
igration of cells in culture dishes. The edges (dotted lines) of cultured HepG2 cells at 0 and 24 h after scratch were
line and the migration line, respectively. The migration area was calculated as regions between the injury and migra
ification of the cell migration rate. The migration rate of HepG2 cells was effectively reduced with the treatment of aP
ardiquimod. The data were the averages of two repeated experiments with quadruplets for each time, *P < 0.05, **P Figure 5: Gardiquimod promotes proliferation and migration of HepG2 cells. (A) HepG2 cells were treated with different
concentration of gardiquimod for 4 h and 24 h respectively, and the proliferation rate was detected by MTT assay. *P < 0.05, **P < 0.01,
***P < 0.001 compared to the untreated control (n = 3). (B) HepG2 cells were treated with different concentration of aPPD for 4 h and
24 h respectively, and the cell mortality was detected by MTT assay. *P < 0.05, **P < 0.01 compared to the untreated control (n = 3). (C) Migration of cells in culture dishes. The edges (dotted lines) of cultured HepG2 cells at 0 and 24 h after scratch were shown as the
injury line and the migration line, respectively. The migration area was calculated as regions between the injury and migration lines. (D)
Quantification of the cell migration rate. The migration rate of HepG2 cells was effectively reduced with the treatment of aPPD compared
with gardiquimod. The data were the averages of two repeated experiments with quadruplets for each time, *P < 0.05, **P < 0.01. DISCUSSION www.impactjournals.com/oncotarget Oncotarget 63864 Because the membrane-bound TLRs share many
properties and similar activation mechanisms, we assume
that the occurrence and progression of HCC lead to
increased translocation or recruitment of all MyD88-
dependent TLRs into lipid rafts, which results in the
constitutive activation of NFκB to produce augmented
proinflammatory cytokines and promote cell proliferation
and survival. Previous reports have shown that lipid rafts
are vital to the inflammatory cytokine-mediated NFκB
activation. For instance, the activation of TNF-α results
TNFR in translocating into lipid rafts, and disrupting
lipid rafts or interfering with the lipid raft composition
could prevent TNF-α-associated TNFR from trafficking
into lipid rafts and reduce the enrollment of RIP into the
engaged receptor, indicating that lipid raft organization is
essential to TNF-α-induced NFκB stimulation [16, 34]. In accordance with these previous reports, we found that
an increase in cholesterol accumulation led to the hyper-
expression of caveolin-1 and flotillin-1, consequently
increased the number of lipid raft microdomains, whereas necessary microenvironment so that certain specialized
signaling events can take place, such as the innate immune
recognition. TLRs as the crucial “sensing” apparatus of
the innate immune system are recruited into lipid raft
microdomains in response to ligands. Subsequently,
sphingolipids, cholesterol and TLRs coalesce together and
form the nanoscale assemblies as signaling platforms to
stabilize and transduce signals that lead to innate immune
activation. Our previous study suggested that the two
typical lipid raft resident proteins, namely, caveolin-1
and flotillin-1were overexpressed in HCC compared to
adjacent noncancerous tissues [22]. The present study
has found direct evidence for the involvement of lipid
rafts in the relationship of enhanced TLR7 signaling and
the occurrence and progression of HCC by increasing
cholesterol enrichment in lipid rafts. With these findings
in mind, we hypothesized that the increased lipid raft
content in HCC might result in enhanced TLR7 trafficking
into rafts and our results appropriately supported this
hypothesis (Figures 4 and 6). Figure 6: Enhanced lipid raft-associated TLR7 signaling results in the activation of downstream NFκB pathway in
HepG2 cells. (A) HepG2 cells were incubated with 10 mM MβCD or 1 μg/ml gardiquimod for 1 h. Cell lysates were harvested and
the proteins were analyzed by immunoblotting with the indicated antibodies. (B) TLR7, MyD88, NFκB were quantified and normalized
to β-actin. Data were presented as mean ± SEM; n = 3. Statistically significant differences between groups were indicated (*P < 0.05). DISCUSSION TLR7 PE-conjugated monoclonal antibody and mouse
IgG2A PE-conjugated isotype control were purchased from
R&D Systems, Inc. (Minneapolis, MN, USA). BV510
anti-human CD14 antibody and BV421 anti-human CD16
antibody were purchased from BD Biosciences (San Jose,
CA, USA). APC/Cy7 anti-human CD3 antibody, PerCP/
Cy5.5 anti-human CD8a antibody, FITC anti-human
CD56 antibody, PE/Cy7 anti-human CD11c antibody
and APC anti-human CD19 antibody were purchased
from Biolegend (San Diego, CA, USA). Protease
inhibitor cocktails were obtained from Roche Molecular
Biochemicals (Mannheim, DE). BCA protein assay kit
and PVDF membranes were purchased from Bio-Rad
(Hercules, CA). MβCD, media (Dulbecco’s modified
Eagle’s medium, DMEM), MTT and other chemicals
were purchased from Sigma (St. Louis, MO, USA). Gardiquimod was purchased from Santa Cruz (California,
USA). aPPD was provided by Shanghai Innovative
Research Centre of Traditional Chinese Medicine
(Shanghai, China). The compound was 97.9% pure as
measured by HPLC analysis. depletion of cholesterol disrupted lipid raft formation. Therefore, cholesterol may promote the formation of lipid
rafts and facilitate the recruitment of TLR7 and its ligands
to lipid rafts, thus contributing to constitutive NFκB
activation. This is one of the many pieces of supportive
evidence for the hypothesis that TLR-driven inflammation
is linked with furthering malignant development. In an
ever-growing field, more research in the TLR signaling
pathways have reflected about our understanding of the
involvement of TLRs in cancer [13, 14, 27]. Our previous study demonstrated that aPPD, the
final metabolite of ginseng saponin, was a high efficient
breaker for lipid rafts. Unlike MβCD, aPPD altered the
contents of resident proteins in the lipid rafts rather than
cholesterol content [24]. Because the structure of aPPD is
more similar to cholesterol, it would not be surprising if it
functions as a stronger raft disruptor through intercalating
itself into the lipid rafts to bring about changes in the
microenvironment of the membrane, which in turn causes
an alteration of the protein constituent of lipid rafts. Our results demonstrated that aPPD inhibited HepG2
cell proliferation and migration. More interestingly, the
migration rate of HepG2 cells induced by gardiquimod
(a TLR7 agonist) was efficiently decreased after
aPPD treatment (Figure 5). These findings suggested
that aPPD may serve as a potent TLR7 antagonist for
pharmacological intervention in tumor therapy. Patients Human liver tissues (formalin-fixed paraffin-
embedded tissues and cryostat tissues) and peripheral
blood were used in this study. The study included patients
with chronic hepatitis B (CHB) (n = 23), HBV-related
liver cirrhosis (LC) (n = 18), HBV-related HCC (n = 68)
and normal liver (n = 21). The patients were enrolled at
Beijing 302 Hospital (Beijing, China) from January 2014
to December 2014. The study was conducted according
to the guidelines of the Declaration of Helsinki. Informed
consent was obtained from each patient, and the study
protocol was approved by the Ethics Committee of Beijing
302 Hospital. The clinical and pathological characteristics
of the patients are listed in Table 1. In conclusion, certain viral products, as TLR
ligands, anchoring in the cell membrane directly activate
TLR7 signaling. Once the binding of TLR7 and ssRNA,
the polymer subsequently aggregates in lipid raft
microdomains at the endosomal membrane and induces
the intracellular signaling cascades which contribute
to activation of NFκB and production and secretion of
proinflammatory cytokines that promote the proliferation
and formation of cancerous cells. To our knowledge,
this study is the first to provide new insights into the
pathogenesis of HCC that lipid rafts promote liver cancer
cell proliferation and migration by up-regulation of TLR7
expression on HCC patients and cell lines. In summary,
our data suggest that inhibition of the signaling pathway
involved raft-associated TLR7 by using antagonists could
serve as a novel approach to prevent the occurrence and
progression of HCC. The diagnostic criteria conformed to “The guideline
of prevention and treatment for chronic hepatitis B” [35]. All LC patients were in Child class B and C by Child-
Pugh score evaluation. HCC patients were confirmed
by liver tissue biopsy. All normal were HBsAg-negative
patients with other liver diseases. MATERIALS AND METHODS Human HepG2 cells were cultured in DMEM
supplemented with 10% FBS, 100 U/ml penicillin
and 100 mg/ml streptomycin, at 37°C in a humidified
atmosphere containing 5% CO2 and fed every 2–3 days. A stock solution of 50 mM aPPD was prepared in 100%
ethanol and diluted to proper concentrations in DMEM
immediately before each experiment. Drug-treatment cells
were pretreated with DMEM for 4 h at 37°C, followed by
the addition of 10 mM MβCD, 1 μg/ml gardiquimod or
10 μM aPPD for 1 h. DISCUSSION (C) HepG2 cells were treated with 10 mM MβCD or 1 μg/ml gardiquimod for 1h and then lipid rafts (fractions 4–6) were pooled, and
immunoprecipitated with anti-flotillin-1 polyclonal antibody, followed by immunoblotting with anti-TLR7 monoclonal antibody. Results
were representative of three independent experiments. (D) Lipid rafts isolated as described in panel C were immunoprecipitated with
anti-TLR7 monoclonal antibody, followed by immunoblotting with anti-flotillin-1 polyclonal antibody. Results were representative of three
independent experiments. pid raft-associated TLR7 signaling results in the activation of downstream NFκB pathway in 6: Enhanced lipid raft-associated TLR7 signaling results in the activation of downstream N Figure 6: Enhanced lipid raft-associated TLR7 signaling results in the activation of downstream NFκB pathway in
HepG2 cells. (A) HepG2 cells were incubated with 10 mM MβCD or 1 μg/ml gardiquimod for 1 h. Cell lysates were harvested and
the proteins were analyzed by immunoblotting with the indicated antibodies. (B) TLR7, MyD88, NFκB were quantified and normalized
to β-actin. Data were presented as mean ± SEM; n = 3. Statistically significant differences between groups were indicated (*P < 0.05). (C) HepG2 cells were treated with 10 mM MβCD or 1 μg/ml gardiquimod for 1h and then lipid rafts (fractions 4–6) were pooled, and
immunoprecipitated with anti-flotillin-1 polyclonal antibody, followed by immunoblotting with anti-TLR7 monoclonal antibody. Results
were representative of three independent experiments. (D) Lipid rafts isolated as described in panel C were immunoprecipitated with
anti-TLR7 monoclonal antibody, followed by immunoblotting with anti-flotillin-1 polyclonal antibody. Results were representative of three
independent experiments. www.impactjournals.com/oncotarget Oncotarget 63865 TLR7 PE-conjugated monoclonal antibody and mouse
IgG2A PE-conjugated isotype control were purchased from
R&D Systems, Inc. (Minneapolis, MN, USA). BV510
anti-human CD14 antibody and BV421 anti-human CD16
antibody were purchased from BD Biosciences (San Jose,
CA, USA). APC/Cy7 anti-human CD3 antibody, PerCP/
Cy5.5 anti-human CD8a antibody, FITC anti-human
CD56 antibody, PE/Cy7 anti-human CD11c antibody
and APC anti-human CD19 antibody were purchased
from Biolegend (San Diego, CA, USA). Protease
inhibitor cocktails were obtained from Roche Molecular
Biochemicals (Mannheim, DE). BCA protein assay kit
and PVDF membranes were purchased from Bio-Rad
(Hercules, CA). MβCD, media (Dulbecco’s modified
Eagle’s medium, DMEM), MTT and other chemicals
were purchased from Sigma (St. Louis, MO, USA). Gardiquimod was purchased from Santa Cruz (California,
USA). aPPD was provided by Shanghai Innovative
Research Centre of Traditional Chinese Medicine
(Shanghai, China). The compound was 97.9% pure as
measured by HPLC analysis. Flow cytometry A total of 6 × 106 − 10 × 106 peripheral blood was
collected. The cells were then stained with mouse IgG2A
anti-human TLR7 PE-conjugated monoclonal antibody,
mouse IgG2A PE-conjugated isotype control, BV510
anti-human CD14 antibody, BV421 anti-human CD16
antibody, APC/Cy7 anti-human CD3 antibody, PerCP/
Cy5.5 anti-human CD8a antibody, FITC anti-human
CD56 antibody, PE/Cy7 anti-human CD11c antibody
and APC anti-human CD19 antibody. Stained cells were
analyzed on a Canto™ II machine (BD Biosciences, San
Jose, CA, USA). Data was analyzed using FlowJo 7.6.1
software (Stanford University, Stanford, CA, USA). Immunoblotting and immunoprecipitation Western blotting was performed using specific
antibodies against: TLR7, caveolin-1 (as lipid raft
marker), flotillin-1 (as lipid raft marker), clathrin
(as non-raft marker), MyD88, NFκB, β-actin. Flotillin-1
was immunoprecipitated with a flotillin-1 polyclonal
antibody from treated HepG2 cells, followed by
immunoblotting with antibody against TLR7. A Pierce
co-immunoprecipitation kit (Thermo Scientific, Rockford,
lL, USA) was used according to the manufacturer’s
protocol. TLR7 was immunoprecipitated with a TLR7
monoclonal antibody and then immunoblotted with
antibody against flotillin-1. Blots were developed using
HRP-linked secondary antibody. Signals were detected
using enhanced chemiluminescence (Perkin-Elmer Life
Sciences) and band intensities were quantified using
Image J software (NIH, Bethesda, MD, USA). Lipid raft isolation The fresh liver tissue samples or HepG2 cells
in five 100-mm dishes were mixed with lysis buffer
(150 mM NaCl, 20 mM Na2HPO4, 2 mM NaH2PO4,
20% v/v glycerol, 2 mM sodium orthovanadate with
protease inhibitors, pH 7.4) and homogenized 30 times
with a tight Dounce homogenizer (Sigma). Samples
were further disrupted by intermittent sonication (six
30s pulses with a 1min cooling period in between) and
then centrifuged at 10 K rpm (Beckman-Coulter Optima
L-90 K ultracentrifuge with a SW55Ti rotor, CA, USA)
for 10 min at 4°C to separate cell debris and nuclear
materials. The supernatant was then centrifuged at 32.5 K
rpm (SW55Ti rotor) for 90 min at 4°C to pellet the plasma
membrane (PM). The PM was suspended and solubilised
in 2 ml solubilising buffer containing 0.5% v/v Triton
X-100 in Mes-buffered saline (MBS: 25 mM Mes, 0.15 M
NaCl, pH 6.5), protease inhibitors and 2 mM sodium
orthovanadate for 15min on ice. Then, 2 ml of solubilised
PM were further diluted with an equal volume of 80%
sucrose in MBS and loaded on the bottom of a 13 ml
ultracentrifuge tube overlaid with 4 ml of 30% sucrose/
MBS. Finally, 4 ml of a 5% sucrose/MBS solution were
added as the top layer of the gradient. The gradient was
centrifuged at 31 K rpm (SW41Ti rotor) for 16 h at 4°C
to isolate the lipid raft and non-raft compartments. The
gradient was then fractionated into 12 fractions [22, 24]. For selected cases, formalin-fixed, paraffin-
embedded liver tissues were characterized at the time of
diagnosis by immunohistochemistry using the following
antibodies: TLR7 rabbit polyclonal (1:150 dilution), Ki-67
rabbit monoclonal (1:100), caveolin-1 mouse monoclonal
(1:300) and flotillin-1 mouse monoclonal (1:300). Stains were performed on an automated stainer using
heat induced epitope retrieval (CC1 standard, Ventana
Benchmark) and 3, 3′- diaminobenzidine detection
(DAB, Ventana). Double-labeling immunohistochemistry
was performed using an antibody cocktail for TLR7
rabbit polyclonal (1:150 dilution) and caveolin-1 mouse
monoclonal (1:300) or flotillin-1 mouse monoclonal
(1:300) using DAB detection for TLR7 and fast red for
detection of caveolin-1 or flotillin-1 (Ventana). The
TLR7
staining
was
evaluated
semi-
quantitatively by two independent pathologists using the
scoring system described in Table 2. Proliferation index
was calculated for each HCC by counting the number of
tumor cells positively staining for nuclear Ki-67 from a
total of 1000 tumor cells (expressed as a percentage). Materials Antibodies to TLR7, caveolin-1, flotillin-1,
MyD88, clathrin, and Ki-67 were purchased from
Abcam, Inc. (Cambridge, MA, USA). NFκB antibody
was purchased from Cell Signaling Technology, Inc. (Boston, MA, USA). β-actin antibody, HRP-conjugated
anti-rabbit, and anti-mouse IgG were purchased from
CWBio, Inc. (Beijing, CN). Mouse IgG2A anti-Human www.impactjournals.com/oncotarget Oncotarget 63866 Table 2: TLR7 immunohistochemistry scoring system
Staining score
Description
0
No staining or (+) membranous staining intensity in less than 1/3rd of cells
1
(+) membranous staining intensity in more than 1/3rd of cells or (++) intensity in less than 2/3rd of cells
2
(++) or (+++) membranous staining intensity in more than 2/3rd of cells Table 2: TLR7 immunohistochemistry scoring system Table 2: TLR7 immunohistochemistry scoring system Description Cholesterol measurement Fifty microliters of each fraction were analyzed with
an Amplex Red cholesterol assay kit (Molecular Probes,
Eugene, OR, USA) according to the manufacturer’s
protocol. www.impactjournals.com/oncotarget Oncotarget 63867 Cell proliferation assay Proliferation was determined by culturing 2.5 × 104/
well of human HCC line HepG2 with different doses of
gardiquimod and aPPD in 96-well plates for 4 h and 24 h,
one day before experiments. 3-[4, 5-dimethylthiazol-2-yl]
-2, 5-diphenyltetrazolium bromide (MTT) (50 μl, 0.5 mg/
ml) was added 4 h before the end of the incubation period. Dimethylsulfoxide was used to dissolve granules, and the
absorbance at 570 nm of each sample was determined
with a microplate autoreader (BioTek, Synergy H4, USA). The proliferation rate was determined as follows: (A of
treatment group/A of PBS group) × 100%. 1. Bruix J, Sherman M, American Association for the Study
of Liver D. Management of hepatocellular carcinoma: an
update. Hepatology. 2011; 53:1020–1022. 2. Lafaro KJ, Demirjian AN, Pawlik TM. Epidemiology of
hepatocellular carcinoma. Surg Oncol Clin N Am. 2015;
24:1–17. 3. Wallace MC, Preen D, Jeffrey GP, Adams LA. The evolving
epidemiology of hepatocellular carcinoma: a global
perspective. Expert Rev Gastroenterol Hepatol. 2015;
9:765–779. 4. McGlynn KA, Petrick JL, London WT. Global
epidemiology of hepatocellular carcinoma: an emphasis on
demographic and regional variability. Clin Liver Dis. 2015;
19:223–238. Cell migration assay HepG2 cells were cultured in six-well culture dishes
to 70–90% confluence, followed by scratch with 1 ml
sterilized pipette tips. After washing with pre-warmed PBS,
cells were cultured in serum-free DMEM for 24 h. Images
were taken at 0 h and 24 h after scratch by Olympus
FV1000 confocal microscope (Olympus, Beijing, China). Images of same spots taken at 0 h and 24 h were overlaid
and adjusted with Photoshop CS2 software (Adobe,
China). The edge of cultured cells was marked by linking
cell bodies of furthest, but continually, related cells from
unscratched regions. The edge at 0 h and 24 h were defined
as the injury line and migration line, respectively. The area
between injury and migration lines was considered as
the migration area and was subject to quantification [24]. Areas of migration on images were measured with Image J. 5. Zhu RX, Seto WK, Lai CL, Yuen MF. Epidemiology of
Hepatocellular Carcinoma in the Asia-Pacific Region. Gut
Liver. 2016; 10:332–339. 6. Lai CL, Ratziu V, Yuen MF, Poynard T. Viral hepatitis B. Lancet. 2003; 362:2089–2094. 7. Lin KJ, Lin TM, Wang CH, Liu HC, Lin YL, Eng HL. Down-regulation of Toll-like receptor 7 expression in
hepatitis-virus-related human hepatocellular carcinoma. Hum Pathol. 2013; 44:534–541. 8. Xu N, Yao HP, Sun Z, Chen Z. Toll-like receptor 7 and 9
expression in peripheral blood mononuclear cells from
patients with chronic hepatitis B and related hepatocellular
carcinoma. Acta Pharmacol Sin. 2008; 29:239–244. 9. Chang J, Block TM, Guo JT. The innate immune response
to hepatitis B virus infection: implications for pathogenesis
and therapy. Antiviral Res. 2012; 96:405–413. Statistical analysis Continuous variables were analyzed with Students’
t-test or ANOVA in case of normal distribution. For
intergroup comparison, Mann-Whitney U-test was used
for the non-parametric analyses and chi-square test. For
correlation, Spearman rank correlation was used. 10. Woltman AM, Op den Brouw ML, Biesta PJ, Shi CC,
Janssen HL. Hepatitis B virus lacks immune activating
capacity, but actively inhibits plasmacytoid dendritic cell
function. PloS One. 2011; 6:e15324. 11. Vincent IE, Zannetti C, Lucifora J, Norder H, Protzer U,
Hainaut P, Zoulim F, Tommasino M, Trepo C, Hasan U,
Chemin I. Hepatitis B virus impairs TLR9 expression and
function in plasmacytoid dendritic cells. PloS One. 2011;
6:e26315. No potential conflicts of interest were disclosed. 13. Wang JQ, Jeelall YS, Ferguson LL, Horikawa K. Toll-Like
Receptors and Cancer: MYD88 Mutation and Inflammation. Front Immunol. 2014; 5:367. ACKNOWLEDGMENTS We are grateful to Roche for the excellent technical
help and fund support. 12. Seki E, Brenner DA. Toll-like receptors and adaptor
molecules in liver disease: update. Hepatology. 2008;
48:322–335. CONFLICTS OF INTEREST No potential conflicts of interest were disclosed. GRANT SUPPORT This work was supported by grants from National
Natural Science Foundation of China (no. 81202832 and
no. 81673654), Chinese National S&T Major Project for
Infectious Diseases Control (no. 2012ZX10002-003),
Clinical High-tech Key Project (no. 2010jxgs099), and
China Postdoctoral Science Foundation (no. 2012M510370). 14. Mohamed FE, Al-Jehani RM, Minogue SS, Andreola F,
Winstanley A, Olde Damink SW, Habtesion A, Malago M,
Davies N, Luong TV, Dhillon AP, Mookerjee RP, Dhar DK,
et al. Effect of toll-like receptor 7 and 9 targeted therapy to
prevent the development of hepatocellular carcinoma. Liver
Int. 2015; 35:1063–1076. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 63868 25. Platta HW, Stenmark H. Endocytosis and signaling. Curr
Opin Cell Biol. 2011; 23:393–403. 15. Pikarsky E, Porat RM, Stein I, Abramovitch R, Amit S,
Kasem S, Gutkovich-Pyest E, Urieli-Shoval S, Galun E,
Ben-Neriah Y. NF-kappaB functions as a tumour promoter in
inflammation-associated cancer. Nature. 2004; 431:461–466. 26. Huang B, Zhao J, Unkeless JC, Feng ZH, Xiong H. TLR
signaling by tumor and immune cells: a double-edged
sword. Oncogene. 2008; 27:218–224. 16. Song L, Gong H, Lin C, Wang C, Liu L, Wu J, Li M, Li J. Flotillin-1 promotes tumor necrosis factor-alpha receptor
signaling and activation of NF-kappaB in esophageal
squamous cell carcinoma cells. Gastroenterology. 2012;
143:995–1005 e1012. 27. Ochi A, Graffeo CS, Zambirinis CP, Rehman A,
Hackman M, Fallon N, Barilla RM, Henning JR, Jamal M,
Rao R, Greco S, Deutsch M, Medina-Zea MV, et al. Toll-
like receptor 7 regulates pancreatic carcinogenesis in mice
and humans. J Clin Invest. 2012; 122:4118–4129. 17. Powers KA, Szaszi K, Khadaroo RG, Tawadros PS,
Marshall JC, Kapus A, Rotstein OD. Oxidative stress
generated by hemorrhagic shock recruits Toll-like receptor
4 to the plasma membrane in macrophages. J Exp Med. 2006; 203:1951–1961. 28. Wolska A, Cebula-Obrzut B, Smolewski P, Robak T. Effects
of Toll-like receptor 7 and Toll-like receptor 9 signaling
stimulators and inhibitors on chronic lymphocytic leukemia
cells ex vivo and their interactions with cladribine. Leuk
Lymphoma. 2013; 54:1268–1278. 18. Triantafilou M, Morath S, Mackie A, Hartung T, Triantafilou K. Lateral diffusion of Toll-like receptors reveals that they are
transiently confined within lipid rafts on the plasma membrane. J Cell Sci. 2004; 117:4007–4014. 29. Li L, Cheng FW, Wang F, Jia B, Luo X, Zhang SQ. The
activation of TLR7 regulates the expression of VEGF,
TIMP1, MMP2, IL-6, and IL-15 in Hela cells. Mol Cell
Biochem. 2014; 389:43–49. 19. Kovarova M, Tolar P, Arudchandran R, Draberova L,
Rivera J, Draber P. Structure-function analysis of Lyn
kinase association with lipid rafts and initiation of early
signaling events after Fcepsilon receptor I aggregation. Mol
Cell Biol. 2001; 21:8318–8328. 30. Li Y, Shi Y, McCaw L, Li YJ, Zhu F, Gorczynski R,
DuncanxGS, Yang B, Ben-David Y, Spaner DE. Microenvironmental interleukin-6 suppresses toll-like
receptor signaling in human leukemia cells through miR-
17/19A. Blood. 2015; 126:766–778. 20. Oakley FD, Smith RL, Engelhardt JF. www.impactjournals.com/oncotarget Lipid rafts and
caveolin-1
coordinate
interleukin-1beta
(IL-1beta)-
dependent activation of NFkappaB by controlling
endocytosis of Nox2 and IL-1beta receptor 1 from the
plasma membrane. J Biol Chem. 2009; 284:33255–33264. 31. Ma F, Zhang J, Zhang J, Zhang C. The TLR7 agonists
imiquimod
and
gardiquimod
improve
DC-based
immunotherapy for melanoma in mice. Cell Mol Immunol. 2010; 7:381–388. 21. Pham LV, Tamayo AT, Yoshimura LC, Lo P, Terry N,
Reid PS, Ford RJ. A CD40 Signalosome anchored in lipid
rafts leads to constitutive activation of NF-kappaB and
autonomous cell growth in B cell lymphomas. Immunity. 2002; 16:37–50. 32. Sheyhidin I, Nabi G, Hasim A, Zhang RP, Ainiwaer J, Ma
H, Wang H. Overexpression of TLR3, TLR4, TLR7 and
TLR9 in esophageal squamous cell carcinoma. World J
Gastroenterol. 2011; 17:3745–3751. 33. Chang S, Kodys K, Szabo G. Impaired expression and
function of toll-like receptor 7 in hepatitis C virus infection
in human hepatoma cells. Hepatology. 2010; 51:35–42. 22. Liu Y, Lv JY, Shi JF, Yang M, Liu SH, Li ZW, Wang HB,
Zhang SG, Liu ZW, Ding JB, Xu DP, Zhao JM. Targeting
the raft-associated Akt signaling in hepatocellular
carcinoma. Biomed Res Int. 2014; 2014:836025. 34. Legler DF, Micheau O, Doucey MA, Tschopp J, Bron C. Recruitment of TNF receptor 1 to lipid rafts is essential
for TNFalpha-mediated NF-kappaB activation. Immunity. 2003; 18:655–664. 23. Rog T, Vattulainen I. Cholesterol, sphingolipids, and
glycolipids: what do we know about their role in raft-like
membranes? Chem Phys Lipids. 2014; 184:82–104. 35. Chinese Society of H, Chinese Society of Infectious
Diseases CMA. [The guideline of prevention and treatment
for chronic hepatitis B (2010 version)]. Zhonghua Liu Xing
Bing Xue Za Zhi. 2011; 32:405–415. 24. Liu Y, Yang G, Bu X, Liu G, Ding J, Li P, Jia W. Cell-type-
specific regulation of raft-associated Akt signaling. Cell
Death Dis. 2011; 2:e145. www.impactjournals.com/oncotarget Oncotarget 63869
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https://openalex.org/W4291170504
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https://digital.csic.es/bitstream/10261/282331/1/soil_paper.pdf
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English
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Written in soil and paper. Investigating environmental transformations of a monastic landscape by combining geoarchaeology and palynology with historical analysis at Samos (Spain)
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Journal of archaeological science: Reports/Journal of archaeological science: reports
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Journal of Archaeological Science: Reports 45 (2022) 103575 Journal of Archaeological Science: Reports 45 (2022) 103575 Contents lists available at ScienceDirect A B S T R A C T Keywords:
OSL
Monastery
Pollen
Clerical texts
Agrarian terraces
Land use change Palaeoenvironmental and historical approaches have often been used separately to investigate past land-use
change, but they are still rarely combined, especially in places where the most suitable archives are sediment
sequences. Here we used a transdisciplinary approach combining a multiproxy palaeoenvironmental study of two
pedosedimentological sequences around a medieval Benedictine abbey at Samos in north-west Spain. A robust
chronology was built using OSL apparent ages, conventional OSL and radiocarbon ages and used to date
geochemical and palynological proxies which were then analysed alongside an exhaustive historical review of
medieval and modern ecclesiastical records. The aims were to reconstruct the agrarian history of the place in a
diachronic way and to deepen understanding of the interplay between palaeoenvironmental and historical
sources. We demonstrate the potential value of using geoarchaeology, palynology and written sources together to
address both the physical and socioeconomic aspects of land-use change. documentary evidence was carried out by palynologists like Hall (2006,
2000), Lomas-Clarke and Barber (2007, 2004), Tipping (2010, 2005)
and Wimble et al. (2000) in the UK and Ireland. These examples,
together with other palaeopalynological research from peatlands and
lakes in France (No¨el et al., 2001), Austria (Breitenlechner et al., 2010),
Germany (Stolz and Grunert, 2010), Norway (Hjelle et al., 2010) and
Iceland (Hallsd´ottir, 1993; Riddell et al., 2018) have shown that intense
environmental transformations were linked to monasteries across
Europe. Monastic orders were implicated in land clearance, the devel
opment of both arable and pastoral landscapes and technological inno
vation such as the introduction of new agricultural species (Tipping,
1997). Despite calls for the increased use of paleoenvironmental evi
dence (Greene, 2017), relatively little work has been carried out in areas
lacking peatlands or lakes in their surroundings. In those cases, the most
common environmental approach relies on archaeobotanical studies
focusing on carpological (Åsen, 2021; McKerracher, 2017; Moffet, 2018) Written in soil and paper. Investigating environmental transformations of a
monastic landscape by combining geoarchaeology and palynology with
historical analysis at Samos (Spain) Written in soil and paper. Investigating environmental transformations of a
monastic landscape by combining geoarchaeology and palynology with
historical analysis at Samos (Spain) Noemí Silva-S´anchez a,b,*, Kinnaird Tim c, Marcos Fern´andez-Ferreiro d, Estefanía L
Sam Turner f, Jos´e-Carlos S´anchez-Pardo d a Institute of Heritage Sciences, Incipit-CSIC., Edificio Font´an, Bloque 4 – Cidade da Cultura – Monte Gai´as, s/n, 15707 Santiago de Compostela, Spain
b Ecopast Research Group, Universidade de Santiago de Compostela, Spain
c School of Earth and Environmental Sciences, University of St, Andrews, UK
d Sincrisis Research Group, Universidade de Santiago de Compostela, Spain
e Grupo de Estudios Territoriales, Universidade da Coru˜na, Spain
f McCord Centre for Landscape, Newcastle University, UK * Corresponding author.
E-mail address: noemi.silva-sanchez@incipit.csic.es (N. Silva-S´anchez). a Institute of Heritage Sciences, Incipit-CSIC., Edificio Font´an, Bloque 4 – Cidade da Cultura – Monte Gai´as, s/n, 15707 Santiago de Compostela, Spain
b Ecopast Research Group, Universidade de Santiago de Compostela, Spain
c School of Earth and Environmental Sciences, University of St, Andrews, UK
d Sincrisis Research Group, Universidade de Santiago de Compostela, Spain
e Grupo de Estudios Territoriales, Universidade da Coru˜na, Spain
f McCord Centre for Landscape, Newcastle University, UK Available online 11 August 2022
2352-409X/© 2022 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
E mail address: noemi.silva sanchez@incipit.csic.es (N. Silva Sanchez).
https://doi.org/10.1016/j.jasrep.2022.103575
Received 8 February 2022; Received in revised form 30 June 2022; Accepted 18 July 2022 * Corresponding author.
E-mail address: noemi.si 1. Introduction Researchers in pre- and proto-history have traditionally been more
open to palaeoecological studies than historians and historical archae
ologists. The reasons for this are on one hand related to the degree of
chronological uncertainty that these disciplines usually tolerate
(Dumayne et al., 1995; Tipping, 2005), and on the other to the degree of
importance that scholars of different time periods have attached to
human-environment interactions. Despite these apparently insur
mountable differences, improvements in chronological control over
environmental archives and the growing interest in environmental his
tory offer a compelling framework for collaboration. In fact, the number
of interdisciplinary papers linking history and palaeoecology to respond
to both historical (e.g Bosi et al., 2009; Izdebski et al., 2016, 2022) and
ecological questions (Graze et al., 2019) has rapidly increased in recent
years,Pioneer and systematic comparison of pollen records and N. Silva-S´anchez et al. Journal of Archaeological Science: Reports 45 (2022) 103575 T1C) were investigated further by applying multiproxy palaeoecological
analyses (Fig. 1). The T2A sequence (260 cm) was collected from an
earth terrace located on the mid-slope of the hillside c. 250 m south-west
of the monastery. The feature is about 100 m in length and is aligned
roughly east–west. Today oak-chestnut woodland extends northwards
from the top of the earthwork and a grassy field extends southwards
from its face, though air photographs show the whole area was in use for
agriculture in the 1950 s. T2A is a polycyclic soil with three phases of
soil formation (Fig. 2). A first layer of large, rounded stones occurred
from 230 to 260 cm over a saprolite layer, with a 3A horizon at
220–230 cm. The second phase of soil formation comprises a second line
of smaller stones from 210 to 220 with a 2A horizon at 190–210 cm. The
last phase of soil formation consists of a 20 cm A horizon followed by a B
horizon from 20 cm to 190 cm. The natural vs anthropogenic formation
process of T2A is discussed below. or, more exceptionally, palynological evidence (Gil García 2004, Roubis
et al., 2008), (which has also yielded some very unusual archives, for
example from peat bricks (Deforce et al., 2007) or dust recovered from
ancient manuscripts (Jankovsk´a, 1995)). 1. Introduction Work on the economic role of rural monasteries has shed light on
their importance as power centres where decisions about the exploita
tion of natural resources were made, from livestock and arable farming
to water management (mills and fishponds), salt production and mining
(Gilchrist, 2014). In north-west Iberia, the monastic control of agrarian
resources has largely been studied through the historical sources. Although some research has considered less well-known sites (S´anchez-
Pardo and Fern´andez Ferreiro, 2019), the majority has focused on major
power centres such as San Mill´an de la Cogolla (García de Cort´azar,
1969), San Salvador and San Santa María de Sobrado (Pallares M´endez,
1979), San Xuli´an de Samos (Arias Cuenllas, 1992; Rodríguez Gonz´alez,
2008) and the cathedral of Santiago de Compostela (L´opez Alsina,
2013). On the opposite, eastern side of the Sarria valley, samples were
collected from a 160 cm profile (T1C) which was excavated at the
eastern end of a short length of retaining wall (c. 15 m) located in the
lower part of a very steep slope (Fig. 1). This small field, which lies
approximately 250 m south-east of the main monastic complex, is
currently used as pasture with a few fruit trees; other terraces have been
built to retain sediment further up the slope. To the east and south is an
extensive oak-chestnut woodland with evidence of many abandoned
terraces; as on the other side of the valley, historic air photos show that
this area was formerly used as farmland. As a typical cultivation soil,
T1C lacks a proper A horizon (Fig. 2). Three different B sub-horizons
with a total thickness of 160 cm over a saprolite layer were distin
guished in the field according to colour and surface characteristics with
limits at 70 cm and 110 cm. B1 appeared a darker brown colour whilst
B3 was characterised by a lustre surface. The retaining stone wall behind
T1C extended from a depth of 60 cm to 128 cm. Recent advances in the archaeology of northern Iberian rural land
scapes have underlined its importance for the study of agrarian history
(e.g. Ballesteros-Arias et al., 2006; Fern´andez Mier et al., 2014;
Fern´andez Mier and Alonso Gonz´alez, 2016; Ferro-V´azquez et al., 2014;
Quir´os Castillo et al., 2014). Nevertheless, this type of archive has un
explored potential for monastic landscapes. 1. Introduction Expanding the focus of
agrarian studies to to sediment sequences in the vicinity of monasteries
will not only enable scholars to gain insights on the role of monastic
communities in the transformation of the environment, but also to
evaluate their role in the creation and maintenance of cultural land
scapes (Zanini et al., 2021). In this paper, we present a diachronic agrarian history of the land
around
Samos
Abbey
(Galicia,
Spain)
by
combining
palae
oenvironmental and text-based approaches. Analysis of an two terraces
located in the surroundings of the Abbey using optically-stimulated
luminescence profiling and dating (OSL-PD), geochemistry and paly
nology are linked to an analysis of texts recorded in two key sources, the
medieval Tumbo de Samos (‘Cartulary of Samos’) and the early modern
Apeos de la feligresía de Samos (‘Demarcation of the parish of Samos’). Our objectives were not only to understand better the agrarian history of
Samos Abbey, but also the relationships between analyses using envi
ronmental proxies and written sources. 2.2. Geochemical analyses A total of 55 samples were studied for granulometry and Loss on
ignition analysis (LOI): 32 samples from section T1C (5 cm intervals)
and 23 samples from section T2A (10–5 cm intervals). Samples were
analysed at the Ecopast facilities in the Biology faculty of the Uni
versidade de Santiago de Compostela. Samples were air dried and sieved
to separate the coarse (>2 mm, gravel) and fine earth (<2 mm) frac
tions. LOI was performed at 550 ◦C over 5 h. As organic matter is the
main soil constituent that volatilises at such temperature LOI gives an
indirect measurement of the organic matter content of a soil/sediment
sample. Subsequently 10 g of ashes were mixed over 20 min in an HCl
1 N suspension to break up mineral concretions of Fe and Al. Suspen
sions were then separated into three fractions by wet sieving:
<2–0.2 mm (coarse sand); <0.2–0.05 mm (fine sand) and < 0.05 mm
(silt + clay). Macrocharcoal > 2 mm was collected when present and dry
until constant weight to determine its concentration in the sediment
(data shown as dry weight per 100 gr of fine earth). 2.1. Site description and sampling Samos is located on the Sarria river valley over a Precambrian slate
bedrock at an elevation of ~ 550 m above sea level. The climate is
temperate and present land use comprises a mosaic environment
comprising river forest, deciduous oak, chestnut woodland and grass
land with small-scale arable and horticulture. The Benedictine abbey at
Samos was first documented in an inscription dating to AD 665 which
records a renovation by Ermefredo, the bishop of Lugo, though the site
may have been founded as early as the 6th century (Arias Cuenllas,
1992). Samos enjoyed royal protection from the 8th century, becoming
one of the richest monasteries in north-west Iberia during the Middle
Ages (Rodríguez Gonz´alez, 2008)). It held many properties across
Galicia and the Bierzo area until the 19th century, when most of these
estates were given to lay owners. y
g
p
gi
Sediment elemental composition was analysed by X-ray Fluores
cence (XRF) at the RIAIDT facilities of the Universidade de Santiago de
Compostela. Concentrations of major and minor (Si, Al, Ti, Ca, K, P and
S), trace lithogenic (Rb, Sr, Zr and Th), redox-sensitive elements (Fe and
Mn) and halogens (Cl and Br) were determined using X-ray fluorescence
dispersive EMMA-XRF analysers (Cheburkin and Shotyk, 1996). The
calibration was performed using 36 certified reference materials, con
sisting of rocks and minerals (GSR6, SG1a, SRM1d, SRM278, SRM2780,
SRM688, 5365, AGV1, DTS1, SRM607, SRM70a), sands and clays
(SRM1413, SRM81a, BCSCRM348, SRM679, SRM97b, SRM98b), ashes
(SRM1633a, SRM1633b, SRM2690, SRM2691), soils and sediments
(SO2, SO3, SRM2586, BCRCRM277b, LKSD1, LKSD2, MAG1, PACS1,
RM8704, SRM1646, SRM1646a, SRM1944, SRM2702, SRM2703) and
industrial sludge (SRM2782). Quantification limits were as follows: Si
(0.05 %), Al (0.2 %), K (0.05 %), S (0.03 %), Ti (0.002 %), P and S
(0.01 %), Mn (30 μg/g), Sr and Th (5 μg/g) and Br (2 μg/g), As (1 μg/g). Seven pedosedimentary sequences were sampled in the surroundings
of Samos Abbey in 2017. Samples were collected manually down-profile
at 10–5 cm intervals, stored in zip-lock plastic bags in the field and
transferred immediately to the laboratory for subsequent analyses. In
order to avoid pollen contamination, the exposed surface of the pedo
sedimentary profile was eliminated immediately before sampling. Samples for OSL analysis were collected and stored in light-safe
conditions. The two sequences with the best chronological resolution (T2A and 2 Journal of Archaeological Science: Reports 45 (2022) 103575 N. Silva-S´anchez et al. 2.4. Dating and age depth modelling Luminescence stratigraphies were generated for the seven investi
gated sections following the methodologies of Kinnaird et al. (2017) and
Turner et al. (2021). In total, 129 samples were appraised in the field
using portable OSL equipment to provide a preliminary assessment of
luminescence behaviour and the means to generate relative lumines
cence stratigraphies. The measurement cycle involved an interleaved
sequence of instrument dark count, infra-red stimulated luminescence
(IRSL) and OSL, similar to that described by Kinnaird et al. (2017). This
method allowed for the calculation of IRSL and OSL net signal in
tensities, IRSL and OSL depletion indices and IRSL: OSL ratios. This
proxy information, plotted versus depth in the profile, provided an
insight on the depositional histories of the sediment, and the means to
relate the sedimentary sequences to the construction of the terrace walls
and earthworks. The luminescence stratigraphies for profiles T1C and
T2A were used to position samples in the sequences for dating purposes:
4 from section T1C and 3 from T2A (Table 1, Fig. 2). 3 samples from T2A
were submitted for AMS dating at Beta Analytic (Table 1, Fig. 2). 2.5. Numerical methods When dealing with a large set of variables, the use of multivariate
statistical approaches helps summarize common patterns of variation
beyond the raw data and to get insights into the underlying environ
mental factors. For geochemical data, principal component analysis
(PCA) was applied using SPSS 26.0, in correlation mode and by applying
a varimax rotation. Prior to analysis, the data were standardized (Z-
scores) to avoid scaling effects and obtain average-centred distributions
(Eriksson et al., 1999). 2.3. Palynology and 15 samples from T2A progressed to this stage. Luminescence sen
sitivities (photon counts per Gy) and stored dose (Gy) were evaluated on
paired aliquots of HF-etched quartz concentrates, using procedures
modified from Burbidge et al. (2007) and Kinnaird et al. (2017). The
measurement cycles involved a readout of the natural signal, followed
by a 1 Gy test dose, then readouts of the regenerated cycles following
nominal doses of 5, 10 and 50 Gy, each with a subsequent 1 Gy test dose. For all, OSL followed a preheat of 220 ◦C, and was measured at 125 ◦C
for 60 s. This dataset reproduced the maxima and trends observed in
field profiling, confirming the inferences drawn from the field profiles,
to the extent that the dynamic ranges observed in OSL signal intensities
are replicated in the stored dose estimates. Thus, the positioning of the
dating samples in profiles T1C and T2A was justified. A total of 26 samples were studied by pollen analysis. The T1C
sequence was sterile below 75 cm depth so only the first 13 samples were
studied. At T2A 13 samples were included in the analysis. Pollen and
non-pollen palynomorph (NPPs) extraction was performed by acetolysis
following Barber (1976) at the School of Geosciences at the University of
Aberdeen. A minimum sum of at least 300 total land pollen (TLP) was
achieved for all sub-samples in order to produce a statistically significant
result (Birks and Birks, 1980). Data are expressed as a percentage of the
TLP, with spores and aquatic taxa excluded from the TLP sum. NPPs
were also counted and they are expressed as a percentage of TLP plus
total NPPs. Rare types are indicated by a cross (+), where one cross is
equal to one pollen grain or NPP. Pollen samples were spiked with
Lycopodium clavatum tablets (Stockmarr, 1971). Pollen identification,
including cereal-type pollen, was aided by reference keys (Fægri and
Iversen, 1989; Moore et al., 1991), and supported by Reille’s (1992)
pollen and spore Atlas and a personal modern type-slide reference
collection. Non-pollen palynomorphs classification follows the Hugo de
Vries (HdV) Laboratory (University of Amsterdam). ii
Age depth modelling was performed using both OSL dates, radio
carbon dates and OSL apparent ages by Clam (Blaauw, 2010), (Fig. 3). Table 1 The second written source, the Apeos de la feligresía de Samos, is
stored in the Spanish National Historical Archive (https://pares.mcu. es/). It was drafted by a royal scribe in 1660 to define the property
that belonged to the monks at the heart of their large jurisdiction in the
parish of Samos. An apeo is a written demarcation of a property that may
comprise one or several agricultural plots and buildings held by a spe
cific owner. The following data were collected from the text: names of
the owner and tenant, the boundaries of the property, the area, the type
of crops, the type and number of trees, and whether there were enclosing
walls. Analysis of this written source not only provides information
about how the farmland was laid out and used in the past, but also makes
it possible to re-imagine its physical characteristics (L´opez-Salas, 2017,
2015). 2.1. Site description and sampling Journal of Archaeological Science: Reports 45 (2022) 103575
Fig. 1. Left panel: location of T1C and T2A sediment sequences, with an indication of other sediment sequences sampled within the same field campaign at Samos. Right panel: detailed map of Samos with an indication of the identification codes given to the fields demarcated in the early modern documentation Apeos de la
feligresía de Samos (‘Demarcation of the parish of Samos’, 1660). N. Silva-Sanchez et al. Fig. 1. Left panel: location of T1C and T2A sediment sequences, with an indication of other sediment sequences sampled within the same field campaign at Samos. Right panel: detailed map of Samos with an indication of the identification codes given to the fields demarcated in the early modern documentation Apeos de la
feligresía de Samos (‘Demarcation of the parish of Samos’, 1660). Fig. 2. T1C and T2A sediment sequences stratigraphy. Fig. 2. T1C and T2A sediment sequences stratigraphy. cobaltic oxide. Following combustion, excess oxygen and oxides of ni
trogen were reduced in a reduction column (reduced copper at 650 ◦C). N2 and CO2 were separated on a GC column (Porapak, 2 m). Acetanilide
standard was used for N and C calibration. Carbon and nitrogen analysis were performed at SAI facilities of the
Universidade de A Coru˜na. Analysis is based on the dynamic combustion
of the sample (EA1108, Carlo Erba Instruments). Samples were weighed
in tin capsules (MX5 microbalance, Mettler-Toledo) and introduced into
a quartz reactor containing chromium oxide and silvered cobaltous/ 3 N. Silva-S´anchez et al. Journal of Archaeological Science: Reports 45 (2022) 103575 2.3. Palynology 2.6. Written sources The Tumbo de Samos is a compilation of donations, transfers and
other succession documents related to all of Abbey possesions in NW
Iberia dating from AD 785–1209 which was compiled at the very
beginning of the thirteenth century (ca. CE 1200). It is currently stored
in the University of Santiago de Compostela Historical Archive (https
://arquivo.usc.es/ahus2/index). Although
the
written
charters
included in the Tumbo are structured in similar ways, the scribes did not
record information systematically. For this reason some lands and
properties are richly described (including detailed information on the
composition of estates, their location, etc) whereas others are simply
listed under the name of their village or parish and by identifying either
the grantor — and former owner — or the tenant who occupied or used
that estate. Information about all properties listed in the Tumbo de Samos
was compiled in a database searchable for specific terms, for example
records of crops such as cereals, fruit trees and flax (for production of
linen). Translation and definitions from the words used in the analysis
are included in Table 2. Prior to formal quartz SAR OSL dating, further characterisation of the
profile samples occurred in the laboratory, with 27 samples from T1C Table 1
OSL and Radiocarbon dating. Dating
technique
Material
Lab ID
Depth
(cm)
Radiocarbon
years
Calendar
years
T2A
OSL
Sediment
T2A 33
33
CE
1870 ± 20
OSL
Sediment
T2A 147
147
CE
1690 ± 30
C14
Sediment
Beta −
588,463
190–195
830 ± 30 BP
CE
1166–1268
C14
Charcoal
Beta −
588,462
200–205
320 ± 30 BP
CE
1484–1644
OSL
Sediment
T2A
OSL1
217
410 ± 220
BCE
C14
Charcoal
Beta −
616,102
225–230
2180 ± 30 BP
364–150
BCE
T1C
OSL
Sediment
T1C 75
75
CE
1630 ± 30
OSL
Sediment
T1C 120
120
CE
1240 ± 60
OSL
Sediment
T1C
OSL1
130
CE
1280 ± 40
OSL
Sediment
T1C
OSL2
148
CE
1230 ± 40 3.1. Charcoal, soil physical properties and elemental composition Charcoal is present throughout profile T2A. A high charcoal accu
mulation occurs at 200–210 cm (2A horizon), and two minor ones at 4 Journal of Archaeological Science: Reports 45 (2022) 103575 Journal of Archaeological Science: Reports 45 (2022) 10357
Fig. 3. Age depth model of T1C and T2A sequences. . Silva-S´anchez et al. N. Silva-S´anchez et al. m
v
p
p
Fig. 3. Age depth model of T
Table 2
Translations and definitions of target and other important words
used in the analysis of Tumbo de Samos book (AD 785-1209). Tritico
Wheat
Panizal
Millet plantation
Senara
Cultivated land
Nogarias
Nut plantation
Ceresiale
Cherry plantation
Perares,
Pear plantation
Sautos/Castaniares
Chestnut plantation
Linaria
Flax crop
Vinea
Vineyard
Pumar
Apple orchard
N. Silva-S´anchez et al. Fig. 3. Age depth model of T1C and T2A sequences Fig. 3. Age depth model of T1C and T2A sequences. matter. T2A PC2 factor scores (FS2, Fig. 4) are positive below 200 cm
(except the basal sample) and in the top 50 cm. Table 2
Translations and definitions of target and other important words
used in the analysis of Tumbo de Samos book (AD 785-1209). Tritico
Wheat
Panizal
Millet plantation
Senara
Cultivated land
Nogarias
Nut plantation
Ceresiale
Cherry plantation
Perares,
Pear plantation
Sautos/Castaniares
Chestnut plantation
Linaria
Flax crop
Vinea
Vineyard
Pumar
Apple orchard At T1C six principal components explain 84.1 % of the variance but
again only the first two (67.1 % of the variance) have high factor
loadings for more than one variable. T1C-PC1 explains 44 % of the
variance and shows high positive factor loadings for N, P, C, Ca, LOI, Mn,
S, and Sr; high negative loadings for pH and K; and moderate negative
loadings for As, Th, Ti and Br. According to these loadings, PC1 is
interpreted as indicative of the organic matter content of the soil. Ca and
Sr adscription to this component may be related to an independent
process, with Ca biocycling and isomorphic Ca-Sr substitution the most
probable explanation. The distribution of PC1 factor scores (FS1, Fig. 5)
shows a clear tipping point at 60 cm. Below 60 cm factor scores remain
fairly constant and negative indicating more mineral composition than
in the top 60 cm where FS1 is positive and the organic content of the soil
is higher. 220–230 (3A horizon) and 25–35 cm (at the top of the B horizon). 3.1. Charcoal, soil physical properties and elemental composition T1C
also reveals the continuous presence of charcoal, but here the higher
values occur in the top 60 cm of the sequence (B1 horizon). T1C-PC2 explains 14.1 % of the variance and shows high positive
loadings for coarse sand and high negative loadings for the silt and clay
fraction, with this component thus reflecting a granulometry turnover. Br is linked to this component with moderate negative loadings, indi
cating an enrichment in the silt and clay fraction at this site. Below
110 cm FS2 scores tend to be negative or around zero; from 60 cm to
110 cm they are generally positive with only one negative sample and in
the top 60 cm FS2 show fluctuating values around zero. Granulometry, LOI, pH and elemental composition variations (Sup
porting information) are summarised according to principal components
(Fig. 4). Five principal components account for 83.6 % of the total
variance at T2A (Supporting Information 2). The first two, which
together explain 58.5 % of the variance, have high factor loadings for
more than one variable and consequently they are discussed in this
paper. The first principal component (PC1) explains 38.1 % of the
variance and shows high factor loadings for As, Mn, K, Silt & Clay and
Fe; moderate positive loadings for Ca, N, LOI and Th; high negative
loadings for Si and Fine sand and moderate negative loadings for
gravels, Al, coarse sand and Br. This analysis suggests that PC1 at T2A
reflects a compositional fractionation according to granulometry, with a
silt and clay fraction enriched in As, Mn, K, Fe, Ca, N and Th and fine
sand and gravel fractions enriched in Si and to a lesser extent Al. The
distribution of PC1 Factor scores (FS1, Fig. 4) shows two clear tipping
points. Below 200 cm FS1 are negative, from 200 cm to 50 cm remains
around zero and in the top 50 cm are positive. T2A PC2 explains 20.4 %
of the variance: C, LOI, N and S show high positive loadings, whereas Br
and S moderate positive loadings. On the negative side, there is Ti with
high negative loadings and Fe with moderate negative loadings. Ac
cording to these results, T2A PC2 reflects enrichment with organic 3.2. Palynology The palynological composition of T1C (Fig. 5A) showed the profile
was palynologically sterile bellow 65 cm, probably directly due to a shift
in pH conditions (data not shown). In fact 60 cm represented a tipping
point in organic matter and granulometry conditions too as reflected by
T1C-PC1 and T1C-PC2. This means that palynologycal information is
only available for the B1 horizon. There, the palynological composition
is quite homogenous, although two different pollen zones (T1C-Pz1 and
T1C-Pz2) are distinguished by CONISS with a limit at 35 cm. The whole
record is characterised by a tree dominance with Castanea being the
dominant pollen type with percentages between 37.5 and 83.1 %. Shrubs are just testimonial (<1.6 %) whereas, among herbs, the signal is 5 5 Journal of Archaeological Science: Reports 45 (2022) 103575 N. Silva-S´anchez et al. ig. 4. Summary diagram representing soil stratigraphy, charcoal record and factor scores of the main principal components extracted from the statistical analysis of
he physical properties and the elemental composition variations at T2A and T1C. Fig. 4. Summary diagram representing soil stratigraphy, charcoal record and factor scores of the main principal components extracted from the statistical analysis of
the physical properties and the elemental composition variations at T2A and T1C. dominated by Poaceae (7.3–30.4 %). Cerealia undiff. and Secale cereale
are present but in small percentages (0.9 ± 1.1 and 0.2 ± 0.3 respec
tively). Percentages of total cereals (considering together Cerealia
undiff. plus Secale cereale) ≥3 % occurs at 22.5 and 32.5 cm (3.5 and
2.9 %), in the T1C-Pz2 pollen zone, coinciding with the lowest Castanea
values. Coprophilous fungi Sordaria and Sporormiella also show their
highest values at these depths. exchanges and recapitulations of assets) where an indication of fruit tree
plantations and other crops exists in the Tumbo de Samos. In some cases,
there was a specific entry for the cultivated land identifying it for a
specific purpose, e. g. the transferred property was a vinea, a senara or a
linaria (Table 2). In other cases, the term referring to a crop appeared in
a more general record, i.e. properties that could include several types of
lands and products, but without providing details (e.g. ‘villa’ or ‘hered
itate’). Definitions and translation for the medieval Latin terms used in
the analysis are shown in Table 2. At T2A, three pollen zones were distinguished (Fig. 5B). 3.2. Palynology In all zones,
the herbs dominate the signal (47.9–87.3 %), with Poaceae
(35.3–64.2 %) the main constituent. However, in the second pollen zone
(T2A-Pz2) at 200–230 cm, trees and shrubs are better represented (~30
and 10 % respectively) than in the T2A-Pz1 and T2A-Pz3 pollen zones
where trees are ~ 10 and 20 % respectively and shrubs are just testi
monial. Among trees, Castanea and deciduous Quercus were the most
important taxa, whereas the shrub signal is dominated by Calluna. Regarding anthropogenic indicators, total cereal percentages are over
3 % both in T2A-Pz1 (4 %) and T2A-Pz3 (6.1–22.7 %). At T2A-Pz2 tree
and shrub content was higher, with total cereals at 0.3 in the lower
sample and 4.7 in the higher one, where a slight increase in trees and
shrubs was already detected. Cophrophilous fungi mirror this behaviour. Fruit was by far the most frequently cited crop in the Tumbo de
Samos. In particular, pumares were mentioned in 101 records and vineas
appeared in 77 records. Just a few mentions were found of sautos/cas
taniares (five records), perares (four records), ceresiale (three records)
and nogaria (one record). Linarias were registered seven times. Regarding cereal cultivation, it is noteworthy that the most common
entries used the word senra or senara (19 occurrences). References to
specific types of cereals such as tritico or panizal are infrequent with a
single entry for each and none for rye, oats or barley. 3.3. Written sources
3.3.1. Tumbo de Samos
Fig. 6 reflects the number of transferred properties (donations, 3.3. Written sources 3.3.1. Tumbo de Samos
Fig. 6 reflects the number of transferred properties (donations, 3.3.2. Apeos de la feligresía de Samos
Table 3 summarises information con Table 3 summarises information concerning lands demarcated in the
Apeos (where T1C and T2A were both located) as well as data related to
neighbouring properties. The field containing T1C is numbered A17,
which was described as an agro that also comprised forest, chousa and
lamelo. It could bear about siete fanegas de centeno, ‘seven bushels of rye’. 6 Journal of Archaeological Science: Reports 45 (2022) 103575 N. Silva-S´anchez et al. Journal of Archaeological Science: Reports 45 (2022) 103
Fig. 5. Palynological diagram of T2A (A) and T1C (B). lva-Sanchez et al. Fig. 5. Palynological diagram of T2A (A) and T1C (B). in the fields of geoarchaeology and palaeoenvironmental studies with
the construction of a detailed chronology and the study of historic
monastic texts allowed us not only to reconstruct the agrarian history of
the study area but also to deepen in the interplay between sources of
information that are rarely considered together. in the fields of geoarchaeology and palaeoenvironmental studies with
the construction of a detailed chronology and the study of historic
monastic texts allowed us not only to reconstruct the agrarian history of
the study area but also to deepen in the interplay between sources of
information that are rarely considered together. Fig. 6. Number of entries for fruit tree plantations and crop fields in the Tumbo
de Samos (AD 785–1209). Blue sections represent entries for possessions defined
by a crop. Orange sections represents other types of possessions (e.g. villa or
hereditate) where the specific type of crop is mentioned (e.g. a villa that is
transferred with all its vineyards, apple orchards, etc). Fig. 6. Number of entries for fruit tree plantations and crop fields in the Tumbo i 4.1. Shaping the landscape: Diachronic evolution of Samos Abbey
surroundings 4.1.1. Pre-monastic activity at Samos: Agrarian activity in the iron age
Linking the OSL profiles, the sedimentological-geochemical and the
palynological information about the formation and development of the
T2A sequences there is a reasonable body of evidence pointing to the
existence of agricultural land modifications and terraces at Samos long
before the first written evidence of the monastery’s foundation in AD
665. The palynological composition of the T2A-3A horizon gives evi
dence of highly anthropized landscape with a reduced forest cover. The
total cereal content is > 3 % (Sum of Secale cereale and Cerealia undiff. equal to 4 % at 225–230 cm) providing evidence of local cultivation
(following L´opez-S´aez, L´opez-Merino, 2005). The presence of significant
amounts of coprophilous fungi Cercophora and Sordaria may be inter
preted as indicators of animal husbandry, though considering the evi
dence for local agriculture they might have been incorporated into the
profile as a type of animal manuring. The presence of charcoal remains
at this cultivated level may be indicating the use of fire related to
agricultural activities. The high total cereal content from the T2A-3A
horizon linked to the regularly-constructed features of the stone
layers, suggest anthropogenic rather than natural origins for the T2A
sequence (Fig. 7). The T2A-3A horizon and the stone layer above were
dated to the Iron Age (364–150 BCE by radiocarbon dating charcoal at
T2A-3A and 410 ± 220 BCE by OSL dating of the stone layer). These data
suggest that T2A should be considered an agrarian terrace established
during the Iron Age. Further evidence for Iron Age activity in the local
area comes from the high density of hillforts (Rodríguez Fern´andez,
1994): in a radius of 4.5 km around the monastery there are 16 known
examples, with the closest one, Castro de Pascais, just 800 m from T2A. h
b
l
h
h
h
f
l Fig. 6. Number of entries for fruit tree plantations and crop fields in the Tumbo
de Samos (AD 785–1209). Blue sections represent entries for possessions defined
by a crop. Orange sections represents other types of possessions (e.g. villa or
hereditate) where the specific type of crop is mentioned (e.g. a villa that is
transferred with all its vineyards, apple orchards, etc). Fig. 6. Number of entries for fruit tree plantations and crop fields in the Tumbo
de Samos (AD 785–1209). Blue sections represent entries for possessions defined
by a crop. 4.1. Shaping the landscape: Diachronic evolution of Samos Abbey
surroundings Orange sections represents other types of possessions (e.g. villa or
hereditate) where the specific type of crop is mentioned (e.g. a villa that is
transferred with all its vineyards, apple orchards, etc). Based on the Dictionary of the Galician Royal Academy, a proper defini
tion of those Galician terms, used to name different types of farmland, is
also included at end of Table 3. We also know that in the fields bordering
A17 where TC1 was located, there were a total of 31 chestnuts, 5 apple
trees, 4 oaks and 1 pear. T2A was located in the demarcated property labelled A117. It was
described as an heredad that could produce about tres anegas de centeno,
‘three bushels of rye’. There is no information about tree plantation on
this land, but we know that neighbouring lands had 32 fruit trees, 30
oaks, 7 chestnuts and 2 pear trees. Table 3 Table 3
Historical data about the demarcated properties where T1C and T2A were located in 1660 and their bordering ones. Sequence
ID*
Property in 1660
Owner
Tenant
Area (sembradura)
Trees
T1C
A17
Farmland (agro), forest (monte), ‘chousa’ and
‘lamelo’
Abbey
Felipa Fern´andez and
Antonio Bald´es
7 bushels of rye (7 fanegas de
centeno)
-
Bordering properties:
A2
‘Corti˜na’ in Outeiro
Abbey
Pedro de Bales
1 bushel of rye (1 fanega de
centeno)
-
A15
Field with a garden inside (Prado con huerto
dentro)
Abbey
Felipa Fern´andez and
Antonio Bald´es
1 bushels of bread (1 anega de
pan)
5 apple trees, 7 chestnuts
A16
Forest (monte)
Abbey
Felipa Fern´andez and
Antonio Bald´es
8 bushels of bread (8 fanegas
de pan)
24 chestnuts, 4 oaks, 1 pear
Sequence
ID
Property in 1660
Owner
Tenant
Area (sembradura)
Trees
T2A
A117
Landed property in Bargado (Heredad de
Bargado)
Abbey
Sebasti´an Cap´on
3 bushels of rye (3 anegas de
centeno)
-
Bordering properties:
A99
Landed property in Bargado with a forest
(Heredad de Bargado con un monte)
Abbey
Juan Davila
2,5 bushels of rye (2,5 anegas
de centeno)
20 oaks, 2 chestnuts
A98
Landed property in Bargado (Heredad de
Bargado)
Abbey
Juan Davila
8 bushels of rye (8 anegas de
centeno)
8 oaks
A26
Landed property in Bargado (Heredad de
Bargado)
Abbey
Francisco L´opez
6 ‘tegas’ of rye (6 tegas de
centeno)
-
A91
Farmland in Bargado (Agro de Bargado que es
vergeo)
Abbey
Eufrasio L´opez and Juan
Davila
2,5 bushels of rye (2,5 fanegas
de centeno)
5 chestnuts, 2 pears, 32 fruit
trees, 2 oaks Table 3
Historical data about the demarcated properties where T1C and T2A were located in 1660 and their bordering ones. Table 3 Sequence
ID*
Property in 1660
Owner
Tenant
Area (sembradura)
Trees
T1C
A17
Farmland (agro), forest (monte), ‘chousa’ and
‘lamelo’
Abbey
Felipa Fern´andez and
Antonio Bald´es
7 bushels of rye (7 fanegas de
centeno)
-
Bordering properties:
A2
‘Corti˜na’ in Outeiro
Abbey
Pedro de Bales
1 bushel of rye (1 fanega de
centeno)
-
A15
Field with a garden inside (Prado con huerto
dentro)
Abbey
Felipa Fern´andez and
Antonio Bald´es
1 bushels of bread (1 anega de
pan)
5 apple trees, 7 chestnuts
A16
Forest (monte)
Abbey
Felipa Fern´andez and
Antonio Bald´es
8 bushels of bread (8 fanegas
de pan)
24 chestnuts, 4 oaks, 1 pear
Sequence
ID
Property in 1660
Owner
Tenant
Area (sembradura)
Trees
T2A
A117
Landed property in Bargado (Heredad de
Bargado)
Abbey
Sebasti´an Cap´on
3 bushels of rye (3 anegas de
centeno)
-
Bordering properties:
A99
Landed property in Bargado with a forest
(Heredad de Bargado con un monte)
Abbey
Juan Davila
2,5 bushels of rye (2,5 anegas
de centeno)
20 oaks, 2 chestnuts
A98
Landed property in Bargado (Heredad de
Bargado)
Abbey
Juan Davila
8 bushels of rye (8 anegas de
centeno)
8 oaks
A26
Landed property in Bargado (Heredad de
Bargado)
Abbey
Francisco L´opez
6 ‘tegas’ of rye (6 tegas de
centeno)
-
A91
Farmland in Bargado (Agro de Bargado que es
vergeo)
Abbey
Eufrasio L´opez and Juan
Davila
2,5 bushels of rye (2,5 fanegas
de centeno)
5 chestnuts, 2 pears, 32 fruit
trees, 2 oaks
*ID is represented in Fig. 1. Translations and definitions of types of agricultural properties
Agro
Farmland or a piece of land for farming
Chousa
An enclosed farmland, generally small and nearby a house, or an enclosed piece of forest that is not very large
Corti˜na
A piece of land closed to a farmhouse, which is usually enclosed, small, and used for growing cereal, vegetables, or potatoes
Heredad
A piece of land which is use for farming and usually belongs to only one person, family or entity
Huerto
Garden or vegetable garden
Lamelo
The word ‘lamelo’ is not currently described by the Dictionary of the Galician Royal Academy. 4. Discussion In north-west Iberia, as elsewhere in Europe, the history of agricul
tural terraces remains poorly understood due largely to problems The methodological approach of combining established techniques 7 Journal of Archaeological Science: Reports 45 (2022) 103575 Journal of Archaeological Science: Reports 45 (2022) 103575 N. Silva-S´anchez et al. Table 3 However, there is the word ‘lameiro’ that means a field with running water
where only grass and some bushes grow
Prado
Field, meadow
Translations and definitions of former Galician units of area
Anega
One ‘anega’ can be considered as one ‘fanega’
Fanega
One ‘fanega’ is about 6,440 m2
Tega
One ‘tega’ is about 584 m2
Translations and definitions of selected crops
Centeno
Rye
Pan
Bread data about the demarcated properties where T1C and T2A were located in 1660 and their bordering ones. Fig. 7. Synthetic figure summarising the main geochemical and palynological properties of T2A and T1C sequences. Fig. 7. Synthetic figure summarising the main geochemical and palynological properties of T2A and T1C sequences. mical and palynological properties of T2A and T1C sequences. et al., 2003) and a possible late Iron Age-Roman terrace at Castro de
Follente (L´opez S´aez et al., 2009). The results presented here for T2A
provide further evidence of prehistoric terrace building in the region. associated with dating their construction and use (Turner et al., 2021). Terracing appears to have reached its apogee in the Middle Ages (Bal
lesteros-Arias, 2010; Turner et al. 2021), and although it is likely to have
been fairly common long before (Grau Mira and P´erez Rodríguez, 2008),
dated examples remain scarce. Notable exceptions in north-west Iberia
include a first generation of man-made terraces dating to 1330 BCE
(2σ 1520–1150 BCE) identified in the Saa valley (F´abregas Valcarce Unfortunately, environmental changes between the Iron Age and the
end of the Middle Ages cannot be traced back as the stratigraphic record
of T2A presents a significant hiatus (from the second stone line to the 2A
horizon), coincident with FS1 and FS2 major changes at 200 cm. 8 Journal of Archaeological Science: Reports 45 (2022) 103575 N. Silva-S´anchez et al. Explanations for this hiatus may include cutting back or erosion caused
by anthropogenic or natural processes. Explanations for this hiatus may include cutting back or erosion caused
by anthropogenic or natural processes. knowledge about the regional vegetation history. In north-western Spain
the typical mesophilous forest is dominated by Quercus and Corylus (e.g. Mighall et al., 2006; Silva-S´anchez et al., 2014). The good agreement
between macro-charcoal increase and Quercus and Corylus pollen
decrease indicates the local presence of these tree species in the sur
roundings of the T2A sequence. Table 3 Although the evidence of terracing at Samos dates back to the Iron
Age, the most intense geomorphological transformations were recorded
in modern times. The 1600 s-1900 s were characterised by intense use of
terracing for cereal cultivation that led to extreme geomorphological
transformations at Samos, especially on the western side of the valley. There, at T2A, an enormous accumulation of sediment took place during
the 17th-19th centuries. The process involved accretion of nearly 2 m of
sediment in only 300 years. According to the OSL net intensities this
accumulation occurred gradually. The high cereal percentages recorded
throughout the T2A-B horizon (6.1–22.7 %) combined with the rela
tively moderate slope here suggest the most likely explanation for this
high sediment accumulation was terracing with episodic fill. On the
eastern side of the monastery, a second phase of terrace construction
was detected in the 17th century CE. This corresponded to the T1C-B2
horizon, which was characterised by a coarser granulometry than the
lower T1C-B3 horizon representing the first stage of terrace building in
the 13th century. Early medieval times could not be investigated by palynology due to
the presence of a sedimentological hiatus in the sequence T2A and the
existence of palynological sterility in the older levels of T1C. Fortu
nately, the monastic texts provide a new source of data for this time.:
Analysis of the Tumbo de Samos provides valuable information about the
agrarian resources of the monastery for the period AD 785–1209. Analysis of entries related to land use at Samos (Fig. 6) suggests the
Benedictine community had a special predilection for pumares (101 ci
tations) and vineas (77 citations) which were recorded both as specific
crops and growing areas, followed by senaras (19) and linarias (seven),
which were recorded only as specific crops. i
When interpreting clerical documents, it is crucial to remember that
the data they provide depends on what the monks thought was most
important to record. The predominance of pumares and vineas in the
documentation may be related to the importance of wine and cider
production. Several Christian monastic orders including the Benedic
tines and Cistercians had strong viticultural and pomological traditions
(Bond, 2017; Fernandes, 2012; Martinez Tom´e, 1991). Table 3 Conversely, the independence of the
charcoal record with the Castanea pollen signal may be revealing that
the low percentages detected here may be the product of dispersed trees
not affected by fire or, more probably, the product of woodland located
at a medium distance from the site. Data from the Apeos de la feligresía de
Samos and the pollen record from T1C on the eastern side of the mon
astery – although only available from the 1800 s – tend to support this
hypothesis. 4.1.2. From monastic foundation to modern times: Terracing develops and
the textual archive begins At Samos, the evidence from the T1C and the T2A sequences pre
served late medieval and early modern signals of terracing respectively
(Fig. 7). The three OSL dates from the T1C-B3 horizon, which is below
and in the basal section of the retaining wall, consistently provide a 13th
century age (1230 ± 40; 1280 ± 40 and 1240 ± 60) with the resumption
of T2A terracing (T2A-2A horizon) dated between CE 1484–1644. An
inherent problem of studying agrarian features is the fact that later ac
tivities can mask earlier transformations (Chouquer, 2007). This could
be the case at T2A where the hiatus in the sediment archive includes the
period of the early Middle Ages. Thus, the lack of evidence for early
medieval terracing in these two profiles does not necessarily mean that
during the monastery’s earlier centuries (6th to 10th century) there was
no terracing at Samos. In fact, according to a study which provided in
formation about 12 agrarian terraces in Galicia (Ballesteros-Arias,
2010), most were established during the early Middle Ages, with just
one late medieval and two modern exceptions. Other remarkable ex
amples of early medieval terrace building in north-west Iberia were also
identified at El Manso (Asturias; [(Fern´andez Mier et al., 2014)]), and at
Torrentejo and Aizarna (Basque Country; Narbarte-Hern´andez et al.,
2020, 2019). Many pollen diagrams from north-west Iberia have recorded an in
crease in chestnut during Roman times that were further intensified
during the Middle Ages (Mighall et al., 2006; Mu˜noz Sobrino et al.,
2014; Silva-S´anchez et al., 2014). As in the case of vine and apple
cultivation it seems that Cistercian and Benedictine monastic orders
would have played a role in the spread and technological evolution of
chestnut management (Lucas ´Alvarez and Lucas Domínguez, 1996a,
1996b; Romaní Martínez, 1989). Table 3 In the Iberian
Peninsula, the production of wine had been established in the 7th cen
tury BCE (Bux´o, 2008), though it was not until the late Middle Ages
when viticulture spread through the country, probably related to the
expansion of Christian kingdoms accompanied by the establishment of
the monastic orders (Cunha et al., 2020). We do not actually know when
and where cider making starts but the first recorded references to cider
date back to the classical periodand during the Middle Ages apple or
chards were common in European monasteries (Bond, 2017). At Samos
the monks recorded a pomar as a type of land valuable enough to be
granted, implying that apples were important for them – perhaps
because they were used to produce cider. Palynological information for modern times at T1C and T2A give
evidence of contrasting land use to the eastand west of the Abbey. At
T1C the palynological signal is dominated by Castanea andcereal per
centages are generally lower than 3 %. Interestingly, the predominance
of chestnut in the western sector was already documented in the Apeos
de la feligresía de Samos, so it is possible that the terrace at T1C was
mainly constructed to keep the slope stable and avoid earth moving
down towards the stream below, rather than directly for cereal culti
vation. Nevertheless, it is likely that arable cultivation occurred further
up the hill where the slope was more gentle and further terraces were
built (S´anchez Pardo et al., forthcoming). Furthermore, the Apeos de la feligresía de Samos and the pollen data
both indicate possible differences in the type of cereal grown on the
eastern and western sides of the monastery. In the pollen data, the
relative content of Secale cereale in relation to the total cereal content is
much higher at T2A than at T1C. A similar pattern was found in the
Apeos de la feligresía de Samos where the land around T2A was assessed in
bushels of rye, whereas the land near T1C was assessed both in bushels
of rye and bushels of bread wheat. 4.1.3. Modern times: Landscape transformation and property inventories 4.1.3. Modern times: Landscape transformation and property inventories
Palynological evidence from the western side of the monastery (T2A)
shows that during modern times a deforested landscape comprised a
mosaic of dispersed deciduous trees, localised agriculture (Sum of Secale
cereale and Cerealia undiff. equal to 4.7 and 9.3 %) and animal hus
bandry. The evidence of fire use at this time was very clear with an
important macrocharcoal level dated to CE 1484–1644. This fire event
would have affected primarily Dec. Quercus (decreases from 18 to
4.5 %), Corylus (from 4.7 to 0.7 %), and Calluna (from 11.4 to 3.8 %)
whereas Castanea appear to have slightly increased their prevalence
(from 6.3 to 7.9 %) (Fig. 7A). This is coherent with the available 4.1.4. Historical and environmental archives: Congruence and divergence
All disciplines and sources of information are to some extent frag
mentary and biased. Even with the application of accurate dating
methods, sediments do not always account for a continuous record since
hiatuses occur in their stratigraphies. In palynology, the taxonomical
degree of identification does not always allow research questions about
certain plant species to be addressed. For example, the possibilities of
distinguishing among different fruits from the Rosaceae family is limited 9 Journal of Archaeological Science: Reports 45 (2022) 103575 N. Silva-S´anchez et al. significant role of fruit tree plantations such as apple orchards (taxo
nomically indistinguishable in pollen analysis), as well as crops such as
vines and flax, which are difficult to identify in the regional palynology. By using both texts and pollen data it is possible to obtain information
about the local vegetation that would be impossible to achieve by
focusing on either source independently. due to frequent hybridation and the presence of polyploid races (Moore
et al., 1991). Cereal identification may also be limited to rye, maize and
undifferentiated Cerealia type. Written sources are generally fragmen
tary because information was not recorded in a systematic way or at
regular intervals. In addition, they were always compiled with specific
uses in mind, and this can provide insights into the priorities and values
of the writers. The interplay between geoarchaeological, palaeoecological and
historical archives helps to illuminate the social and economic di
mensions of environmental management. CRediT authorship contribution statement Noemí Silva-S´anchez: Conceptualization, Writing – original draft,
Writing – review & editing. Kinnaird Tim: Conceptualization, Writing –
original draft. Marcos Fern´andez-Ferreiro: Writing – review & editing. Estefanía L´opez-Salas: Writing – review & editing. Sam Turner:
Conceptualization, Writing – review & editing. Jos´e-Carlos S´anchez-
Pardo: Conceptualization, Funding acquisition, Writing – review &
editing. Acknowledgements This research was funded by TERPOMED (2016-PG065) and ECO
LOC (EUR2021-122009) projects funded by the Galician and Spanish
governments. Innes Audrey, support staff at University of Aberdeen,
assisted with pollen extracts preparation. Dr Aayush Srivastava per
formed OSL apparent doses determinations at the University of St
Andrews. Fernando de Alba S´aenz Tejada, Pablo Comenero Crispín and
Alicia Gonz´alez Míguez, students at the University of Santiago de
Compostela (Ecopast research group), are thanked for their support in
the geochemistry laboratory. Carlos Otero Vilari˜no, Rebeca Tall´on
Armada, Mario Fern´andez and Celtia Rodríguez helped during field
sampling. We also wish to thank Dr Cruz Ferro V´azquez and Prof. Antonio Martínez Cortizas for constructive discussions at different
stages in the research. Prof. Anna Maria Mercuri and another anony
mous reviewer are thanked for their helpful and constructive comments. Noemí Silva S´anchez is funded by a Juan de la Cierva-Formaci´on Grant
from the Spanish Government (ref: FJC2018-036266-I). l
Even so, the medieval documentation at Samos still makes an
important contribution to understanding the Abbey’s agrarian history
for two reasons. Firstly, there was a hiatus in pollen data from the soil
archive examined at both T1C and T2A. The OSL apparent ages showed
that at T1C sediment was deposited in the Middle Ages but pollen was
not preserved, whereas at T2A sediment dating between the Iron Age
and later Middle Ages was absent. Secondly, some of the most
economically significant species recorded in the texts could not be
distinguished or occur only rarely in the environmental archives. Declaration of Competing Interest The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper. 5. Conclusions Even though the possessions of many monasteries were located kil
ometres away from the abbey buildings, the concept of monastic land
scapes are still rather limited and geographically restricted. The study of
ecclesiastical texts rarely explores aspects relevant to land management
in a systematic way and archaeological studies of monasteries rarely
include palaeoecological approaches. In this study of two sediment se
quences from the surroundings of Samos Abbey, results from sedimen
tology, geochemistry and palynology (with robust chronological control
using OSL and radiocarbon dating) were combined with detailed study
of agrarian resources from medieval and early modern documentation. Intense environmental and geomorphological transformations
involving forest clearance, cereal cultivation and very likely also
terracing have occurred at Samos since at least the Iron Age. Environ
mental changes at the time the Abbey was founded were not preserved
in the two pedosedimentological sequences studied, but successive ep
isodes of terracing during late medieval and early modern times were
identified. The palynological analysis provided evidence of a mosaic
pattern of land management with cereal fields and chestnut forest pre
dominant at those times. By analysing medieval and early modern texts
associated with the monastery it was possible to appreciate the Even though the possessions of many monasteries were located kil
ometres away from the abbey buildings, the concept of monastic land
scapes are still rather limited and geographically restricted. The study of
ecclesiastical texts rarely explores aspects relevant to land management
in a systematic way and archaeological studies of monasteries rarely
include palaeoecological approaches. In this study of two sediment se
quences from the surroundings of Samos Abbey, results from sedimen
tology, geochemistry and palynology (with robust chronological control
using OSL and radiocarbon dating) were combined with detailed study
of agrarian resources from medieval and early modern documentation. 4.1.3. Modern times: Landscape transformation and property inventories The systematic application of
this approach to other monastic environments would provide valuable
information about past monastic landscapes and provide further insights
on the role of monastic communities in the creation of cultural
landscapes. In our example, Modern documentation provides snapshots in time
that are geographically limited to the surroundings of the Abbey. Nevertheless, in some respects it agreed with with palynological data,
for example in both types of showing reflecting tge predominance of
chestnut plantations on the western side of the Abbey. By contrast the
medieval documentation comprised a compilation of documents pro
duced over centuries and covering a large area. Here, a discrepancy
emerged between the documents and soil archives in relation to cereals
and chestnuts. Before the introduction of maize and potato from the
Americas, native cereals and chestnuts were crucial for early medieval
societies, a fact which is s reflected clearly in the pollen record (Adua,
1999; Conedera et al., 2004). Documents from the monastery of San
Vicenzo de Pombeiro suggest that the economic productivity of chestnut
was second only to vines (Lucas ´Alvarez and Lucas Domínguez, 1996a). However, in terms of the number of entries, medieval records of chest
nut are minimal at both Pombeiro and Samos. By contrast, the medieval
documentation of Samos provides much more detail of apple, grape and
flax management which is of great value given both that apple pollen is
not easily discernible from other Rosaceae undiff., and that Vitis pollen is
generally very uncommon (for a further discussion see Montecchi and
Mercuri 2018) and its dimorphism may further hinder its identification
(Mercuri et al., 2021). The central importance of vines and flax to me
dieval society is underscored by references in the Galician documents to
vinatarios and lenzarios, two classes of servile agrarian peasants linked to
the production of these crops (Fern´andez Ferreiro, 2021). In the Samos
documents only a lenzarios was recorded even though vines are more
commonly recorded than flax in the Tumbo de Samos. Arias Cuenllas, M., 1992. Historia del monasterio de San Juli´an de Samos. Diputaci´on
Provincial de Lugo, Samos.
Å Adua, M., 1999. The sweet chestnut throughout history from the miocene to the third
millennium. Acta Hortic. https://doi.org/10.17660/ActaHortic.1999.494.2. Ballesteros-Arias, P., Criado-Boado, F., Andrade Cernadas, J.M., 2006. Formas y fechas
de un paisaje agrario de ´epoca medieval: a Cidade da Cultura en Santiago de
Compostela. Arqueol. Espac. Journal of Archaeological Science: Reports 45 (2022) 103575 John Wiley & Sons,
Chichester. L´opez-S´aez, J.A., L´opez-Merino, L., 2005. Precisiones metodol´ogicas acerca de los
indicios paleopalinol´ogicos de agricultura en la Prehistoria de la Península Ib´erica. Portugalia 26, 53–64. Fernandes, R., 2012. Descriç˜ao do terreno ao redor de Lamego duas l´eguas. [1531-1532]. Casal de Cambra: Caleidosc´opio — Ediç˜ao e Artes Gr´aficas. L´opez-Salas, E., 2015. San Juli´an de Samos-Lugo, estudio e interpretaci´on del dise˜no
mon´astico y su evoluci´on. Universidade da Coru˜na. PhD Thesis. L´
S l
E
2017 D
di
h
l
i
l
f h
i
b
d
l f i
Fern´andez Ferreiro, M., 2021. O estatus servil na Alta Idade Media. Escravitude e
servidume na sociedade galego-portuguesa (s´eculos IX-XII). Santiago de Compostela:
Universidade, 2021 [thesis dissertation unpublised]. L´opez-Salas, E., 2017. Decoding the planning rules of the monastic urban and rural forms
around Samos Abbey. In: Abel, M. (Ed.), Medieval Urban Planning: The Monastery
and Beyond. Cambridge Scholars Publishing, Newcastle upon Tyne, pp. 46–74. ´ Fern´andez Mier, M., Alonso Gonz´alez, P., 2016. Medieval north-west Spain: What can
agrarian archaeology tell us about living rural landscapes?, in: Kl´apˇstˇe, J., Ruralia, X. (Eds.), Agrarian Technology in the Medieval Landscape. Brepols publishers,
Smolenice, pp. 291–308. https://doi.org/10.1484/m.ruralia-eb.5.110473. Lucas ´Alvarez, M., Lucas Domínguez, P., 1996a. El priorato benedictino de San Vicenzo
de Pombeiro y su colecci´on diplom´atica en la Edad Media. Ed. Do Castro., A Coru˜na. ´ Lucas ´Alvarez, M., Lucas Domínguez, P., 1996b. El monasterio de San Clodio do Ribeiro
en la Edad Media: estudio y, documentos. Ed. Do Castro, A Coru˜na. Fern´andez Mier, M., Fern´andez, J.F., Alonso Gonz´alez, P., Antonio L´opez S´aez, J., P´erez
Díaz, S., Hern´andez Beloqui, B., 2014. The investigation of currently inhabited
villages of medieval origin: Agrarian archaeology in Asturias (Spain). Quat. Int. 346,
41–55. https://doi.org/10.1016/j.quaint.2014.01.032. Martinez Tom´e, A., 1991. El monasterio cisterciense en el origen de los vinos espa˜noles. Servicio de Extensi´on Agraria, Madrid. Mercuri, A.M., Torri, P., Florenzano, A., Cl`o, E., Lippi, M.M., Sgarbi, E., Bignami, C.,
2021. Sharing the agrarian knowledge with archaeology: First evidence of the
dimorphism of vitis pollen from the middle bronze age of n italy (terramara santa
rosa di poviglio). Sustainability 13, 1–20. https://doi.org/10.3390/su13042287. Ferro-V´azquez, C., Martínez-Cortizas, A., N´ovoa-Mu˜noz, J.C., Ballesteros-Arias, P.,
Criado-Boado, F., 2014. 1500 years of soil use reconstructed from the chemical
properties of a terraced soil sequence. Quat. Int. 346, 28–40. https://doi.org/
10.1016/j.quaint.2014.03.023. Mighall, T.M., Martínez Cortizas, A., Biester, H., Turner, S.E., 2006. Journal of Archaeological Science: Reports 45 (2022) 103575 Journal of Archaeological Science: Reports 45 (2022) 103575 N. Silva-S´anchez et al. Bond, J., 2017. Production and consumption of food and drink in the Medieval
Monastery. In: Keevill, G., Aston, M., Hall, T. (Eds.), Monastic Archaeology: Papers
on the Study of Medieval Monasteries. Oxbow Books. through time. Quat. Int. 220, 133–146. https://doi.org/10.1016/j. quaint.2009.09.023. through time. Quat. Int. 220, 133–146. https://doi.org/10.1016/j. quaint.2009.09.023. Izdebski, A., Holmgren, K., Weiberg, E., Stocker, S.R., Büntgen, U., Florenzano, A.,
Gogou, A., Leroy, S.A.G., Luterbacher, J., Martrat, B., Masi, A., Mercuri, A.M.,
Montagna, P., Sadori, L., Schneider, A., Sicre, M.A., Triantaphyllou, M., Xoplaki, E.,
2016. Realising consilience: How better communication between archaeologists,
historians and natural scientists can transform the study of past climate change in the
Mediterranean. Quat. Sci. Rev. 136, 5–22. https://doi.org/10.1016/j. quascirev.2015.10.038. Bosi, G., Mercuri, A.M., Guarnieri, C., Bandini Mazzanti, M., 2009. Luxury food and
ornamental plants at the 15th century A.D. Renaissance court of the Este family
(Ferrara, Northern Italy). Veg. Hist. Archaeobot. 18, 389–402. https://doi.org/
10.1007/s00334-009-0220-z. Breitenlechner, E., Hilber, M., Lutz, J., Kathrein, Y., Unterkircher, A., Oeggl, K., 2010. The impact of mining activities on the environment reflected by pollen, charcoal and
geochemical analyses. J. Archaeol. Sci. 37, 1458–1467. https://doi.org/10.1016/j. jas.2010.01.006. q
Izdebski, A., Guzowski, P., Poniat, R., Masci, L., Palli, J., Vignola, C., Bauch, M.,
Cocozza, C., Fernandes, R., Ljungqvist, F.C., Newfield, T., Seim, A., Abel-Schaad, D.,
Alba-S´anchez, F., Bj¨orkman, L., Brauer, A., Brown, A., Czerwi´nski, S., Ejarque, A.,
Fiłoc, M., Florenzano, A., Fredh, E.D., Fyfe, R., Jasiunas, N., Kołaczek, P., Kouli, K.,
Koz´akov´a, R., Kupryjanowicz, M., Lagerås, P., Lamentowicz, M., Lindbladh, M.,
L´opez-S´aez, J.A., Luelmo-Lautenschlaeger, R., Marcisz, K., Mazier, F., Mensing, S.,
Mercuri, A.M., Milecka, K., Miras, Y., Nory´skiewicz, A.M., Novenko, E.,
Obremska, M., Panajiotidis, S., Papadopoulou, M.L., Pędziszewska, A., P´erez-
Díaz, S., Piovesan, G., Pluskowski, A., Pokorny, P., Poska, A., Reitalu, T., R¨osch, M.,
Sadori, L., S´a Ferreira, C., Sebag, D., Słowi´nski, M., Stanˇcikait˙e, M., Stivrins, N.,
Tunno, I., Veski, S., Wacnik, A., Masi, A., 2022. Palaeoecological data indicates land-
use changes across Europe linked to spatial heterogeneity in mortality during the
Black Death pandemic. Nat. Ecol. Evol. 6, 297–306. https://doi.org/10.1038/
s41559-021-01652-4. Burbidge, C.I., Sanderson, D.C.W., Housley, R.A., Allsworth Jones, P., 2007. Survey of
Palaeolithic sites by luminescence profiling, a case study from Eastern Europe. Quat. Geochronol. 2, 296–302. https://doi.org/10.1016/j.quageo.2006.05.024. Bux´o, R., 2008. The agricultural consequences of colonial contacts on the Iberian
Peninsula in the first millennium b.c. Veg. Hist. Journal of Archaeological Science: Reports 45 (2022) 103575 Archaeobot. 17, 145–154. https://
doi.org/10.1007/s00334-007-0133-7. g
Chouquer, G., 2007. Quels sc´enarios pour l’histoire du paysage? Orientations de
r´echerche pour l’arch´eog´eographie. Estudos Arqueol´ogicos das Universidades de
Coimbra e Porto, Coimbra/Porto. Conedera, M., Krebs, P., Tinner, W., Pradella, M., Torriani, D., 2004. The cultivation of
Castanea sativa (Mill.) in Europe, from its origin to its diffusion on a continental
scale. Veg. Hist. Archaeobot. 13, 161–179. https://doi.org/10.1007/s00334-004-
0038-7. Jankovsk´a, V., 1995. Pollen analysis of dust preserved in four medieval books. Folia
Geobot. 30, 97–100. https://doi.org/10.1007/BF02813224. Cunha, J., Ib´a˜nez, J., Teixeira-Santos, M., Braz˜ao, J., Fevereiro, P., Martínez-Zapater, J. M., Eiras-Dias, J.E., 2020. Genetic Relationships Among Portuguese Cultivated and
Wild Vitis vinifera L. Germplasm. Front. Plant Sci. 11, 1–14. https://doi.org/
10.3389/fpls.2020.00127. Kinnaird, T., Bol`os, J., Turner, A., Turner, S., 2017. Optically-stimulated luminescence
profiling and dating of historic agricultural terraces in Catalonia (Spain). J. Archaeol. Sci. 78, 66–77. https://doi.org/10.1016/j.jas.2016.11.003. Lomas-Clarke, S.H., Barber, K.E., 2004. Palaeoecology of human impact during the
historic period: palynology and geochemistry of a peat deposit at Abbeyknockmoy,
Co., Galway, Ireland. The Holocene 14, 721–731. https://doi.org/10.1191/
0959683604hl750rp. Deforce, K., Bastiaens, J., Ameels, V., 2007. Peat re-excavated at the abbey of Ename
(Belgium): Archaeobotanical evidence for peat extraction and long distance
transport in Flanders around 1200 AD. Environ. Archaeol. 12, 87–94. https://doi. org/10.1179/174963107x172750. Lomas-Clarke, S.H., Barber, K.E., 2007. Human impact signals from peat bogs - a
combined palynological and geochemical approach. Veg. Hist. Archaeobot. 16,
419–429. https://doi.org/10.1007/s00334-006-0085-3. Dumayne, L., Stoneman, R., Barber, K., Harkness, D., 1995. Problems associated with
correlating calibrated radiocarbon-dated pollen diagrams with historical events. The
Holocene 5, 118–123. Eriksson, L., Johansson, E., Kettaneh-Wold, N., Wold, S., 1999. Introduction to multi- and
megavariate data analysis using projection methods (PCA & PLS). Umetrics AB,
Umea. L´opez Alsina, F., 2013. La ciudad de Santiago de Compostela en la Alta Edad Media, 2nd,
Editio. ed. Consorcio de Santiago, Santiago de Compostela. L´opez S´aez, J.A., L´opez Merino, L., P´erez Díaz, S., Parcero-Oubi˜na, C., Criado-Boado, F.,
2009. Contribuci´on a la caracterizaci´on de los espacios agrarios castre˜nos:
documentaci´on y an´alisis palinol´ogico de una posible terraza de cultivo en el castro
de Follente (Caldas de Reis, Pontevedra). Trab. Prehist. 66, 23–34. https://doi.org/
10.3989/tp.2009.09020. F´abregas Valcarce, R., Martínez Cortizas, A., Blanco Chao, R., Chesworth, W., 2003. Environmental change and social dynamics in the second-third millennium BC in
NW Iberia. J. Archaeol. Sci. 30, 859–871. https://doi.org/10.1016/S0305-4403(02)
00264-9. Fægri, K., Iversen, J., 1989. Textbook of Pollen Analysis, 4th Edition. Journal of Archaeological Science: Reports 45 (2022) 103575 Proxy climate and
vegetation changes during the last five millennia in NW Iberia: pollen and non-
pollen palynomorph data from two ombrotrophic peat bogs in the North Western
Iberian Peninsula. Rev. Palaeobot. Palynol. 141, 203–223. https://doi.org/10.1016/
j.revpalbo.2006.03.013. j q
García de Cort´azar, J.A., 1969. El dominio del monasterio de San Mill´an de la Cogolla
(siglos x al xiii). Ediciones Universidad de Salamanca, Salamanca. Gilchrist, R., 2014. Monastic and church archaeology. Annu. Rev. Anthropol. 43,
235–250. https://doi.org/10.1146/annurev-anthro-102313-025845. p
g
Gil-García, M.J., 2004. Estudio polínico de la huerta del Monasterio del Paular
(Rascafría, Madrid). Universidad de Alcal´a de Henares, Madrid. Moffet, L., 2018. The archaeobotany of Late Medieval plant remains: The resource and
the research. In: The Oxford Handbook of Later Medieval Archaeology in Britain. Gerrad, C and Guti´errez A. (Eds). Oxford Handbooks Online.McKerracher M 2017. Seeds and status: the archaeobotany of monastic Lyminge. In: Early medieval
monasticism in the North Sea Zone (Anglo-Saxon Studies in Archaeology and History
20). Thomas, G, Knox, A (Eds). University of Oxford School of Archaeology. Grace, M., Resit Akçakaya, H., Bennett, E., Hilton-Taylor, C., Long, B., Milner-Gulland, E. J., Young, R., Hoffmann, M., 2019. Using historical and palaeoecological data to
inform ambitious species recovery targets. Philos. Trans. R. Soc. B Biol. Sci. 374
https://doi.org/10.1098/rstb.2019.0297. p
g
Grau Mira, I., P´erez Rodríguez, V., 2008. Aproximaci´on arqueol´ogica al estudio de los
paisajes aterrazados en el ´area central valenciana. LVCENTVM 33–50. Moore, P.D., Webb, J.A., Collinson, M.E., 1991. Pollen Analysis, 2nd ed. Blackwell,
Oxford. Greene, J.P., 2017. Strategies for future research and site investigation, in: Keevill, G. (Ed.), Monastic Archaeology: Papers on the Study of Medieval Monasteries. Oxbow
books, pp. 4–8. Mu˜noz Sobrino, C., García-Moreiras, I., Castro, Y., Martínez Carre˜no, N., De Blas, E.,
Fernandez Rodríguez, C., Judd, A., García-Gil, S., 2014. Climate and anthropogenic
factors influencing an estuarine ecosystem from NW Iberia: New high resolution
multiproxy analyses from San Sim´on Bay (Ría de Vigo). Quat. Sci. Rev. 93, 11–33. https://doi.org/10.1016/j.quascirev.2014.03.021. Hall, V.A., 2000. Pollen analytical investigations of the Irish landscape ad 200–1650. Peritia 14, 342–371. Narbarte-Hern´andez, J., Iriarte, E., Rad, C., Carrancho-Alonso, ´A., Gonz´alez-
Samp´eriz, P., Pe˜na-Chocarro, L., Quir´os-Castillo, J.A., 2019. On the origin of rural
landscapes: Looking for physico-chemical fingerprints of historical agricultural
practice in the Atlantic Basque Country (N Spain). Sci. Total Environ. 681, 66–81. https://doi.org/10.1016/j.scitotenv.2019.05.082. Hall, V.A., 2006. p
g
j
Narbarte-Hern´andez, J., Iriarte, E., Rad, C., Tejerizo, C., Eraso, J.F., Quir´os-Castillo, J.A.,
2020. Long-term construction of vineyard landscapes in the Ebro Valley: The References Adua, M., 1999. The sweet chestnut throughout history from the miocene to the third
millennium. Acta Hortic. https://doi.org/10.17660/ActaHortic.1999.494.2. Arias Cuenllas, M., 1992. Historia del monasterio de San Juli´an de Samos. Diputaci´on
Provincial de Lugo, Samos. Å Åsen, P.A., 2021. Medieval monastery gardens in iceland and norway. Religions 12. https://doi.org/10.3390/rel12050317. Intense environmental and geomorphological transformations
involving forest clearance, cereal cultivation and very likely also
terracing have occurred at Samos since at least the Iron Age. Environ
mental changes at the time the Abbey was founded were not preserved
in the two pedosedimentological sequences studied, but successive ep
isodes of terracing during late medieval and early modern times were
identified. The palynological analysis provided evidence of a mosaic
pattern of land management with cereal fields and chestnut forest pre
dominant at those times. By analysing medieval and early modern texts
associated with the monastery it was possible to appreciate the Ballesteros-Arias, P., 2010. La arqueología rural y la construcci´on de un paisaje agrario
medieval: el caso de Galicia, in: Kirchner, H. (Ed.), Por Una Arqueología Agraria. Perspectivas de Investigaci´on Sobre Espacios de Cultivo En Las Sociedades
Medievales Hisp´anicas. Archaeopress, Oxford, pp. 25–39. Ballesteros-Arias, P., Criado-Boado, F., Andrade Cernadas, J.M., 2006. Formas y fechas
de un paisaje agrario de ´epoca medieval: a Cidade da Cultura en Santiago de
Compostela. Arqueol. Espac. Barber, K.E., 1976. History of vegetation. In: Chapman, S.B. (Ed.), Methods in Plant
Ecology. Blackwell, Oxford, pp. 5–83. Birks, H.J.B., Birks, H.H., 1980. Quaternary Palaeoecology. Arnold, y
gy
Blaauw, M., 2010. Methods and code for “classical” age-modelling of radiocarbon
sequences. Quat. Geochronol. 5, 512–518. https://doi.org/10.1016/j. quageo.2010.01.002. 10 Journal of Archaeological Science: Reports 45 (2022) 103575 The vegetation history of monastic and secular sites in the midlands of
Ireland over the last two millennia. Veg. Hist. Archaeobot. 15, 1–12. https://doi.org/
10.1007/s00334-005-0072-0. Hallsd´ottir, M., 1993. Frj´oranns´okn ´a m´osniðum úr Viðey. Raunvísindastofnun
H´ask´olans, Reykjavík. Narbarte-Hern´andez, J., Iriarte, E., Rad, C., Tejerizo, C., Eraso, J.F., Quir´os-Castillo, J.A.,
2020. Long-term construction of vineyard landscapes in the Ebro Valley: The Hjelle, K.L., Halvorsen, L.S., Overland, A., 2010. Heathland development and
relationship between humans and environment along the coast of western Norway 11 Journal of Archaeological Science: Reports 45 (2022) 103575 Journal of Archaeological Science: Reports 45 (2022) 103575 N. Silva-S´anchez et al. deserted village of Torrentejo (Basque Country, Spain). Catena 187, 104417. https://
doi.org/10.1016/j.catena.2019.104417. Salvador de Bande y Santiago de Barbadelo. Stud. Hist. Hist. Mediev. 37, 105. https://doi.org/10.14201/shhme2019371105135. deserted village of Torrentejo (Basque Country, Spain). Catena 187, 104417. https://
doi.org/10.1016/j.catena.2019.104417. Salvador de Bande y Santiago de Barbadelo. Stud. Hist. Hist. Mediev. 37, 105. https://doi.org/10.14201/shhme2019371105135. Silva-S´anchez, N., Martínez Cortizas, A., L´opez-Merino, L., 2014. Linking forest cover,
soil erosion and mire hydrology to late-Holocene human activity and climate in NW
Spain. Holocene 24, 714–725. https://doi.org/10.1177/0959683614526934. No¨el, H., Garbolino, E., Brauer, A., Lallier-Verg`es, E., De Beaulieu, J.L., Disnar, J.R.,
2001. Human impact and soil erosion during the last 5000 yrs as recorded in
lacustrine sedimentary organic matter at Lac d’Annecy, the French Alps. J. Paleolimnol. 25, 229–244. https://doi.org/10.1023/A:1008134517923. Stockmarr, J., 1971. Tablets with spores used in absolute pollen analysis. Pollen Spores
13, 614–621. Pallares M´endez, M.C., 1979. El monasterio de Sobrado: un ejemplo del protagonismo
mon´astico en la Galicia medieval. Diputaci´on Provincial, A Coru˜na. ´ Stolz, C., Grunert, J., 2010. Late Pleistocene and Holocene landscape history of the
central Palatinate forest (Pf¨alzerwald, south-western Germany). Quat. Int. 222,
129–142. https://doi.org/10.1016/j.quaint.2009.08.022. Quir´os Castillo, J.A., Nicosia, C., Polo-Díaz, A., Ruiz del ´Arbol, M., 2014. Agrarian
archaeology in northern Iberia: Geoarchaeology and early medieval land use. Quat. Int. 346, 56–68. https://doi.org/10.1016/j.quaint.2013.10.003. Tipping, R., 2005. Palaeoecology and political history: evaluating driving forces in
historic landscape change in southern Scotland. In: Whyte, I., Winchester, A.J.L. (Eds.), Society, Landcape and Environment in Upland Britain. Society for Landscape
Studies, Birmingham, pp. 11–21. https://doi.org/10.1016/B978-0-323-60984-
5.00062-7. Reille, M., 1992. Pollen et Spores d’Europe et d’Afrique du Nord, 2nd ed. Laboratoire de
Botanique Historique et Palynologie, Marseille. Riddell, S.J., Erlendsson, E., Eddud´ottir, S.D., Gíslad´ottir, G., Kristj´ansd´ottir, S., 2018. Pollen, Plague & Protestants: The Medieval Monastery of Þingeyrar
(Þingeyraklaustur) in Northern Iceland. Environ. Archaeol. 1–18. Journal of Archaeological Science: Reports 45 (2022) 103575 https://doi.org/
10.1080/14614103.2018.1531191. ii Tipping, R., 2010. Bowmont: An Environmental History ofthe Bowmont Valley and the
Northern Cheviot Hills, 10000 BC-AD 2000. Society of Antiquaries of Scotland,
Edinburgh. Rodríguez Fern´andez, T., 1994. El fin del mundo fortificado y la aparici´on de las «aldeas
abiertas» : la evidencia del Centro-Oriente de Lugo (Samos y Sarria). Espac. Tiempo y
Forma. Ser. I, Prehist. y Arqueol. 153–189. https://doi.org/10.5944/
etfi.7.1994.4606. Tipping, R., 1997. Medieval Woodland History from the Scottish Southern Uplands: Fine
Spatial-Scale Pollen Data from a Small Woodland Hollow., in: Smout, C. (Ed.),
Scottish Woodland History,. Scottish Cultural Press, Dalkeith, pp. 51–74. i
Rodríguez Gonz´alez, M.C., 2008. San Xuli´an de Samos. Unha instancia de poder na Idade
Media, in: Folgar de la Calle, M.C. (Ed.), San Xuli´an de Samos: Historia e Arte Nun
Mosteiro. Opus Monasticorum III. Consellería de Innovaci´on e Industria, Santiago de
Compostela, pp. 49–72. Turner, S., Kinnaird, T., Varinlio˘glu, G., S¸erifo˘glu, T.E., Koparal, E., Demirciler, V.,
Athanasoulis, D., Ødegård, K., Crow, J., Jackson, M., Bol`os, J., S´anchez-Pardo, J.C.,
Carrer, F., Sanderson, D., Turner, A., 2021. Agricultural terraces in the
Mediterranean: medieval intensification revealed by OSL profiling and dating. Antiquity 95, 773–790. https://doi.org/10.15184/aqy.2020.187. Romaní Martínez, M., 1989. A colecci´on diplom´atica do mosteiro cisterciense de Santa
María de Oseira (Ourense). T´orculo, Santiago de Compostela. Wimble, G., Wells, C.E., Hodgkinson, D., 2000. Human impact on mid- and late Holocene
vegetation in south Cumbria. UK. Veg. Hist. Archaeobot. 9, 17–30. https://doi.org/
10.1007/BF01295011. Roubis, D., Sogliani, F., Lazzari, M., Mercuri, A.M., 2008. Climate, geoarchaeological and
bioarchaeological practices, landscape archaeology: data to evaluate the use and the
transformation of a monastic site (Jure Vetere, Italy). Boulletin Geol. Soc. Greece 42,
49–60. Zannini, P., Frascaroli, F., Nascimbene, J., Persico, A., Maxwell Halley, J., Stara, K.,
Midolo, G., Chiarucci, A., 2021. Sacred natural sites and biodiversity conservation: a
systematic review. Biodivers. Conserv. 30, 3747–3762. https://doi.org/10.1007/
s10531-021-02296-3. S´anchez-Pardo, J.C., Fern´andez Ferreiro, M., 2019. Monasterios familiares y espacios
agrarios en la Galicia del a˜no mil. Un estudio comparativo de los dominios de San 12
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J-CO: A Platform-Independent Framework for Managing Geo-Referenced JSON Data Sets
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Electronics
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Keywords: collections of JSON documents; geo-referenced data sets; platform-independent frame-
work for managing JSON data sets Received: 19 December 2020
Accepted: 27 February 2021
Published: 7 March 2021 Giuseppe Psaila *
and Paolo Fosci Giuseppe Psaila *
and Paolo Fosci Giuseppe Psaila *
and Paolo Fosci Department of Management, Information and Production Engineering, University of Bergamo,
24044 Dalmine (BG), Italy; paolo.fosci@unibg.it
* Correspondence: giuseppe.psaila@unibg.it; Tel.: +39-035-205-2355 Department of Management, Information and Production Engineering, University of Bergamo,
24044 Dalmine (BG), Italy; paolo.fosci@unibg.it
* Correspondence: giuseppe.psaila@unibg.it; Tel.: +39-035-205-2355 Abstract: Internet technology and mobile technology have enabled producing and diffusing massive
data sets concerning almost every aspect of day-by-day life. Remarkable examples are social media
and apps for volunteered information production, as well as Open Data portals on which public
administrations publish authoritative and (often) geo-referenced data sets. In this context, JSON
has become the most popular standard for representing and exchanging possibly geo-referenced
data sets over the Internet.Analysts, wishing to manage, integrate and cross-analyze such data sets,
need a framework that allows them to access possibly remote storage systems for JSON data sets,
to retrieve and query data sets by means of a unique query language (independent of the specific
storage technology), by exploiting possibly-remote computational resources (such as cloud servers),
comfortably working on their PC in their office, more or less unaware of real location of resources. In this paper, we present the current state of the J-CO Framework, a platform-independent and
analyst-oriented software framework to manipulate and cross-analyze possibly geo-tagged JSON
data sets. The paper presents the general approach behind the J-CO Framework, by illustrating
the query language by means of a simple, yet non-trivial, example of geographical cross-analysis. The paper also presents the novel features introduced by the re-engineered version of the execution
engine and the most recent components, i.e., the storage service for large single JSON documents and
the user interface that allows analysts to comfortably share data sets and computational resources
with other analysts possibly working in different places of the Earth globe. Finally, the paper reports
the results of an experimental campaign, which show that the execution engine actually performs in
a more than satisfactory way, proving that our framework can be actually used by analysts to process
JSON data sets.
Citation: Psaila, G.; Fosci, P. J-CO: A
Platform-Independent Framework for
Managing Geo-Referenced JSON
Data Sets. Electronics 2021, 10, 621. https://doi.org/10.3390/
electronics10050621
Academic Editor: Guillermo López
Taboada
Received: 19 December 2020
Accepted: 27 February 2021
Published: 7 March 2021 Citation: Psaila, G.; Fosci, P. J-CO: A
Platform-Independent Framework for
Managing Geo-Referenced JSON
Data Sets. Electronics 2021, 10, 621.
https://doi.org/10.3390/
electronics10050621 electronics 1. Introduction Unfortunately, when different storage technologies are involved, analysts wishing
to integrate data sets encounter objective obstacles, such as proprietary or missing query
languages, different computational capabilities, restricted or impossible access to JSON
stores in writing mode, and different user interfaces. In other words, we observed the lack
of a unifying framework and of a unifying query language suitable for people without
(or with basic) programming skills. Due to the distribution of data storage systems on
different servers, possibly located far away each other, as well as to the availability of the
necessary computational resources on remote servers (like cloud servers), analysts who
share data sets with other investigation teams located in various parts of the Earth globe
should exploit those computational resources remotely, comfortably working in their office. This perspective further complicates the panorama. These considerations became clear in our mind while participating to the Urban Nexus
project [7], a joint effort of geographers and computer scientists to develop models and tools
for studying mobility on the basis of Big Data, Open Data and Social Media. The project
involved three European research institutions, i.e., University of Bergamo (Italy), Ecole
Polytechnique Fédérale de Lausanne (EPFL) (Switzerland) and Angila Ruskin University of
Cambridge (UK). So, in 2017 we conceived a new framework, named J-CO (that stands for
JSON COllections). The original goal of the framework was to provide a high-level query
language able to natively manipulate collections of JSON documents possibly exploiting
their geo-tagging, so as to allow geographers to perform complex analysis tasks. Although started as part of the Urban Nexus project, now it is an autonomous project. Currently, the goal of the framework is twofold: it has to provide a platform-independent
view of several (possibly remote) data storage services, storing possibly geo-tagged JSON
data sets; it has to provide a query language, named J-CO-QL, for manipulating sets of
JSON documents, by natively supporting spatial representations and operations. y
y
pp
g p
p
p
This paper presents an extensive review of the organization of the framework, by
discussing how the integration of the concept of platform independence (with respect to
storage technology) and of the loosely-coupled approach has led us to devise a powerful
framework for providing data analysts with a tool for cross-analyzing heterogeneous
JSON data sets. 1. Introduction Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. Internet technology and mobile technology have enabled the massive production of
information concerning more or less any aspect of day-by-day life. In fact, social media,
mobile devices and apps for volunteered information production have contributed to gather
huge volumes of possibly geo-referenced data sets. Nevertheless, public administrations are
producing a very large number of publicly available data sets concerning the administrated
territory, known as Open Data, on dedicated portals. Often, Open Data are geo-referenced
data sets describing authoritative information about territories, such as boundaries of
municipalities, roads, public transportation lines, and so on and so forth. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article
distributed
under
the
terms
and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/). p
p
p
The most popular standard for distributing these data sets is JSON (acronym for
JavaScript Object Notation) [1]: its success is due to the flexibility it provides, because it is
a simple serialized representation of main-memory objects managed by object-oriented
programming languages; with respect to XML (eXtensible Mark-up Language) [2], it is
simpler and faster to manage JSON data sets than XML documents. The GeoJSON format [3,
4] is a remarkablGIS (Geographical Information Systems), which is based on JSON. https://www.mdpi.com/journal/electronics Electronics 2021, 10, 621. https://doi.org/10.3390/electronics10050621 Electronics 2021, 10, 621 2 of 35 Currently, many technologies are available or are under development to store and
manage large JSON data sets, including NoSQL (that stands for Not only SQL) database
systems [5], which are becoming quite popular, such as MongoDB [6]. However, as usual,
one single technology is not enough to fully deal with every kind of data set; furthermore,
we cannot think that a unique and central storage system could store all data sets to manage,
especially when complex cross-analysis tasks must be performed by analysts. We are also
envisioning virtual JSON stores, i.e., abstractions of JSON stores obtained by aggregating
links of JSON data sets of interest available on the Internet (for example, published by Open
Data portals); we figure out that such virtual JSON stores could provide only a read-only
access method. 2. Background and Related Work We now illustrate the background of our research. First, we present the motivations of
our proposal. Then, we present related research work. 1. Introduction With respect to our previous publications [8–10] the main contribution
of the paper is to provide an organic and comprehensive description of the current state
of the framework, which, after re-engineering the J-CO-QL Engine (the execution engine
of J-CO-QL) and adding novel components, has assumed the nature of a loosely-coupled
and platform-independent framework for manipulating and transforming (possibly geo-
tagged) JSON data sets. Specifically, the novel components are the storage service for large
single JSON documents (named J-CO-DS) and the user interface (named J-CO-UI), which
allows analysts to comfortably share data sets and computational resources with other
analysts possibly working in different places of the Earth globe. The J-CO Framework is
not a database system, but a tool that provides a platform-independent query language
and processing capabilities to retrieve data sets from various JSON stores (not necessarily
DBMSs, i.e., DataBase Management Systems), cross-analyze them and possibly store results
into its own storage service J-CO-DS (in place of other JSON stores that could provide only
a read-only access mode). As a final contribution, for the first time the paper analyzes
performance of the execution engine; the results show that it performs quickly with compact Electronics 2021, 10, 621 3 of 35 3 of 35 data sets and scales properly when the size of data sets increases. Consequently, we can
claim that the J-CO Framework is ready to be actually used by analysts. data sets and scales properly when the size of data sets increases. Consequently, we can
claim that the J-CO Framework is ready to be actually used by analysts. The paper is organized as follows. Section 2 presents the background of our project
and related work. Section 3 introduces the J-CO Framework, specifically focusing on the
novel components and on the main features of the re-engineered J-CO-QL Engine. Section 4
introduces the J-CO-QL language, by means of a simple, yet non-trivial, cross-analysis
example, by means of which key instructions of the language are presented. Section 5
presents the latest component added to the framework, i.e., the user interface named
J-CO-UI. Section 6 reports the experimental campaign and discusses the results. Finally,
Section 7 draws the conclusions. 2.1. Background of the Proposal The first step towards the idea of a loosely-coupled and platform-independent framework
for managing JSON data sets was suggested after the work on the FollowMe project [11,12]:
in that project, we had the idea of exploiting social media to trace movements of users. We
chose the name “FollowMe” because we traced travelers that posted geo-located messages on
Twitter; travelers were detected in a pool of 30 airports connected with the airport of Bergamo
(northern Italy), since we wanted to study the relevance of this airport in relation to EXPO
2015, held in Milan (only 40 km separate Milan from Bergamo). Then [13], we integrated
the FollowMe project within a framework for analyzing trips of Twitter users; in particular
(see [14]), we developed a clustering technique for identifying common paths followed by
travelers during their trips. In this preliminary work, trips of Twitter users were represented as
JSON documents; we had to analyze them on the basis of a multi-paradigmatic approach (see
the Urban Nexus project [7]). While facing this analysis task, we experienced the limitations of
current SQL and NoSQL technology when managing heterogeneous geo-located data in the
form of JSON documents. As mentioned above, we had the idea of the J-CO Framework while working in the
Urban Nexus project [7]. The goal of this project was to study how city users (inhabitants
and commuters) live the city, by means of Big Data, Open Data and any kind of Web source. The project integrated authoritative data sets, statistical data sets and data from social
media. In fact, we argued that novel information sources and Big Data (intended as variety
of data sets and sources, on the basis of the Seven V’s model [15]) can be beneficial to foster
geographical studies about how people live territories. In this project, we realized that
data come from many sources as JSON or GeoJSON [3] data sets; we also realized that
geographers and analysts ask for high-level tools to work on data sets. These considerations
inspired the research work on the J-CO Framework. 2.2. Related Work (on NoSQL Databases and on Query Languages for JSON Documents) The advent of XML in the late 1990s as the standard format for representing semi-
structured documents, for exchanging them through the Internet, gave researchers the
idea to investigate the development of database technology for storing XML documents. A large number of proposals came out from this research topic: the reader can refer
to [16–18] for some surveys. The common feature of all these proposals is that they are
XML-native databases, i.e., databases that natively store XML documents (independently
of the underlying technology); thus, they constitute an early investigation towards NoSQL
databases, because they abandoned the relational data model. XML-native databases had
impact on data mining research as well: in fact, the ability of XML to represent both raw
data and mined patterns opened the perspective of using XML and XML-native databases
to store both data and mined patterns within a unifying representation framework [19,20]. However, some practical obstacles, concerned with the complexity of managing XML Electronics 2021, 10, 621 4 of 35 documents and their excessive verbosity, have limited XML to become “the representation
format” for the Internet. In contrast, JSON has been favored in this sense. documents and their excessive verbosity, have limited XML to become “the representation
format” for the Internet. In contrast, JSON has been favored in this sense. JSON has become the most popular standard for exchanging data sets between dif-
ferent layers of the same web information system and, very often, between different
information systems; furthermore, many web services and social-media APIs (Application
Programming Interfaces) adopt JSON to exchange data sets. Consequently, the area of
NoSQL databases started developing NoSQL DBMSs to natively store JSON data sets. An
interesting survey on NoSQL databases is [5], where several systems are cataloged and
classified. One common feature of most NoSQL databases is that they abandon the ACID (Atom-
icity, Consistency, Isolation, Durability) properties of transactions (as provided by relational
DBMSs) in favor of the Base (Basically Available, Soft state, Eventual consistency) model
[21,22] to deal with concurrency. In particular, since JSON data are generically called
“documents” [23], a DBMS able to natively store JSON documents falls into the category
of document databases. One of the most famous representatives is MongoDB [6]. Readers
interested in NoSQL DBMSs evaluation can refer to [5,22]. Obviously, the diffusion of JSON and of JSON-native DBMS technology asks for
suitable and high-level query languages. 2.2. Related Work (on NoSQL Databases and on Query Languages for JSON Documents) Before defining our query language named J-
CO-QL [9,10] (see Section 4 for a synthetic introduction), we studied previous proposals. Hereafter, we present the most relevant languages. We compare their features with those
that characterize J-CO-QL; Table 1 synthetically reports the results of the comparison; the
table considers only those languages that operate on JSON documents; rows report the
features we considered. Specifically, they are introduced below. 1. Adaptability to heterogeneity. This feature refers to the ability of the language to
manipulate heterogeneous documents with one single instruction. In fact, usually
query operators have sets of documents as operands; sets of documents may contain
heterogeneous documents, i.e., documents having (possibly many) different structures
from each other; if the operators are able to work on documents with totally different
structure with one single instruction, i.e., processing them all together, the feature
called Adaptability to heterogeneity is met. In contrast, if operators deal with one
single structure at a time, this feature is not met. 2. Spatial operations. This feature refers to the capability of performing spatial opera-
tions on geo-tagged documents. g
gg
3. Platform independence. This feature refers to the fact that the query language is
independent of a specific platform, typically JSON document stores. 4. Re-use of intermediate results. When complex transformations are performed, inter-
mediate results could be precious for next processing steps. With this feature, we refer
to the capability of the query language to explicitly handle and re-use intermediate
results, to avoid re computations as well as to avoid storing intermediate results
temporarily within JSON databases. 5. Orientation to Analysts. This feature refers to the fact that operators of the query
language are oriented to analysts, without specific procedural programming skills. 5. Orientation to Analysts. This feature refers to the fact that operators of the query
language are oriented to analysts, without specific procedural programming skills. 6. Orientation to Programmers. This feature refers to the fact that operators of the
query language are oriented to computer programmers, having strong computer
programming skills. 6. Orientation to Programmers. This feature refers to the fact that operators of the
query language are oriented to computer programmers, having strong computer
programming skills. Table 1. Comparison of query languages for JSON data. Query Language
J-CO-QL
Jaql
SQL++
JSONiq
MongoDB
1. Adaptability to heterogeneity
YES
NO
NO
NO
NO
2. Spatial operations
YES
NO
NO
NO
YES
3. 2.2. Related Work (on NoSQL Databases and on Query Languages for JSON Documents) Platform independence
YES
NO
YES
YES
NO
4. Re-use of intermediate results
YES
NO
NO
NO
NO
5. Orientation to Analysts
YES
NO
YES
NO
NO
6. Orientation to Programmers
NO
YES
NO
YES
YES Table 1. Comparison of query languages for JSON data. Electronics 2021, 10, 621 5 of 35 After this premise, we can start presenting various languages for querying JSON
data sets. Jaql was proposed in [21] to help Hadoop (the popular Map-Reduce framework,
see [24,25]) programmers develop complex transformations, when writing Map-Reduce
programs on Big Data. It is designed to be flexible and independent of the physical repre-
sentation of data. Furthermore, since it is thought for the Map-Reduce paradigm, where
complex computations are modeled as sequences of Map-Reduce phases, Jaql strongly
relies on the concept of “pipe”. Considering the features reported in Table 1, Jaql does
not support heterogeneity of documents, does not provide spatial operations, is not plat-
form independent, does not provide re-usability of intermediate results and is oriented
to programmers. The language that we repute the best proposal before J-CO-QL is SQL++ [26]. Its semi-
structured data model is designed as an extension of both JSON and the relational data
model; consequently, the language is derived from the classical SELECT statement by adding
a limited number of language extensions. In this sense, [27] reports the case of N1QL for
Analytics, an interesting implementation of SQL++ for the Couchbase Server. Considering the
features reported in Table 1, SQL++ is not specifically designed to work with heterogeneous
documents in an explicit way, as well as spatial operations are not considered at all. It is
platform independent, but it does not provide re-usability of intermediate results and it is
oriented to analysts (because it derives from SQL). y
JSONiq [28] is a query language for JSON documents derived from XQuery [29]. The
functional style of the language, the semantics of comparisons in case of data heterogeneity
and the declarative snapshot-based updates are taken from XQuery, while other specific
features are not suitable for querying JSON documents, such as management of mixed
content, because they are peculiar of XML. However, like XQuery, it can be hardly used by
inexperienced users. 2.2. Related Work (on NoSQL Databases and on Query Languages for JSON Documents) In contrast, it is not designed to work with large
JSON data sets, while our framework (and its query language) is oriented to provide data
analysts with a tool for managing data about real entities, described by large sets of JSON
documents and/or large GeoJSON documents. The overall J-CO Framework (and the J-CO-QL query language, in particular) relies on
our previous research work on collections of complex spatial data [35,36]. In those works,
we proposed a database model capable to deal with heterogeneous collections of possibly
nested spatial objects; the proposal encompassed an algebra to query complex spatial
data, derived from classical relational algebra. In J-CO we follow a different approach: we
have not defined a database model, because J-CO relies on JSON data sets. Furthermore,
J-CO-QL abandons the classical syntax of relational algebra, in favor of a more flexible
syntax, suitable to specify complex transformation processes in a more intuitive and flexible
way; in fact, we applied the experience we made in [37], where we defined a language for
manipulating clusters of web search results performed through a mobile device. The J-CO Framework is specifically designed to be loosely-coupled with underlying
JSON document stores: it seamlessly accesses any kind of JSON document stores (provided
that a connector is available) and provides its own computational capabilities; consequently,
J-CO-QL is platform independent (feature 3 in Table 1). This is why we say that it is a
“multi-store framework”. In effect, when we defined the approach, we had the concept of
PolyStore DBMS in mind; it denotes database management systems that deal with several
DBMSs at the same time, each of them possibly providing a different logical model, such
as relational, graph, JSON, pure-text, images and videos. It appears that BigDAWG [38] is
the first polystore system in the literature; nevertheless, it is not an isolated case and the
work on polystores is going on. For example, [39] reports about Polystore++, a polystore
system for analytic applications. However, the definition of polystore systems requires
the development of techniques for building query plans (as in [40]), as well as to define
extensions of the classical relational algebra (as in [41]). These previous workson polystores clearly adopted a pure database approach, in
which it is assumed that the DBMSs integrated within the polystore provide their own
and fully exploitable query languages. 2.2. Related Work (on NoSQL Databases and on Query Languages for JSON Documents) Considering the features reported in Table 1, JSONiq is not able to
work on heterogeneous documents and does not provide spatial operations; it is platform
independent, but it does not provide re-usability of intermediate results and it is not
oriented to analysts, while it is oriented to programmers. y
p
g
The query language provided by MongoDB [5,30], probably the most popular JSON
document DBMS, is certainly considered the reference language (URL: https://docs. mongodb.com/manual/, accessed on 3 March 2021). If we consider the features reported
in Table 1, it is not designed to manipulate totally heterogeneous documents. It provides
spatial operations, but they can be performed only on documents that are materialized
in the database (because they must be previously indexed by means of spatial indexes);
spatial operations cannot be performed on temporary data. It is not platform indepen-
dent, because it is specific of MongoDB; re-usability of intermediate results is not natively
supported, because they must be saved into the database. The syntax, which is based
on object-oriented programming, makes it not specifically oriented to analysts; however,
complex transformations can be performed only by writing JavaScript functions within the
query, thus this makes the language definitely oriented to programmers. Other approaches to manipulate heterogeneous big data recognize the importance of
a declarative query language [31,32], although they do not work on JSON data sets. The
advantage is to simplify coding of complex transformations, in particular in the context of
Map-Reduce programming. An interesting example is Spark SQL [33], introduced within
the Apache Spark framework, one of the two most-popular frameworks supporting the
Map-Reduce paradigm for processing Big Data. It is designed to allow for specifying
complex manipulations of data sets without using Map-Reduce primitives (the framework
transforms Spark SQL queries into low-level Map-Reduce primitives). However, Spark SQL
still does not consider at all spatial operations. p
p
A proposal concerned with spatial operations is GeoSPARQL [34]. It is a Geographic
Query Language for RDF data, proposed as a standard by the OGC (Open Geospatial
Consortium—URL: https://www.ogc.org/, accessed on 3 March 2021), for querying
geospatial data on the Semantic Web. GeoSPARQL is designed to accommodate sys- Electronics 2021, 10, 621 6 of 35 tems based on qualitative spatial reasoning and systems based on quantitative spatial
computation, to ease data analysis. 3. A Framework for Manipulating Geo-Referenced Data 3. A Framework for Manipulating Geo-Referenced Data
In this section, we introduce the J-CO Framework. In this section, we introduce the J-CO Framework. 2.2. Related Work (on NoSQL Databases and on Query Languages for JSON Documents) However, an interesting sentence from [41] says:
“Polystores seek to provide a mechanism to allow applications to transparently achieve
the benefits of diverse databases while insulating applications from the details of these
databases”. The J-CO Framework is not a polystore, since it relies on a unique data model
and does not push computation to DBMSs, in order to be platform independent; however,
it certainly has been designed in order to make applications insulated from the specific
JSON document store technology. gy
The loosely-coupled design of the J-CO Framework, motivated by the fact that JSON
document stores could not provide the necessary processing capabilities or permissions
to execute queries, could appear a strange decision to database experts. Currently, the
J-CO-QL Engine executes queries only in main memory. However, the world of database
systems is more and more getting populated by solutions that manage large data sets
in main memory. A field in which this approach could appear paradoxical is Business
Intelligence. However, there exist systems (see [42]) that perform Business Intelligence
tasks on large volumes of data to aggregate in main memory. The Qlik suite [43,44] is a quite
famous suite for Business Intelligence that operates in main memory: it first collects data
from data sources into main memory, then it performs aggregations requested by users. 3.1. Organization of the Framework The framework we conceived for integrating and transforming geo-referenced JSON
data is named J-CO (acronym for JSON COllections) and comprehends several components. 7 of 35 Electronics 2021, 10, 621 •
One or more NoSQL storage systems, powered by MongoDB, ElasticSearch [45] and, in
the future, other similar systems (for example, AWS Amazon DB, https://aws.amazon. com/documentdb/, accessed on 3 March 2021). •
J-CO-DS is a simplified storage system for sets of JSON documents; since it is based
on the file-system, it is able to store very large JSON documents that would not be
managed by other systems, like MongoDB. It is not a DBMS, since it neither provides a
query language nor supports OLTP operations. q
y
g
g
pp
p
•
J-CO-QL is the query language around which the entire framework is built. The
language allows for specifying complex transformations, possibly based on spatial op-
erations. •
The J-CO-QL Engine executes J-CO-QL queries by retrieving data stored in one or
many JSON storage systems. y
g
y
•
A User Interface, named J-CO-UI, provides users with a powerful tool to write complex
queries step-by-step, by possibly inspecting temporary results of the process. The overall framework relies on a unique data model. The overall framework relies on a unique data model. The overall framework relies on a unique data model. Definition 1. Data Model. A “collection” is a set of JSON documents, where documents are
heterogeneous, i.e., possibly have their own structure, without limitations. A “database” is a set of
collections; neither predefined structures nor schemas for documents in collections are considered. Hereafter, we give a more detailed description of two key components, specifically
J-CO-DS and J-CO-QL Engine. J-CO-QL will be presented in Section 4, while J-CO-UI will
be introduced in Section 5. Now, we present the layer-based view of the framework we reported in Figure 1,
which motivates the loosely-coupled design. We considered three layers, i.e., the Data Layer,
the Engine Layer and the Interface Layer. •
The Data Layer includes all data stores, managed by any kind of NoSQL storage
system (currently, MongoDB, ElasticSearch and J-CO-DS). Since each single data store
can contain a relevant piece of information to integrate, they must be viewed in a
seamless way by users: they do not have to take care of the specific storage technology. •
The Engine Layer encompasses one or many instances of the J-CO-QL Engine. 3.1. Organization of the Framework Why
more than one instance? Because it could be installed on several servers, in order to
provide many different users, possibly located in different areas of the Earth globe,
with the necessary computational power. y
p
p
•
The Interface Layer encompasses one or more installations of J-CO-UI: in fact, users
(typically, analysts) write queries and develop transformation processes through this
tool. Since J-CO-UI is a desktop application (see Section 5), its instances are installed
on analysts’ PCs. Consequently, we can say that the loosely-coupled design of the J-CO Framework
allows for creating an eco-system of data, computational resources and human resources
distributed around the world, working together on the same investigation problem. Each single computational resource (user interfaces, J-CO-QL Engine and JSON storage
systems) communicate through the Internet and can be easily added to or removed from
the eco-system. Each single computational resource (user interfaces, J-CO-QL Engine and JSON storage
systems) communicate through the Internet and can be easily added to or removed from
the eco-system. 8 of 35 Electronics 2021, 10, 621 Figure 1. Application Layers for the J-CO Framework. Figure 1. Application Layers for the J-CO Framework. Discussion. Thinking about JSON stores, we do not have to think that they are necessarily
DBMSs that work on JSON documents instead of relational data; in fact, people usually
think that they provide a query language able to transform data items in a way similar to
what SQL does (through the INSERT, UPDATE, DELETE and SELECT statements). The scenario
is quite different, for various reasons. First of all, we discuss the topic of query languages. •
Some JSON storage systems provide query languages, in a way similar to traditional
DBMSs; however, these languages do not provide operations that are familiar to SQL
users, like “joins”. An example is MongoDB, in which to perform queries equivalent
to “joins” it is often necessary to write procedural JavaScript code, to overcome the
limitations of its query language. •
Other JSON storage systems do not provide any query language or capability to trans-
form stored data. ElasticSearch falls into this category: it is an engine for information
retrieval, which receives and provides data in JSON format through its API. However,
it is not a DBMS: when a document is received, it is indexed by means of an inverted
index, that allows keyword-based searches to retrieve indexed documents. 3.2. J-CO-DS We decided to develop this storage service when we encountered some limitations
exposed by available NoSQL storage systems, in particular, MongoDB and ElasticSearch. •
MongoDB [6] is a true DBMS for JSON data. Its data model is the same we introduced
in Definition 1: a “database” is a set of collections, where a “collection” is a set of
JSON documents. Neither predefined structures nor schemas must be defined: a
collection can store heterogeneous JSON documents, with no limitations. However,
MongoDB is designed to manage very large collections of small documents: it does not
accept JSON documents whose internal binary representation (the BSON format) is
larger than 16MB. This limitation makes impossible to store large GeoJSON documents
within MongoDB databases: in fact, a GeoJSON document is a single document that can
be giant; in case of complex geometries, the BSON representation can easily exceed
16 MB. Consequently, MongoDB is actually unable to manage very large documents. MongoDB is designed to manage very large collections of small documents: it does not
accept JSON documents whose internal binary representation (the BSON format) is
larger than 16MB. This limitation makes impossible to store large GeoJSON documents
within MongoDB databases: in fact, a GeoJSON document is a single document that can
be giant; in case of complex geometries, the BSON representation can easily exceed
16 MB. Consequently, MongoDB is actually unable to manage very large documents. q
y
g
y
g
y
g
•
On the other side, ElasticSearch [45] is a very popular tool for performing information
retrieval operations. It is not designed to be a pure DBMS, but since it receives and
provides JSON data sets through its API, it can be assimilated to the database view. However, it is not good to store very large GeoJSON documents into ElasticSearch,
because it has to index the whole document, including geometries; this would cause a
significant waste of time (during indexing) and would negatively affect results pro-
vided by ElasticSearch at retrieval time, because coordinates would be considered for
text-based retrieval as text fragments, not as numbers denoting geo-tagging. Elastic-
Search does not provide any query language, in the sense of database query languages:
stored data cannot be manipulated as it is possible to do, e.g., within MongoDB. Obviously, its API allows for uploading, getting and deleting data sets. 3.1. Organization of the Framework Clearly, it
has not to provide a query language as intended for DBMSs, because it is not a DBMS. •
In principle, any Open Data portal could be a read-only JSON store: if we had
a tool able to provide a database-like abstraction of Open Data portals, we could
consider it as a valid JSON store from which data can be read for further processing. Obviously, we cannot expect any processing functionality (in terms of query language)
by such sources. Another point to consider when thinking about JSON storage systems is whether they
provide a concurrency model. •
A strong concurrency model is one of the key success factors of relational DBMSs. Some JSON store systems provide strong concurrency models, like CouchDB [25,46],
that, for this reason, has been chosen as the DBMS for HyperLedger Fabric [47], the
well-known block-chain platform developed by Linux Foundation. •
A strong concurrency model is one of the key success factors of relational DBMSs. Some JSON store systems provide strong concurrency models, like CouchDB [25,46],
that, for this reason, has been chosen as the DBMS for HyperLedger Fabric [47], the
well-known block-chain platform developed by Linux Foundation. Electronics 2021, 10, 621 9 of 35 9 of 35 •
Originally, MongoDB provided very limited concurrency control and transaction
management. Now, as long as it becomes more and more adopted, new versions enrich
support to transactions; consequently, it is becoming a true DBMS for JSON documents. pp
q
y
g
•
Other JSON storage systems do not provide any concurrency control or support to
transactions. This is the case, for instance, of ElasticSearch. 3.2. J-CO-DS Through the user interface, usual basic management operations can be performed,
such as database creation and deletion, collection creation and deletion and upload of Through the user interface, usual basic management operations can be performed,
such as database creation and deletion, collection creation and deletion and upload of Through the user interface, usual basic management operations can be performed,
such as database creation and deletion, collection creation and deletion and upload of
JSON documents into collections. J-CO-DS does not support transactions and does
not provide explicit locking mechanisms, because they are not necessary; only an
implicit locking mechanism on collections, when either a read or a write operation is
performed, is provided to serialize access to single collections. Through the user interface, usual basic management operations can be performed,
such as database creation and deletion, collection creation and deletion and upload of
JSON documents into collections. J-CO-DS does not support transactions and does
not provide explicit locking mechanisms, because they are not necessary; only an
implicit locking mechanism on collections, when either a read or a write operation is
performed, is provided to serialize access to single collections. p
JSON documents into collections. J-CO-DS does not support transactions and does
not provide explicit locking mechanisms, because they are not necessary; only an
implicit locking mechanism on collections, when either a read or a write operation is
performed, is provided to serialize access to single collections. 3.3. J-CO-QL Engine The J-CO-QL Engine is the core software component of the framework, because it
executes J-CO-QL queries. Recently, it has been re-engineered, in order to be compliant
with the multi-layer view depicted in Figure 1. It mainly operates as a service on a specific
TCP port. It actually executes J-CO-QL queries, by connecting to data storage services to
get and save data sets (collections of JSON documents). Since its re-engineering, it provides
two different operating modes: •
Batch Mode: this mode can be used to execute complex and long queries, previously
written by analysts. •
Batch Mode: this mode can be used to execute complex and long queries, previously
written by analysts. y
y
•
Interactive Mode: this mode can be used to write queries step-by-step, so as to build
complex transformation processes, in an interactive manner. In particular, when operating in Interactive Mode, the service provides the follow-
ing features: In particular, when operating in Interactive Mode, the service provides the follow-
ing features: •
Long-Term Query Process: the query process lives for a long time, the process state is
not lost while waiting for new instructions; •
Step-by-step Issuing: instructions are provided one at a time; if the instruction generates
an error (lexical, syntactic or semantic error) simply the process state does not change; •
Roll-back: the user can roll-back the execution of the process to a previous execu-
tion state. 3.2. J-CO-DS So, we argued that a data storage service for large JSON (possibly GeoJSON) docu-
ments was missing and we decided to develop J-CO-DS. J-CO-DS provides the same data model as MongoDB: a database is a set of collections,
and a collection can contain any kind of (possibly huge) JSON documents. No query
language is provided, because J-CO-DS is not meant to be a DBMS: it is a simple JSON
storage system, which does not support OLTP operations at document level. J-CO-DS
had to be easily developed and fast in retrieving and storing documents: consequently,
we adopted a lightweight approach by design, i.e., collections are managed through the
file-system. No extra features are provided, neither indexes nor spatial indexes, because
J-CO-DS is designed neither for processing queries not for supporting operational activities. In fact, J-CO-DS is thought to be a simple storage system for collections of JSON documents,
able to manage large single documents; it can be used by analysts to organize data sets,
without the complexity of a pure DBMS, like MongoDB. Note that this approach is perfectly
coherent with the rest of the framework, which is designed to be platform independent as
far as storage systems are concerned. Obviously, a managing interface is provided. Hereafter, we present the components
of J-CO-DS. •
J-CO-DS Service is the true storage service that can be contacted through a TCP port,
either by J-CO-QL Engine instances or by any other application wishing to exploit
its services. •
J-CO-DS Manager is a user interface that allows for interactively managing the data
store; it is an application that can be installed on administrator’s PC and provides two
execution modes: Electronics 2021, 10, 621 10 of 35 10 of 35 –
Stand-alone mode: in this execution mode, the application uses a text-based inter-
face, that can be accessed through a classical text-based shell; –
Stand-alone mode: in this execution mode, the application uses a text-based inter-
face, that can be accessed through a classical text-based shell; –
Web-based mode: when launched in this mode, the application provides a web-based
interface, by means of an internal web server packaged in the executable file. –
Web-based mode: when launched in this mode, the application provides a web-based
interface, by means of an internal web server packaged in the executable file. These features are controlled by J-CO-UI (see Section 5). In particular,
the connector to MongoDB has to encode/decode JSON documents to/from the BSON rep-
resentation. As far as the management of geometries is concerned, we used theJTS—Java Topology
Suite library (URL: https://github.com/locationtech/jts, accessed on 3 March 2021). The
library is able to parse GeoJSON geometries represented as GeometryCollection types
(see [3]), so as to obtain its own internal representation, suitable to easily perform spatial
operations. The library is able to re-generate GeoJSON representations of geometries from
its own internal representation. Currently, all collections are processed only in main memory, without using mass
memory. In order to overcome limitations in memory size, as well as to accelerate spatial
operations, we plan to introduce hybrid processing methods (main and mass memory) as
well as to create (spatial) indexes on the fly. Discussion. In the application context of our framework, decoupling data source environ-
ments from the processing environment is an essential feature. First of all, it is due to the
fact that JSON storage systems may not provide any capability to transform data (see the
discussion in Section 3.1). As an effect, the J-CO-QL Engine remains independent of actual
processing capability (if any) provided by JSON stores (the situation in which no JSON
store providing processing capability is connected is possible). Furthermore, the J-CO-QL
Engine could implement J-CO-QL operators that cannot be supported by query languages
(for example, operators for performing data mining and machine learning activities, that
we are planning as future work). A second, but not less important, reason is concerned with integration of collections
coming from different JSON stores: to integrate them, data sets should be moved from
one store to another. We do not want users to move data from one store to another
(provided that they provide the necessary computational power), because this would limit
the flexibility we want to provide analysts with. y
p
y
In fact, the user can be substantially unaware of where and how a database is managed,
because, as depicted in Figure 2, queries and the engine seamlessly operate on collections
and integrate them, irrespective of their actual source (Section 4 will highlight this aspect). Thus, we can see the J-CO-QL Engine as a service built on top of other services, specifically
JSON stores and NoSQL DBMSs. Thus, it could be a way to somehow “federate” JSON
stores. These features are controlled by J-CO-UI (see Section 5). These features are controlled by J-CO-UI (see Section 5). Platform Independence. One of the main goal of the J-CO Framework is being able to
access several data sources, managed by different storage technologies. The reason for
developing a novel query language (the J-CO-QL query language, presented in Section 4),
is to provide a uniform language that is independent of the specific storage platform. In fact, due to the significant differences that characterize capabilities of query languages
provided by data storage systems, as well as the possible absence of query languages
and processing capabilities, the J-CO-QL Engine cannot transfer any processing activity
to data sources. This is beneficial for the evolution of J-CO-QL, in which we plan to add
instructions for complex tasks such as machine learning. The following features are related
with platform independence. Platform Independence. One of the main goal of the J-CO Framework is being able to
access several data sources, managed by different storage technologies. The reason for
developing a novel query language (the J-CO-QL query language, presented in Section 4),
is to provide a uniform language that is independent of the specific storage platform. •
Multiple Connectors. The J-CO-QL Engine is equipped with a specific connector for
each single data storage system we have considered so far, i.e., MongoDB, ElasticSearch
and J-CO-DS. These connectors provide a uniform interface to the code of the J-CO-QL
Engine towards different data storage systems. g
g
y
•
Retrieving/storing Collections. The J-CO-QL Engine interacts with data storage systems
only to retrieve the content of collections and to store new collections. No data
processing is asked to data storage systems because input collections are transferred to
the J-CO-QL Engine and output collections are transferred to the data storage systems. J
Q
g
p
g
y
•
Loosely Coupling. As a result, the J-CO-QL Engine is loosely coupled to data storage
systems: all processing activities (i.e., execution of J-CO-QL transformation processes) Electronics 2021, 10, 621 11 of 35 11 of 35 are performed within the J-CO-QL Engine on the local copy of collections, not on the
source databases. are performed within the J-CO-QL Engine on the local copy of collections, not on the
source databases. The J-CO-QL Engine is implemented in Java standard edition. This choice further
achieves platform independence, in this case as far as the operating system is concerned. Connectors to storage systems invoke specific APIs provided by them. 4. J-CO-QL: The Query Language of the J-CO Framework In this section, we introduce J-CO-QL, the query language of the J-CO Framework. First
of all, in Section 4.1 we introduce the main features of the language. Then, in Section 4.2
we describe some key instructions of the language by means of an illustrating example. These features are controlled by J-CO-UI (see Section 5). Nevertheless, the J-CO-QL Engine is not a NoSQL DBMS: it simply retrieves data
from and store data into databases managed by JSON stores and NoSQL DBMSs, which
continue providing their specific functionality: for example, MongoDB serves information
systems for operational activities, while ElasticSearch is a document search engine. Thus,
the J-CO Framework enables to create a sort of “side federation” of JSON stores currently
used for other purposes; this is also a sort of “light federation”, because JSON stores are
“federated” only during the time the J-CO-QL Engine is connected to them. At the best of
our knowledge, we have found no similar proposals. 12 of 35 12 of 35 Electronics 2021, 10, 621 Figure 2. General approach to process collections (represented as rectangles): no processing activities on data sources. ch to process collections (represented as rectangles): no processing activities on data sources. Figure 2. General approach to process collections (represented as rectangles): no processing activities on data sources. Listing 2. Excerpt of collection BikeLanes.
Listing 2 Excerpt of collection BikeLanes. Collection BikeLanes: { "City" : "Milan",
"Name" : "VIA ROBERTO KOCH",
"Trunks" : 2,
"geometry":
{ "type" : "GeometryCollection",
"geometries" : [
{ "type" : "MultiLineString",
"coordinates" : [
[
[9.11455186249295, 45.4446414404081], ... ] ] },
{ "type" : "MultiLineString",
"coordinates" : [
[
[9.11469823062049, 45.4446837789302],
... ] ] }
] } },
{ "Properties" : { "City" : "Milan",
"Name" : "VIA COL MOSCHIN",
"Trunks" : 1 },
"geometry":
{ "type" : "GeometryCollection",
"geometries" : [
{ "type" : "MultiLineString",
"coordinates" : [
[
[9.18457781600816, 45.4519411253397],
... ] ] }
] } }
... { "type" : "MultiLineString", "coordinates" : [
[
[9.11455186249295, 45.4446414404081], ... ] ] },
{ "t
"
"M ltiLi
St i
" "coordinates" : [
[
[9.11455186249295, 45.4446414404081], ... ] ] },
{ "type" : "MultiLineString", { "type" : "MultiLineString", yp
g
"coordinates" : [
[
[9.11469823062049, 45.4446837789302],
... ] ] }
] } } ] } },
Properties" : { "City" : "Milan", { "Properties" : { "City" : "Milan", y
"Name" : "VIA COL MOSCHIN", "Trunks" : 1 }, { "type" : "GeometryCollection", "geometries" : [ { "type" : "MultiLineString", yp
g
"coordinates" : [
[
[9.1845778160081 "coordinates" : [
[
[9.18457781600816, 45.4519411253397],
... ] ] }
] } } ] } } 4.1. J-CO-QL Main Features
J-CO-QL is designed to work with collections of JSON documents (see Definition 1). In a document, fields can be either simple fields (numbers or strings) or complex fields (i.e.,
As a final remark, notice that coordinates (longitude and latitude) are expressed based
on the WGS84 (World Geodetic System) standard, our Coordinate Reference System (CRS);
currently no other CRS is supported. ,
p
(
g )
p
(
,
nested documents) or vectors (of numbers, strings and documents). Since J-CO-QL has to natively deal with geo-references, its data model must natively
encompass them. However, JSON does not consider geo-references. We decided to rely on
th G
JSON t
d
d [3 4] fild d
ibi
t i
l
JSON
t d d
y
pp
Since the first version of the language [8], J-CO-QL instructions have met the closure
property, that is, they get collections and generate collections. Furthermore, the language
provides the key features reported hereafter. 4.1. J-CO-QL Main Features J-CO-QL is designed to work with collections of JSON documents (see Definition 1). In a document, fields can be either simple fields (numbers or strings) or complex fields (i.e.,
nested documents) or vectors (of numbers, strings and documents). Since J-CO-QL has to natively deal with geo-references, its data model must natively
encompass them. However, JSON does not consider geo-references. We decided to rely on
the GeoJSON standard [3,4]: fields describing geometries are complex JSON nested docu-
ments that can describe points, lines and polygons, as well as collections of them; for this
purpose, the GeoJSON standard provides a specific data type named GeometryCollection. In J-CO-QL, geo-references play a special role, since the language provides operators and
constructs to handle them. In order to give this special role to geo-references, the J-CO-QL
data model considers the special ~geometry field: if present at the root level of a document,
this means that the document is geo-referenced (or geo-tagged). The name ~geometry is
fully compliant with JSON naming rules, but ~ is rarely used in field names: in our model,
it is used to denote a special role of the field. The value of the ~geometry field is based on
the GeometryCollection type of the GeoJSON standard. As an example, consider Listings 1 and 2, which report the excerpts of two collections
of documents: Listing 1 reports the excerpt of the collection named Districts, which
describes districts of cities in Northern Italy; Listing 2 reports the excerpt of the collection
named BikeLanes, which describes bike lanes in Northern Italy. These two collections are
inspired by real data sets published on the Open Data portal of Milan (Italy) City Council
(URL: https://dati.comune.milano.it/, accessed on 3 March 2021). p
The reader can notice that the documents in the BikeLanes collection (Listing 2) have
different structures, although they describe the same type of geographical entity, i.e., bike
lanes; we will see how J-CO-QL is able to deal with heterogeneous collections. Furthermore, notice that documents in the Districts collection (Listing 1) are already
compliant with the J-CO-QL data model, since they have the root-level ~geometry field. In contrast, documents in the BikeLanes collection (Listing 2) do not have the ~geometry
field, but the geometry field (without ~, because they are possibly derived from a GeoJSON
document). Consequently, J-CO-QL should provide constructs to align geometries to the
J-CO-QL data model. 13 of 35
ork. Collection Districts: Collection Districts: { "ID": 74,
"Properties": {
"Name": "ROSERIO",
"City": "Milan" },
"~geometry": {
"type": "Polygon",
"coordinates": [
[ [ [9.13005657492143, 45.5229429902529], ...,
[9.13003510901344, 45.5229853369611] ] ] ] } },
{ "ID": 82,
"Properties": {
"Name": "COMASINA",
"City": "Milan" },
"~geometry": {
"type": "Polygon",
"coordinates": [
[ [ [9.16950766784049, 45.5251391141947], ...,
[9.16920281509976, 45.5253660742429] ] ] ] } },
... 13 of 39 Listing 2. Excerpt of collection BikeLanes. Listing 2 Excerpt of collection BikeLanes. 4.1. J-CO-QL Main Features First
ction 4 2 Electronics 2021, 10, 621 Listing 1. Excerpt of collection Districts. Listing 1 Excerpt of collection Distric Listing 2. Excerpt of collection BikeLanes.
Listing 2 Excerpt of collection BikeLanes. the GeoJSON standard [3,4]: fields describing geometries are complex JSON nested docu-
ments that can describe points, lines and polygons, as well as collections of them; for this
purpose, the GeoJSON standard provides a specific data type named GeometryCollection. In J-CO-QL, geo-references play a special role, since the language provides operators and
•
J-CO-QL instructions are able to deal with documents with different structure with
one single instruction. This is the feature named Adaptability to heterogeneity in
Table 1. Electronics 2021, 10, 621 14 of 35 14 of 35 •
J-CO-QL instructions allow for specifying complex transformations oriented to analyze
data. This is the feature named Orientation to Analysts in Table 1. •
J-CO-QL instructions allow for specifying complex transformations oriented to analyze
data. This is the feature named Orientation to Analysts in Table 1. •
J-CO-QL instructions directly deal with geo-references (through the ~geometry field)
and spatial operations. This is the feature named Spatial operations in Table 1. •
J-CO-QL instructions directly deal with geo-references (through the ~geometry field)
and spatial operations. This is the feature named Spatial operations in Table 1. J-CO-QL queries (or transformation processes) are sequences of instructions [10,48]. The execution process of queries is based on the concept of state of the query process (or
query-process state, in the rest of the paper), which is a pair s = (tc, IR), where tc is a
collection named Temporary Collection, while IR is a database named Intermediate Results
database. Each instruction ij starts from a given query-process state s(j−1) and generates a new
query-process state sj. A specific instruction asks the J-CO-QL Engine to store tc (the
Temporary Collection) into IR (the Intermediate Results database), so that the collection could
be taken as input by a subsequent instruction. p
y
q
The J-CO-QL Engine executes each query process in isolation: several users can use
the same instance of the engine in a concurrent way. Thus, the goal of the IR database,
one for each query process, is twofold. First, it temporarily stores intermediate results
of the process, so that they do not have to be stored into persistent databases (since they
are intermediate results). This is the feature named Re-use of intermediate results in
Table 1. Second, it ensures isolation of query process execution as far as intermediate
results are concerned. 4.2. Language by Example In this section, we briefly illustrate J-CO-QL, by showing a short example of cross
analysis. Suppose that three data storage systems are involved in the activity: a J-CO-
DS server having 10.0.0.11 as (toy) IP address; two MongoDB servers, whose (toy) IP
addresses are 10.0.0.12 and 10.0.0.13. Specifically, we have three databases. The first one is named Boundaries; it is hosted
by the J-CO-DS server and stores authoritative data sets about administrative boundaries
(since they are usually very large, it could not be possible to store them within MongoDB
databases, see Section 3.2); in particular, this database contains the Districts collection
(that contains JSON documents describing districts of cities in Northern Italy, such as
Milan). Recall that Listing 1 shows an excerpt of this collection. The second database is named RegionInfo: it is managed by the MongoDB server
with IP address 10.0.0.12; it contains collections related with territory. In particular, we
have the BikeLanes collection, which contains documents describing the path of bike lanes
in Northern Italy. Listing 2 reports an excerpt of this collection (recall that documents have
heterogeneous structure). The third database is managed by the MongoDB server having IP address 10.0.0.13:
its name is toShare, because it contains data sets that the analysis team has to share with
other teams. The team of analysts has the following (sample) goal: discovering the bike lanes in
the city of Milan that cross at least two districts of the city of Milan. The J-CO-QL query for
performing the analysis is reported in Listings 3 and 4. Hereafter, we will explain it, by
briefly introducing each single instruction. The reader can exploit Figure 3, which reports
the execution trace of the process: the trace starts with the empty query-process state
([], {}) taken as input by the first GET COLLECTION instruction (line 4 of Listing 3); the
query-process state produced by each single instruction is reported (for simplicity, we do
not report the actual content of temporary collections, but only an identifier ti, where i is
the line that generates the temporary collection ti). 15 of 35 Electronics 2021, 10, 621 Figure 3. Execution trace for the sample transformation process written with J-CO-QL
ronics 2021, 1, 1
15 of 39 Figure 3. Execution trace for the sample transformation process written with J-CO-QL. Figure 3. Execution trace for the sample transformation process written with J-CO-QL. p
p
J
Q
Listing 3. Sample transformation process written by means of J-CO-QL (part 1).
Listing 3 Sample transformation process written by means of J-CO-QL (part 1).
1:
USE DB Boundaries
ON SERVER jcods ’http://10.0.0.11:17017’;
2:
USE DB RegionInfo
ON SERVER MongoDB ’http://10.0.0.12:27017’;
3:
USE DB ToShare
ON SERVER MongoDB ’http://10.0.0.13:27017’;
4:
GET COLLECTION Districts@Boundaries;
5:
FILTER
CASE
WHERE WITH .Properties.City
AND .Properties.City="Milan"
GENERATE
{.ID, .Name:
.Properties.Name}
KEEPING GEOMETRY
DROP OTHERS;
6:
SET INTERMEDIATE AS Milan_Districts;
7:
GET COLLECTION BikeLanes@RegionInfo;
8:
FILTER
CASE
WHERE WITH .City
AND .City="Milan"
GENERATE {.Name}
SETTING GEOMETRY .geometry
WHERE WITH .Properties.City
AND .Properties.City="Milan"
GENERATE
{.Name:
.Properties.Name}
SETTING GEOMETRY .geometry
DROP OTHERS;
9:
SET INTERMEDIATE AS Milan_BikeLanes; Listing 3. Sample transformation process written by means of J-CO-QL (part 1). Listing 3 Sample transformation process written by means of J-CO-QL (part 1). 1:
USE DB Boundaries
ON SERVER jcods ’http://10.0.0.11:17017’;
2:
USE DB RegionInfo
ON SERVER MongoDB ’http://10.0.0.12:27017’;
3:
USE DB ToShare
ON SERVER MongoDB ’http://10.0.0.13:27017’;
4:
GET COLLECTION Districts@Boundaries;
5:
FILTER
CASE
WHERE WITH .Properties.City
AND .Properties.City="Milan"
GENERATE
{.ID, .Name:
.Properties.Name}
KEEPING GEOMETRY
DROP OTHERS;
6:
SET INTERMEDIATE AS Milan_Districts;
7:
GET COLLECTION BikeLanes@RegionInfo;
8:
FILTER
CASE
WHERE WITH .City
AND .City="Milan"
GENERATE {.Name}
SETTING GEOMETRY .geometry
WHERE WITH .Properties.City
AND .Properties.City="Milan"
GENERATE
{.Name:
.Properties.Name}
SETTING GEOMETRY .geometry
DROP OTHERS;
9:
SET INTERMEDIATE AS Milan_BikeLanes; Listing 3. Sample transformation process written by means of J-CO-QL (part 1). Listing 3 Sample transformation process written by means of J-CO-QL (part 1). 16 of 35
16 of 39 16 of 35
16 of 39 Electronics 2021, 10, 621
Electronics 2021, 1, 1 Listing 4. Sample transformation process written by means of J-CO-QL (part 2). Listing 4 Sample transformation process written by means of J-CO-QL (p Listing 4. Sample transformation process written by means of J-CO-QL (part 2). Listing 4 Sample transformation process written by means of J-CO-QL (p Listing 4. Sample transformation process written by means of J-CO-QL (part 2).
Listing 4 Sample transformation process written by means of J CO QL (part 2).
10: SPATIAL JOIN OF COLLECTIONS
Milan_Districts AS D, Milan_BikeLanes AS B
ON INTERSECT
SET GEOMETRY INTERSECTION
CASE
WHERE WITH .D.ID, .B.Name
GENERATE {.DistrictName:
.D.Name,
.BikeLaneName:
.B.Name}
KEEPING GEOMETRY
DROP OTHERS
REMOVE DUPLICATES;
11: GROUP
PARTITION
WITH .BikeLaneName
BY .BikeLaneName
INTO .DistrictSegs
DROP GROUPING FIELDS
DROP OTHERS;
12: FILTER
CASE
WHERE WITH .BikeLaneName, .DistrictSegs
GENERATE {.BikeLaneName,
.NumOfDistricts:
COUNT(.DistrictSegs)}
SETTING GEOMETRY Aggregate(.DistrictSegs)
DROP OTHERS;
13: FILTER
CASE
WHERE WITH .BikeLaneName, .NumOfDistricts
AND.NumOfDistricts >= 2
GENERATE {.BikeLaneName}
KEEPING GEOMETRY
DROP OTHERS;
14: SAVE AS BikeLanesCrossingDistricts@ToShare; 13: FILTER
CASE
WHERE WITH .BikeLaneName, .NumOfDistricts
AND.NumOfDistricts >= 2
GENERATE {.BikeLaneName}
KEEPING GEOMETRY
DROP OTHERS; DROP OTHERS; 14: SAVE AS BikeLanesCrossingDistricts@ToShare; 14: SAVE AS BikeLanesCrossingDistricts@ToShare; •
The GET COLLECTION instruction at line 4 gets the Districts collection from the
Boundaries database: the collection becomes the new temporary collection t4 of the
query process (as depicted in Figure 3). •
The GET COLLECTION instruction at line 4 gets the Districts collection from the
Boundaries database: the collection becomes the new temporary collection t4 of the
query process (as depicted in Figure 3). q
y p
p
g
•
The FILTER instruction at line 5 actually filters documents that describe districts of
Milan. It takes the current temporary collection t4 and applies the CASE WHERE clause,
which is a document selection condition. In the instruction, the condition relies on
the WITH predicate: this predicate is true if the document under selection contains the
specified field (or fields, if a list of field names is specified). In the specific case, the
condition is true if the document under selection contains the City field within the
root-level Properties field and if its value is "Milan". •
The FILTER instruction at line 5 actually filters documents that describe districts of
Milan. It takes the current temporary collection t4 and applies the CASE WHERE clause,
which is a document selection condition. In the instruction, the condition relies on
the WITH predicate: this predicate is true if the document under selection contains the
specified field (or fields, if a list of field names is specified). In the specific case, the
condition is true if the document under selection contains the City field within the
root-level Properties field and if its value is "Milan". For each document that meets the condition, the GENERATE action inserts a restructured
version of the document into the output temporary collection t5, such that the ID field
is reported as it is, while a new root-level Name field is derived from the previously
nested one (specified by the expression .Name:
.Properties.Name). The ~geometry
field is kept as it is, as specified by the KEEPING GEOMETRY directive. The final DROP OTHERS option specifies that documents that do not meet the selection
condition must be discarded and will not appear in the output collection (the language
admits an alternative KEEP OTHERS option, which would keep not-selected documents
into the output collection). We introduced this option to make J-CO-QL adaptive to
heterogeneity (feature 1 of Table 1). The output collection becomes the new temporary collection t5. 14: SAVE AS BikeLanesCrossingDistricts@ToShare; This execution model derives from the semantics of relational algebra, where an oper-
ator produces a table that is taken as input by the next operator; in relational algebra, the
inverted syntax of unary operators (operands are behind the operators) and the infix syntax
of binary operators make writing queries somehow unnatural. In J-CO-QL, instructions
implicitly start from the query-process state possibly generated by the previous instruction
and generate a new query-process state, which contains a possibly modified version of the
temporary collection; thus, a query is a kind of “pipe” of instructions, that are written in
the same order as they are thought. In fact, J-CO-QL is neither procedural nor imperative:
no variables and no instructions to control the execution flow (like cycles) are provided. Instructions specify complex transformations on documents in a declarative way, following
the same approach behind the SELECT statement of SQL, which is the reason why SQL is
widely used by people without programming skills to query and analyze data. Let us start describing the first part of the query, reported in Listing 3. Let us start describing the first part of the query, reported in Listing 3. •
First of all, it is necessary to specify which databases to connect to. The three USE DB
instructions at lines 1–3 do this work. Notice that the ON clauses specify the connection
strings necessary to connect to the desired servers. These instructions do not change •
First of all, it is necessary to specify which databases to connect to. The three USE DB
instructions at lines 1–3 do this work. Notice that the ON clauses specify the connection
strings necessary to connect to the desired servers. These instructions do not change Electronics 2021, 10, 621 17 of 35 17 of 35 the query-process state; consequently, both temporary collection and Intermediate
Results database IR remain empty; this is why in Figure 3 we start depicting the query
process form line 4. the query-process state; consequently, both temporary collection and Intermediate
Results database IR remain empty; this is why in Figure 3 we start depicting the query
process form line 4. the query-process state; consequently, both temporary collection and Intermediate
Results database IR remain empty; this is why in Figure 3 we start depicting the query
process form line 4. 14: SAVE AS BikeLanesCrossingDistricts@ToShare; An excerpt is reported
in the upper part of Listing 5, where the restructured version of documents shown in
Listing 1 is reported. g
p
In the CASE WHERE clause and in the GENERATE action, fields are referred to by means
of a dot notation like .A.B.C, in order to deal with nested documents (the sample dot
notation would access the C field nested within a B field, in turn nested within the A
field at the root level of the document). •
The temporary collection t5 produced by line 5 contains only districts of Milan. Line 6
saves this temporary collection into the Intermediate Results database IR with name
Milan_Districts, for further processing during the same query process; this is the
feature Re-use of intermediate results reported in Table 1. In Figure 3, notice how
the IR database is no longer empty in the query-process state produced by line 6. •
Lines 7 to 9 basically perform, on bike lanes, the same task performed by lines 4 to 6. Specifically, line 7 gets the BikeLanes collection from the RegionInfo database; this
collection becomes the new temporary collection t7. p
y
•
The FILTER instruction at line 8 selects bike lanes of Milan. However, recall from
Listing 2 that documents describing bike lanes are heterogeneous: for this reason,
the CASE clause contains two WHERE selection conditions, each one followed by a cor-
responding GENERATE action. When a document is evaluated, if the first selection
condition is not true, the second one is evaluated and, if true, the document is re-
structured according to the corresponding GENERATE action. If none of the selection
conditions is true, the document is discarded from the output collection, according to
the DROP OTHERS option. p
Notice that line 8 is an example of one of the innovative features introduced by J-CO-
QL, i.e., the capability of dealing with heterogeneous collections (feature Adaptability
to heterogeneity in Table 1). In fact, the collection can contain documents with several
structures (as in the example); by means of multiple WHERE conditions and the contri-
bution of the KEEP OTHERS/DROP OTHERS options, the same instruction is able to filter
documents with different structures, focusing on some of them, possibly unifying
the structure of output documents as well as possibly generating documents with
different structures, with one single instruction. Collection t4 saved as Milan_Districts into the IR database: 14: SAVE AS BikeLanesCrossingDistricts@ToShare; Electronics 2021, 10, 621 18 of 35 18 of 35 Notice that the two GENERATE actions restructure the selected documents in order to
have the same structure. The two documents shown in Listing 2 are restructured as
reported in the lower part of Listing 5. Furthermore, the two GENERATE actions have to adapt geo-tagging to the J-CO-QL data
model: the SETTING GEOMETRY .geometry option specifies that the new ~geometry
special field must be derived from the geometry field formerly present in the source
selected document (in the lower part of Listing 5, which reports the output collection,
it is possible to see that, now, documents have the ~geometry field). This field is inter-
nally managed by means of the representation provided by the JTS—Java Topology
Suite library; consequently, the SETTING GEOMETRY option asks the J-CO-QL Engine to
convert a pure JSON field (the former geometry field) into the internal representation
adopted to represent the ~geometry field. The SETTING GEOMETRY option was introduced to explicitly deal with geometries; it is
accompanied by the alternative KEEPING GEOMETRY and DROPPING GEOMETRY options
that, respectively, actually keep and drop the ~geometry field coming from the source
document. In fact, this field has a special meaning for documents; consequently, we
thought that the language had to force users to always consider this field in a special
way, without confusing it with other fields. y
g
Line 9 saves the resulting temporary collection t8 (containing only bike lanes of Milan)
into the Intermediate Results database IR, with name Milan_BikeLanes: this can be
clearly seen in Figure 3, where the IR database in the output query-process state of
line 9 now contains two collections. 18 of 39 y
g
Line 9 saves the resulting temporary collection t8 (containing only bike lanes of Milan)
into the Intermediate Results database IR, with name Milan_BikeLanes: this can be
clearly seen in Figure 3, where the IR database in the output query-process state of
line 9 now contains two collections. 18 of 39 Listing 5. Excerpt of collections Milan_Districts (upper part) and Milan_BikeLanes (lower part)
saved into the Intermediate Results database IR. Listing 5 Excerpt of collections Milan_Districts (upper part) and Milan_BikeLanes
(lower part) saved into the Intermediate Results database IR. Listing 5. Excerpt of collections Milan_Districts (upper part) and Milan_BikeLanes (lower part)
saved into the Intermediate Results database IR. 14: SAVE AS BikeLanesCrossingDistricts@ToShare; Recall that the Milan_Districts collection describes
districts in Milan: each document in the collection contains a field named ~geometry,
which, in this case, describes the boundary of the district as a polygon. This collection
is aliased as D in the instruction. The second collection is Milan_BikeLanes: it is
aliased as B and the ~geometry field of each document, in this case, represents the
full path of a single bike lane. These two collections are taken from the Intermediate
Results database, in that they are intermediate results obtained by previous instruc-
tions in the query (when no database is specified after a collection name, it is retrieved
from the IR database). This is how J-CO-QL completes the support to the feature
Re-usability of intermediate results reported in Table 1. •
The query carries on at line 10 with the SPATIAL JOIN instruction. This is the key
instruction provided by J-CO-QL in order to perform complex transformations con-
cerned with geo-referenced data. Recall that the Milan_Districts collection describes
districts in Milan: each document in the collection contains a field named ~geometry,
which, in this case, describes the boundary of the district as a polygon. This collection
is aliased as D in the instruction. The second collection is Milan_BikeLanes: it is
aliased as B and the ~geometry field of each document, in this case, represents the
full path of a single bike lane. These two collections are taken from the Intermediate
Results database, in that they are intermediate results obtained by previous instruc-
tions in the query (when no database is specified after a collection name, it is retrieved
from the IR database). This is how J-CO-QL completes the support to the feature
Re-usability of intermediate results reported in Table 1. The SPATIAL JOIN instruction computes pairs of documents in the two collections,
such that the spatial-join condition specified in the ON clause is satisfied. Specifically, a
new document is built if the geometries of the two paired documents intersect. The SET
GEOMETRY clause specifies the geometry to assign to the document obtained by pairing
the two original ones: we specify that we want the intersection of the two geometries,
i.e., the fragment of polyline that describes the portion of bike lane crossing the given
district. The upper part of Listing 6 reports an excerpt of the documents resulting
from the generation of pairs that satisfy the spatial-join condition. 14: SAVE AS BikeLanesCrossingDistricts@ToShare; Notice the D field,
which contains the original document coming from the collection aliased as D, the B
field, which contains the original document coming from the collection aliased as B,
and the ~geometry field resulting from the intersection of the two original geometries. g
y
g
g
g
The subsequent CASE WHERE clause is necessary to restructure the documents, remov-
ing nesting; it behaves as the CASE WHERE clause already seen in the FILTER instruction. The specific WHERE selection condition in the instruction uses the WITH predicate, so
as to select documents having the desired fields; then, the GENERATE action specifies
how to restructure each document that satisfies the condition; the KEEPING GEOMETRY
option specifies that we maintain the geometry obtained by the spatial join (i.e., the The subsequent CASE WHERE clause is necessary to restructure the documents, remov-
ng nesting; it behaves as the CASE WHERE clause already seen in the FILTER instruction. The specific WHERE selection condition in the instruction uses the WITH predicate, so
as to select documents having the desired fields; then, the GENERATE action specifies
how to restructure each document that satisfies the condition; the KEEPING GEOMETRY
option specifies that we maintain the geometry obtained by the spatial join (i.e., the
intersection of the two original geometries). The lower part of Listing 6 reports an
excerpt of the temporary collection t10 (as reported in Figure 3) resulting from the
SPATIAL JOIN; notice how the documents in the upper part of the listing are restruc-
tured by the GENERATE action. y
The final REMOVE DUPLICATES option asks for removing duplicates from the output
temporary collection. This option is available in many instructions (in particular
FILTER): when it is not specified, removal of duplicates is not performed. The SPATIAL JOIN could be done on the temporary collection as well, by using the
collection name TEMPORARY. This is not usually possible in JSON DBMSs, which enable
spatial queries only on materialized documents, because they have to build spatial
indexes. In contrast, J-CO-QL is able to spatially join collections obtained from two
different databases, possibly managed by two different storage services, without
forcing analysts to transfer them from one storage to another. g
y
g
Furthermore, if we explicitly consider MongoDB, currently its query language is not at
all able to perform something similar to the SPATIAL JOIN without writing JavaScript
code, i.e., procedural and imperative code. 14: SAVE AS BikeLanesCrossingDistricts@ToShare; Listing 5 Excerpt of collections Milan_Districts (upper part) and Milan_BikeLanes
(lower part) saved into the Intermediate Results database IR. Collection t4 saved as Milan_Districts into the IR database: Collection t4 saved as Milan_Districts into the IR database:
{ "ID": 74,
"Name": "ROSERIO",
"~geometry":
{ "type": "MultiPolygon",
"coordinates": [
[ [ [9.13005657492143, 45.5229429902529], ...,
[9.13003510901344, 45.5229853369611] ] ] ] } },
{ "ID": 82,
"Name": "COMASINA",
"~geometry":
{ "type": "MultiPolygon",
"coordinates": [
[ [ [9.16950766784049, 45.5251391141947], ...,
[9.16920281509976, 45.5253660742429] ] ] ] } },
... Collection t8 saved as Milan_BikeLanes into the IR database:
{ "Name": "VIA ROBERTO KOCH",
"~geometry": {
"type" : "GeometryCollection",
"geometries" : [
{ "type" : "MultiLineString",
"coordinates" : [ [ [ 9.11455186249295, 45.4446414404081],
[ 9.11469823062049, 45.4446837789302] ] ] },
{ "type" : "MultiLineString",
"coordinates" : [ [ [9.11469823062049, 45.4446837789302],
[9.11475944738880
45.4447021324387], ... ] ] },
... { "ID": 74,
"Name": "ROSERIO",
"~geometry":
{ "type": "MultiPolygon",
"coordinates": [
[ [ [9.13005657492143, 45.5229429902529], ...,
[9.13003510901344, 45.5229853369611] ] ] ] } },
{ "ID": 82,
"Name": "COMASINA",
"~geometry":
{ "type": "MultiPolygon",
"coordinates": [
[ [ [9.16950766784049, 45.5251391141947], ...,
[9.16920281509976, 45.5253660742429] ] ] ] } }, Collection t8 saved as Milan_BikeLanes into the IR database: { "Name": "VIA ROBERTO KOCH",
"~geometry": {
"type" : "GeometryCollection",
"geometries" : [
{ "type" : "MultiLineString",
"coordinates" : [ [ [ 9.11455186249295, 45.4446414404081],
[ 9.11469823062049, 45.4446837789302] ] ] },
{ "type" : "MultiLineString",
"coordinates" : [ [ [9.11469823062049, 45.4446837789302],
[9.11475944738880
45.4447021324387], ... ] ] },
... { "Name": "VIA ROBERTO KOCH",
"~geometry": {
"type" : "GeometryCollection",
"geometries" : [
{ "type" : "MultiLineString",
"coordinates" : [ [ [ 9.11455186249295, 45.4446414404081],
[ 9.11469823062049, 45.4446837789302] ] ] },
{ "type" : "MultiLineString",
"coordinates" : [ [ [9.11469823062049, 45.4446837789302],
[9.11475944738880
45.4447021324387], ... ] ] },
... { "type" : "MultiLineString", 19 of 35 19 of 35 Electronics 2021, 10, 621 The result of the first part of the process, so far illustrated, is sketched in Listing 5;
Figure 4 depicts both districts (black lines) and bike lanes (red lines) of Milan. These two
collections are the starting point for the second part of the process, reported in Listing 4. •
The query carries on at line 10 with the SPATIAL JOIN instruction. This is the key
instruction provided by J-CO-QL in order to perform complex transformations con-
cerned with geo-referenced data. 14: SAVE AS BikeLanesCrossingDistricts@ToShare; For each identified group of documents (i.e.,
documents having the same value for the BikeLaneName field), a new document is
put into the output collection, such that all common fields are reported and a new
field, an array of grouped documents named DistrictSegs (as specified by the INTO
clause) is added. The DROP GROUPING FIELDS option specifies that grouping fields (in
this case, only the BikeLaneName field) are removed from grouped documents. y
)
g
p
An excerpt of the temporary collection t11, as numbered in Figure 3, is reported in the
upper part of Listing 7. y
g
p
An excerpt of the temporary collection t11, as numbered in Figure 3, is reported in the
upper part of Listing 7. Once documents are grouped, it is necessary (line 12) to add a field to be assigned
with the number of elements that are present in the DistrictSegs array, so as to know
how many districts are crossed by each bike lane. w many districts are crossed by each bike lane. The FILTER instruction at line 12 selects all the documents and restructures them by
adding the field named NumOfDistricts. The lower part of Listing 7 reports the tem-
porary collection t12, as numbered in Figure 3, resulting from the FILTER instruction. It is worth noticing the SETTING GEOMETRY option: it derives the ~geometry field
of each output document by aggregating single geometries of documents within
the DistrictSegs array (remember that these geometries represent the fragment
of bike lane that intersect the specific district); specifically, the Aggregate function
unites single geometries, obtaining either a GeometryCollection or a MultiPoint, or
a MultiLineString, or a MultiPolygon (see the specification of GeoJSON [3]) as value
of the ~geometry field. The FILTER instruction at line 12 selects all the documents and restructures them by
adding the field named NumOfDistricts. The lower part of Listing 7 reports the tem-
porary collection t12, as numbered in Figure 3, resulting from the FILTER instruction. •
The FILTER instruction at line 13 is necessary to actually select documents whose value
of the NumOfDistricts field is no less than 2. This is done by the CASE WHERE clause. Then, the GENERATE action further restructures selected documents, in order to get
the final structure: in particular, only the BikeLaneName field is kept, as well as the
geometry obtained by the FILTER instruction at line 12. 14: SAVE AS BikeLanesCrossingDistricts@ToShare; This is why, in Table 1, we said that
MongoDB is not Oriented to Analysts but is Oriented to Programmers; in contrast,
J-CO-QL is fully oriented to analysts, because its instructions are declarative and no
procedural integration is allowed. Furthermore, if we explicitly consider MongoDB, currently its query language is not at
all able to perform something similar to the SPATIAL JOIN without writing JavaScript
code, i.e., procedural and imperative code. This is why, in Table 1, we said that
MongoDB is not Oriented to Analysts but is Oriented to Programmers; in contrast,
J-CO-QL is fully oriented to analysts, because its instructions are declarative and no
procedural integration is allowed. At this point (line 11), it is necessary to group documents resulting from the SPATIAL
JOIN, in order to count the number of districts that are crossed by each bike lane. In
fact, if a bike lane crosses several districts, several documents having the same value At this point (line 11), it is necessary to group documents resulting from the SPATIAL
JOIN, in order to count the number of districts that are crossed by each bike lane. In
fact, if a bike lane crosses several districts, several documents having the same value Electronics 2021, 10, 621 20 of 35 20 of 35 for the BikeLaneName field are in the output of the SPATIAL JOIN instruction (line 10). In the GROUP instruction, the goal of the PARTITION clause is to select documents (from
the temporary collection t10 produced by the previous instruction) that have some
common fields or characteristics. In line 11 of Listing 4, we select documents having
the BikeLaneName field. In practice, we specify a partition of the full set of documents
in the temporary collection; the DROP OTHERS option at the end of line 11 specifies
that documents not selected for the specified partition are discarded from the output
temporary collection. This is again to meet the feature Adaptability to heterogeneity
reported in Table 1; in particular, since many PARTITION clauses are allowed, many
different grouping tasks on many different document structures could be specified
within the same instruction. At this point, documents in the partition are then grouped based on the BikeLaneName
field, as specified by the BY clause. 14: SAVE AS BikeLanesCrossingDistricts@ToShare; The lower part of Listing 7
reports an excerpt of the resulting temporary collection t13. p
p
g
p
y
•
The query terminates (line 14) by saving the final temporary collection t13 into a per-
sistent database. In particular, the SAVE AS instruction saves the temporary collection
into the toShare database with name BikeLanesCrossingDistricts. p
p
g
p
y
•
The query terminates (line 14) by saving the final temporary collection t13 into a per-
sistent database. In particular, the SAVE AS instruction saves the temporary collection
into the toShare database with name BikeLanesCrossingDistricts. With respect to previous works [9,10], J-CO-QL has been slightly modified. So, this
section is not a mere summary of the language, but presents the latest state of its evolution. In particular, the GROUP instruction has been provided with the DROP GROUPING FIELDS
option (that allows for removing grouping fields from within grouped documents). Another
major improvement is the novel REMOVE DUPLICATES option, which has been added to the
FILTER, SPATIAL JOIN, JOIN and MERGE instructions (the latter two instructions are not
used in this paper), in order to let the user decide whether to force removal of duplicates. Furthermore, many minor and very detailed lexical and syntactic changes were made. 21 of 35 Electronics 2021, 10, 621 Figure 4. Districts (black lines) and bike lanes (red lines) in Milan (Italy), described by documents in collections
Milan_Districts and Milan_BikeLanes sketched in Listing 6. Figure 4. Districts (black lines) and bike lanes (red lines) in Milan (Italy), described by documents in collections
Milan_Districts and Milan_BikeLanes sketched in Listing 6. We make some considerations about the general philosophy behind the J-CO-QL
language. Its instructions are declarative, so analysts without any specific procedural
programming skills can effectively use them (feature Orientation to Analysts in Table 1). Furthermore, even though queries can be seen as procedures, they are not procedural
programs; in contrast, they are “piped” (sequential) applications of declarative instructions. In this sense, we say that J-CO-QL is a high-level language, compared with procedural
programming languages. Notice that avoiding procedural programming was a design requirement, because
our target users are analysts and geographers. For example, consider the SPATIAL JOIN
instruction (just to cite one): the user can perform such a non-trivial operation simply
by specifying how to spatially join documents and then by specifying how to restructure
joined documents. Within SPATIAL JOIN (line 10) and before CASE WHERE: [{"D": {"ID": 83,
"Name": "BRUZZANO",
"~geometry": {"type": "MultiPolygon",
"coordinates": [ ... ] } },
"B": {"Name": "River-side",
"~geometry": {"type": "LineString",
"coordinates": [ ... ] } },
"~geometry": {"type": "LineString",
"coordinates": [...]}
},
{"D": {"ID": 74, "Name": "SACCO",
"~geometry": {"type": "MultiPolygon",
"coordinates": [ ... ] } },
"B": {"Name": "River-side",
"~geometry": {"type": "LineString",
"coordinates": [ ... ] } },
"~geometry": {"type": "LineString",
"coordinates": [...] }
...] "coordinates": [ ... ] } }, yp
g
"coordinates": [...]} yp
yg
"coordinates": [ ... ] } }, 14: SAVE AS BikeLanesCrossingDistricts@ToShare; In [10], we introduced a large variety of spatial predicates for this
purpose. The user can easily exploit them, in a totally independent way with respect to
actual processing/querying capability provided by specific JSON storage systems: no
pre-indexation of collections is necessary; spatial joins can be performed on the fly, when it
is necessary, on temporary collections. 22 of 35
of 39 Electronics 2021, 10, 621
Electronics 2021, 1, 1 Listing 6. Excerpt of documents generated by SPATIAL JOIN (line 10). Listing 6 Excerpt of documents generated by SPATIAL JOIN (line 1 At the end of SPATIAL JOIN at line 10, temporary collection t10 : [{"DistrictName": "BRUZZANO",
"BikeLaneName": "River-side",
"~geometry": {"type": "LineString",
"coordinates": [...] }},
{"DistrictName": "SACCO",
"BikeLaneName": "River-side",
"~geometry": {"type": "LineString",
"coordinates": [...] }},
...] [{"DistrictName": "BRUZZANO",
"BikeLaneName": "River-side",
"~geometry": {"type": "LineString",
"coordinates": [...] }},
{"DistrictName": "SACCO",
"BikeLaneName": "River-side",
"~geometry": {"type": "LineString",
"coordinates": [...] }},
...] Listing 7. Excerpt of documents generated by the GROUP instruction (line 11) and by the FILTER
instruction at line 13 and saved into the persistent database toShare (line 14). Listing 7 Excerpt of documents generated by the GROUP operator (line 11) and by the FILTER
operator at line 13 and saved into the persistent database toShare (line 14). At the end of GROUP (line 11), temporary collection t11: : [{"BikeLaneNameName": "River-side",
"DistrictSegs": [
{"DistrictName": "BRUZZANO",
"~geometry": {"type": "LineString",
"coordinates": [...] }},
{"DistrictName": "SACCO",
"~geometry": {"type": "LineString",
"coordinates": [...] }}]
},
...] },
...] Temporary collection t13 (line 13) and persistent collection BikeLanesCrossingDistricts:
[{"BikeLaneName": "River-side",
"~geometry": {"type": "MultiLineString",
"coordinates": [...] }
},
...] Temporary collection t13 (line 13) and persistent collection BikeLanesCrossingDistricts:
[{"BikeLaneName": "River-side",
"~geometry": {"type": "MultiLineString",
"coordinates": [...] }
},
...] Temporary collection t13 (line 13) and persistent collection BikeLanesCrossingDistricts: Electronics 2021, 10, 621 23 of 35 23 of 35 5. J-CO-UI: The User Interface of the J-CO Framework The most recent component of the J-CO Framework that we are introducing in this
paper is named J-CO-UI: it is the user interface that analysts can use to write complex
integration and transformation processes. J-CO-UI is a stand-alone application written in
Java; it is installed on analysts’ PCs, so that they can comfortably work on remotely-stored
data sets sitting in their office. Figure 5 reports a screenshot of the main window. The lower text area allows for
writing J-CO-QL instructions to execute; the Execute button sends the content of the text
area to the J-CO-QL Engine service which the application is connected to; if no error is
signaled by the engine, the executed instructions are appended to the above text area
(not editable). In the screenshot, we connected to a database named MyDB on a MongoDB
server pre-configured as mongo1; the GET COLLECTION instruction retrieved the collection
named SampleCollection from the database; the FILTER instruction selected only those
documents with the Approved field; finally, the SET INTERMEDIATE instruction saved the
temporary collection into the Intermediate Results database IR. p
y
The Save button allows for saving the overall sequence of executed instructions into a
text file; the Show Console button allows for showing the console with the trace of commu-
nication between the application and the J-CO-QL Engine. Figure 5. Main window of the J-CO-UI application. Figure 5. Main window of the J-CO-UI application. Figure 5. Main window of the J-CO-UI application. The current query-process state can be inspected by pressing the Inspect state button,
that opens the window (named Inspection Window) reported in Figure 6. In the left-hand
pane, we find buttons to inspect the current temporary collection tc, as well as collections
stored within the Intermediate Results database IR. If the user chooses to inspect the Electronics 2021, 10, 621 24 of 35 temporary collection or selects a collection in the IR database, its content is shown in the
right-hand pane. Buttons on the bottom of the window acts on the content of the right-hand
side area: the Expand button shows the full structure of documents (as in this case); the
Save button saves the documents into a text file. Figure 6. Inspection window of the J-CO-UI application. Figure 6. Inspection window of the J-CO-UI application. 5. J-CO-UI: The User Interface of the J-CO Framework If the user understands that the query process took a non-satisfactory way, the process
can be backtracked (or rolled-back): by pressing the Backtrack button in the main window
(Figure 5), the last executed instruction is removed from the process and the query-process
state before its execution is restored. This way, complex processes can be written by
adopting a trial-and-error approach, which is coherent with data-analysis tasks, where the
final goal of the process often is not known in advance. The analysts can explore more than
one solution, possibly rolling back when the followed way is not satisfactory. This functionality exploits the Interactive Mode provided by the J-CO-QL Engine (presented in
Section 3.3), which would be ineffective and useless without a user interface specifically designed. We conclude this section with a final consideration. Instead of developing a stand-
alone application, an alternative solution for developing J-CO-UI could be a web appli-
cation. When we designed J-CO-UI, we thought that this solution is less flexible: in fact,
a web server must be set up and kept running on a dedicated computational resource. Furthermore, an additional communication tier would be added (instead of a direct com-
munication between analyst’s PC and J-CO-QL Engine instance, the web server would be in
the middle). In contrast, the stand-alone application allows for a free composition of user
interfaces and computational resources. However, web applications are very familiar for
and appreciated by users; thus, we are considering to develop a web user interface. 6. Performance Evaluation In order to assess the real possibility of using the J-CO Framework to deal with large
collections of JSON documents, we performed an experimental evaluation. The goal is to
evaluate its capability to scale with increasing size of collections. The critical component of the framework is the J-CO-QL Engine, that actually executes
J-CO-QL instructions. Electronics 2021, 10, 621 25 of 35 25 of 35 We decided to build the data sets to analyze scalability by moving from the same data
sets used to build the running example, i.e., the Districts collection and the BikeLanes
collection. Then, by using a multiplication factor denoted as f, we generated the other data
sets: given a value for f, a collection that contains f times the districts in the Districts
collection and a collection that contains f times the bike lanes in the BikeLanes collection
were created. Table 2 reports the full list of data sets we created for the experiments. The same query presented in Section 4 is used to perform experiments. We decided to
adopt this strategy to build data sets for various reasons. First of all, this way the query
always obtains the same results, independently of the size of the source data sets, because
redundant copies of documents are removed by the SPATIAL JOIN instruction (remember
the REMOVE DUPLICATES option); second, the number of document pairs created by the
SPATIAL JOIN instruction before removing duplicates is not random, but it can be obtained
by multiplying the pairs obtained for the DS_1 data set by f 2 (this way, we exactly control
how execution time should scale). Table 2. Data sets used for analyzing scalability. Data Set Id
Factor f
N. of Districts
N. of Bike Lanes
DS_1
1
88
422
DS_10
10
880
4220
DS_25
25
2200
10,550
DS_50
50
4400
21,100
DS_75
75
6600
31,650
DS_100
100
8800
42,200
DS_150
150
13,200
63,300
DS_200
200
17,600
84,400 Table 2. Data sets used for analyzing scalability. Experiments were conducted on a PC powered by a Processor Intel quad-Core i7-8550-
U, running at 1.80 GHz, equipped with 16 GB RAM and 250 GB Solid State Drive. The
version of the Java Virtual Machine (JVM) was 1.8.0_251. 6.1. Scalability of the J-CO-QL Engine 6.1. Scalability of the J-CO-QL Engine First of all, we tested the execution of the query five times on the basic data set DS_1,
in order to check for the stability of the J-CO-QL Engine. Table 3 reports the measured
execution times. Rows from 1 to 14 correspond to each single instruction in the query
(see Listings 3 and 4) , while the bottom row reports the overall execution times. The Avg. column reports the average values of the 5 executions, while columns from Exec. 1 to
Exec. 5 report times measured during each single execution. We can notice that the overall query has an average execution time of about 1.8 s,
which is acceptable if we consider the complexity of dealing with geometries of districts,
which are not simple. In fact, the most time consuming instruction is the SPATIAL JOIN, as
we expected, which has an average execution time of 0.7 s (on the overall time of 1.8 s). The other time-consuming instructions are GET COLLECTION, SET INTERMEDIATE and
SAVE AS, although their order of magnitude is at least 2/3 times less; in this sense, observe
that the SPATIAL JOIN instruction gets collections from the IR DB, which is managed
though the file system, so we can consider that 0.2 s on 0.7 s are necessary to get the two
input collections into main memory. Comparing column Exec. 1 with columns Exec. 2 to Exec. 5, we can notice how the
first execution of the query is significantly slower than the next ones, i.e., more than 3.9 s
vs. 1 s (for Exec. 5). This fact is mitigated by the average, but it is surprising: we think it is
the Java Virtual Machine that during the first execution has to do extra work that is not
performed during subsequent executions. Electronics 2021, 10, 621 26 of 35 Table 3. Execution times (in ms) for data set DS_1 on one single process of the J-CO-QL Engine. Instruction
Avg. Exec. 1
Exec. 2
Exec. 3
Exec. 4
Exec. 5
1. USE DB
0.31
0.99
0.31
0.16
0.04
0.03
2. USE DB
0.03
0.05
0.02
0.03
0.03
0.03
3. USE DB
0.02
0.03
0.02
0.03
0.02
0.02
4. GET COLLECTION
306.55
1,088.70
138.00
122.97
99.64
83.46
5. FILTER
2.43
9.99
0.86
0.30
0.50
0.51
6. SET INTERMEDIATE
164.39
247.16
172.25
126.64
162.62
113.25
7. 6.1. Scalability of the J-CO-QL Engine GET COLLECTION
59.27
100.33
64.47
63.80
31.84
35.90
8. FILTER
220.29
409.07
241.38
150.78
143.52
156.72
9. SET INTERMEDIATE
99.56
157.43
103.85
94.21
74.21
68.09
10. SPATIAL JOIN
706.82
1,417.74
677.38
489.00
507.97
442.00
11. GROUP
5.82
18.64
3.20
2.34
2.31
2.62
12. FILTER
133.85
302.74
111.68
99.64
89.16
66.03
13. FILTER
1.58
2.72
1.70
1.46
0.93
1.06
14. SAVE AS
98.17
172.54
84.16
91.87
81.15
61.12
Total (ms)
1,799.08
3,928.10
1,599.28
1,243.23
1,193.96
1,030.84 Table 3. Execution times (in ms) for data set DS_1 on one single process of the J-CO-QL Engine. Consequently, we decided to repeat the experiment by using the DS_10 data set (see
Table 2), in order to check if the above-mentioned phenomenon still appears. Table 4
reports the measured execution times. We can notice that the first execution (the Exec. 1
column) is still significantly slower than next executions. However, typically users perform
the same query only once, so we decided to run next experiments in a different way: we
considered again 5 executions of the query, but each time the J-CO-QL Engine was shut
down and restarted, so as to have the JVM in the same initial state. With such a setting, we
obtained stable execution times for all 5 repetitions of the same query. Table 4. Execution times (in ms) for data set DS_10 on one single process of the J-CO-QL Engine. Instruction
Avg. Exec. 1
Exec. 2
Exec. 3
Exec. 4
Exec. 5
1. USE DB
0.04
0.08
0.04
0.03
0.03
0.02
2. USE DB
0.03
0.02
0.02
0.02
0.03
0.03
3. USE DB
0.02
0.02
0.02
0.01
0.02
0.01
4. GET COLLECTION
0.01
0.02
0.01
0.01
0.02
0.01
5. FILTER
5.47
20.14
3.01
1.34
1.49
1.36
6. SET INTERMEDIATE
800.51
1075.87
874.02
671.30
670.68
710.68
7. GET COLLECTION
214.74
437.30
167.66
151.11
147.97
169.66
8. FILTER
1196.96
2173.38
989.40
904.84
927.73
989.45
9. SET INTERMEDIATE
486.41
713.83
417.10
403.20
416.89
481.03
10. SPATIAL JOIN
11,475.88
16,998.58
10,088.89
10,032.71
10,074.85
10,184.37
11. GROUP
4.12
12.07
2.96
2.10
1.45
2.03
12. FILTER
70.84
128.41
56.46
56.87
56.32
56.11
13. FILTER
0.65
1.02
0.56
0.53
0.55
0.57
14. SAVE AS
82.80
144.41
87.49
58.31
57.59
66.22
Total (ms)
14,338.48
21,705.16
12,687.65
12,282.40
12,355.62
12,661.57 Table 4. Execution times (in ms) for data set DS_10 on one single process of the J-CO-QL Engine. 6.1. Scalability of the J-CO-QL Engine After the preliminary experiment previously discussed, we performed the experiments
for all the data sets reported in Table 2, i.e., DS_1, DS_10, DS_25, DS_50, DS_75, DS_100,
DS_150 and DS_200. Table 5 reports the measured execution times (average of five executions, performed
shutting down the J-CO-QL Engine after each execution). For each test, the total execution
time is reported in ms in the Total (ms) row; the Total (min) row reports the same times
converted to minutes (for example, 1.25 min), while the Total (min′s′′) row converts them
to the form minutes and seconds (for example, 1′15′′). Finally the SPATIAL JOIN % row
reports the percentage of execution time taken by the SPATIAL JOIN instruction on the
overall query execution time, while the Rest of the query % row reports the percentage of
query execution time without the SPATIAL JOIN. q
y
First of all, the reader can notice that instructions from 11 to 14, that are executed
after the SPATIAL JOIN, are not affected by the increasing size of data sets. This is due to
the fact that documents in the source collections are obtained by duplicating the original
documents in the DS_1 data set. So, the spatial join would generate multiple copies of
the same documents; however, the REMOVE DUPLICATES option (see Listing 4) removes Electronics 2021, 10, 621 27 of 35 27 of 35 duplicate documents from the output temporary collection generated by the SPATIAL
JOIN instruction; thus, this temporary collection is always the same, independently of the
data set. s (in ms) for data sets DS_1, DS_10, DS_25, DS_50, DS_75, DS_100, DS_150 and DS_200. Table 5. Execution times (in ms) for data sets DS_1, DS_10, DS_25, DS_50, DS_75, DS_100, DS_150 and DS_200. Instruction
DS_1
DS_10
DS_25
DS_50
DS_75
DS_100
DS_150
DS_200
1. USE DB
2
1
1
1
1
1
1
1
2. USE DB
0
0
0
0
0
0
0
0
3. USE DB
0
0
0
0
0
0
0
0
4. GET COLLECTION
951
1,598
2,368
3,726
5,177
5,733
7,966
9,831
5. FILTER
8
22
36
52
55
57
59
66
6. SET INTERMEDIATE
214
952
1,885
3,362
4,338
5,201
8,180
10,505
7. GET COLLECTION
83
374
893
1,020
1,368
1,747
2,566
3,911
8. FILTER
324
1,841
2,938
5,340
8,344
9,872
15,747
20,460
9. SET INTERMEDIATE
136
654
1,230
2,340
3,669
3,958
6,349
8,657
10. 6.1. Scalability of the J-CO-QL Engine The reader can clearly see how the quadratic complexity of
the SPATIAL JOIN instruction dominates as long as the size of data sets increases. 1 10
25
50
75
100
150
200
0
10
20
30
40
50
60
70
80
90
100
Datest
Percentage weight (%)
SPATIAL JOIN
Rest of the query
Figure 8. Percentage weight of SPATIAL JOIN on the overall query. SPATIAL JOIN
Rest of the query Percentage weight (%) Figure 8. Percentage weight of SPATIAL JOIN on the overall query. Nevertheless, the reader can notice that the J-CO-QL Engine performs quite well,
even with large data sets. In fact, DS_200 contains 17,600 documents in the Districts
collection and 84,400 documents in the BikeLanes collection; this means that the SPATIAL
JOIN instruction has to compute the intersection of geometries for 1,485,440,000 pairs and
remove duplicates accordingly; it takes 50 min, which is a quite acceptable execution time,
considering the size of the data set. We obtained this good performances by exploiting
multi-threading, so as to exploit all the cores in the CPU. In the near future, we plan to
investigate the adoption of spatial indexes computed on the fly, i.e., immediately before
the execution of the SPATIAL JOIN instruction; in particular, we will have to understand
the threshold above which it becomes advantageous to spend time to build spatial indexes,
so as to introduce some heuristics to decide whether to use them. We conclude this part of the experimental evaluation by considering the behavior of
the FILTER instruction. To this end, we rely again on Table 5. First of all, consider the first two FILTER instructions at line 5 and line 8, where
the former selects districts, while the latter selects bike lanes. They both scale linearly. Furthermore, the execution times remain very low for the FILTER instruction at Line 5
(from 8 ms to 66 ms), while the FILTER instruction at Line 8 takes longer (from 324 ms to
more than 20 s). This is due to many reasons: the first one is the fact that the BikeLanes
collection contains more than 4 times documents than those in collection Districts; the
second reason is that the FILTER instruction at line 8 builds the internal representation of
the ~geometry field (based on the JTS Java Topology Suite library), which is expensive to
do. 6.1. Scalability of the J-CO-QL Engine SPATIAL JOIN
857
13,095
56,513
207,209
445,508
737,696
1,652,120
3,021,693
11. GROUP
9
8
11
12
14
13
14
11
12. FILTER
228
92
125
130
121
134
103
121
13. FILTER
3
1
1
1
1
1
1
1
14. SAVE AS
153
126
130
126
127
174
118
124
Total (ms)
2,966
18,764
66,132
223,319
468,722
764,587
1,693,224
3,075,381
Total (min)
0.05
0.31
1.10
3.72
7.81
12.74
28.22
51.26
Total (min′s′′)
0′03′′
0′19′′
1′06′′
3′43′′
7′49′′
12′45′′
28′13′′
51′15′′
SPATIAL JOIN %
28.90%
69.79%
85.45%
92.79%
95.05%
96.48%
97.57%
98.25%
Rest of the query %
71.10%
30.21%
14.55%
7.21%
4.95%
3.52%
2.43%
1.75% Figure 7 plots the execution times, with respect to the size of the data sets. On the x
axis, we report the multiplication factor f we used to generate each single data set. Three
lines are plotted: the red line shows the overall execution times; the blue line shows the
execution time taken by the SPATIAL JOIN instruction; the green line shows the overall
execution time without the time taken by the SPATIAL JOIN instruction. The three lines
clearly show how the execution time is dominated by the SPATIAL JOIN instruction. In fact,
the rest of the query (green line) scales very well and its contribution becomes substantially
negligible for the large data sets. 1 10
25
50
75
100
150
200
0
500,000
1,000,000
1,500,000
2,000,000
2,500,000
3,000,000
Datest
Execution time (ms)
Overall query
SPATIAL JOIN
Rest of the query
Figure 7. Overall query vs. SPATIAL JOIN vs. Rest of the query (execution times). This behavior is further highlighted by Figure 8, which plots two curves: the blue
li
h
th
t
f
ti
ti
t k
b
th SPATIAL JOIN i
t
ti
th Execution time (ms) Figure 7. Overall query vs. SPATIAL JOIN vs. Rest of the query (execution times). This behavior is further highlighted by Figure 8, which plots two curves: the blue
line shows the percentage of execution time taken by the SPATIAL JOIN instruction; the This behavior is further highlighted by Figure 8, which plots two curves: the blue
line shows the percentage of execution time taken by the SPATIAL JOIN instruction; the Electronics 2021, 10, 621 28 of 35 28 of 35 green line shows the percentage of the overall execution time without the time taken by
the SPATIAL JOIN instruction. 6.1. Scalability of the J-CO-QL Engine In fact, the FILTER instruction at line 5 does not have to do that, since documents in the
Districts collection already have the ~geometry field, so that their internal representation Electronics 2021, 10, 621 29 of 35 29 of 35 is built during the GET COLLECTION instruction at line 4. The reader can see that the GET
COLLECTION instruction at line 7 is much faster than the one at line 4, because it does not
have to build the internal representation of the geometry, which is done during the GET
COLLECTION instruction at Line 4. is built during the GET COLLECTION instruction at line 4. The reader can see that the GET
COLLECTION instruction at line 7 is much faster than the one at line 4, because it does not
have to build the internal representation of the geometry, which is done during the GET
COLLECTION instruction at Line 4. We now consider the FILTER instructions at Lines 12 and 13. Due to the fact that all
data sets are obtained by cloning documents in the original DS_1 data set, the SPATIAL
JOIN instruction at Line 10 produces exactly the same documents for each data set, because
it removes duplicates. Consequently, the two FILTER instructions at Lines 12 and 13
should be independent of the data set. While the latter is stable and very fast (the FILTER
instruction selects a very small number of very simple documents), we cannot say the same
for the former one. We noticed this behavior and we investigated it. It depends on the way
the Java Virtual Machine (JVM) manages main memory; strangely, it is slower with the
smallest data set (228 ms) and faster with DS_10 (92 ms), while it does not exceed 134 ms
in the worst case (excluded DS_1). We can conclude this analysis stating that two main factors affect the execution times
of the FILTER instruction. They are the size of the input temporary collection (as far as
the number of documents is concerned) and the fact that the internal representation of
geometries must be built or not. Nevertheless, its execution times are always negligible if
compared with the SPATIAL JOIN instruction. 6.2. J-CO-DS vs. MongoDB Another test we performed is comparing performance of J-CO-DS, the storage service
developed as an integral part of the framework, and MongoDB. Table 6 reports the times
needed to execute the GET COLLECTION instruction to retrieve the Districts collection,
from the various data sets generated for the experimental campaign; once loaded, this
temporary collection was saved (by means of the SAVE AS instruction) to a MongoDB
database as well as to a J-CO-DS database. Table 6 reports the measured execution times. The From MongoDB row and the From J-CO-DS row report the execution times (in ms)
to perform the GET COLLECTION instruction from the MongoDB database and from the
J-CO-DS database, respectively; Figure 9 depicts the results (the green bars correspond
to tests performed on MongoDB, while the blue bars correspond to tests performed on
J-CO-DS). Similarly, in Table 6 the To MongoDB row and the To J-CO-DS row report the
execution times to perform a SAVE AS instruction to the MongoDB database and to the
J-CO-DS database, respectively; Figure 10 depicts the results (green bars and blue bars
correspond to tests performed on MongoDB and on J-CO-DS, respectively). Table 6. Performance comparison between MongoDB and J-CO-DS. Dataset
DS_1
DS_10
DS_25
DS_50
DS_75
DS_100
DS_150
DS_200
From MongoDB
922
847
979
1,915
2,686
3,713
5,535
7,537
From J-CO-DS
173
954
1,529
3,039
4,550
6,327
9,222
12,435
To MongoDB
100
655
1,324
2,728
4,033
5,303
8,050
11,034
To J-CO-DS
180
858
1,325
2,480
3,784
4,856
7,547
10,154 Table 6. Performance comparison between MongoDB and J-CO-DS. From Figure 9, we notice that execution times to get collections from MongoDB for
the DS_1, DS_10 and DS_25 data sets are not affected by their size; probably, this is due
to the time needed to connect to the database. With the other (and larger) data sets, the
execution time increases linearly, as we expected. In contrast, getting collections from the
J-CO-DS database behaves in a more regular way, being substantially linear for all the data
sets, because J-CO-DS is based on the file system and the connection time is negligible. However, as the size of the collection increases, J-CO-DS becomes slower than MongoDB,
because J-CO-DS stores collections as text files, which are larger than the BSON binary
representation used by MongoDB. 7. Conclusions We can now conclude the paper. First of all, we report the summary of the paper. Second, we discuss possible future work. 6.2. J-CO-DS vs. MongoDB 30 of 35 Electronics 2021, 10, 621 DS_1
DS_10
DS_25
DS_50
DS_75
DS_100
DS_150
DS_200
0
1,000
2,000
3,000
4,000
5,000
6,000
7,000
8,000
9,000
10,000
11,000
12,000
13,000
Dataset
Execution time (ms)
From MongoDB
From J-CO-DS
Figure 9. Performance comparison between MongoDB and J-CO-DS (for getting collections). Figure 9. Performance comparison between MongoDB and J-CO-DS (for getting collections). Figure 10 depicts the results for writing collections to the databases. Both the databases
show a regular behavior. Notice that as long as the size of the collection increases, J-CO-DS
becomes a little faster. We think that this is due to the fact that documents in the collection
to save to MongoDB must be converted into the BSON binary format before sending them,
while J-CO-DS receives documents in the JSON text format. Figure 10 depicts the results for writing collections to the databases. Both the databases
show a regular behavior. Notice that as long as the size of the collection increases, J-CO-DS
becomes a little faster. We think that this is due to the fact that documents in the collection
to save to MongoDB must be converted into the BSON binary format before sending them,
while J-CO-DS receives documents in the JSON text format. So, the BSON representation gives a little advantage to MongoDB for reading collec-
tions, but at the same time it gives a little disadvantage for writing collections. Nevertheless,
we can see that there are no substantial differences between the two storage systems; con-
sequently, J-CO-DS can be effectively used in place of MongoDB to store large collections of
JSON documents, without the limitation that characterizes MongoDB (single documents
cannot have a BSON representation larger than 16 MByte). 31 of 35 Electronics 2021, 10, 621 DS_1
DS_10
DS_25
DS_50
DS_75
DS_100
DS_150
DS_200
0
1,000
2,000
3,000
4,000
5,000
6,000
7,000
8,000
9,000
10,000
11,000
12,000
13,000
Dataset
Execution time (ms)
To MongoDB
To J-CO-DS
Figure 10. Performance comparison between MongoDB and J-CO-DS (for saving collections). Figure 10. Performance comparison between MongoDB and J-CO-DS (for saving collections). 7.1. Summary In this paper, we presented J-CO, a platform-independent framework for managing
JSON data sets. The platform-independent approach is motivated by the large variety of
data storage systems that could be involved in analysis activities performed by analysts
(such as geographers) based on Big Data and Open Data sources, as well as social media
data and volunteered information. Very often, such information is geo-tagged, because
it describes features concerning territories, movements of people on the Earth globe, and
so on. Consequently, the framework is oriented to (spatial) analysts and is built around a
language, named J-CO-QL, that natively deals with spatial representation and provides
spatial operations. When we say that the J-CO Framework is oriented to analysts, we also mean that it is
designed to support integration and cross-analysis of data coming from different JSON
stores possibly powered by different technologies with various processing capabilities. It is
not at all designed to support operational OLTP activities, like a classical DBMS. In fact, the
J-CO Framework is not at all a database management system: it is a unifying framework
for analysis activities, since it provides a platform-independent view of JSON stores and
JSON data sets. Components in the J-CO Framework are loosely-coupled; storage systems,
execution engines and user interfaces are possibly delocalized over the Internet and are Electronics 2021, 10, 621 32 of 35 32 of 35 connected each other when it is necessary. This loosely-coupled design provides flexibility
and ease of cooperation among people all around the world working together by sharing
data sets. In this paper, we presented the current state of development of the J-CO Framework,
which has finally become exploitable by analysts, thanks to the addition of two novel
components, i.e., the data-storage service for large single JSON documents named J-CO-
DS and the user interface named J-CO-UI, as well as thanks to the re-engineering of the
J-CO-QL Engine. Furthermore, for the first time, we report the results of an experimental
evaluation that we conducted to test scalability of the execution engine. The results show
that the J-CO-QL Engine performs quite fast with small data sets and scales well with large
data sets, being able to process a query based on a SPATIAL JOIN instruction on the largest
data set we built in about 50 min. 7.2. Future Work Even though the reader could think that we are at the end of the development of the
J-CO Framework, this is not true. In fact, we plan to work on the framework by following
many development directions. First of all, we will further improve the J-CO-QL Engine. The first topic we are going
to address is concerned with performance. In fact, with the current stage of development
we demonstrated the feasibility of the idea. Now, we are going to devise and implement
specific optimizations, in particular for spatial operations: in fact, we think that the adoption
of spatial indexes computed on the fly could improve the capability of dealing with very
large data sets; however, due to the loosely-coupled approach (that makes the J-CO-QL
Engine able to process data sets coming from JSON stores having no processing capability),
as well as due to the fact that the SPATIAL JOIN can be performed on temporary collections,
it is not possible to rely on pre-built spatial indexes; the engine will have to build them on
the fly, task that needs additional execution time; thus, it will be important to understand
when to adopt spatial indexes. p
p
We will also investigate the possibility to integrate the J-CO-QL Engine with a Map-
Reduce platform, such as Spark, that we successfully experimented for building a blind
querying engine for Open Data sets [49] and for JSON data sets stored within JSON
document stores [50]. In fact, in order to process actual Big Data, this solution appears
to be promising; nonetheless, we will maintain the loosely-coupled approach, so as to
keep user interfaces independent of computational resources actually adopted to process
J-CO-QL queries. The second topic we are going to address is the evolution of J-CO-QL. Apart from the
definition of new instructions for specific analysis tasks (such as operators for perform-
ing data mining and/or machine learning tasks on JSON data sets) and location-based
queries [51,52], we will try to make J-CO-QL to meet the data-independence principle. In
fact, a weakness of the current proposed instructions is that users need to be aware of the
structure of JSON documents; in fact, the current level of J-CO-QL is higher than tradi-
tional procedural programming languages, since its instructions are declarative, although
composed in a “piped” way; so its level is not sufficiently high. 7.1. Summary This fact allows us to claim that the J-CO Framework is
actually suitable to work on data sets whose size is large, although we cannot talk about Big
Data. We also compared J-CO-DS and MongoDB, as far as reading and writing collections
are concerned: we discovered that the two storage systems have comparable performances,
both when reading collections and when writing collections; consequently, since J-CO-DS
does not pose any limitation to the size of documents, it is a complementary tool for storing
huge JSON documents; furthermore, since J-CO-DS provides a simple interface (in that it
does not provide a query language), it can be easily adopted by users that need a simple
tool to store JSON data sets. References 1. Bray, T. The Javascript Object Notation (JSON) Data Interchange Format. 2014. Available online: https://www.rfc-editor.org/
rfc/rfc7159.txt (accessed on 3 March 2021). 1. Bray, T. The Javascript Object Notation (JSON) Data Interchange Format. 2014. Available online: https://www.rfc-editor.org/
rfc/rfc7159.txt (accessed on 3 March 2021). 2. Bray, T.; Paoli, J.; Sperberg-McQueen, C.M.; Maler, E.; Yergeau, F. Extensible markup language (XML) 1.0
2000. Available online: https://www.w3.org/TR/xml/ (accessed on 25 February 2021). p
g
y
3. Butler, H.; Daly, M.; Doyle, A.; Gillies, S.; Hagen, S.; Schaub, T. The geojson format. Internet Engineering Task Force (IETF); 2016. Available online: https://tools.ietf.org/html/rfc7946 (accessed on 25 February 2021). 3. Butler, H.; Daly, M.; Doyle, A.; Gillies, S.; Hagen, S.; Schaub, T. The geojson format. Internet Engin
Available online: https://tools.ietf.org/html/rfc7946 (accessed on 25 February 2021). 3. Butler, H.; Daly, M.; Doyle, A.; Gillies, S.; Hagen, S.; Schaub, T. The geojson format. Internet Engineering Task Force (IETF); 2016. Available online: https://tools.ietf.org/html/rfc7946 (accessed on 25 February 2021). y
y
g
g
j
Available online: https://tools.ietf.org/html/rfc7946 (accessed on 25 February 2021). p
g
y
4. Chow, T.E. Geography 2.0: A mashup perspective. In Advances in Web-based GIS, Mapping Services Furthermore, Applications; CRC
Press: Boca Rato, FL, USA, 2011; pp. 15–36. 4. Chow, T.E. Geography 2.0: A mashup perspective. In Advances in Web-based GIS, Mapping
Press: Boca Rato, FL, USA, 2011; pp. 15–36. 4. Chow, T.E. Geography 2.0: A mashup perspective. In Advances in Web-based GIS, Mapping Services Furthermore, Applications; CRC
Press: Boca Rato, FL, USA, 2011; pp. 15–36. pp
5. Cattell, R. Scalable SQL and NoSQL data stores. ACM Sigmod Rec. 2011, 39, 12–27. [CrossRef] 6. Chodorow, K. MongoDB: The Definitive Guide; O’Reilly Media, Inc.: Sebastopol, CA, USA, 2013. 7. Burini, F.; Cortesi, N.; Gotti, K.; Psaila, G. The Urban Nexus Approach for Analyzing Mobility in the Smart City: Towards the
Identification of City Users Networking. Mob. Inf. Syst. 2018, 2018, 6294872. [CrossRef] 8. Bordogna, G.; Capelli, S.; Psaila, G. A big geo data query framework to correlate open data with social network geotagged posts. In Proceedings of the Annual International Conference on Geographic Information Science, Wageningen, The Netherlands, 9–12
May 2017; pp. 185–203. 9. Bordogna, G.; Ciriello, D.E.; Psaila, G. A flexible framework to cross-analyze heterogeneous multi-source geo-referenced
information: The J-CO-QL proposal and its implementation. In Proceedings of the International Conference on Web Intelligence,
Leipzig, Germany, 23–26 August 2017; pp. 499–508. Leipzig, Germany, 23–26 August 2017; pp. 499–508. 7.2. Future Work In our next evolution of
J-CO-QL, we aim at defining two layers of instructions: the user-layer will provide instruc-
tions that will be directly invoked by users; the hidden layer will provide instructions that
will be automatically invoked by translating instructions in the user layer by a mediator. Electronics 2021, 10, 621 33 of 35 33 of 35 As defined in [53], “A mediator is a software module that exploits encoded knowledge
about certain sets or subsets of data to create information for a higher layer of applications”:
sources are encapsulated by wrappers (which access their data sources in a transparent
way to mediators) to present data in the form needed by a mediator. Finally, we argued that web sources that publish JSON data sets as well as GeoJSON
documents, such as Open Data portals, could play the role of read-only JSON stores. In
fact, we are planning to develop a new component to add to the framework, that will create
virtual JSON stores by connecting to web sources of interest. The J-CO-QL Engine will
connect to these virtual JSON stores in a seamless way: in fact, such virtual JSON stores
will not have any computational capability, thus the platform-independent design will be
effective for them too. Author Contributions: Conceptualization and methodology, G.P.; software, P.F.; writing—original
draft preparation, G.P.; writing—review and editing, G.P. and P.F. All authors have read and agreed
to the published version of the manuscript. Funding: This research received no external funding. Data Availability Statement: https://github.com/zunstraal/J-Co-Project/ (accessed on 7 March 2021). Data Availability Statement: https://github.com/zunstraal/J-Co-Project/ (accessed on 7 March 2021). Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. 17.
Gou, G.; Chirkova, R. Efficiently querying large XML data repositories: A survey.
IEEE Trans. Knowl. Data Eng. 2007,
19, 1381–1403. [CrossRef] References 31
Doulkeridis C ; NØrvåg K A survey of large-scale analytical query processing in MapReduce VLDB J The Int J V 30. Arora, R.; Aggarwal, R.R. Modeling and querying data in mongodb. Int. J. Sci. Eng. Res. 2013, 4, 141 144. 31. Doulkeridis, C.; NØrvåg, K. A survey of large-scale analytical query processing in MapReduce. VLDB J. The Int. J
[C
f] ,
;
gg
,
g
q
y
g
g
J
g
, ,
31. Doulkeridis, C.; NØrvåg, K. A survey of large-scale analytical query processing in MapReduce. VLDB J. The Int. J. Very Large
Data Bases 2014, 23, 355–380. [CrossRef] 31. Doulkeridis, C.; NØrvåg, K. A survey of large-scale analytical query processing in MapReduce. VLD
Data Bases 2014, 23, 355–380. [CrossRef] 32. Goyal, V.; Soni, D. Survey paper on Big Data Analytics using Hadoop Technologies. Int. J. Curr. Eng. Sci. Res. (IJCESR) 2006,
3, 2394–2697. 33. Armbrust, M.; Xin, R.S.; Lian, C.; Huai, Y.; Liu, D.; Bradley, J.K.; Meng, X.; Kaftan, T.; Franklin, M.J.; Ghodsi, A.; et al. Spark sql:
Relational data processing in spark. In Proceedings of the 2015 ACM SIGMOD International Conference on Management of Data,
Melbourne, VIC, Australia, 31 May–4 June 2015; pp. 1383–1394. y
pp
34. Battle, R.; Kolas, D. Geosparql: Enabling a geospatial semantic web. Semant. Web J. 2011, 3, 355–370. [CrossRef] 35. Bordogna, G.; Pagani, M.; Psaila, G. Database model and algebra for complex and heterogeneous spatial entities. In Progress in
Spatial Data Handling; Springer: Berlin, Germany, 2006; pp. 79–97. 36. Psaila, G. A database model for heterogeneous spatial collections: Definition and algebra. In Proceedin
Conference on Data and Knowledge Engineering (ICDKE); IEEE: New York, NY, USA, 2011; pp. 30–35. 37. Bordogna, G.; Campi, A.; Psaila, G.; Ronchi, S. An interaction framework for mobile web search. In Proceedings of the 6th
International Conference on Advances in Mobile Computing and Multimedia, Linz, Austria, 24–26 November 2008; pp. 183–191. 38. Duggan, J.; Elmore, A.J.; Stonebraker, M.; Balazinska, M.; Howe, B.; Kepner, J.; Madden, S.; Maier, D.; Mattson, T.; Zdonik, S. The
BIGDAWG polystore system. ACM Sigmod Rec. 2015, 44, 11–16. [CrossRef] 39. Singhal, R.; Zhang, N.; Nardi, L.; Shahbaz, M.; Olukotun, K. Polystore++: Accelerated Polystore System for Heterogeneous
Workloads. In Proceedings of the 2019 IEEE 39th International Conference on Distributed Computing Systems (ICDCS), Dallas,
TX, USA, 7–10 July 2019; pp. 1641–1651. y
pp
40. Hamadou, H.B.; Gallinucci, E.; Golfarelli, M. References p g
y
g
pp
10. Bordogna, G.; Capelli, S.; Ciriello, D.E.; Psaila, G. A cross-analysis framework for multi-source volunteered, crowdsourced, and
authoritative geographic information: The case study of volunteered personal traces analysis against transport network data. Geo-Spat. Inf. Sci. 2018, 21, 257–271. [CrossRef] 11. Cuzzocrea, A.; Psaila, G.; Toccu, M. Knowledge discovery from geo-located tweets for supporting advanced big data analytics: A
real-life experience. In Model and Data Engineering, Rhodes, Greece; Springer: Berlin, Germany, 2015; pp. 285–294. 12. Cuzzocrea, A.; Psaila, G.; Toccu, M. An innovative framework for effectively and efficiently supporting big data analytics over
geo-located mobile social media. In Proceedings of the 20th International Database Engineering & Applications Symposium,
Montreal, QC, Cananda, 11–13 July 2016. y
13. Bordogna, G.; Cuzzocrea, A.; Frigerio, L.; Psaila, G.; Toccu, M. An interoperable open data framework for discovering popular
tours based on geo-tagged tweets. Int. J. Intell. Inf. Database Syst. 2017, 10, 246–268. [CrossRef] g
gg
f
y
14. Bordogna, G.; Frigerio, L.; Cuzzocrea, A.; Psaila, G. Clustering geo-tagged tweets for advanced big data analytics. In Proceedings
of the 2016 IEEE International Congress on Big Data (BigData Congress), San Francisco, CA, USA, 27 June–2 July 2016; pp. 42–51. 14. Bordogna, G.; Frigerio, L.; Cuzzocrea, A.; Psaila, G. Clustering geo-tagged tweets for advanced big data analytics. In Proceedings
of the 2016 IEEE International Congress on Big Data (BigData Congress), San Francisco, CA, USA, 27 June–2 July 2016; pp. 42–51. 15. Uddin, M.F.; Gupta, N. Seven V’s of Big Data understanding Big Data to extract value. In Proceedings of the 2014 Zone 1
Conference of the American Society for Engineering Education, Bridgeport, CT, USA, 3–5 April 2014; pp. 1–5. g
g
(
g
g
),
,
,
,
J
J
y
; pp
15. Uddin, M.F.; Gupta, N. Seven V’s of Big Data understanding Big Data to extract value. In Proceedings of the 2014 Zone 1
Conference of the American Society for Engineering Education, Bridgeport, CT, USA, 3–5 April 2014; pp. 1–5. 16. Feng, J.H.; Qian, Q.; Liao, Y.G.; Li, G.L.; Ta, N.; Zhou, L.Z. Survey of research on native xml databases. Appl. Res. Comput. 2006,
6, 1–7. 34 of 35 34 of 35 Electronics 2021, 10, 621 18. Haw, S.C.; Lee, C.S. Data storage practices and query processing in XML databases: A survey. Knowl. Based Syst. 2011,
24, 1317–1340. [CrossRef] 19. Kurgan, L.A.; Musilek, P. References A survey of Knowledge Discovery and Data Mining process models. Knowl. Eng. Rev. 2006, 21, 1–24. [CrossRef] [
]
20. Meo, R.; Psaila, G. An XML-based database for knowledge discovery. In Proceedings of the International Conference on
Extending Database Technology, Munich, Germany, 26–31 March 2006; pp. 814–828. 21. Nayak, A.; Poriya, A.; Poojary, D. Type of NOSQL databases and its comparison with relational databases. Int. J. Appl. Inf. Syst. 2013, 5, 16–19. 22. Hecht, R.; Jablonski, S. Nosql evaluation: A us case oriented survey. In Proceedings of the CSC-2011 International Conference on
Cloud and Service Computing, Hong Kong, China, 12–14 December 2011; pp. 336–341. 23. Han, J.; Haihong, E.; Le, G.; Du, J. Survey on NoSQL database. In Proceedings of the 2011 6th Inte
Pervasive Computing and Applications, Chengdu, China, 11–13 May 2011; pp. 363–366. g
g
y
24. Beyer, K.S.; Ercegovac, V.; Gemulla, R.; Balmin, A.; Eltabakh, M.; Kanne, C.C.; Ozcan, F.; Shekita, E.J. Jaql: A scripting language
for large scale semistructured data analysis. Proc. VLDB Endow. 2011, 4, 1272–1283. [CrossRef] g
y
25. Anderson, J.C.; Lehnardt, J.; Slater, N. CouchDB: The Definitive Guide: Time to Relax; O’Reilly Media, Inc.: Sebastopol, CA, USA,
2010. 26. Ong, K.W.; Papakonstantinou, Y.; Vernoux, R. The SQL++ unifying semi-structured query language, and an expressiveness
benchmark of SQL-on-Hadoop, NoSQL and NewSQL databases. arXiv 2014, arXiv:1405.3631. 27. Chamberlin, D. SQL++ For SQL Users: A Tutorial. 2018. Available online: Amazon.com (accessed on 3 March 2021). 28. Florescu, D.; Fourny, G. JSONiq: The history of a query language. IEEE Internet Comput. 2013, 17, 86–90. [CrossRef] 27. Chamberlin, D. SQL++ For SQL Users: A Tutorial. 2018. Available online: Amazon.com (accessed on 3 March 2021). 28. Florescu, D.; Fourny, G. JSONiq: The history of a query language. IEEE Internet Comput. 2013, 17, 86–90. [CrossRef] 28. Florescu, D.; Fourny, G. JSONiq: The history of a query language. IEEE Internet Comput. 2013, 17, 86–90. [CrossRef] 29. Chamberlin, D. XQuery: An XML query language. IBM Syst. J. 2002, 41, 597–615. [CrossRef] y
q
y
g
g
y
, R.; Aggarwal, R.R. Modeling and querying data in mongodb. Int. J. Sci. Eng. Res. 2013, 4, 141–144. 30. Arora, R.; Aggarwal, R.R. Modeling and querying data in mongodb. Int. J. Sci. Eng. Res. 2013, 4, 30. Arora, R.; Aggarwal, R.R. Modeling and querying data in mongodb. Int. J. Sci. Eng. Res. 2013, 4, 141–144. 47.
Bortnikov, E.A.A.B.V.; Konstantinos, C.C.; Enyeart, C.A.D.C.D.; Laventman, C.F.G.; Manevich, Y.; Muralidharan, S.; Murthy, C.;
Nguyen, B.; Sethi, M.; Singh, G.; et al. Hyperledger Fabric: A Distributed Operating System for Permissioned Blockchains; In
Proceedings of the 13th EuroSys Conference, Porto, Portugal, 23–26 April 2018. ,
;
,
;
,
Q
pp
J
f
,
,
[
]
51.
Bordogna, G.; Pagani, M.; Pasi, G.; Psaila, G. Evaluating uncertain location-based spatial queries. In Proceedings of the 2008
ACM Symposium on Applied Computing, Ceara, Brazil, 16–20 March 2008; pp. 1095–1100. 53.
Wiederhold, G. Mediators in the architecture of future information systems. Computer 1992, 25, 38–49. y
g
p
g
y
pp
49.
Pelucchi, M.; Psaila, G.; Toccu, M. Hadoop vs. Spark: Impact on Performance of the Hammer Quer
Corpora. Algorithms 2018, 11, 209. [CrossRef] y
p
pp
p
g
pp
52.
Bordogna, G.; Pagani, M.; Pasi, G.; Psaila, G. Managing uncertainty in location-based queries. Fuzzy Sets
[CrossRef] p
g
50.
Marrara, S.; Pelucchi, M.; Psaila, G. Blind Queries Applied to JSON Document Stores. Information 2019, 10, 291. [CrossRef]
51
Bordogna G ; Pagani M ; Pasi G ; Psaila G Evaluating uncertain location-based spatial queries In Proceedings of the 2008 p
g
50.
Marrara, S.; Pelucchi, M.; Psaila, G. Blind Queries Applied to JSON Document Stores. Information 2019, 10, 291. [CrossRef] p
g
50.
Marrara, S.; Pelucchi, M.; Psaila, G. Blind Queries Applied to JSON Document Stores. Information 2019, 10, 291. [CrossRef]
51.
Bordogna, G.; Pagani, M.; Pasi, G.; Psaila, G. Evaluating uncertain location-based spatial queries. In Proceedings of the 2008
ACM Symposium on Applied Computing Ceara Brazil 16 20 March 2008; pp 1095 1100 g
y
g
p
Hubert, G.; Cabanac, G.; Sallaberry, C.; Palacio, D. Query operators shown beneficial for improving search re
Conference on Theory and Practice of Digital Libraries; Springer: Berlin, Germany; 2011, pp. 118–129. Conference on Theory and Practice of Digital Libraries; Springer: Berlin, Germany; 2011, pp. 118 129.
49.
Pelucchi, M.; Psaila, G.; Toccu, M. Hadoop vs. Spark: Impact on Performance of the Hammer Query Engine for Open Data
Corpora Algorithms 2018 11 209 [CrossRef] References Answering GPSJ queries in a polystore: A dataspace-based approach. In Proceedings
of the International Conference on Conceptual Modeling, Salvador, Brazil, 4–7 November 2019; pp. 189–203. 41. Jananthan, H.; Zhou, Z.; Gadepally, V.; Hutchison, D.; Kim, S.; Kepner, J. Polystore mathematics of relational algebra. In
Proceedings of the 2017 IEEE International Conference on Big Data (Big Data), Boston, MA, USA, 11–14 December 2017; pp. 3180–3189. 42. Rantung, V.P.; Kembuan, O.; Rompas, P.T.D.; Mewengkang, A.; Liando, O.E.S.; Sumayku, J. In-memory business intelligence:
Concepts and performance. In IOP Conference Series: Materials Science and Engineering; IOP Publishing: Sebastopol, CA, USA,
2018; Volume 306, p. 012129. p
43. Shukla, A.; Dhir, S. Tools for data visualization in business intelligence: Case study using the tool Qlikvi
Design and Intelligent Applications; Springer: Berlin, Germany, 2016; pp. 319–326. Tools for data visualization in business intelligence: Case study using the tool Qlikview. In Information Systems
t Applications; Springer: Berlin, Germany, 2016; pp. 319–326. 44. Mora, J.M.L. Qlik Sense Implementation: Dashboard Creation and I
Master’s Thesis, Universidade Nova de Lisboa, Lisbon, Portugal, 2020. 44. Mora, J.M.L. Qlik Sense Implementation: Dashboard Creation and Implementation of the Test Performance Methodology. Master’s Thesis, Universidade Nova de Lisboa, Lisbon, Portugal, 2020. g
45. Gormley, C.; Tong, Z. Elasticsearch: The Definitive Guide: A Distributed Real-Time Search and Analytics Engine; O’Reilly Media, Inc.:
Sebastopol, CA, USA, 2015. 46. Manyam, G.; Payton, M.A.; Roth, J.A.; Abruzzo, L.V.; Coombes, K.R. Relax with CouchDB—Into the non-relational DBMS era of
bioinformatics. Genomics 2012, 100, 1–7. [CrossRef] 35 of 35 Electronics 2021, 10, 621 35 of 35 Conference on Theory and Practice of Digital Libraries; Springer: Berlin, Germany; 2011, pp. 118–129. 49. Pelucchi, M.; Psaila, G.; Toccu, M. Hadoop vs. Spark: Impact on Performance of the Hammer Query Engine for Open Data
Corpora. Algorithms 2018, 11, 209. [CrossRef] p
g
50. Marrara, S.; Pelucchi, M.; Psaila, G. Blind Queries Applied to JSON Document Stores. Information 2019, 10, 291. [CrossRef]
51. Bordogna, G.; Pagani, M.; Pasi, G.; Psaila, G. Evaluating uncertain location-based spatial queries. In Proceedings of the 2008
ACM Symposium on Applied Computing, Ceara, Brazil, 16–20 March 2008; pp. 1095–1100. p
0. Marrara, S.; Pelucchi, M.; Psaila, G. Blind Queries
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English
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On the origin and evolution of a new anchialine stygobitic &lt;i&gt;Microceratina&lt;/i&gt; species (Crustacea, Ostracoda) from Christmas Island (Indian Ocean)
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Journal of micropalaeontology
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INTRODUCTION GEOLOGICAL AND HYDROLOGICAL SETTING
Christmas Island lies in the Indian Ocean, about 350 km south-
west of Java and 1200 km west of the Australian continent. Grimes (2001) reviewed the sparse knowledge of the geomor-
phology, geology and genesis of the cave systems on the island
and Humphreys & Eberhard (1998, 2001) have discussed the
natural history of the island as it pertains to the subterranean
fauna. Christmas Island, which is the tip of an isolated
seamount rising 4.5 km from the ocean floor in the Wharton
Basin of the Indian Plate, is the only member exposed above
sea-level of a series of volcanoes that formed from the mid-
Cretaceous, starting about 80 Ma. As it is drifting northwards
(70–80 mm a1) it will descend into the Java Trench in about
four million years (Grimes, 2001). The island, which reaches an
altitude of 361 m, is small (135 km2) and is capped by a sequence
of Tertiary limestones up to 250 m thick (Pettifer & Polak, 1979)
that overlie the basaltic rocks that form the core of the island. Because the island is rising c. 140 mm ka1 (Woodroffe, 1988) it
is flanked by a series of emergent limestone terraces. Volcanism
was reactivated in the Eocene (35–40 Ma) from which date there
is a series of poorly exposed carbonates, the main exposures
being of Late Oligocene to Middle Miocene age. The exposed
landmass would never have been substantially larger than at
present due to the steep submarine slopes on the island, but at
times the island could have been both much smaller (even
submerged) and fragmented, if, as suggested by Barrie (1967), an
atoll was present. The main limestones contain fossils laid down
in shallow water (Adams & Belford, 1974) in the Late Oligocene
(26 Ma). Deposition of the main limestones ceased at the end of
the Middle Miocene (c. 14 Ma) as a result of the global fall in
sea-level and exposed limestones were then subjected to several Ostracods of the family Cytheruridae G.W. Müller are exclu-
sively marine and brackish water-dwelling species which inhabit
a wide range of coastal and deep-sea habitats (Kempf, 1986;
Hartmann, 1989). On the origin and evolution of a new anchialine stygobitic Microceratina species (Crustacea,
Ostracoda) from Christmas Island (Indian Ocean) On the origin and evolution of a new anchialine stygobitic Microceratina species (Crustacea,
Ostracoda) from Christmas Island (Indian Ocean) TADEUSZ NAMIOTKO1, KAREL WOUTERS2, DAN L. DANIELOPOL3 & WILLIAM F. HUMPHREYS4
1University of Gdan¯sk, Department of Genetics & Cytology, Kladki 24, 80-822 Gdan¯sk, Poland (e-mail: namiotko@biotech.univ.gda.pl). 2Royal Belgian Institute of Natural Sciences, Department of Invertebrates, Vautierstraat 29, B-1000 Brussels and Department of Biology,
K.U. Leuven, Leuven, Belgium (e-mail: karel.wouters@naturalsciences.be). 3 ABSTRACT – Marine species of the ostracod genus Microceratina Swanson (Cytheruridae, Eucytheruri-
nae) were until now known only from their hard parts, the valves and carapaces, as no living animals have
been described. Here we report the first living population, from a tropical anchialine cave. The description
of the limbs and hard parts of this new taxon, M. martensi sp. nov., enhances our understanding of the
origin and evolution of the cave-dwelling Microceratina – the new species and M. pseudoamfibola
(Barbeito-Gonzalez) from an anchialine cave in Southern Italy – and clarifies their affinities with other
Eucytherurinae species. Microceratina is known from both Recent and fossil species (Quaternary, Tertiary
and Late Cretaceous) from shelf and deep-sea habitats and/or sedimentary facies, located in the Pacific
Ocean (along the Australian and New Zealand coasts), the Mediterranean (Greece and Italy), the North
Atlantic (British Isles) and the Baltic Sea (Rügen Island). This suggests that the Microceratina group
spread through the expanding Tethys Ocean. The morphological traits of the two cave-dwelling species
reflect their ecological conditions. Cave-dwelling Microceratina species appear to have originated from
epigean shallow water species predisposed to colonize subterranean habitats. J. Micropalaeontol. 23(1):
49–59, May 2004. Journal of Micropalaeontology, 23: 49–59. Journal of Micropalaeontology, 23: 49–59. 0262-821X/04 $15.00 2004 The Micropalaeontological Society CHARACTERISTICS OF THE SAMPLING SITE The new species was found amongst roots in an anchialine pool
in a small, unnamed cave CI-54, situated in the North East Point
of Christmas Island (Fig. 1). The cave is probably connected to
two adjacent anchialine caves, Runaway Cave (CI-2) and The
19th Hole (CI-19), and these three caves are treated here as a
single anchialine system. The caves open on the lowest terrace, close to the coast. Waters within the caves are affected by marine tides, have
different surface salinities and show the salinity stratification
typical of anchialine waters (Table 1) (Humphreys & Eberhard,
1998). Cave CI-54 lies between Runaway Cave and the coast,
therefore, the salinity is likely to be greater than the brackish
water (salinity c. 9–18 ppt) in Runaway Cave (Table 1), but not
substantially greater as the ostracod was collected amongst
submerged roots of rainforest plants. After heavy rain the
uppermost water layer will probably be dominated by under-
ground
freshwater
drainage
from
the
plateau
(TDS
of
195–280 mg dm3; Polak, 1976). The cave waters are well
oxygenated but have not been sampled at any depth either for
their biota or for physico-chemical parameters. It is not known,
therefore, whether the deeper water becomes suboxic as reported
for many other anchialine systems (Sket, 1996; Humphreys,
1999; Iliffe, 2000). Fig. 1. Map of Christmas Island with the location of the sampling sites
(black star). million years of karst development. The lack of extensive
Pliocene or Pleistocene limestone deposition suggests that the
island has not been extensively submerged since the Miocene
(Grimes, 2001). The rainfall (annual mean 2109 mm: Falkland, 1986) is highly
variable owing to the impact on the island of the El Niño/
Southern Oscillation (ENSO) but supports rainforest covering
the island. Typical of karst drainage, rainfall infiltrates the
limestone and then mostly drains down the slope of the under-
lying basalt until reaching the sea in caves and through coastal
and offshore springs (Pettifer & Polak, 1979). A low lens of fresh
groundwater occurs in some areas (Barrett, 1985). )
Although cave CI-54 was not sampled thoroughly for fauna,
the following aquatic taxa accompanied the new ostracod
species: Syllidae indet. (Polychaeta); Nerilla sp. (Archiannelida:
Nerillidae); Nitocrella/Nitokra complex (Harpacticoida: Ameiri-
dae); Bryocyclops muscicola (Menzel) (Cyclopoida: Cyclopini-
dae); a new copepod genus (Calanoida: Arietellidae); Leucothoe
sp. (Amphipoda: Leucothoidae); Alpheidae indet. CHARACTERISTICS OF THE SAMPLING SITE (Decapoda);
Parahippolyte
uveae
Borradaile
(Decapoda:
Hippolytidae);
Antecaridina lauensis (Edmondson) (Decapoda: Atyidae; found
also in CI-2 and CI-19); Macrobranchium lar (Fabricius) (Deca-
poda: Palaemonidae); Gobioidae indet. (Pisces: Perciformes). In
CI-2 Procaris sp. nov. (Decapoda: Procarididae) and Eleotridae
indet. (Pisces: Perciformes) were encountered. Waters on the plateau are fresh (Total Dissolved Solids
(TDS) 195–280 mg dm3: Polak, 1976) but mix with seawater
near the coast and so will accelerate karst formation through the
effects of mixing corrosion (Ford & Williams, 1989) to develop
salt–freshwater interface caves (e.g. from the Bahamas see Vogel
et al., 1990; Mylroie et al., 1991). Because sea-level stands during
the Pleistocene were predominantly much lower (Chappell &
Thom, 1977) these caves are likely to extend at least 80 m below
the present sea-level, notwithstanding an uplift of the island of
14–20 m in the last 124 ka (Woodroffe, 1988). Speleothems INTRODUCTION Representatives of the genus Microceratina
Swanson, 1980 are known from the Southern Hemisphere at
sites close to the Australian and New Zealand coasts (Swanson,
1980; Whatley & Downing, 1983; Yassini & Jones, 1995) and
from the Northern Hemisphere, in the Mediterranean (Greece
and Italy; Mazzini & Gliozzi, 2000). Microceratina is a cytheru-
rid genus recorded from the Late Cretaceous (Herrig, 1991) to
Recent. However, until now, the nine species attributed to this
genus have been described from empty carapaces and/or valves,
the material coming from open marine environments, with the
exception of Microceratina pseudoamfibola (Barbeito-Gonzalez,
1971), which was described from an anchialine cave in southern
Italy (Mazzini & Gliozzi, 2000). The discovery of a living stygobitic population of a new
Microceratina species within the karstic system of tropical
Christmas Island in the Indian Ocean is important as it repre-
sents the first Microceratina species for which the appendages
can be described. Furthermore, it presents the opportunity to
reconstruct the origin and evolution of this phylogenetic lineage
which has produced a clearly stygobitic species. Pursuit of this
goal is worthwhile because there are few well-documented cases
where both living and fossil records of ostracods have been used
to reconstruct the pattern of colonization and the manner of
their adaptation to the subterranean environment. One of the
most complete reconstructions of the evolutionary history
resulting in stygobitic species is of the Danielopolina group
(Danielopol et al., 2000). 49 T. Namiotko et al. Fig. 1. Map of Christmas Island with the location of the sampling sites
(black star). drowned by at least 6 m provide evidence of lower sea-level
(Humphreys & Eberhard, 1998) and submarine springs have
been reported at a depth of 200 m in Flying Fish Cove (Pettifer
& Polak, 1979). Description of female Valves. (Fig. 2a–b; Pl. 1, figs 1–7) Very small, somewhat
translucent and elongate. Dorsal margin straight, ventral margin
weakly sinuous; dorsal and ventral margins weakly tapering
towards the posterior. Anterior margin evenly rounded, with a
large flange, posterior margin evenly rounded. Lateral surface
inflated, forming a mid-ventral and postero-ventral alar process. Carapace in dorsal view broad, with nearly parallel lateral
margins; anterior and posterior extremities pointed. Ornamen-
tation consisting of a distinct reticulate pattern of polygonal
meshes, each mesh being subdivided into a number of smaller
meshes surrounded by fine polygonal muri; two to three longi-
tudinal ventral striae near the expansion of the alar process. Pore clusters of the loophole type with three subcircular to oval
holes. Anterior and posterior inner lamellae wide. Anterior
vestibulum narrow, with nine broad marginal pore canals, some
of them false, with a central bulbous widening. Posterior vesti-
bulum shallow and elongate, with five to six broad marginal
pore canals. Hinge weakly developed, merodont, with very
weakly crenulated (difficult to see), small and elongate terminal
teeth and in the right valve a shallow central groove. Muscle scar
pattern consisting of an oblique row of four elongate adductor
scars and a large V- to U-shaped frontal scar. The imprints of
the muscle scars are visible on the external side of the valves as
small protuberances of nearly the same shape as the scars. Dissected specimens were mounted in glycerine on slides, with
valves stored dry in micropalaeontological cells, whereas entire
specimens were preserved in 97% ethanol in a glass tube. Valve
dimensions were measured to the nearest 0.01 mm under the
stereomicroscope at 500. Valves and appendages were exam-
ined and drawn using a microscope fitted with a drawing tube at
magnifications of 500, 800 or 2000 (oil immersion). Selected valves were prepared for the SEM and studied at the
SEM laboratory of the Royal Belgian Institute of Natural
Sciences in Brussels. MATERIAL AND METHODS Groundwaters of caves CI-54, Runaway Cave (CI-2) and 19th
Hole (CI-19) were sampled directly but other groundwaters on Table 1. Some physical and chemical parameters of groundwaters from anchialine caves adjacent to the sampling site Cave CI-54, recorded on 5–6
April 1998. CAVE
Dissolved oxygen
Conductivity
Temperaturea
pH
mg dm3
%
mS cm1
(C
Runaway Cave (CI-2)
surface
7.06
97.7
5.5
26.9
7.99
2 m depth
6.67
96.6
8.1
26.5
7.50
The 19th Hole (CI-19)
surface
6.60
94.0
31.2
27.0
7.60
2 m depth
5.05
73.0
34.4
27.4
7.35
From Humphreys & Eberhard (1998)
aThe air temperature in CI-19 was 27(C. Table 1. Some physical and chemical parameters of groundwaters from anchialine caves adjacent to the sampling site Cave CI-54, recorded on 5–6
April 1998. 50 New stygobitic ostracod species from Christmas Island 28912), one female carapace (C 28913), one female carapace
with disarticulated valves (C 28914), and 15 adult females and 9
juveniles preserved in ethanol (C 28915), deposited in the
collections of the Western Australian Museum, Perth. the island were sampled through springs and boreholes. Samples
were taken variously by hand-net following visual sighting of
fauna, by plankton nets of appropriate dimensions hauled
through the water column, with or without pre-baiting, and/or
by the use of baited traps left in place overnight. Ostracods were
collected in cave CI-54 by S. M. Eberhard on 13 April 1998. Physico-chemical parameters of groundwaters were measured in
situ using electronic instruments (Watts & Humphreys, 2000):
pH using a WTW pH 320 meter with a SenTix 97T pH-
combined electrode with integrated temperature sensor and
redox probe and dissolved oxygen using a WTW Oxi 320 meter
and a CellOx 325 oxygen sensor (Wissenschaftlich-Technisch
Werkstatten GmbH, Weilheim, Germany). Conductivity was
measured with a TPS Model LC 84 conductivity meter (TPI
Electronics, Springwood, Queensland, Australia). All were
calibrated as specified using the recommended standards. Type locality. Cave CI-54 (unnamed) opening on the lowest
terrace, close to the coast, c. 1.5 km south from the North East
Point
of
Christmas
Island,
Indian
Ocean
(geographical
co-ordinates: c. 10(25’20” S, 105(42’02” E; altitude: entrance c. 10 m above sea-level; water at sea-level). For details, see the
previous section. SYSTEMATIC DESCRIPTION SYSTEMATIC DESCRIPTION
Order Podocopida G.W. Müller, 1894
Suborder Podocopina Sars, 1866
Superfamily Cytheroidea Baird, 1850
Family Cytheruridae G.W. Müller, 1894
Subfamily Eucytherurinae Puri, 1974
(emend. Maddocks & Steineck, 1987)
Genus Microceratina Swanson, 1980 Type species. Microceratina quadrata Swanson, 1980 (by original
designation). Type species. Microceratina quadrata Swanson, 1980 (by original
designation). Microceratina martensi sp. nov. (Pl. 1, figs 1–7, Figs 2–4) Size of carapace. Holotype length (L) 0.31 mm, height (H)
0.15 mm; paratypes (n=6) L 0.31–0.33 mm, H 0.13–0.15 mm. Derivation of name. Named after our colleague and friend Koen
Martens (Royal Belgian Institute of Natural Sciences, Brussels),
in recognition of his important contributions to the study of
ostracods. Antennule. (Fig. 3a) Consisting of five functional, generally
slender podomeres (2-segmented base and 3-segmented ramus),
although the equivalents of seven podomeres can be recognized. First podomere dorso-distally with a row of thin setulae. Second
and third podomeres not differentiated, without any visible
suture. The ventro-median point of insertion of the long seta
(reaching beyond the distal end of fifth podomere) marks the
former boundary between these two podomeres. Both of these
podomeres, forming one compound segment, set dorsally with
brushes consisting of long setulae. Fourth podomere (the first
podomere of the ramus) with one dorso-distal seta. Fifth and
sixth podomeres incompletely differentiated, joined by an indis-
tinct, inflexible suture. Fifth podomere bearing three setae: two
unequal dorsal setae, and one ventral seta, while sixth podomere
set with four setae: two very long and one short dorsal setae, and Diagnosis. A small species of Microceratina with well-developed
valve reticulation and distinct ventro-lateral ridges along alar
process; dorsal and ventral margins weakly tapering towards the
posterior; pore clusters of the loophole type, with three sub-
circular to oval holes. Body unpigmented, no visible ocular
structures. Holotype. A female with valves stored dry and dissected limbs
preserved in a sealed glycerine preparation (C 28908), deposited
in the collections of the Western Australian Museum, Perth. Paratypes. Four females with valves stored dry and dissected
limbs preserved in a sealed glycerine preparation (C 28909–C 51 T. Namiotko et al. additional seta on the ventro-distal corner. Seventh
omere absent. The terminal (eighth) podomere distally with
e setae and one aesthetasc The latter as long as the ramus
Antenna. (Fig. 3b) Four podomeres and a large exopo
(spinneret seta) extending almost to the tips of the term
claws First endopodal podomere sub-quadrangular with
anation of Plate 1. Microceratina martensi sp. nov., Christmas Island. All figured specimens are females. fig. 1. Left valve, external v
ype (C 28910), 250. fig. 2. Right valve, external view, paratype (C 28909), 250. fig. 3. Right valve, internal view, holotype (C 28908),
. Right valve, internal view, holotype, 310. fig. 5. Type species. Microceratina quadrata Swanson, 1980 (by original
designation). Left valve, dorsal view, holotype, 250. fig. 6. Right valve, detail of ornamentation
, paratype (C 28909), 7600. fig. 7. Right valve, detail of ornamentation, paratype (C 28909), 900. Explanation of Plate 1. Microceratina martensi sp. nov., Christmas Island. All figured specimens are females. fig. 1. Left valve, external view,
paratype (C 28910), 250. fig. 2. Right valve, external view, paratype (C 28909), 250. fig. 3. Right valve, internal view, holotype (C 28908), 250. fig. 4. Right valve, internal view, holotype, 310. fig. 5. Left valve, dorsal view, holotype, 250. fig. 6. Right valve, detail of ornamentation and
pores, paratype (C 28909), 7600. fig. 7. Right valve, detail of ornamentation, paratype (C 28909), 900. one additional seta on the ventro-distal corner. Seventh
podomere absent. The terminal (eighth) podomere distally with
three setae and one aesthetasc. The latter, as long as the ramus,
is fused at the base with one adjoining seta and the point of their
insertion is located ventrally compared to both remaining setae. Antenna. (Fig. 3b) Four podomeres and a large exopodite
(spinneret seta) extending almost to the tips of the terminal
claws. First endopodal podomere sub-quadrangular, with two
unequal ventro-distal setae and a brush of fine long setulae
inserted on the external surface. Second endopodal podomere 52 New stygobitic ostracod species from Christmas Island Fig. 2. Microceratina martensi sp. nov., Christmas Island, female speci-
mens: (a) right valve, internal view, holotype (C 28908); (b) left valve,
internal view, paratype (C 28910). Scale: 100 µm. First walking leg. (Fig. 4a) Slender and 4-segmented. First
podomere with one long dorso-median seta, one shorter dorso-
distal knee-seta and two long ventral setae inserted in the first
half of the podomere. The edge of the joint between first and the
second podomere with a row of setulae. Second podomere
elongated, bearing one antero-distal seta which extends about to
the two third of the length of the next (third) podomere. Terminal podomere with a long, slender and regularly curved
claw; this claw slightly longer than two last podomeres
combined. Second walking leg. (Fig. 4b) Has the same chaetotaxial arma-
ment as in the preceding limb, although with longer podomeres
and a claw. Third walking leg. (Fig. 4c) 4-segmented, with much larger first
and second podomeres than those of the second walking leg. First podomere with only one ventral seta and one knee-seta. Second podomere very elongated and slender with one distal
seta. Tegumentary pigments. Not visible. Tegumentary pigments. Not visible. Ocular structures. Undeveloped. Mandible. (Fig. 3c)
Consisting
of
coxa
and
indistinctly
4-segmented palp. Coxa long and stout with one relatively long
seta. The masticatory edge of coxa well developed, armed with
five or six strong teeth, alternating with spines and followed
by at least one hirsute seta. First palp podomere with two
unequal,
internally
directed
setae
and
a
vibratory
plate
(exopodite) bearing only one very long seta. Second palp
podomere dorsally with one long seta, one peculiarly bent
ventro-distal plumose seta, and two distal setae orientated
internally. Third palp podomere in the dorsal two-thirds
with five externally directed setae (three long setae reaching
tips of the claws on the terminal podomere and two shorter
setae), medio-distally with one internally projected seta, and
ventro-distally with one long and one short setae (short seta not
seen on the left mandible). Terminal palp podomere (about a
half
of
the
penultimate
podomere)
with
three
sub-equal
claw-like setae. Type species. Microceratina quadrata Swanson, 1980 (by original
designation). Absolute lengths of the penultimate and the terminal
podomeres as well as of a claw comparable with those of the
second leg. Fig. 2. Microceratina martensi sp. nov., Christmas Island, female speci-
mens: (a) right valve, internal view, holotype (C 28908); (b) left valve,
internal view, paratype (C 28910). Scale: 100 µm. Caudal rami (furca) and posterior end of the body. (Fig. 4d)
Caudal ramus with three setae. Genital operculum ovate. Abdominal extremity conical with obtuse end and with a brush
of caudal setulae. long and narrow, bearing one seta inserted at two-thirds of the
length of the dorsal margin, two sub-equal ventro-median setae
as well as one ventro-distal seta. Terminal podomere minute, set
with two strong claws associated with externally situated thick
seta (aesthetasc). Microceratina sp. 1 Whatley & Downing, 1983. Mazzini &
Gliozzi (2000) noted that, because they have open pores, M.
punctata, M. quadratamacroreticulata and Microceratina sp. 1
should not be included in the genus Microceratina. Further
study is needed to establish the generic status of each of these
species. Microceratina costata should be assigned to the genus
Cytherura Sars (subfamily Cytherurinae G.W. Müller) owing to
the outline and ornamentation of the valves. Microceratina sp. 1 Whatley & Downing, 1983. Mazzini &
Gliozzi (2000) noted that, because they have open pores, M.
punctata, M. quadratamacroreticulata and Microceratina sp. 1
should not be included in the genus Microceratina. Further
study is needed to establish the generic status of each of these
species. Microceratina costata should be assigned to the genus
Cytherura Sars (subfamily Cytherurinae G.W. Müller) owing to
the outline and ornamentation of the valves. Males unknown Remarks. Nine species of Microceratina were recognized before
this study solely on the morphology of the valves. Five of these
species, M. pseudamfibola (Barbeito-Gonzalez, 1971), M. poligo-
nia (Colalongo & Pasini, 1980), M. quadratamicroreticulata
Yassini & Jones, 1995, M. aequabilis Herrig, 1991 and Micro-
ceratina sp. 2 Whatley & Downing, 1983, have only faint
reticulation and, therefore, can be easily distinguished from the
present species. Furthermore, M. pseudoamfibola is a larger
species (L 0.40 mm) with parallel dorsal and ventral margins; M. poligonia is also a larger species (L 0.51 mm), which, apart from
the reticulation, resembles M. martensi sp. nov.; M. quadratami-
croreticulata is a smaller species (L 0.28 mm) with nearly parallel
dorsal and ventral margins; Microceratina sp. 2 is somewhat
larger (L 0.39 mm) and has a wide ventro-lateral alar process;
and M. aequabilis is also larger (L 0.44 mm) with parallel dorsal
and ventral margins. Four other species exhibit a strongly
developed reticulation and, therefore, resemble M. martensi sp. nov. They can, however, be distinguished from the new species. M. quadrata is larger (L 0.47 mm) with parallel ventral and
dorsal margins; M. amfibola (Barbeito-Gonzalez, 1971) is also
larger (L 0.42 mm) with a strongly developed and wide ventro-
lateral alar process; M. reticulata (Bonaduce et al., 1975) is
slightly larger (L 0.39 mm) with wedge-shaped valves in lateral Maxillula. (Fig. 3d) Elongated respiratory plate, three endites
and 2-segmented palp. The former carrying at least 16 normal
plumous rays on its posterior rim and two aberrant setae
pointed downward towards the mouth. First palp podomere
bearing at least three long dorso-distal setae. Second palp
podomere with 2–3 ventro-median setae and distally with one
claw and at least one seta. Endites with unknown exact number
of distal setae. These setae being closely packed together and
lying on top of one another. 53 3. Microceratina martensi sp. nov., Christmas Island, female holotype (C 28908): (a) antennule; (b) antenna; (c) mandible; (d) maxillula. Scale
m. T. Namiotko et al T. Namiotko et al. Fig. 3. Microceratina martensi sp. nov., Christmas Island, female holotype (C 28908): (a) antennule; (b) antenna; (c) mandible; (d) maxillula. Scale:
50 µm. Microceratina sp. 1 Whatley & Downing, 1983. Mazzini &
Gliozzi (2000) noted that, because they have open pores, M. punctata, M. quadratamacroreticulata and Microceratina sp. 1
should not be included in the genus Microceratina. Males unknown Further
study is needed to establish the generic status of each of these
species. Microceratina costata should be assigned to the genus
Cytherura Sars (subfamily Cytherurinae G.W. Müller) owing to
the outline and ornamentation of the valves. view because of the strongly tapering dorsal and ventral
margins; and M. foveolata (Colalongo & Pasini, 1980) is dis-
tinctly larger (L 0.51 mm) with oblong valves and a convex
dorsal margin. Other species assigned to the genus Microceratina are not
considered by the present authors as belonging to that genus. These are M. punctata Whatley & Zhao, 1987, M. quadratama-
croreticulata Yassini & Jones, 1995, M. costata Herrig, 1991 and 54 Fig. 4. Microceratina martensi sp. nov., Christmas Island, female holotype (C 28908): (a) first walking leg; (b) second walking leg; (c) third walk
eg; (d) posterior end of the body and furca. Scale: 50 µm. New stygobitic ostracod species from Christmas Island New stygobitic ostracod species from Christmas Island New stygobitic ostracod species from Christmas Island Fig. 4. Microceratina martensi sp. nov., Christmas Island, female holotype (C 28908): (a) first walking leg; (b) sec
leg; (d) posterior end of the body and furca. Scale: 50 µm. Fig. 4. Microceratina martensi sp. nov., Christmas Island, female holotype (C 28908): (a) first walking leg; (b) second walking leg; (c) third walking
leg; (d) posterior end of the body and furca. Scale: 50 µm. PRESENT AND PAST GEOGRAPHICAL DISTRIBUTION
OF MICROCERATINA SPECIES 2 Whatley &
Downing, 1983; 10, M. martensi sp. nov. (this paper). The distributions are based on the papers in which the species were described and others
(Breman, 1975; Ciampo, 1976, 1986; Mostafawi, 1989; Peeters, 1994; Coles et al., 1996; Aiello et al., 2000; Mazzini & Gliozzi, 2000; Aiello & Barra,
2001; Arbulla et al., 2001). Circles, Recent; triangles, Quaternary; star, Pliocene; squares, Miocene; diamond, Late Cretaceous (see text for additional
information). Fig. 5. Distribution of Microceratina-species. 1, M. quadrata Swanson, 1980; 2, M. pseudoamfibola (Barbeito-Gonzalez, 1971); 3, M. amfibola
(Barbeito-Gonzalez, 1971); 4, M. poligonia (Colalongo & Pasini, 1980); 5, M. reticulata (Bonaduce, Ciampo & Masoli, 1975); 6, M. foveolata
(Colalongo & Pasini, 1980); 7, M. quadratamicroreticulata Yassini & Jones, 1995; 8, M. aequabilis Herrig, 1991; 9, Microceratina sp. 2 Whatley &
Downing, 1983; 10, M. martensi sp. nov. (this paper). The distributions are based on the papers in which the species were described and others
(Breman, 1975; Ciampo, 1976, 1986; Mostafawi, 1989; Peeters, 1994; Coles et al., 1996; Aiello et al., 2000; Mazzini & Gliozzi, 2000; Aiello & Barra,
2001; Arbulla et al., 2001). Circles, Recent; triangles, Quaternary; star, Pliocene; squares, Miocene; diamond, Late Cretaceous (see text for additional
information). interest is the recent discovery by Mazzini & Gliozzi (2000) of
M. pseudoamfibola from a submarine cave in the Palinuro Bay in
the Tyrrhenian Sea, SW Italy. Finally, M. reticulata was sam-
pled in the Bay of Naples and in the Adriatic Sea (Bonaduce
et al., 1975). Neogene representative of the genus. Microceratina poligonia
has the widest known stratigraphical and geographical range of
the genus and flourished for a very long time over a wide area. This species also occurred in Sicily throughout the Pliocene
(Aiello & Barra, 2001) and Pleistocene (Aiello et al., 2000). In
the Mediterranean it is also known from the Early Pleistocene of
Calabria, SW Italy (Colalongo & Pasini, 1980) and from Recent
sediments in the Adriatic Sea (as ?Eucytherura sp. in Breman,
1975). Finally, M. poligonia was reported from two sites in the
NE Atlantic: late Pleistocene deposits SW of Ireland and
(Sub-)Recent sediments west of Scotland (Coles et al., 1996). In
addition to M. poligonia, three other species of the genus have
been recorded from the Mediterranean Early Pleistocene: M. foveolata from Calabria, SW Italy (Colalongo & Pasini, 1980),
M. pseudoamfibola from Campania, SW Italy (Ciampo, 1976)
and M. PRESENT AND PAST GEOGRAPHICAL DISTRIBUTION
OF MICROCERATINA SPECIES temperate fauna (Kaufmann, 1973). There are no records of the
genus from Palaeogene deposits. One of the Neogene repre-
sentatives of the genus Microceratina (M. sp. 2 Whatley &
Downing) is known from Middle Miocene deposits in SE
Australia (Whatley & Downing, 1983). Two Recent species,
M. quadrata and M. quadratamicroreticulata, occur today in
coastal sediments offSE Australia (Yassini & Jones, 1995) and
southern New Zealand (Swanson, 1980). However, the most
detailed data on the geographical and eco-stratigraphical distri-
bution of Neogene to modern species of Microceratina are from
the Mediterranean Basin. The oldest specimens from that area,
from the Miocene of southern (Sicily) and northern (Piedmont)
Italy, are M. poligonia (Ciampo, 1986), which is the other temperate fauna (Kaufmann, 1973). There are no records of the
genus from Palaeogene deposits. One of the Neogene repre-
sentatives of the genus Microceratina (M. sp. 2 Whatley &
Downing) is known from Middle Miocene deposits in SE
Australia (Whatley & Downing, 1983). Two Recent species,
M. quadrata and M. quadratamicroreticulata, occur today in
coastal sediments offSE Australia (Yassini & Jones, 1995) and
southern New Zealand (Swanson, 1980). However, the most
detailed data on the geographical and eco-stratigraphical distri-
bution of Neogene to modern species of Microceratina are from
the Mediterranean Basin. The oldest specimens from that area,
from the Miocene of southern (Sicily) and northern (Piedmont)
Italy, are M. poligonia (Ciampo, 1986), which is the other Figure 5 illustrates the known distribution of fossil and Recent
Microceratina species. The oldest known species of the genus, M. aequabilis Herrig,
1991, was found in Upper Maastrichtian erratic boulders on the
Island of Rügen (Germany). The boulders probably originated
in the middle to eastern Baltic Sea area where the source rocks
were deposited during the Cretaceous in the relatively shallow
seas of the Danish-Polish Furrow (Herrig, 1992). This area,
situated at that time in the North European Province belonging
to the North-temperate Realm, was close to the boundary of the
Tethyan Realm and predominantly comprised warm to mid- 55 T. Namiotko et al. Fig. 5. Distribution of Microceratina-species. 1, M. quadrata Swanson, 1980; 2, M. pseudoamfibola (Barbeito-Gonzalez, 1971); 3, M. amfibola
(Barbeito-Gonzalez, 1971); 4, M. poligonia (Colalongo & Pasini, 1980); 5, M. reticulata (Bonaduce, Ciampo & Masoli, 1975); 6, M. foveolata
(Colalongo & Pasini, 1980); 7, M. quadratamicroreticulata Yassini & Jones, 1995; 8, M. aequabilis Herrig, 1991; 9, Microceratina sp. ORIGIN AND EVOLUTION OF MICROCERATINA
MARTENSI SP. NOV. ORIGIN AND EVOLUTION OF MICROCERATINA
MARTENSI SP. NOV. There is considerable evidence (Larwood & Whatley, 1993;
Larwood et al., 1996; Allmon & Ross, 2001) that shallow
benthic habitats of oceanic islands, including seamounts, were
passively colonized from remote sources in the Tertiary to the
Recent by a rich and diverse ostracod fauna. Even marine
interstitial ostracods could have arrived by passive drift to
volcanic islands like the Galapagos (Danielopol & Bonaduce,
1990b). Therefore, the colonization of Christmas Island by a
Microceratina species could have occurred from the Tertiary to
the Recent, with cave colonization allowed by the karstification
of the limestone during the Neogene. Most of the accompanying
fauna sampled in the karstic system where M. martensi sp. nov. was found comprises epigean species, which could have reached
the island by passive dispersal at any time, even recently. However, Procarididae appear to be restricted to anchialine
caves where they are sympatric with one or more species of atyid
shrimps. These co-occurrences of apparently ancient caridean
families have been proffered as support for the contention that
crevicular habitats have served as faunal refuges (Kensley &
Williams, 1986). The occurrence of procaridid, alpheid, hippo-
lytid and atyid shrimps in the same anchialine system on
Christmas Island is similar to that found on Bermuda (Hart &
Manning, 1981), renowned for its diversity of anchialine fauna
(Iliffe, 1994). This, therefore, could be an argument for an old
colonization of the subterranean habitats of this island by
crustaceans, including also the Microceratina species described
here. Because Recent Microceratina species exist in open marine
habitats, the new species is not a relic (sensu Humphreys, 2000),
which survived by using the Christmas Island karst system as a
refugium, the argument used by Kensley & Williams (1986) for
the occurrence of decapod procaridids from other anchialine
systems. Other seamount islands have endemic ostracod species
belonging to other Eucytherurinae groups, as, for example
Eucytherura on Ita Mai Tai and Horizon guyots in the Pacific This new ostracod species is a stygobiont and displays the
typical troglomorphic traits known in other interstitial and/or
cave-dwelling cytheroids, lacking ocular structures in possessing
no eyes or ocular tubercles on the valves (cf. Hartmann, 1973;
Danielopol & Hartmann, 1985). In contrast, shallow water
epibenthic species of the related genus Eucytherura G.W. PRESENT AND PAST GEOGRAPHICAL DISTRIBUTION
OF MICROCERATINA SPECIES reticulata from Rhodos Island, Greece (Mostafawi,
1989). Microceratina still occurs in the Mediterranean. In
addition to M. poligonia, three other species were sampled from
Recent Mediterranean sediments. Microceratina amfibola was
found at sites close to Naxos Island in the Aegean Sea (Barbeito-
Gonzalez, 1971) and from the Adriatic Sea (Bonaduce et al.,
1975). Microceratina pseudoamfibola occurs in coastal sediments
offCorsica (Peeters, 1994), NE Sardinia (Arbulla et al., 2001)
and Naxos Island (Barbeito-Gonzalez, 1971). Of particular Prior to this paper, the geographical distribution of Microcer-
atina species (Fig. 5) represented a disjunct distribution pattern,
from Cretaceous to Recent, in warm to temperate zones on both
sides of the tropics but not in tropical environments. The
discovery of the new Microceratina species in the Indian Ocean
is remarkable, therefore, being the first record of the genus in the
tropics. There may be different explanations for this. First, other
Microceratina species may occur in tropical seas and oceans but
are yet unfound. It is known from both palaeontological studies
and from studies on Recent ostracod shells, that very small
species (smaller than 0.45 mm and termed ‘micro-ostracods’ by
Herrig (1998)), are absent from or are poorly represented in the
fossil record, being either overlooked, unrecognized or consid-
ered as juveniles. Recent publications, however, show that
micro-ostracod fossil associations are occasionally preserved
(Herrig, 1988, 1991, 1992, 1993, 1994, 1998; Pokorny, 1989;
Danielopol et al., 1991; Danielopol & Wouters, 1992; Herrig
et al., 1997; Swain, 2000). For instance, Herrig (1998) described
new micro-ostracods from Maastrichtian erratic boulders of the 56 New stygobitic ostracod species from Christmas Island Baltic region. He observed remarkable morphological simi-
larities between Late Cretaceous ostracods and Quaternary and
Recent ostracods from the western Indo-Pacific Ocean in a
number of genera, such as Puncia Hornibrook, Manawa Horni-
brook, Pelecocythere Athersuch, Saida Hornibrook, Jankeij-
cythere McKenzie, Heinia Bold, Hanaicythere Yajima and
Miracythere Hornibrook, as well as in Microceratina. He
concluded that “the occurrence of morphologically similar
species in the boreal Upper Cretaceous of the Danish–Polish
Furrow and in Tertiary and Quaternary sediments of the
Indo-Pacific realm confirms the hypothesis of a close biogeo-
graphic correspondence, in spite of the huge spatial and tem-
poral distances” (Herrig, 1998, p. 2). that in which M. martensi sp. nov. was collected because in
both systems the bottom is covered by rich organic matter,
respectively plant detritus and plant roots. PRESENT AND PAST GEOGRAPHICAL DISTRIBUTION
OF MICROCERATINA SPECIES The surface ornamentation and the degree of calcification of
the carapaces of Microceratina show considerable variation. This ranges from the weakly ornate and thinly calcified valves of
M. pseudoamfibola and M. martensi sp. nov., to the more
strongly ornate valves of M. amfibola and Microceratina sp. 2
Whatley & Downing. None of the known Microceratina species
have ocular tubercules on the valves, possibly owing to the
poor development of the inner ocular structures as seen in
M. martensi sp. nov. M. martensi sp. nov. has an extremely small carapace with
elongate and barely ornate valves, similar to those typical of
marine interstitial ostracods (Hartmann, 1973). This mor-
phology suggests that M. martensi sp. nov. may also live outside
the cave in the sublittoral interstitial sandy habitats of Christmas
Island or even other oceanic islands. Tuberoloxoconcha tuberosa
Hartmann occurs both in marine caves and in sublittoral
interstitial habitats (Danielopol & Bonaduce, 1990a) close to the
locations where M. pseudoamfibola has been found (Mazzini &
Gliozzi, 2000). This suggests that the colonization of the anchia-
line caves in both southern Italy and on Christmas Island
may have been by ostracods predisposed to inhabit aphotic
subterranean habitats. The Danish–Polish Furrow was part of the Tethyan Ocean
during the Cretaceous and Microceratina can, therefore, be
called a Tethyan genus with species dispersed along the northern
margin of the Tethys. This is another example of the importance
of the Tethyan corridor for ostracod distribution mainly during
the Cretaceous and the Palaeogene, as emphasized by McKenzie
(1986). Possibly, the Microceratina lineage developed a disjunct
distribution later, but judging from the fossil evidence presented
here, the distribution of the genus was already disjunct in the
Miocene. REFERENCES Adams, C.G. & Belford, D.J. 1974. Foraminiferal biostratigraphy of the
Oligocene–Miocene limestones of Christmas Island (Indian Ocean). Palaeontology, 17: 457–506. Falkland, A. 1986. Christmas Island (Indian Ocean): water resources
study in relation to proposed development at Waterfall. Department of
Territories, Hydrology and Water Resources Unit. Report., 86/19: 123
pp. Aiello, G. & Barra, D. 2001. Pliocene ostracod assemblages at the M Pl
3-M Pl 4 boundary in the Cape Rossello borehole (Agrigento, Sicily). Bolletino della Societa Paleontologica Italiana, 40: 97–107. Ford,
D.C. &
Williams,
P.W. 1989. Karst
Geomorphology
and
Hydrology. Unwin Hyman, London, 601 pp. Aiello, G., Barra, D. & Bonaduce, G. 2000. Systematics and biostratig-
raphy of the Ostracoda of the Plio-Pleistocene Monte S. Nicola
section (Gela, Sicily). Bolletino della Societa Paleontologica Italiana,
39: 83–112. Grimes, K.G. 2001. Karst features of Christmas Island (Indian Ocean). Helictite, 37: 41–58. Hart, C.W. & Manning, R.B. 1981. The cavernicolous caridean shrimps
of Bermuda (Alpheidae, Hippolytidae, and Atyidae). Journal of
Crustacean Biology, 1: 441–456. Allmon, W.A. & Ross, R.M. 2001. Nutrients and evolution in the
marine realm. In: Allmon, W.A. & Bottjier, D.J. (Eds), Evolutionary
Paleoecology. The Ecological context of Macroevolutionary Change. Columbia University Press, New York, 105–148. Hartmann, G. 1973. Zum gegenwärtigen Stand der Erforschung der
Ostracoden
interstitieller
Systeme. Annales
de
Spéléologie,
28:
417–426. Arbulla, D., Pugliese, N. & Russo, A. 2001. Ostracodi del Golfo Saline
(Sardegna nord-orientale). Studi Trentini di Scienze Naturali, Acta
Geologica, 77: 25–35. Hartmann, G. 1989. Ostracoda. In: Bronns, H.G. (Ed.), Klassen und
Ordnungen des Tierreichs, 5, Sect 1. G. Fischer Verlag, Jena,
787–1067. Barbeito-Gonzalez, P.J. 1971. Die Ostracoden des Küstenbereichs von
Naxos
(Griechenland)
und
ihre
Lebensbereiche. Mitteilungen
aus dem Hamburgischen Zoologischen Museum und Institut, 67:
255–326. Herrig, E. 1988. Punciidae Ostrakoden aus der höheren Oberkreide der
Mittleren Ostsee. Geschiebekunde Aktuell, 4: 33–37. Herrig, E. 1991. Neue Ostrakoden aus verkieselten Kalksteinen der
höhern
Oberkreide
(Ober-Maastricht)
der
Dänisch–Polnischen
Furche/Ostsee. II. Geologisches Jahrbuch, A, 128: 99–115. Barrett, F.J. 1985. Christmas Island water resources summary report. Internal Report, Christmas Island Phosphate Company, 20/2/1985,
Christmas Island, 39 pp. (unpublished). Herrig, E. 1992. Neue Ostrakoden aus verkieselten Kalksteine der
höheren
Oberkreide
(Ober-Maastricht)
der
Dänisch–Polnischen
Furche/Ostsee. I. Zeitschrift für Geologische Wissenschaften, 20:
27–49. Barrie, J. 1967. The geology of Christmas Island. Bureau of Mineral
Resources Record, Canberra, 1967/37, 45 pp. (unpublished). Bonaduce, G., Ciampo, G. & Masoli, M. 1975. Distribution of Ostra-
coda in the Adriatic Sea. ORIGIN AND EVOLUTION OF MICROCERATINA
MARTENSI SP. NOV. Müller
have strongly developed ocular structures in which the pig-
mented eyes project into well-developed ocular fossae on the
carapace and which are visible from the outside as ocular
tubercles (see the illustrations of G.W. Müller, 1894, table 20,
figs 19 and 20 for E. gibbera and E. alata). Note, however,
that bathyal and abyssal species of Eucytherura also lack
ocular structures. In addition, the antennule and antenna of
M. martensi sp. nov. display well-developed distal aesthetascs
(Fig. 3a, b). In contrast, instead of aesthetascs only ‘simple’
setae occur in the other Eucytherurinae from open marine
habitats for which appendages have been described. For
example, species of the genera Eucytherura (e.g. E. gibbera
G.W. Müller) and Xylocythere (e.g. X. pointillissima Maddocks
& Steineck). The ecological distribution and the morphological details of
the carapace of the Microceratina lineage suggest that this is a
very flexible group evolutionarily. Species have adapted to a
wide range of habitats such as sandy sediments at 20–50 m depth
(M. quadrata from Port Pegasus Stewart Island: Swanson,
1980), sandy/silty sediments at depths no greater than 50–60 m
(M. amfibola and M. pseudoamfibola in the Mediterranean:
Barbeito-Gonzalez, 1971; Bonaduce et al., 1975), frigid con-
ditions of the deep sea at depths of 650–750 m (M. poligonia in
the North Atlantic from Recent and Quaternary facies: Coles et
al., 1996) and anchialine caves in Southern Italy and Christmas
Island. In Southern Italy M. pseudoamfibola is found in an
anchialine cave habitat (Mazzini & Gliozzi, 2000) comparable to 57 T. Namiotko et al. Ocean (Larwood & Whatley, 1993; Boomer & Whatley, 1996;
Larwood et al., 1996). Microceratina martensi sp. nov. appears
to be endemic to Christmas Island. Chappell, J. & Thom, B.G. 1977. Sea levels and coasts. In: Allen, J.,
Golson, J. & Jones, R. (Eds), Sunda and Sakul: Prehistoric studies in
southeast Asia, Melanesia & Australia. Academic Press, London,
275–291. Ocean (Larwood & Whatley, 1993; Boomer & Whatley, 1996;
Larwood et al., 1996). Microceratina martensi sp. nov. appears
to be endemic to Christmas Island. Ciampo, G. 1976. Ostracodi pleistocenici di Cala Bianca (Marina di
Camerota, Salerno). Bolletino della Societa Paleontologica Italiana,
15: 3–23. Manuscript received 30 January 2003
Manuscript accepted 23 May 2003 Danielopol, D.L., Baltanás, A. & Humphreys, W.F. 2000. Danielopolina
kornickeri sp. n. (Ostracoda, Thaumatocypridoidea) from a western
Australian anchialine cave: morphology and evolution. Zoologica
Scripta, 29: 1–16. Manuscript received 30 January 2003 Manuscript received 30 January 2003
Manuscript accepted 23 May 2003 ACKNOWLEDGEMENTS Ciampo, G. 1986. Ostracodi del limite Tortoniano/Messiniano in alcune
sezione italiane. Bolletino della Societa Paleontologica Italiana, 24:
29–110. TN was supported by a grant of the University of Gdan¯sk (no
BW/1030-5-0054-2) as well as by the Scientific Exchange
Programme of the Polish Academy of Sciences (PAN) and the
Austrian Academy of Sciences (ÖAW). We thank I. Mazzini
(Forschungsinstitut Senckenberg, Frankfurt/Main) for useful
information pertaining to Microceratina pseudoamfibola in the
Italian cave. For examining various taxa we thank R. Wilson
(Melbourne Museum: Annelida), G. A. Boxshall (The Natural
History Museum, London: Copepoda Harpacticoida and Cyclo-
poida), G. L. Pesce (University di l’Aquila, Italy: Copepoda
Calanoida),
J. Lowry
(Australian
Museum:
Amphipoda),
J. Short (Queensland Museum: Alpheidae, Procarididae, Hippo-
lytidae), S. C. Choy (Water Resources Queensland: Atyidae,
Palaemonidae). S. Eberhard’s (Caveworks, Western Australia)
skill in the water and observations greatly enhanced the work on
Christmas Island. The field work on Christmas Island was
funded (WFH) under contract to Parks Australia North (now
Environment Australia). Colalongo, M.L. & Pasini, G. 1980. La ostracofauna plio-pleistocenica
della sezione Vrica in Calabria (con considerazioni sul limite
Neogene/Quaternario). Bolletino della Societa Paleontologica Italiana,
19: 44–126. Coles, G.P., Ainsworth, N.R., Whatley, R.C. & Jones, R.W. 1996. Foraminifera and Ostracoda from Quaternary carbonate mounds
associated with gas seepage in the Porcupine Basin, offshore Western
Ireland. Revista Española de Micropaleontologia, 28: 113–151. Danielopol, D.L. & Bonaduce, G. 1990a. The colonisation of subsurface
habitats by the Loxoconchidae Sars and the Psammocytheridae Klie. In: Whatley, R. & Maybury, C. (Eds), Ostracoda and Global Events. Chapman & Hall, London, 437–458. Danielopol, D.L. & Bonaduce, G. 1990b. The origin and distribution of
the interstitial Ostracoda of the species group Xestoleberis arcturi
Triebel (Crustacea). Courier Forschungsinstitut Senckenberg, 123:
69–86. Danielopol, D.L. & Hartmann, G. 1985. Ostracoda. In: Botosaneanu, L. (Ed.), Stygofauna Mundi. Brill, Leiden, 265–294. Danielopol, D.L. & Wouters, K. 1992. Evolutionary (palaeo) biology of
marine interstitial ostracods. Geobios, 25: 207–211. Danielopol, D.L., Piller, W.E. & Huber, T. 1991. Pseudolimnocythere
hainburgensis n. sp. (Ostracoda, Loxoconchidae) aus dem Miozän
(Badenium) des Wiener Beckens. Neues Jahrbuch für Geologie und
Paläontologie Monatshefte, 8: 458–469. Manuscript received 30 January 2003 New stygobitic ostracod species from Christmas Island Mostafawi, N. 1989. Limnische und marine Ostracoden aus dem Neo-
gen der Insel Rhodos (Griechenland). Courier Forschungs-Institut
Senckenberg, 113: 117–157. Herrig, E., Frenzel, P. & Reich, M. 1997. Zur Mikrofauna einer
Ober-Campan-Scholle
von
der
Halbinsel
Wittow
(NW
Rügen/Ostsee). Freiberger Forschungsheft, C468: 129–169. g
)
g
g
f
Humphreys, W.F. 1999. Physico-chemical profile and energy fixation in
Bundera Sinkhole, an anchialine remiped habitat in north-western
Australia. Journal of the Royal Society of Western Australia, 82:
89–98. Mylroie, J.E., Carew, J.L., Sealey, N.E. & Mylroie, J.T. 1991. Cave
development on New Providence Island and Long Island, Bahamas. Cave Science, 18: 139–151. Müller, G.W. 1894. Die Ostracoden des Golfes von Neapel und der
angrenzenden Meeresabschnitte. Fauna und Flora des Golfes von
Neapel, 21: 1–404. Humphreys, W.F. 2000. Relict faunas and their derivation. In: Wilkens,
H., Culver, D.C. & Humphreys, W.F. (Eds), Ecosystems of the World. Subterranean Ecosystems, 30. Elsevier, Amsterdam, 417–433. Peeters, M. 1994. Een systematische en actuopaleontologische studie van
ostracoden in sedimenten van de Baai van Calvi (Corsica). Unpub-
lished licentiate thesis. University of Leuven, 104 pp. Humphreys, W.F. & Eberhard, S.M. 1998. Assessment of the ecological
values and management options for cave use on Christmas Island:
Project 97/002. Unpublished report prepared for Parks Australia
North, 134 pp. Pettifer, G.R. & Polak, E.J. 1979. Christmas Island (Indian Ocean),
geophysical survey for groundwater, 1976. Bureau of Mineral
Resources, Geology and Geophysics Record 1979/33, Canberra, 47
pp. (unpublished). pp
Humphreys, W.F. & Eberhard, S.M. 2001. Subterranean fauna of
Christmas Island, Indian Ocean. Helictite, 37: 59–74. Iliffe, T.M. 1994. Bermuda. In: Juberthie, C. & Decu, V. (Eds),
Encyclopaedia Biospeologica, 3. Laboratoire Souterrain du CNRS,
Moulis, France, 417–424. Pokorny, V. 1989. Pussella and Saipanetta (Ostracoda, Crustacea) in the
Lower Turonian of Bohemia, Czechoslovakia. Casopis pro Miner-
alogii a Geologii, 34: 225–237. Iliffe, T.M. 2000. Anchialine cave ecology. In: Wilkens, H., Culver, D.C. & Humphreys, W.F. (Eds), Ecosystems of the World. Subterranean
Ecosystems, 30. Elsevier, Amsterdam, 59–76. Polak, E.J. 1976. Christmas Island (Indian Ocean), geophysical survey
for groundwater, 1973. Bureau of Mineral Resources, Geology and
Geophysics Record 1976/100, Canberra, 37 pp. (unpublished). Kaufmann, E.G. 1973. Cretaceous Bivalvia. In: Hallam, A. (Ed.), Atlas
of Biogeography. Elsevier Scientific Publishing Company, Amsterdam,
353–383. Sket, B. 1996. The Ecology of anchialine caves. Trends in Ecology and
Evolution, 11: 221–255. Swain, F.M. 2000. Micro-Ostracoda from the Eocene of North Caro-
lina:
a
suggested
interstitial
faunule. Revista
Española
de
Micropaleontologia, 32: 411–420. REFERENCES Pubblicazione della Stazione Zoologica di
Napoli, 40: 1–304. Herrig,
E. 1993. Gleiche
Ostracodenarten
aus
Oberkreide
und
Tertiär/Quartär. Zitteliana, 20: 349–359. Boomer, I.D. & Whatley, R.C. 1996. Ostracod endemism on mid-Pacific
guyots from ODP legs 143 and 144. In: Moguilevsky, A. & Whatley,
R. (Eds), Microfossils and Oceanic Environments. University of Wales
Press, Aberystwyth, 403–412. Herrig, E. 1994. Polycopidae (Crustacea, Ostracoda) aus der borealen
Oberkreide des mittleren und südlichen Ostseeraumes. Paläontolo-
gisches Zeitschrift, 68: 351–359. f
Herrig,
E. 1998. Mikroostrakoden
(Cytheracea)
des
Ober-
Maastrichtium aus dem Ostsee-Raum (Dänisch–Polnische Furche). Courier Forschungsinstitut Senckenberg, 204: 1–29. y
y
Breman, E. 1975. Distribution of ostracodes in the bottom sediments of the
Adriatic Sea. PhD thesis. Vrije Universiteit Amsterdam, 165 pp. 58 New stygobitic ostracod species from Christmas Island Kempf, E.K. 1986. Index and bibliography of marine Ostracoda. 1,
Index A. Sonderveröffentlichungen des Geologischen Instituts der Uni-
versität zu Köln, 50: 1–762. Swanson, K.M. 1980. Five new species of Ostracoda from Port Pegasus,
Stewart Island. New Zealand Journal of Marine and Freshwater
Research, 14: 205–211. Kensley, B. & Williams, D. 1986. New shrimps (families Procarididae
and Atyidae) from a submerged lava tube in Hawaii. Journal of
Crustacean Biology, 6: 417–437. Vogel, P.N., Mylroie, J.E. & Carew, J.L. 1990. Limestone petrology and
cave morphology on San Salavador Island, Bahamas. Cave Science,
17: 19–30. Larwood, J. & Whatley, R.C. 1993. Tertiary to Recent evolution of
Ostracoda in isolation on seamounts. In: McKenzie, K.G. & Jones,
P.J. (Eds), Ostracoda and Life Sciences. Balkema, Rotterdam,
531–549. Watts, C.H.S. & Humphreys, W.F. 2000. Six new species of Nirridessus
and Tjirtudessus (Dytiscidae; Coleoptera) from underground waters in
Australia. Records of the South Australian Museum, 33: 127–144. Larwood, J., Whatley, R.C. & Boomer, I.D. 1996. Ostracod evolution
on seamounts: evidence from Horizon Guyot, Central Pacific Ocean
(DSDP sites 44 & 171) and the Ninetyeast Ridge, East Indian Ocean
(DSDP Site 214). In: Moguilevsky, A. & Whatley, R.C. (Eds),
Microfossils and Oceanic Environments. University of Wales Press,
Aberystwyth, 385–401. Whatley, R. & Downing, S. 1983. Middle Miocene Ostracoda from
Victoria, Australia. Revista Española de Micropaleontologia, 15:
347–406. Whatley, R. & Zhao, Q. 1987. Recent Ostracoda of the Malacca Straits. Part I. Revista Española de Micropaleontologia, 19: 327–366. y
y
Maddocks, R.F. & Steineck, P.L. 1987. Ostracoda from experimental
wood-island habitats in the deep sea. Micropaleontology, 33: 318–355. Woodroffe, C.D. 1988. Vertical movement of isolated oceanic islands at
plate margins: evidence from emergent reefs in Tonga (Pacific Ocean),
Cayman Islands (Caribbean Sea) and Christmas Island (Indian
Ocean). Zeitschrift für Geomorphologie, Suppl., 69: 17–39. Mazzini, I. & Gliozzi, E. 2000. Occurrence of fossil and Recent
Microceratina Swanson 1980 (Ostracoda, Eucytherurinae) in the
Mediterranean. Micropaleontology, 46: 143–152. Yassini, I. & Jones, B.G. 1995. Foraminiferida and Ostracoda from
estuarine and shelf environments on the southeastern coast of Australia. University of Wollongong Press, Wollongong, 485 pp. p
y
McKenzie, K.G. 1986. Ostracoda: new aspects of their biogeography. In: Gore, R.H. & Heck, K.L. (Eds), Crustacean biogeography. Crus-
tacean Issues, 4. Balkema, Rotterdam, 257–277. 59 59
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General practitioners' views on reattribution for patients with medically unexplained symptoms: a questionnaire and qualitative study
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BMC Family Practice Open Access BioMed Central BioMed Central BioMed Central Open Ac
Research article
General practitioners' views on reattribution for patients with
medically unexplained symptoms: a questionnaire and qualitative
study
Christopher Dowrick1, Linda Gask*2, John G Hughes1, Huw Charles-Jones3,
Judith A Hogg4, Sarah Peters5, Peter Salmon6, Anne R Rogers2 and
Richard K Morriss7 Address: 1Division of Primary Care, School of Population, Community and Behavioural Sciences, University of Liverpool, Liverpool L69 3GB, UK,
2National Primary Care Research and Development Centre, 5th Floor Williamson Building, University of Manchester, Oxford Road, Manchester
M13 9PL, UK, 3The Lache Health Centre, Hawthorn Road, Chester, CH4 8HX, UK, 4Division of Psychiatry, School of Population, Community and
Behavioural Sciences, University of Liverpool, Liverpool L69 3GB, UK, 5Division of Psychology, School of Psychological Sciences, University of
Manchester, Manchester M13 9PL, UK, 6Division of Clinical Psychology, School of Population, Community and Behavioural Sciences, University
of Liverpool, Liverpool L69 3GB, UK and 7Division of Psychiatry, School of Community Health Sciences, University of Nottingham, South Block,
A Floor, Queen's Medical School, Nottingham, NG7 2UH, UK Email: Christopher Dowrick - cfd@liverpool.ac.uk; Linda Gask* - Linda.Gask@manchester.ac.uk; John G Hughes - jghughes@liv.ac.uk;
Huw Charles-Jones - huw.charlesjones@nhs.net; Judith A Hogg - Judith.hogg@ntlworld.com; Sarah Peters - sarah.peters@manchester.ac.uk;
Peter Salmon - psalmon@liv.ac.uk; Anne R Rogers - anne.rogers@manchester.ac.uk; Richard K Morriss - richard.morris@nottingham.ac.uk
* Corresponding author * Corresponding author Received: 17 April 2008
Accepted: 19 August 2008 This article is available from: http://www.biomedcentra This article is available from: http://www.biomedcentral.com/1471-2296/9/46 © 2008 Dowrick et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ;
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Early studies suggested reattribution had promise as a
simple and effective intervention that GPs could employ Early studies suggested reattribution had promise as a
simple and effective intervention that GPs could employ simple and effective intervention that GPs could employ
Content of the Reattribution Intervention
Figure 1
Content of the Reattribution Intervention. ȱ
Stage
Content
Feeling understood
Elicit
physical
symptoms,
psychosocial
problems, mood state, beliefs held by patient
about
their
problem,
relevant
physical
examination and investigations
Broadening the agenda
Summarise
physical
and
psychosocial
findings. Negotiate these findings with
patient
Making the link
Give explanation relating physical symptom
to psychosocial problems of lifestyle because
of link in time or physiology
Negotiating further treatment
Arrange follow up or treatment of symptoms,
psychosocial problems or mental disorder Abstract Background: The successful introduction of new methods for managing medically unexplained
symptoms in primary care is dependent to a large degree on the attitudes, experiences and
expectations of practitioners. As part of an exploratory randomised controlled trial of
reattribution training, we sought the views of participating practitioners on patients with medically
unexplained symptoms, and on the value of and barriers to the implementation of reattribution in
practice. Methods: A nested attitudinal survey and qualitative study in sixteen primary care teams in north-
west England. All practitioners participating in the trial (n = 74) were invited to complete a
structured survey. Semi-structured interviews were undertaken with a purposive sub-sample of
survey respondents, using a structured topic guide. Interview transcripts were used to identify key
issues, concepts and themes, which were grouped to construct a conceptual framework: this
framework was applied systematically to the data. Results: Seventy (95%) of study participants responded to the survey. Survey respondents often
found it stressful to work with patients with medically unexplained symptoms, though those who
had received reattribution training were more optimistic about their ability to help them. Interview
participants trained in reattribution (n = 12) reported that reattribution increased their confidence
to practice in a difficult area, with heightened awareness, altered perceptions of these patients,
improved opportunities for team-building and transferable skills. However general practitioners
also reported potential barriers to the implementation of reattribution in routine clinical practice,
at the level of the patient, the doctor, the consultation, diagnosis and the healthcare context. Page 1 of 10
(page number not for citation purposes) BMC Family Practice 2008, 9:46 http://www.biomedcentral.com/1471-2296/9/46 http://www.biomedcentral.com/1471-2296/9/46 Conclusion: Reattribution training increases practitioners' sense of competence in managing
patients with medically unexplained symptoms. However, barriers to its implementation are
considerable, and frequently lie outside the control of a group of practitioners generally
sympathetic to patients with medically unexplained symptoms and the purpose of reattribution. These findings add further to the evidence of the difficulty of implementing reattribution in routine
general practice. Background on a routine basis for patients with MUS [11-14]. How-
ever the results of recent randomised trials indicate that,
whilst the techniques of reattribution can successfully be
taught to GPs, and do have a measurable impact on prac-
titioner behaviour in consultations about MUS, it is more
difficult to identify tangible or lasting benefits in terms of
improved outcomes for patients [15-17]. g
Approximately 20% of patients present physical symp-
toms in primary care which general practitioners (GPs)
are unable to explain by physical disease [1,2]. These
patients frequently receive extensive investigation, referral
and treatment for medically unexplained symptoms
(MUS). However such interventions are often ineffective
[3-6] and these clinical encounters can lead to dissatisfac-
tion on the part of both doctors and patients [7]. For
patients it may create a frustrating dependence on primary
care consultations which generates ambivalence, aliena-
tion and unhappiness with medical contact [8]. The reasons for the apparently limited efficacy of reattri-
bution in routine clinical practice are complex. They
include: the impact of contextual factors within the organ-
isation and delivery of primary care [9]; and the expecta-
tions of patients with MUS about the type of care they
should receive from GPs. Patients may have concerns that
their needs for treatment, explanation and support will
not adequately be met by doctors who appear to be look-
ing for psychosocial rather than medical explanations for
their symptoms [18]. Reattribution is a structured intervention, designed to pro-
vide a simple explanation of the mechanism of a patient's
MUS, through negotiation and other features of patient-
centred communication, and to be delivered during rou-
tine consultations [9]. It has four stages: enabling the
patient to feel understood; broadening the agenda beyond
physical symptoms; making the link with psychosocial
issues; and negotiating further treatment [10] (See Figure
1). The attitudes, experiences and expectations of GPs them-
selves are also important, since they are essential to the
successful implementation of any new method for man-
aging MUS in primary care. It is therefore necessary to
ascertain what GPs value about reattribution as an inter-
vention for MUS, and the feasibility of implementing it in
everyday clinical practice. In order to explore these issues,
we conducted a questionnaire survey and undertook qual-
itative interviews with GPs who were taking part in an
exploratory RCT of reattribution training in north-west
England [10]. Page 2 of 10
(page number not for citation purposes) http://www.biomedcentral.com/1471-2296/9/46 http://www.biomedcentral.com/1471-2296/9/46 BMC Family Practice 2008, 9:46 of work at a time of their choosing. Respondents were
remunerated for taking part. of work at a time of their choosing. Respondents were
remunerated for taking part. included some inner city areas. The GPs were mostly aged
35 to 50 (n = 45, 60%), with 10 (13%) under the age of
35 years and 19 (25%) over the age of 50 years. Thirty-
eight (51%) were male. There were no differences
between the training groups in terms of GP or practice
characteristics. included some inner city areas. The GPs were mostly aged
35 to 50 (n = 45, 60%), with 10 (13%) under the age of
35 years and 19 (25%) over the age of 50 years. Thirty-
eight (51%) were male. There were no differences
between the training groups in terms of GP or practice
characteristics. Interviews were audio-taped and transcribed verbatim. Transcripts were read and re-read by JGH, CD and SP to
familiarize and immerse the researchers in the data. Pat-
terns and themes within the transcripts were identified
and notes made within the margins of transcripts. Follow-
ing principles of grounded theory research, meetings were
held with the whole research team to discuss and identify
key issues, concepts and themes arising from the data, and
to group them thematically to construct a conceptual
framework [19]. The thematic framework was applied sys-
tematically to the data. When devising the conceptual
framework the research team were mindful of the aims of
the research. All transcripts were indexed by JGH and then
collated together within each code. The range and dimen-
sions of each category and sub-category were identified. Conceptual connectors were sought to understand how
categories and subcategories were linked at the level of
properties and dimensions. All 74 practitioners were invited to participate in an attitu-
dinal survey. Practitioners who had received reattribution
training (RT) were asked for their views on: patients with
persistent MUS (three statements); managing patients
with MUS (four statements); the process of RT (five state-
ments); and putting RT into practice (six statements). Practitioners who had not received RT were given only the
first two sets of statements. Ten statements were framed
negatively, and seven positively. Responses were in the
form of a 5 point Likert scale. Attitudinal Survey Seventy (95%) of the 74 practitioners in the sample
responded to the survey. Of these, half had received RT. The attitudinal statements, and range of responses, are
presented in Table 1. Respondents tended not to enjoy consultations with
patients with persistent MUS. Although most did not
think these patients took up too much of their time, a sub-
stantial majority reported that they often caused them
considerable stress. They thought these patients are worth
trying to help, and that they present interesting diagnostic
and therapeutic challenges. However, most respondents
reported that they often do not know how to help these
patients. Twenty four interviews were conducted, by MT and JGH,
between August 2005 and May 2006, up to 31 months
after practitioners were recruited into the study. Sixteen
interviewees were women. Three were aged under 35, 14
were between 35 and 50, and seven were over 50. Nine
worked in inner city practices, 14 in suburban practices,
and one was from a rural practice. They had recruited
between 0 and 11 patients to the trial (mean 3.3; median
3). Twelve were in the training arm of the trial and 12 were
in the control arm. The selected sample represented the
full range of responses to the two identified survey ques-
tions. There were two significant differences in responses
between practitioners who had received RT and those who
had not, when analysed using one way ANOVA. Practi-
tioners who had received RT were more likely to report
that patients with MUS take up too much time (mean
scores 3.26 vs. 3.83, F = 4.062, p = 0.048). However they
were less likely to report that they often did not know how
to help these patients (mean scores 3.11 vs. 2.29, F =
9.188, p = 0.003). Amongst respondents who had received RT, most
reported that they found the training enjoyable, and disa-
greed that it had not taught them anything new. However
most thought it was not easy to find time to concentrate
on RT. Few thought that lectures or payment would have
improved their experience of or engagement with RT. A topic guide invited participants' views on patients with
persistent MUS and their management of MUS. For those
who had been trained in reattribution, further prompts
invited views on the value and benefits of reattribution,
and also on any barriers to its implementation in practice. http://www.biomedcentral.com/1471-2296/9/46 Responses were analysed
using SPSS version 15.0, first generically and then to iden-
tify significant differences between practitioners who had
or had not received RT. Qualitative interviewees were purposively selected by MT
and JGH (with advice from JAH, SP, LG and CD) from
questionnaire respondents, in order to generate as wide a
variation of views as possible. The sampling criteria
included: respondents from each practice (to ensure all
participating practices were represented); the practice set-
ting (rural, suburban; inner city); the number of patients
recruited by GPs into the trial; entry into training or con-
trol arm of the trial; gender and age of the GP; and partic-
ipants' responses to two attitudinal questions about
patients with MUS: 'I think patients with MUS take up too
much time, which I could use more productively with other
patients'; and 'There's a lot of patients for whom reattribution
does not work'. Methods
Th
i The setting for this study was 16 practices in the north-
west of England. The study sample was composed of the
practitioners (73 GPs and one nurse prescriber (NP)) tak-
ing part in an exploratory randomised control trial of the
effects of reattribution training on GP communication
behaviour with patients presenting with persistent MUS. The six hour (two session) training programme was deliv-
ered by a health facilitator to all members of the practice
team [10]. The study was approved by the North West
multi-centre research ethics committee. Practices had a median of four (range two to 10) GPs. Three practices served inner city populations, one a rural
population and 12 practices urban populations that Content of the Reattribution Intervention
Figure 1
Content of the Reattribution Intervention. Page 2 of 10
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(page number not for citation purposes) BMC Family Practice 2008, 9:46 http://www.biomedcentral.com/1471-2296/9/46 Table 1: Attitudinal survey of practitioners participating in MUST Table 1: Attitudinal survey of practitioners participating in MUST
Statement
Agree completely
Agree partly
Unsure
Disagree partly
Disagree completely
missing
about patients with PMUS* N = 70
N (%)
N (%)
N (%)
N (%)
N (%)
I enjoy consultations with patients who have PMUS
3 (4)
17 (24)
14 (20)
30 (43)
6 (9)
-
I think patients with PMUS take up too much of my
time, which I could use more productively with
other patients
4 (6)
15 (21)
5 (7)
31 (44)
15 (21)
-
I find that patients with PMUS often cause me
considerable stress
8 (11)
43 (61)
3 (4)
11(16)
5 (7)
-
I don't think it's worth trying to do much with
patients who have PMUS
0 (0)
3 (4)
5 (7)
28 (40)
33 (47)
1 (1)
I find that patients with PMUS present me with
interesting diagnostic challenges
13 (19)
38 (54)
5 (7)
12 (17)
1 (1)
-
I find that patients with PMUS present me with
interesting therapeutic challenges
18 (26)
40 (57)
3 (4)
7 (10)
1 (1)
1 (1)
I often don't know how to help patients who have
PMUS
8 (11)
33 (47)
3 (4)
21 (30)
4 (6)
1 (1)
about reattribution training N = 35
The reattribution training programme did not really
teach me anything new
2 (6)
11 (31)
4 (11)
12 (34)
6 (17)
-
It was easy to find time to concentrate on the
training programme, despite the pressure of clinical
work in my practice
4 (11)
10 (29)
1(3)
14 (40)
6 (17)
-
The training programme would have been better if it
included more formal lectures
0 (0)
2 (6)
6 (17)
8 (23)
19 (54)
-
I would have been more inclined to engage with the
training programme if I'd been paid to attend the
sessions
3 (9)
5 (14)
6 (17)
11 (31)
10 (29)
-
In general, I enjoyed the training programme
15 (43)
15 (43)
3 (9)
2 (6)
0 (0)
-
I have found it easy to put reattribution into practice
6 (17)
20 (57)
3 (6)
6 (17)
0 (0)
-
I have already forgotten some of the reattribution
stages
4 (11)
20 (57)
1 (3)
5 (14)
5 (14)
-
I often need several consultations with patients to
achieve all the reattribution stages
18 (51)
14 (40)
1 (3)
2 (6)
0 (0)
-
There are lots of patients with whom reattribution
does not work
5 (14)
12 (34)
13 (37)
4 (11)
1 (3)
-
In general, putting reattribution into practice makes
my consultations with these patients more enjoyable
7 (20)
13 (37)
9 (26)
6 (17)
0 (0)
-
In general, putting reattribution into practice makes
my consultations with these patients quicker
1 (3)
6 (17)
10 (29)
13 (37)
5 (14)
- Agree completely
Agree partly
Unsure
Disagree partly
Disagree completely
missing Limitations and benefits of reattribution Trained respondents reported that putting RT into practice
was fairly easy, and that it generally made consultations
with MUS patients more enjoyable, although not quicker. Attitudinal Survey However, most agreed that they had already forgotten
some of the reattribution stages. Almost all considered
that it often needed several consultations to complete all
stages of reattribution, and very few disagreed with the
statement that there are lots of patients for whom reattri-
bution does not work. Participants voiced some criticisms of the training in reat-
tribution. Some thought they were already doing it: 'it
didn't give me anything new' [PGP11]. Concerns were also
expressed about the time commitment, aspects of the for-
mat and content of the training programme and – com-
monly – the lack of reinforcement: 'supervision would have
been nice' [PGP12]. However, they described both direct
and indirect benefits from learning about reattribution
(see Figure 2). Qualitative Interviews Many indicated that training in reattribution increased
their awareness and altered their perception of patients
with MUS: In this section of the paper we focus on the responses of
twelve GPs who had received training in reattribution to
questions about: a) limitations and benefits of reattribu-
tion; and b) barriers to its implementation in routine clin-
ical practice. It er, helped one to sort of spot the situations perhaps a bit,
bit better, increased one's awareness, and you know, Page 4 of 10
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x Increased awareness of patients with MUS
x Altered perceptions of patients with MUS
x Greater confidence in treating patients with MUS
x Reflection on management of patients with MUS Barriers to implementing reattribution Respondents described many barriers to implementing
reattribution in routine general practice. We categorise
these as arising from the patient, the doctor, the consulta-
tion, the diagnosis, and the context of care (see Figure 3). It's made me stop and think why I am referring this person
yet again. I think there are not an awful lot of examples,
since we've been doing the training come to mind referring
here there and everywhere, but just in general because of
the training I've thought lets just stop and look, what are we
actually achieving [PGP02] Indirect benefits
x Comparing consultation skills
x Cross-over into chronic disease management Indirect benefits
x Comparing consultation skills
x Cross-over into chronic disease management GPs views of benefits of reattribution training
Figure 2
GPs views of benefits of reattribution training. difficult to do to discuss the physiology of anxiety and how
it might produce physical symptoms [PGP09] difficult to do to discuss the physiology of anxiety and how
it might produce physical symptoms [PGP09] increased perhaps just really the, the trying to explain to
patients the, the way the symptoms developed if you like
from, well often from anxiety or other causes [PGP23] Respondents also reported indirect benefits from reattri-
bution training, which were unrelated to their manage-
ment of MUS. As well as 'recharging the batteries' (PGP11)
and doing something new 'to put on the CV' (PGP18), the
training programme was seen as a valuable opportunity
for GPs to compare consultation skills with colleagues
within their own practice. Some respondents had also
used reattribution in their consultations with non-MUS
patients: altered my perception a bit, its easy to get stale and view
that group of patients as difficult or troublesome or irksome
at times because we're not always at our best every time
[PGP11] Some found that reattribution training had a positive
impact on their consultations, increasing their confidence
in discussing MUS, and enabling them to reflect on their
management decisions: Some found that reattribution training had a positive
impact on their consultations, increasing their confidence
in discussing MUS, and enabling them to reflect on their
management decisions: The emphasis it puts on explanation and so on, I think that,
you can carry across into other areas....I think some of the
chronic diseases most probably been some cross-over
[PGP23] I'd like to think that I do go a little bit more into other agen-
das, other issues that might be fuelling the symptoms that
they've got and try and approach those other problems
rather than just focusing on a prescription for something for
pain [PGP13] Patient barriers The attitudes of patients with MUS were seen as important
barriers by many GPs. They frequently described how
patients may have entrenched views that their symptoms
have an organic basis: Some GPs valued the additional structure provided by
reattribution: I think it did, it did formalise it but I think it wasn't some-
thing that I would have been embarrassed about or found it Well it's their mindset isn't it? It's their belief that there is
a physical cause [PGP18] Page 5 of 10
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(page number not for citation purposes) BMC Family Practice 2008, 9:46 http://www.biomedcentral.com/1471-2296/9/46 http://www.biomedcentral.com/1471-2296/9/46 about getting benefits or support from the university or sup-
port from key people in their lives [PGP22] Patient barriers x Entrenched views of symptom causation x Learned behaviours Doctor barriers
Respondents were aware of variations in communication
skills and the ability to deliver the stages of reattribution
in routine clinical settings: x Secondary gain from unexplained symptom x Variations in skills in managing patients with MUS x Prior expectations of patients It's like basic training. Sort of communication skills, history
taking.....some people are better than others [PGP18] It's like basic training. Sort of communication skills, history
taking.....some people are better than others [PGP18] x Concern about dependence x Emotional state during consultation Consultation barriers They were aware that their prior expectations of the
patient could influence how likely they were to attempt
reattribution. x Patients choosing lack of continuity x Communicating difficult experiences x Doctors’ responses to patients with MUS With certain people, before they've come in, you've already
perceived it's going to be difficult because that's your prior
expectation, your prior knowledge of them.....I mean and
they could be coming with something totally different, but
your initial reaction is "oh my god" [PGP16] x Complex presentations x Nebulous definitions of MUS x Time x Quality Outcomes Framework GPs expressed concern that getting involved with reattri-
bution may lead to the patient becoming dependent on
them: x Impact of secondary care x Medico-legal framework of primary care Not that the relationship has broken down but you don't
want to have such a close relationship with them that
they're relying on you [PGP09] GPs' views
patients w
Figure 3 GPs' views of barriers to implementation of reattribution for
patients with MUS
Figure 3
GPs' views of barriers to implementation of reattri-
bution for patients with MUS. GPs views of barriers to implementation of reattribution for
patients with MUS
Figure 3
GPs' views of barriers to implementation of reattri-
bution for patients with MUS. Two respondents acknowledged that their own emotional
state or mood affected the likelihood of using reattribu-
tion. Respondents expressed various and occasionally contra-
dictory opinions about the impact of gender, intelligence,
class and ethnicity on the likelihood of patients' holding
fixed beliefs in physical causation of MUS. However they
all considered the presence of such beliefs to be a funda-
mental barrier to successful reattribution. Diagnostic barriers Many GPs commented that patients do not present solely
with MUS, but rather have numerous symptoms, some
with an organic basis and some with a psychological
underpinning. The presence of organic and medically
unexplained symptoms can make it harder for GPs and
patients to disentangle the causes of their symptoms. It does tend to be that if, if they are often in secondary care
that they are given a physical diagnosis whether there is one
or not....That can support their belief in a physical problem
[PGP21] Some respondents saw it as their role to protect patients
from this potential source of harm: Most patients I find have medically unexplained and med-
ically explainable symptoms, its very complicated and in my
experience its very rare to have somebody just coming in
with unexplained symptoms because nowadays if we're
diagnosing more people with hypertension than diabetes
and whatever, so we are giving people labels for certain con-
ditions anyway [PGP09] You see these people getting referred to the hospital with
back pain and the next thing you know some bright spark is
going to operate on them and you think 'What!' ....Maybe
we're here in a way as a gateway to try and prevent harm
as well as anything else [PGP06] The difficulty of labelling patients as 'definitely' having
MUS was also seen as a potential barrier in deciding who
and when to use reattribution: However others were aware of the potentially punitive
medico-legal framework within which they operate,
which pushes them towards an over-emphasis on the
identification or exclusion of physical illness However others were aware of the potentially punitive
medico-legal framework within which they operate,
which pushes them towards an over-emphasis on the
identification or exclusion of physical illness This is a bit more nebulous, you've got to think more deeply
about it and it's not as though the patient is coming in and
you catch the MUS and label the MUS and that maybe why
its higgilty piggilty cos I can't label it and say right that's
your MUS and this is what we are going to do. [it depends] how you're feeling that day [PGP04] [it depends] how you're feeling that day [PGP04] I try [to reattribute] on a good day. On a bad day I just try
and give the least damaging medication and do the least
number of referrals [PGP08] Many GPs considered that patients' belief in organic
causes of MUS could be perpetuated by family members
since childhood, becoming a learnt behaviour or a strat-
egy for coping with unhappiness. Consultation barriers
Respondents considered that patients' consulting behav-
iour can make it difficult to apply reattribution, particu-
larly if they choose to consult with different GPs within a
practice. These are patients who've learnt to present their unhappi-
ness in physical ways and they may have been in a family
where they went to the doctor with every little bit of pain so
their mothers might have been frequent attenders and its
been a sort of learnt behaviour....I think they must have
learnt that some behaviour is advantageous [PGP01] If they're at the more difficult end of the spectrum they will
often deliberately pick off a locum doctor or a registrar to get
what they want, they have an agenda [PGP11] However, GPs were not always critical of their patients'
unwillingness or inability to communicate with their doc-
tor sufficiently to allow them to broaden the agenda. Respondents thought patients could derive benefit from
their symptoms, in relation to work, state benefits or fam-
ily support: [There are] occasions where you do have a very good rela-
tionship but something is too, either too painful or too pri-
vate to share with you [PGP16] The patient might have gains from their, from their symp-
toms as well. So they might have, there might be gains Page 6 of 10
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(page number not for citation purposes) BMC Family Practice 2008, 9:46 http://www.biomedcentral.com/1471-2296/9/46 http://www.biomedcentral.com/1471-2296/9/46 Its difficult because sometimes you can see..., I've got one
patient that all her symptoms started after the break up of
her marriage and she can't see the link, and because of the
type of person she is, she's extremely proud and you don't
want to keep banging on about it [PGP04] Some GPs thought that the Quality Outcomes Framework
(QOF) compounded these time pressures, and detracted
from their ability to reattribute. It's probably impacted on every consultation in the fact that
you need to collect data. Diagnostic barriers And that
makes it more difficult, you know whom we will we start to
reattribute, whom wants to get to think, to think what
causes their symptoms and how early we do it [PGP02] You're never criticised for over-diagnosing and inappropri-
ately over-treating patients but you can lose your job for
missing a diagnosis, so the whole thing tips completely the
wrong way, and not in the patients favour in that sense
[PGP08] [it depends] how you're feeling that day [PGP04] So often it's difficult to spend the
time pursuing things that you might have pursued before,
because actually you've got to record their height, weight,
body mass, or whether they smoke and all practical stuff,
and that detracts from being able to pick up problems. ...So
I think it does actually alter consultations in those situations
[PGP22] Some GPs acknowledged that their own responses to
patients during consultations could impede reattribution: Some GPs acknowledged that their own responses to
patients during consultations could impede reattribution: I find some patients quite easy to get a rapport with, and it's
the ones that you really have little in common with, or not
even, you can get on with people that you have nothing in
common with but when there's just something that brushes
you up the wrong way and I find that quite hard to turn
around [PGP04] There was a common view that contact with secondary
care was unhelpful, with an increase in inappropriate
physical diagnoses, and greater entrenchment in symp-
toms. Discussion
Summary of findings Responses to the questionnaire survey indicated that these
practitioners were generally sympathetic to patients with
MUS, but often found them a source of stress and difficult
to help. Although respondents who had received RT
tended to be less positive in their views about patients
with MUS, they were more confident that they knew how
to help them. Respondents generally enjoyed the process
of training, but often forgot key elements of reattribution,
and presented mixed views about the ease and practicality
of its implementation in practice. Strengths and limitations We have previously demonstrated that this group of GPs
is more sympathetic towards patients presenting with
MUS, and places greater value on their own psychosocial
skills in relation to such patients, than GPs who do not
wish to take part in research of this kind [20]. Further-
more, we have shown in this study that they see substan-
tial direct and indirect benefits of reattribution. They may
therefore be taken as a group of doctors who tend to be
well disposed towards the implementation of reattribu-
tion within routine clinical practice in primary care. For
these reasons, the many and various concerns they express
about the limited scope for implementing reattribution
deserve serious consideration – coming from a group of
'critical friends' rather than from neutral or even hostile
observers. We see this as a major strength of this study. Most of the barriers described by these GPs, however,
indicate a more nuanced and reflective response to the
problems of managing patients with MUS, including a
ready acknowledgement not only of the difficult diagnos-
tic and organisational context within which they have to
operate, but also of the significance of their own attitudes
and personal responses. Although they value the use of
reattribution for patients with MUS, they do not always
feel able to take on the work involved, or to shoulder the
burden of responsibility that may ensue [25]. In this sense
at least, the barriers they describe may not merely be
obstacles to success, but may sometimes have a protective
function for doctors acting within a difficult arena. By definition, therefore, a limitation of this study is that
the views of these GPs cannot be assumed to be represent-
ative of their discipline: the relatively high proportion of
female participants in the qualitative survey, compared to
the proportion of female GPs in England as a whole also
indicates unrepresentativeness. Secondly, while most of
the barriers they discuss are common across all healthcare
settings, there are several organisational constraints that
are specific to healthcare in the UK, such as ten minute
consultations and the Quality and Outcomes Framework
[21], and may not be generalisable elsewhere. We also There are important differences and synergies between GP
and patient perspectives. GPs considered patients with
MUS to have fixed physical attributions. However this
view is not supported by recent evidence from primary
care [26]. Comparisons with existing literature This is the first study to describe the views of GPs about
the benefits and barriers to implementing reattribution in
routine clinical practice. Our methods, combining struc-
tured attitudinal responses with semi-structured inter-
views with a purposive sample of GPs, were chosen to
allow as wide a range of perspectives as possible to
emerge. This paper is therefore takes forward previous
research in this field, which has demonstrated changes in
GPs' attitudes to patients with MUS following reattribu-
tion by quantitative means [11,15,23], but has not com-
bined this with qualitative methods to gather their
detailed views about reattribution and how it may work in
practice. In apparent contrast to our quantitative finding,
Danish GPs who had been trained in reattribution
reported less concern about time spent with MUS patients
[23]: however this study reported within-subject differ-
ences over time, whereas we compared attitudes of trained
and non-trained respondents. These GPs described a complex and interlinking set of bar-
riers to the implementation of reattribution. Some of
these observations could be regarded as stereotypical and
partial: the frequent tendency to blame time constraints
for difficulties in applying reattribution in practice can be
seen as an example of a 'culturally honourable' excuse, a
means of mitigating responsibilities when behaviour is
questioned [24]. http://www.biomedcentral.com/1471-2296/9/46 BMC Family Practice 2008, 9:46 http://www.biomedcentral.com/1471-2296/9/46 The participants in the qualitative study who had received
RT described direct benefits from reattribution, including
a greater sense of confidence and coherence when consult-
ing with patients with MUS, and also indirect benefits
including sharing consultation skills and application of
new skills in the management of chronic disease. How-
ever they also reported multiple interlinking barriers to
the successful implementation of reattribution in routine
practice. Barriers at the patient level included the percep-
tion of entrenched views about physical causes of symp-
toms that were not amenable to change, compounded by
learned behaviours and secondary gains. Doctor barriers
included lack of skill, negative expectations of the patient,
concern about encouraging dependence and personal
emotional states. Barriers within the consultation
included patients choosing not to consult with a regular
doctor or being unwilling to share private information,
and GPs' responses to particular patients. Diagnostic bar-
riers were the frequent combination of explained and
unexplained symptoms, and the nebulous definitions of
MUS. Barriers in the healthcare context included time,
organisational requirements, concerns about the negative
impact of secondary care and the fear of the medico-legal
consequences of missing physical diagnoses. Addition-
ally, they report the complexity of working with degrees of
uncertainty which are difficult to resolve. acknowledge that the metaphor of 'barriers' used by the
research team is based on the assumed ability of partici-
pants to implement change, while operating within a
highly complex set of diagnostic and organisational con-
texts [22]. Page 8 of 10
(page number not for citation purposes) Barriers in the healthcare context Respondents commonly reported insufficient time to deal
effectively with MUS patients, with busy surgeries and
brief appointments pushing them away from reattribu-
tion and towards short-term solutions. Time pressure is such that you're looking at certain quick
fixes, you may not be consciously looking outside the box
[PGP02] Page 7 of 10
(page number not for citation purposes) Page 7 of 10
(page number not for citation purposes) http://www.biomedcentral.com/1471-2296/9/46 http://www.biomedcentral.com/1471-2296/9/46 Competing interests components of their illness beliefs [27], which is a further
barrier to GPs attempts to reattribute. It appears that both
GPs and patients consider the problem to be complex,
while believing that the other party holds a more simplis-
tic view than their own. Despite recognising the value in
preventing further contact with secondary resources, GPs
admitted reluctance in abandoning pathology investiga-
tions. A driving force for this, from GPs' perspectives, is
the medico-legal framework within which they work. Elsewhere patients describe pursuing a similar agenda;
also out of fear of missing disease, either now or if pre-
senting with future problems [27]. p
g
The authors declare that they have no competing interests. p
g
The authors declare that they have no competing interests. Authors' contributions CD designed and analysed the questionnaire and drafted
the manuscript. JAH organised and collated the attitudi-
nal survey. JGH undertook qualitative interviews and ini-
tiated the qualitative analysis. CD, JGH and SP completed
the qualitative analysis. RKM, LG, CD, PS, SP and ARR
conceived the study, and participated in its design and
coordination. All authors read and approved the final
manuscript. Funding
M di
l R g
Medical Research Council (Grant reference number
G0100809), Mersey Care NHS Trust and Mersey Primary
Care R&D Consortium. It is important to recognise that both patients with MUS,
and the GPs they encounter are heterogeneous groups,
with competing attributions on one side [28], and varying
degrees of empathy and skill on the other. A key question,
therefore, is how to establish a common understanding of
illness and treatment expectations. Our findings indicate
the need for greater specificity, with regard to the patients
and circumstances in which the techniques of reattribu-
tion may successfully be applied. This is of relevance both
to future research and to current practice. Ethical approval: This study was approved by the North
West multi-centre research ethics committee. Acknowledgements We thank the following practices and their patients that took part in the
study: Bousfield Health Centre; Brownhill Surgery; Brownlow Group Prac-
tice; Ellesmere Medical Centre; Heaton Medical Centre; Manor Health
Centre; Moore Street Surgery; Pendleside Medical Practice; Primrose Bank
Medical Centre; Roe Lee Surgery; Somerville Group Practice; Stonehill
Medical Centre; Unsworth Medical Centre; Victoria Park Health Centre;
Villa Medical Centre; Westmoreland General Practice. Some of the barriers to reattribution reported by GP
respondents were seen as immutable, such as the coexist-
ence of unexplained and explained symptoms and (per-
haps) the fixed beliefs of patients. It may therefore be
useful for educators to focus on addressing those barriers
to psycho-social interventions which appear more amena-
ble to change, such as the need for increased skill, or for
different medical attitudes towards patients with MUS. A
stepped care approach, assuming only a basic level of
knowledge, interest and skill amongst the majority of
GPs, may also address the diagnostic and organisational
barriers reported by our respondents [29]. We also thank Nigel Crompton, Marie Evans, Elaine McVeigh and Victoria
Wilson, who undertook reattribution training; Maria Towey (MT) who
undertook some of the qualitative interviews; and Francis Creed who
helped to develop the protocol and was a grantholder. Finally, we thank Kurt Fritzsche and Marianne Rosendal for their reviews of
a previous version of this paper. Strengths and limitations Rather, patients choose to present physical attri-
butions to their GP whilst withholding psychological Page 8 of 10
(page number not for citation purposes) Page 8 of 10
(page number not for citation purposes) BMC Family Practice 2008, 9:46 http://www.biomedcentral.com/1471-2296/9/46 http://www.biomedcentral.com/1471-2296/9/46 http://www.biomedcentral.com/1471-2296/9/46 References 1. Peveler R, Kilkenny L, Kinmoth AL: Medically unexplained physi-
cal symptoms in primary care: a comparison of self-report
screening questionnaires and clinical opinion. Journal of Psycho-
somatic Research 1997, 42:245-252. 1. Peveler R, Kilkenny L, Kinmoth AL: Medically unexplained physi-
cal symptoms in primary care: a comparison of self-report
screening questionnaires and clinical opinion. Journal of Psycho-
somatic Research 1997, 42:245-252. One way forward would be to take the ideal type of cir-
cumstances for the delivery of reattribution as described
by these practitioners: patients whose MUS are associated
with fluid causative beliefs, have no family expectations of
illness and see nothing to gain from their symptoms; in
consultations with regular GPs who feel well disposed
towards their patients and comfortable in themselves, are
confident in their consultation skills, can tolerate diagnos-
tic uncertainty, and feel concerned neither about time
pressures nor punitive organisational or medico-legal
constraints. These appear to be the circumstances in
which reattribution is most likely to prove successful. It
may therefore be wise to demonstrate efficacy in relatively
calm conditions first, before braving the elements and
exploring more troubled waters. 2. Weijden van der T, Velsen van M, Dinant G-J, Hasselt van CM, Grol
R: Unexplained complaints in general practice: prevalence,
patients' expectations, and professionals' test-ordering
behavior. Medical Decision Making 2003, 23:226-231. g
,
3. Kouyanou K, Pither C, Rabe-Hesketh , Wessely S: A comparative
study of iatrogenesis, medication abuse and psychiatric mor-
bidity in chronic pain patients with and without medically
explained symptoms. Pain 1998, 76:417-426. g
,
3. Kouyanou K, Pither C, Rabe-Hesketh , Wessely S: A comparative
study of iatrogenesis, medication abuse and psychiatric mor-
bidity in chronic pain patients with and without medically
explained symptoms. Pain 1998, 76:417-426. 4. Stanley IM, Peters S, Salmon P: A primary care perspective on
prevailing assumptions about persistent medically unex-
plained physical symptoms. International Journal of Psychiatry in g
3. Kouyanou K, Pither C, Rabe-Hesketh , Wessely S: A comparative
study of iatrogenesis, medication abuse and psychiatric mor-
bidity in chronic pain patients with and without medically
explained symptoms. Pain 1998, 76:417-426. explained symptoms. Pain 1998, 76:417 426. 4. Stanley IM, Peters S, Salmon P: A primary care perspective on
prevailing assumptions about persistent medically unex-
plained physical symptoms. International Journal of Psychiatry in
Medicine 2002, 32:125-140. explained symptoms. Pain 1998, 76:417 426. 4. http://www.biomedcentral.com/1471-2296/9/46 http://www.biomedcentral.com/1471-2296/9/46 26. 7. Salmon P, Peters S, Stanley I: Patients' perceptions of medical
explanations for somatisation disorders: qualitative analysis. BMJ 1999, 318:372-376. 26. Salmon P, Peters S, Rogers A, Gask L, Clifford R, Iredale W, Dowrick
C, Morriss R: Peering through the barriers in GPs' explana-
tions for declining to participate in research: the role of pro-
fessional autonomy and the economy of time. Family Practice
2007, 24:269-275. J
8. Nettleton S: 'I just want permission to be ill': Towards a soci-
ology of medically unexplained symptoms. Social Science and
Medicine 2006, 62:1167-1178. 27. 27. Peters S, Rogers A, Salmon P, Gask L, Towey M, Clifford R, Dowrick
C, Morriss R: Qualitative analysis of patient experiences of GP
attempts to manage medically unexplained symptoms: why
did improvement in doctor communication by reattribution
training not translate into patient improvement? Journal of
General Internal Medicine in press. 9. Little P, Everitt H, Williamson I, Warner G, Moore M, Gould C:
Observational study of effect of patient centredness and pos-
itive approach on outcomes of general practice consulta-
tions. BMJ 2001, 323:908-911. J
10. Morriss R, Dowrick C, Salmon P, Peters S, Rogers A, Dunn G, Lewis
B, Charles-Jones H, Hogg J, Clifford R, Rigby C, Gask L: Turning the-
ory into practice: rationale, feasibility and external validity of
an exploratory randomized controlled trial of training family
practitioners in reattribution to manage patients with med-
ically unexplained symptoms (MUST. General Hospital Psychiatry
2006, 28:343-351. 28. Rief W, Nanke A, Emmerich J, Bender A, Zech T: Causal illness
attributions in somatoform disorders: associations with
comorbidity and illness behavior. Journal of Psychosomatic
Research 2004, 57:367-371. 29. Smith RC, Lein C, Collins C, Lyles JS, Given B, Dwamena FC, Coffey
J, Hodges A, Gardiner JC, Goddeeris J, Given CW: Treating
patients with medically unexplained symptoms in primary
care. Journal of General Internal Medicine 2003, 18:478-89. 11. Morriss RK, Gask L, Ronalds C, Downes-Grainger E, Thompson H,
Leese B, Goldberg D: Cost-effectiveness of a new treatment for
somatized mental disorder taught to GPs. Family Practice 1998,
15:119-125. Pre-publication history 12. Morriss RK, Gask L, Ronalds C, Downes-Grainger E, Thompson H,
Goldberg D: Clinical and patient satisfaction outcomes of a
new treatment for somatized mental disorder taught to gen-
eral practitioners. British Journal of General Practice 1999,
49:263-267. The pre-publication history for this paper can be accessed
here: http://www.biomedcentral.com/1471-2296/9/46/prepub http://www.biomedcentral.com/1471-2296/9/46/prepub 13. Morriss RK, Gask L: Treatment of patients with somatized
mental disorder: effects of reattribution training on out-
comes under the direct control of the family doctor. Psycho-
somatics 2002, 43:394-399. 14. Blankenstein AH: Somatising patients in general practice reat-
tribution, a promising approach. In PhD thesis Netherlands: Vrije
Universiteit; 2001. 15. Larisch A, Schweickhardt A, Wirsching M, Fritzsche K: Psychosocial
interventions for somatizing patients by the general practi-
tioner: a randomized controlled trial. Journal of Psychosomatic
Research 2004, 57:507-514. 16. Rosendal M, Olesen F, Fink P, Toft T, Sokolowski I, Bro F: A rand-
omized controlled trial of brief training in the assessment
and treatment of somatisation in primary care: effects on
patient outcome. General Hospital Psychiatry 2007, 29:364-373. p
p
y
y
17. Morriss R, Dowrick C, Salmon P, Peters S, Dunn G, Rogers A, Lewis
B, Charles-Jones H, Hogg J, Clifford R, Gask L: Cluster randomised
controlled trial of training practices in reattribution for med-
ically unexplained symptoms. British Journal of Psychiatry 2007,
191:531-542. 18. Salmon P, Ring A, Dowrick CF, Humphris GM: What do general
practice patients want when they present medically unex-
plained symptoms, and why do their doctors feel pressu-
rised? Journal of Psychosomatic Research 2005, 59:255-260. J
y
19. Corbin J, Strauss A: Grounded theory research: Procedures,
canons, and evaluative criteria. Qualitative Sociology 1990,
13:3-21. 20. Salmon P, Peters S, Clifford R, Iredale W, Gask L, Rogers A, Dowrick
C, Hughes J, Morriss R: Why do general practitioners decline
training to improve management of medically unexplained
symptoms. Journal of General Internal Medicine 2007, 22:565-571. y
p
J
21. BMA and NHS Confederation: The Blue Book: Investing in Gen-
eral Practice. The New General Medical Services Contract. London; 2003. References Stanley IM, Peters S, Salmon P: A primary care perspective on
prevailing assumptions about persistent medically unex-
plained physical symptoms. International Journal of Psychiatry in
Medicine 2002, 32:125-140. 5. Ring A, Dowrick CF, Humphris GM, Davies J, Salmon P: The soma-
tizing effect of clinical consultation: what patients and doc-
tors say and do not say when patients present medically
unexplained physical symptoms. Social Science and Medicine
2005, 61:1505-1515. 6. Barsky AJ, Orav EJ, Bates DW: Somatization increases medical
utilization and costs independent of psychiatric and medical
comorbidity. Archives of General Psychiatry 2005, 62:903-910. Barsky AJ, Orav EJ, Bates DW: Somatization increases medical
utilization and costs independent of psychiatric and medical
comorbidity. Archives of General Psychiatry 2005, 62:903-910. Page 9 of 10
(page number not for citation purposes) Page 9 of 10
(page number not for citation purposes) BMC Family Practice 2008, 9:46 http://www.biomedcentral.com/1471-2296/9/46 Pre-publication history Publish with BioMed Central and every
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(page number not for citation purposes) Publish with BioMed Central and every
scientist can read your work free of charge 22. Checkland K, Harrison S, Marshall M: Is the metaphor of 'barriers
to change' useful in understanding implementation? Evi-
dence from general medical practice. Journal of Health Services
Research and Policy 2007, 12:95-100. y
23. Rosendal M, Bro F, Sokolowski I, Fink P, Toft T, Olesen F: A ran-
domised controlled trial of brief training in assessment and
treatment of somatisation: effects on GPs' attitudes. Family
Practice 2005, 22:419-427. 24. Salmon P, Dowrick C, Ring A, Humphris G: Voiced but unheard
agendas: qualitative analysis of the psychosocial cues that
patients with unexplained symptoms present to general
practitioners. British Journal of General Practice 2004, 54:171-176. p
J
25. Cocksedge S, May C: Pastoral relationships and holding work in
primary care: affect, subjectivity and chronicity. Chronic Illness
2005, 1:157-163.
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https://openalex.org/W4387344367
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https://www.nature.com/articles/s41598-023-44031-8.pdf
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English
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Impact of changes in physical activity and incident fracture after acute ischemic stroke
|
Scientific reports
| 2,023
|
cc-by
| 7,234
|
Dae young Cheon 1,5, Kyung‑Do Han 2,5, Jeen Hwa Lee 1, Kyung‑Ho Yu 3, Bo Young Choi 4* &
Minwoo Lee3* Dae young Cheon 1,5, Kyung‑Do Han 2,5, Jeen Hwa Lee 1, Kyung‑Ho Yu 3, Bo Young Choi 4* &
Minwoo Lee3* Stroke survivors are at an increased risk of falls and fractures. Physical activity is inversely associated
with the fracture risk in the general population. However, the association between incident fracture
risk and changes in habitual physical activity before and after an index stroke remains unclear. This
study attempted to explore the association between incident fracture risk and changes in physical
activity after stroke. Using the claims database of the National Health Insurance Program in Korea,
participants with their first ischemic stroke between 2010 and 2016 were enrolled in the study. The
participants were divided into four groups according to changes in physical activity habits evaluated
using two consecutive self-reported questionnaires before and after stroke, if available: persistent
non-exercisers, exercise dropouts, new exercisers, and persistent exercisers. The primary outcome
was a composite of vertebral, hip, and other fractures. We performed multivariable Cox proportional
hazard regression analysis and provided adjusted hazard ratios and 95% confidence intervals for each
outcome. Among 202,234 participants included from 1,005,879 datasets, 16,621 (8.22%) experienced
any type of fracture as the primary outcome. After multivariable adjustment, exercise dropouts
(n = 37,106), new exercisers (n = 36,821), and persistent exercisers (n = 74,647) had a significantly
reduced risk of any type of fracture (aHR 0.932, 95% CI 0.893–0.973; aHR 0.938, 95% CI 0.900–0.978;
aHR 0.815, 95% CI 0.780–0.852, respectively) compared to the persistent non-exercisers (n = 53,660). Furthermore, regardless of pre-stroke exercise status, those who exercised ≥ 1000 metabolic
equivalents of tasks post-stroke had a significantly reduced risk of fractures. Initiating or continuing
moderate-to-vigorous regular physical activity after acute ischemic stroke was associated with a
significantly lower risk of incident fractures, including hip, vertebral, and other types. Stroke is a leading cause of disability, and stroke survivors are at an increased risk of fractures due to reduced
bone mineral density and an elevated risk of falls. Previous studies have suggested that stroke increases the risk of
fracture by up to 4 times compared with the general population1–3. More than two-thirds of stroke survivors with
mild-to-moderate disability experience falls during the first 6 months after acute stroke4. www.nature.com/scientificreports www.nature.com/scientificreports Data source and study population Data source and study population y p p
We utilized a nationwide medical dataset from the Korean National Health Insurance Service (K-NHIS), a single
insurer administered in Korea that covers more than 97% of the Korean population. The K-NHIS provides a bien-
nial general health examination to all individuals aged 40 years and older, which includes demographic, clinical,
and laboratory workups and a standard questionnaire regarding lifestyle behaviors, such as exercise and drinking
and smoking habits10. Furthermore, the K-NHIS retains medical claims data, including inpatient and outpatient
usage of medical services, prescription records, and diagnostic codes according to International Classification of
Diseases (ICD)-10. The study was approved by the Institutional Review Board (IRB) of the Dongtan Sacred Heart
Hospital (IRB number: HDT 2023-01-010). The participants who underwent national health checkups provided
written informed consent for the use of their data for research purposes. This study was designed and conducted
according to the Strengthening the Reporting of Observational Studies in Epidemiology Reporting Guidelines. h
d
b
f h
l
ll
d d
f
h From the K-NHIS database of the entire Korean population, we collected data from participants who were
newly diagnosed with acute ischemic stroke between January 2010 and December 2016. Acute ischemic stroke
was defined using the 10th edition diagnostic codes of the ICD-10 I63 and I64 upon admission, combined
with brain imaging using either computed tomography or magnetic resonance imaging. This definition was
validated with high accuracy in previous studies11–14. Among the 1,005,879 participants, those who did not
undergo a general health examination with a standard questionnaire 2 years before (541,463 participants) and
after (199,777 participants) the index stroke were excluded. Among the remaining 264,639 patients, those with
missing questionnaires (6855 participants), those younger than 40 years (5264 participants), those with previous
diagnoses of any fracture (44,038 participants), and those who experienced any fracture within 1 year after their
second examination (6248 participants) were excluded to reduce potential reverse causality. Ultimately, 202,234
participants were included in the analysis. (Fig. 1). Definition of exercise habit changes i
g
Information on exercise habits was obtained from two consecutive standardized self-report questionnaires on
the intensity and frequency of physical activity over the past 12 months. This questionnaire was based on the
International Physical Activity Questionnaire, developed by the World Health Organization and validated for
the Korean population15. The physical activity section of the questionnaire included three questions regarding
the frequency of light, moderate, and vigorous exercise per week. Light-intensity exercise was defined as walking
or sweeping carpets for ≥ 30 min. Moderate intensity included bicycle riding, walking at a brisk pace, or playing
sports, such as tennis, for ≥ 30 min. Vigorous-intensity exercise was defined as running, climbing, quick bicycle
riding, or aerobic workouts for ≥ 20 min. Regular physical activity was defined as moderate or vigorous exercise
performed at least once a week. The participants were then assigned to one of the following four groups accord-
ing to changes in exercise habits before and after the index stroke: (1) persistent non-exercisers, (2) exercise
dropouts, (3) new exercisers, and (4) persistent exercisers. To evaluate the influence of energy expenditure on
outcomes, we stratified regular physical activity groups according to the degree of energy expenditure using
metabolic equivalents of tasks (METs). The multipliers for light, moderate and vigorous-intensity exercise were
2.9, 4.0, and 7.0, respectively for the calculation of energy expenditure. Total energy expenditure, defined as the
summation of METs multiplied by the frequency of each intensity exercise with the minimum duration, was
divided into < 1000 and ≥1000 Met-min/week for subgroup analysis16. Dae young Cheon 1,5, Kyung‑Do Han 2,5, Jeen Hwa Lee 1, Kyung‑Ho Yu 3, Bo Young Choi 4* &
Minwoo Lee3* Post-stroke fractures
not only increase the degree of disability and mortality, but also increase the burden on the family and society.i Physical activity is one of the most important modifiable factors associated with fracture risk, along with
other lifestyle behaviors5. Previous studies have shown that initiating or maintaining regular exercise may prevent
fractures in older patients6,7. Furthermore, regular exercise after stroke is an important modality that reduces
multiple risk factors for post-stroke fractures. Thus, regular exercise is recommended for stroke survivors to
maintain the bone mineral density and manage falls8,9. However, it is still not well known whether initiating or
continuing exercise after stroke can prevent fracture risk via the aforementioned mechanisms.t gt
p
In the present study, we investigated the association between changes in physical activity after acute ischemic
stroke and incident fracture risk using the Korean National Health Insurance Services (K-NHIS) database. 1Division of Cardiology, Department of Internal Medicine, Dongtan Sacred Heart Hospital, Hwaseong,
Korea. 2Department of Statistics and Actuarial Science, Soongsil University, Seoul, Korea. 3Department of
Neurology, Hallym University Sacred Heart Hospital, Anyang, Korea. 4Department of Physical Education, Hallym
University, Chuncheon, Korea. 5These authors contributed equally: Dae young Cheon and Kyung-Do Han. *email:
bychoi@hallym.ac.kr; neuromlee@hallym.or.kr | https://doi.org/10.1038/s41598-023-44031-8 Scientific Reports | (2023) 13:16715 www.nature.com/scientificreports/ Results A total of 202,234 patients (mean age, 64.52 ± 10.42; male, 54.48%) were included in our analyses. The number
and percentages of persistent non-exercisers, exercise dropouts, new exercisers, and persistent exercisers were
74,467 (36.9%), 37,106 (18.3%), 36,821 (18.2%), and 53,660 (26.5%), respectively. The baseline and demographic
characteristics of each group are presented in Table 1. Compared with persistent non-exercisers, persistent exer-
cisers tended to be younger, more likely to be male, alcohol users, and less likely to have diabetes, hypertension,
or chronic kidney disease. There were no clinically significant differences in laboratory results among the groups. yh
y
gif
y
g
g
p
During a mean follow-up of 4.13 ± 2.03 years, there were 16,621 (8.22%) primary composite outcomes (any
types of fractures). Furthermore, 8399 (4.15%) and 2356 (1.16%) participants had vertebral and hip fractures,
respectively. Compared with persistent non-exercisers, those who initiated or continued regular exercise after
stroke had a significantly lower risk of all types of fractures (new exercisers, aHR 0.932; 95% CI 0.893–0.973,
persistent exercisers, aHR 0.815; 95% CI 0.780–0.852, Table 2). Furthermore, those who failed to continue their
exercise habits and those who dropped out had a mild but significantly lower risk of all types of fractures than
those who did not. (aHR 0.938; 95% CI 0.900–0.978). Consistent findings were observed for both vertebral and
hip fractures, except that the new exerciser group did not show a significantly reduced risk of hip fractures. Kaplan–Meier curves demonstrated significant differences in primary and secondary outcomes between the
groups according to the status of exercise habits changes. (Fig. 2).i g
p
g
g
g
Subgroup analyses were performed according to age, sex, and comorbidities. Although consistent findings
were observed throughout the age and comorbidity subgroups, a significant interaction was observed between
the sex groups. (Table 3 and Supplemental Table S1). The beneficial effects of exercise were not prominent in
female groups; only persistent exercisers showed a significantly reduced risk of all types of fractures, and new
exercisers or exercise dropouts were not associated with a reduced risk of incident fractures. Additional analyses
were performed to determine the effect of the degree of exercise on the risk of fractures. Overall, regardless of
pre-stroke exercise habits, exercising ≥ 1000 METs-min/week after acute stroke was associated with a lower risk
of fractures compared with no exercise or < 1000 METs-min/week. www.nature.com/scientificreports/ including smoking and drinking habits, were also recorded. Smoking status was divided into never smokers,
ex-smokers, and current smokers based on their responses to the questionnaire. Alcohol consumption status
was divided into alcohol users (alcohol consumption) and non-alcohol users. Comorbidities were operationally
defined based on medical claims according to the ICD-10 codes (hypertension, I10–I13 or I15; diabetes mellitus,
E11–E14; dyslipidemia, E7817; chronic kidney diseases: glomerular filtration rate, calculated by the Modification
of Diet in Renal Disease, lower than 60 ml/min/1.73 m2). Those who were medical benefit beneficiaries and were
included in the lowest income level quartile were defined as low-income level. Furthermore, systolic/diastolic
blood pressure and laboratory results, including random glucose, glomerular filtration rate, and total cholesterol,
were obtained during ambulatory health examination visits after the index stroke.ht g
yt
The primary composite outcome of this study was the incidence of any fracture after ischemic stroke, includ-
ing the vertebral, hip, and other types of fractures. Vertebral fracture was defined when both ICD codes (S12.0,
S12.1, S12.2, S22.0, S22.1, S32.0, M48.4, or M48.5) and at least 2-time outpatient visits were confirmed18–20. Hip fracture was defined when both ICD codes (S72.0, S72.1, or 72.2) and at least one hospital admission were
confirmed19,21–23. Other fractures were defined when ICD codes (S42.0, S42.2, S42.3, S52.5, S52.6, S82.3, S82.5,
and S82.6) and at least 2-time outpatient visits were confirmed20. The secondary outcomes included the occur-
rence of vertebral and hip fractures. The date of the second health examination was defined as the index date,
and the participants were followed up until the occurrence of the primary outcome or December 31, 2019,
whichever came first. Statistical analysis y
We used a complete case analysis approach. Participants with missing or incomplete data were excluded from
the study. The baseline and demographic characteristics were presented using the mean ± standard deviation for
continuous variables and numbers with frequencies for categorical variables. Differences between study groups
were compared using one-way analysis of variance or the Chi-square test, as appropriate. The annual incidence
of the outcome variables was calculated by dividing the number of outcome events by 1,000 person-years. Multivariable Cox proportional hazard regression analyses were conducted to estimate the crude and adjusted
hazard ratios (HR) and 95% confidence intervals (CI) for the association between changes in exercise habits
and the incidence of fractures. The covariates adjusted for the analysis were determined according to the priori
knowledge with sequential steps as follows: Model 1 for age and sex and Model 2 for age, sex, comorbidities
(hypertension, dyslipidemia, diabetes mellitus, and chronic kidney diseases), and smoking, alcohol consumption,
and low-income status. The persistent non-exercisers group were used as the reference group. Subgroup analyses
were performed according to age (40–65, 65 + years), sex, and the presence of other comorbidities. To identify
whether the degree of exercise was associated with incident fractures, we stratified the post-stroke exercise groups
into two subgroups by MET-min/week with a cutoff of 1000.t g
p
yf
Statistical analyses were performed using SAS 9.4 software (SAS Institute, Cary, NC, USA), and 2-sided
p < 0.05 were considered statistically significant. Ethical approvalh pp
The study was approved by the Institutional Review Board (IRB) of the Dongtan Sacred Heart Hospital (IRB
umber: HDT 2023-01-010). Covariates and outcome variables nformation on all covariates was collected based on the second health examination. We obtained demographic
ata, including age, sex, years of education, height, weight, and waist circumference. Lifestyle risk behaviors 2
ports | (2023) 13:16715 |
https://doi.org/10.1038/s41598-023-44031-8
Figure 1. Flowchart of study. Scientific Reports | (2023) 13:16715 | https://doi.org/10.1038/s41598-023-44031-8 https://doi.org/10.1038/s41598-023-44031-8 www.nature.com/scientificreports/ Results Number of patients
Number of events
IR (per 1000 PY)
aHR (95% CI) Model 1
aHR (95% CI)
Model 2
All-cause fracture
Persistent non-exerciser
74,647
7611
25.22
1 (Ref)
1 (Ref)
Exercise dropouts
37,106
3117
20.34
0.933 (0.895, 0.973)
0.938 (0.900, 0.978)
New exerciser
36,821
2925
18.88
0.925 (0.887, 0.966)
0.932 (0.893, 0.973)
Persistent exerciser
53,660
2968
13.15
0.800 (0.766, 0.836)
0.815 (0.780, 0.852)
Vertebral fracture
Persistent non-exerciser
74,647
4093
13.18
1 (Ref)
1 (Ref)
Exercise dropouts
37,106
1593
10.13
0.913 (0.861, 0.967)
0.918 (0.866, 0.973)
New exerciser
36,821
1443
9.08
0.894 (0.841, 0.949)
0.900 (0.847, 0.956)
Persistent exerciser
53,660
1270
5.52
0.703 (0.659, 0.750)
0.716 (0.671, 0.764)
Hip fracture
Persistent non-exerciser
74,647
1201
3.77
1 (Ref)
1 (Ref)
Exercise dropouts
37,106
425
2.65
0.848 (0.759, 0.948)
0.861 (0.771, 0.963)
New exerciser
36,821
390
2.41
0.881 (0.785, 0.989)
0.904 (0.805, 1.014)
Persistent exerciser
53,660
340
1.46
0.726 (0.641, 0.822)
0.768 (0.678, 0.870) Table 1. Results Baseline characteristics and incident fractures according to the changes in exercise habit in patients
Total (n = 202,234)
Persistent non-exerciser
(n = 74,647)
Exercise dropouts
(n = 37,106)
New exerciser (n = 36,821)
Persistent exerciser
(n = 53,660)
P-value
Baseline characteristics
Age at stroke onset
63.59 ± 10.42
65.86 ± 10.28
64.29 ± 10.2
62.82 ± 10.24
60.45 ± 10.01
< 0.001
Sex, male
110,171 (54.48)
34,950 (46.82)
20,443 (55.09)
19,905 (54.06)
34,873 (64.99)
< 0.001
Current smoker
25,434 (12.58)
9402 (12.6)
4492 (12.11)
4737 (12.86)
6803 (12.68)
0.013
Any alcohol consumption
52,796 (26.11)
14,261 (19.1)
8584 (23.13)
10,082 (27.38)
19,869 (37.03)
< 0.001
Low income
33,434 (16.96)
13,186 (18.13)
6137 (16.98)
6263 (17.42)
7848 (15.01)
< 0.001
Obesity
78,947 (39.04)
29,124 (39.02)
14,386 (38.77)
14,478 (39.32)
20,959 (39.06)
0.498
Body mass index
24.28 ± 3.11
24.22 ± 3.27
24.24 ± 3.11
24.32 ± 3.08
24.34 ± 2.9
< 0.001
Diabetes mellitus
51,307 (25.37)
20,067 (26.88)
9761 (26.31)
9158 (24.87)
12,321 (22.96)
< 0.001
Hypertension
130,097 (64.33)
50,290 (67.37)
24,213 (65.25)
23,371 (63.47)
32,223 (60.05)
< 0.001
Dyslipidemia
112,013 (55.39)
41,121 (55.09)
20,684 (55.74)
20,578 (55.89)
29,630 (55.22)
0.030
Chronic kidney disease
25,012 (12.37)
10,943 (14.66)
4785 (12.9)
4243 (11.52)
5041 (9.39)
< 0.001
Random glucose
105.64 ± 29.06
106.36 ± 31.04
106.29 ± 29.67
105.07 ± 28.13
104.58 ± 26.23
< 0.001
Total cholesterol
181.17 ± 41.59
182.18 ± 41.84
180.69 ± 41.68
180.87 ± 41.73
180.31 ± 41.03
< 0.001
Glomerular filtration rate
84.19 ± 43.79
82.84 ± 38.78
83.7 ± 42.96
84.86 ± 45.49
85.97 ± 49.33
< 0.001
Systolic blood pressure
126.95 ± 15.26
127.7 ± 15.76
127.13 ± 15.4
126.69 ± 15.08
125.97 ± 14.5
< 0.001
Diastolic blood pressure
77.16 ± 9.85
77.13 ± 10.01
77.13 ± 9.91
77.21 ± 9.77
77.19 ± 9.65
0.522
Incident fracture
All types of fracture
16,621 (8.22)
7611 (10.2)
3117 (8.4)
2925 (7.94)
2968 (5.53)
< 0.001
Vertebral fracture
8399 (4.15)
4093 (5.48)
1593 (4.29)
1443 (3.92)
1270 (2.37)
< 0.001
Hip fracture
2356 (1.16)
1201 (1.61)
425 (1.15)
390 (1.06)
340 (0.63)
< 0.001 g
g
p
with ischemic stroke. Table 2. Risk for All-cause fracture, vertebral fracture and hip fracture according to the changes in exercise
habit. IR incidence rate, aHR adjusted hazard ratio, PY person-years. Results # Model 1: age and sex-adjusted, Model
2: Model 1 + smoking status, alcohol status, low income, history of diabetes, hypertension, dyslipidemia and
chronic kidney disease adjusted. Number of patients
Number of events
IR (per 1000 PY)
aHR (95% CI) Model 1
aHR (95% CI)
Model 2
All-cause fracture
Persistent non-exerciser
74,647
7611
25.22
1 (Ref)
1 (Ref)
Exercise dropouts
37,106
3117
20.34
0.933 (0.895, 0.973)
0.938 (0.900, 0.978)
New exerciser
36,821
2925
18.88
0.925 (0.887, 0.966)
0.932 (0.893, 0.973)
Persistent exerciser
53,660
2968
13.15
0.800 (0.766, 0.836)
0.815 (0.780, 0.852)
Vertebral fracture
Persistent non-exerciser
74,647
4093
13.18
1 (Ref)
1 (Ref)
Exercise dropouts
37,106
1593
10.13
0.913 (0.861, 0.967)
0.918 (0.866, 0.973)
New exerciser
36,821
1443
9.08
0.894 (0.841, 0.949)
0.900 (0.847, 0.956)
Persistent exerciser
53,660
1270
5.52
0.703 (0.659, 0.750)
0.716 (0.671, 0.764)
Hip fracture
Persistent non-exerciser
74,647
1201
3.77
1 (Ref)
1 (Ref)
Exercise dropouts
37,106
425
2.65
0.848 (0.759, 0.948)
0.861 (0.771, 0.963)
New exerciser
36,821
390
2.41
0.881 (0.785, 0.989)
0.904 (0.805, 1.014)
Persistent exerciser
53,660
340
1.46
0.726 (0.641, 0.822)
0.768 (0.678, 0.870) Table 2. Risk for All-cause fracture, vertebral fracture and hip fracture according to the changes in exercise
habit. IR incidence rate, aHR adjusted hazard ratio, PY person-years. # Model 1: age and sex-adjusted, Model
2: Model 1 + smoking status, alcohol status, low income, history of diabetes, hypertension, dyslipidemia and
chronic kidney disease adjusted. Results However, among those who exercised < 1000
METs-min/week, exercising ≥ 1000 METs-min/week post-stroke was not particularly beneficial compared to
working < 1000 METs-min/week post-stroke (Supplemental Table S2). Scientific Reports | (2023) 13:16715 | https://doi.org/10.1038/s41598-023-44031-8 www.nature.com/scientificreports/ www.nature.com/scientificreports/
Discussion
In this nationwide cohort study, we observed the following findings: first, during a median of 4.13 years after
stroke, 8.22% of the patients experienced incident fractures. As expected, the incidence of fractures was higher
in the female patients. Second, both the initiation and continuation of regular exercise after acute stroke were
significantly associated with a reduced risk of all types of fractures Exercise dropouts after stroke were also
Table 1. Baseline characteristics and incident fractures according to the changes in exercise habit in patients
with ischemic stroke. Results Total (n = 202,234)
Persistent non-exerciser
(n = 74,647)
Exercise dropouts
(n = 37,106)
New exerciser (n = 36,821)
Persistent exerciser
(n = 53,660)
P-value
Baseline characteristics
Age at stroke onset
63.59 ± 10.42
65.86 ± 10.28
64.29 ± 10.2
62.82 ± 10.24
60.45 ± 10.01
< 0.001
Sex, male
110,171 (54.48)
34,950 (46.82)
20,443 (55.09)
19,905 (54.06)
34,873 (64.99)
< 0.001
Current smoker
25,434 (12.58)
9402 (12.6)
4492 (12.11)
4737 (12.86)
6803 (12.68)
0.013
Any alcohol consumption
52,796 (26.11)
14,261 (19.1)
8584 (23.13)
10,082 (27.38)
19,869 (37.03)
< 0.001
Low income
33,434 (16.96)
13,186 (18.13)
6137 (16.98)
6263 (17.42)
7848 (15.01)
< 0.001
Obesity
78,947 (39.04)
29,124 (39.02)
14,386 (38.77)
14,478 (39.32)
20,959 (39.06)
0.498
Body mass index
24.28 ± 3.11
24.22 ± 3.27
24.24 ± 3.11
24.32 ± 3.08
24.34 ± 2.9
< 0.001
Diabetes mellitus
51,307 (25.37)
20,067 (26.88)
9761 (26.31)
9158 (24.87)
12,321 (22.96)
< 0.001
Hypertension
130,097 (64.33)
50,290 (67.37)
24,213 (65.25)
23,371 (63.47)
32,223 (60.05)
< 0.001
Dyslipidemia
112,013 (55.39)
41,121 (55.09)
20,684 (55.74)
20,578 (55.89)
29,630 (55.22)
0.030
Chronic kidney disease
25,012 (12.37)
10,943 (14.66)
4785 (12.9)
4243 (11.52)
5041 (9.39)
< 0.001
Random glucose
105.64 ± 29.06
106.36 ± 31.04
106.29 ± 29.67
105.07 ± 28.13
104.58 ± 26.23
< 0.001
Total cholesterol
181.17 ± 41.59
182.18 ± 41.84
180.69 ± 41.68
180.87 ± 41.73
180.31 ± 41.03
< 0.001
Glomerular filtration rate
84.19 ± 43.79
82.84 ± 38.78
83.7 ± 42.96
84.86 ± 45.49
85.97 ± 49.33
< 0.001
Systolic blood pressure
126.95 ± 15.26
127.7 ± 15.76
127.13 ± 15.4
126.69 ± 15.08
125.97 ± 14.5
< 0.001
Diastolic blood pressure
77.16 ± 9.85
77.13 ± 10.01
77.13 ± 9.91
77.21 ± 9.77
77.19 ± 9.65
0.522
Incident fracture
All types of fracture
16,621 (8.22)
7611 (10.2)
3117 (8.4)
2925 (7.94)
2968 (5.53)
< 0.001
Vertebral fracture
8399 (4.15)
4093 (5.48)
1593 (4.29)
1443 (3.92)
1270 (2.37)
< 0.001
Hip fracture
2356 (1.16)
1201 (1.61)
425 (1.15)
390 (1.06)
340 (0.63)
< 0.001
Table 2. Risk for All-cause fracture, vertebral fracture and hip fracture according to the changes in exercise
habit. IR incidence rate, aHR adjusted hazard ratio, PY person-years. # Model 1: age and sex-adjusted, Model
2: Model 1 + smoking status, alcohol status, low income, history of diabetes, hypertension, dyslipidemia and
chronic kidney disease adjusted. Discussion
h In this nationwide cohort study, we observed the following findings: first, during a median of 4.13 years after
stroke, 8.22% of the patients experienced incident fractures. As expected, the incidence of fractures was higher
in the female patients. Second, both the initiation and continuation of regular exercise after acute stroke were
significantly associated with a reduced risk of all types of fractures. Exercise dropouts after stroke were also
mildly but significantly associated with a reduced risk of fracture. Third, in female groups, the impact of post-
stroke exercise was less prominent and exercise dropouts or new exercisers were not associated with a reduced https://doi.org/10.1038/s41598-023-44031-8 Scientific Reports | (2023) 13:16715 | www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 2. Incidence probability of all-cause fracture (A, P < 0.001), vertebral fracture (B, P < 0.001) and hip
fracture (C, P < 0.001) after ischemic stroke according to change of physical activity habits. *Adjusted for age,
sex, alcohol consumption, smoking, income, diabetes, hypertension, dyslipidemia and chronic kidney disease. Figure 2. Incidence probability of all-cause fracture (A, P < 0.001), vertebral fracture (B, P < 0.001) and hip
fracture (C, P < 0.001) after ischemic stroke according to change of physical activity habits. *Adjusted for age,
sex, alcohol consumption, smoking, income, diabetes, hypertension, dyslipidemia and chronic kidney disease. Table 3. Subgroup analyses according to the age and sex group and risk for all-cause fracture. IR incidence
rate, aHR adjusted hazard ratio, PY person-years. # Model 1: age and sex adjusted, Model 2: Model 1 + smoking
status, alcohol status, low income, history of diabetes, hypertension, dyslipidemia and chronic kidney disease
adjusted. Exercise habit change status
Number of patients
Number of events of all-
cause dementia
IR (per 1000 PY)
aHR (95% CI) Model 1
aHR (95% CI) Model 2
Male
Persistent non-exerciser
34,950
2349
16.90
1 (Ref)
1 (Ref)
Exercise dropouts
20,443
1118
13.40
0.844 (0.786, 0.906)
0.851 (0.793, 0.914)
New exerciser
19,905
1053
12.64
0.863 (0.802, 0.928)
0.871 (0.810, 0.937)
Persistent exerciser
34,873
1386
9.41
0.733 (0.686, 0.784)
0.752 (0.703, 0.804)
Female
Persistent non-exerciser
39,697
5262
32.32
1 (Ref)
1 (Ref)
Exercise dropouts
16,663
1999
28.63
0.981 (0.932, 1.033)
0.985 (0.936, 1.038)
New exerciser
16,916
1872
26.12
0.956 (0.907, 1.008)
0.963 (0.913, 1.015)
Persistent exerciser
18,787
1582
20.18
0.845 (0.798, 0.894)
0.856 (0.809, 0.906)
p for interaction
< 0.001
0.002
40–64 years
Persistent non-exerciser
34,950
1,254
8.87
1 (Ref)
1 (Ref)
Exercise dropouts
20,443
595
7.02
0.862 (0.782, 0.950)
0.873 (0.792, 0.963)
New exerciser
19,905
547
6.47
0.881 (0.797, 0.975)
0.892 (0.807, 0.987)
Persistent exerciser
34,873
600
4.02
0.655 (0.594, 0.722)
0.675 (0.612, 0.745)
≥ 65 years
Persistent non-exerciser
39,697
2,839
16.78
1 (Ref)
1 (Ref)
Exercise dropouts
16,663
998
13.77
0.940 (0.875, 1.011)
0.943 (0.877, 1.013)
New exerciser
16,916
896
12.07
0.897 (0.832, 0.967)
0.900 (0.835, 0.971)
Persistent exerciser
18,787
670
8.29
0.742 (0.681, 0.808)
0.747 (0.686, 0.814)
p for interaction
0.198
0.341 Table 3. Subgroup analyses according to the age and sex group and risk for all-cause fracture. IR incidence
rate, aHR adjusted hazard ratio, PY person-years. www.nature.com/scientificreports/ Furthermore, specific neurological impairments such as hemineglect and attention deficits may increase the risk
of falls and subsequent fractures27,28. Considering this, proper physical activity and rehabilitation after a stroke
may not only improve functional outcomes, but also reduce the chances of falls among the stroke population. Our results regarding the reduced risk of fracture in physically active stroke survivors are consistent with the
findings of previous studies. However, most studies have only assessed the status of physical activity at a single
time point, rather than the changes before and after the index stroke. Furthermore, no randomized clinical trials
have been conducted with fracture as an outcome because they require a large sample size and follow-up dura-
tion, as fracture incidence is not as high29. Several studies have been conducted to determine whether exercise
could reduce the risk factors for fractures, such as fall frequency, balance, muscle mass, and bone mineral density,
which may reflect future fracture risk30–34. Furthermore, even for stroke survivors, the benefits of physical activity
may outweigh the potential injuries caused by exercise. In our study, moderate-to-vigorous exercise after stroke
was associated with a reduced risk of fracture. Furthermore, regular physical activity before stroke onset also
had protective effects, even when participants failed to continue regular physical activity after the stroke. These
findings are in line with those of previous studies in the general population without any history of stroke, which
report that habitual exercise changes are associated with incident fractures6.hiff The identified sex differences in fracture incidence and reduced protective effects of exercise were consistent
with those of previous studies. Females have a higher prevalence of osteoporosis35 and tend to have more severe
neurological deficits and less recovery from stroke36. Furthermore, even with regular physical activity, the degree
of increase in muscle mass and bone density is limited in females. As muscle mass and bone density play major
roles in protecting bones after falls or trauma, physical activity may have less of an impact on female patients37,38. p
gt
p y
y
y
p
p
Our study had several limitations. First, as our database was based on the claims dataset of the national insur-
ance system, we lacked important clinical variables associated with ischemic stroke, including stroke severity,
antithrombotic use, and discharge status, which may affect the risk of fractures after stroke. References 1. Brown, D. L., Morgenstern, L. B., Majersik, J. J., Kleerekoper, M. & Lisabeth, L. D. Risk of fractures after stroke. Cerebrovasc. Dis
25, 95–99. https://doi.org/10.1159/000111997 (2008).t p
g
2. Dennis, M. S., Lo, K. M., McDowall, M. & West, T. Fractures after stroke: Frequency, types, and associations. Stroke. 33, 728–734
https://doi.org/10.1161/hs0302.103621 (2002).t p
g
2. Dennis, M. S., Lo, K. M., McDowall, M. & West, T. Fractures after stroke: Frequency, types, and associations. Stroke. 33, 728–734. https://doi.org/10.1161/hs0302.103621 (2002).t p
g
(
)
3. Pouwels, S. et al. Risk of hip/femur fracture after stroke: A population-based case-control study. Stroke. 40, 3281–3285. https://
doi.org/10.1161/STROKEAHA.109.554055 (2009). p
g
3. Pouwels, S. et al. Risk of hip/femur fracture after stroke: A population-based case-control study. Stroke. 40, 3281–3285. https://
doi.org/10.1161/STROKEAHA.109.554055 (2009). g
4. Forster, A. & Young, J. Incidence and consequences of falls due to stroke: A systematic inquiry. BMJ. 311, 83–86. https://doi.org/
10.1136/bmj.311.6997.83 (1995). j
5. Coupland, C., Wood, D. & Cooper, C. Physical inactivity is an independent risk factor for hip fracture in the elderly. J. Epidemiol. Community Health. 47, 441–443. https://doi.org/10.1136/jech.47.6.441 (1993). j
5. Coupland, C., Wood, D. & Cooper, C. Physical inactivity is an independent risk factor for hip fracture in the elderly. J. Epidemiol. Community Health. 47, 441–443. https://doi.org/10.1136/jech.47.6.441 (1993). y
p
g
j
6. Han, S. et al. Changes in physical activity and risk of fracture: A Korean nationwide population-based cohort study. Sci. Rep. 10,
16266. https://doi.org/10.1038/s41598-020-73495-1 (2020).f 6. Han, S. et al. Changes in physical activity and risk of fracture: A Korean nationwide population-based cohort study. Sci. Rep. 10,
16266. https://doi.org/10.1038/s41598-020-73495-1 (2020).f p
g
(
)
7. Kemmler, W., Haberle, L. & von Stengel, S. Effects of exercise on fracture reduction in older adults: A systematic review and m
analysis. Osteoporos Int. 24, 1937–1950. https://doi.org/10.1007/s00198-012-2248-7 (2013). p
g
(
)
7. Kemmler, W., Haberle, L. & von Stengel, S. Effects of exercise on fracture reduction in older adults: A systematic review and meta-
analysis. Osteoporos Int. 24, 1937–1950. https://doi.org/10.1007/s00198-012-2248-7 (2013). y
p
p
g
8. Billinger, S. A. et al. Physical activity and exercise recommendations for stroke survivors: A statement for healthcare professionals
from the American Heart Association/American Stroke Association. Stroke. 45, 2532–2553. https://doi.org/10.1161/STR.00000
00000000022 (2014). 8. Billinger, S. A. et al. Physical activity and exercise recommendations for stroke survivors: A statement for healthcare professionals
from the American Heart Association/American Stroke Association. Stroke. 45, 2532–2553. Conclusion Initiating or continuing moderate-to-vigorous regular physical activity after acute ischemic stroke is associated
with a significantly lower risk of incident fractures, including hip, vertebral, and other types. The promotion of
exercise among ambulatory stroke survivors may reduce the risk of fractures. www.nature.com/scientificreports/ Second, as our study
only recruited participants who were both willing and able to attend ambulatory health examinations before and
after stroke, our study population may be limited to participants with mild stroke with functional independ-
ence. Furthermore, most participants in our study were ambulatory post-stroke and capable of completing the
questionnaires. This baseline functional capability suggests that they might have engaged in healthier lifestyle
behaviors compared to the broader stroke survivor population, introducing a potential for bias. However, given
the expansive scale of our study population, any such bias is anticipated to be minimal. Even so, considering the
consistent functional status across our cohort, it’s imperative to highlight that our findings might not fully extend
to all stroke survivors, especially those with greater functional impairments. Third, exercise habits in our study
were based on self-reports, which may not provide an exact representation of the actual duration and intensity
of the exercise. This method might also not specify whether the exercise was part of a structured rehabilitation
program or initiated independently by the participants. Nonetheless, we regard rehabilitation exercises as a subset
of physical activity. Given that dedicated participation in rehabilitation exercises is postulated to aid in fracture
prevention following a stroke, we chose not to differentiate explicitly between general physical activity and tar-
geted rehabilitation exercises. Despite these inherent limitations, this large-scale nationwide cohort study used
real-world data to reveal the effect of changes in exercise habits on the risk of subsequent fractures after stroke. Data availabilityh The data that support the findings of this study are available from National Health Insurance Sharing Service of
Korea but restrictions apply to the availability of these data, which were used under license for the current study,
and so are not publicly available. Data are however available from the corresponding author Minwoo Lee, upon
reasonable request and with permission of National Health Insurance Sharing Service of Korea. Received: 21 July 2023; Accepted: 3 October 2023 www.nature.com/scientificreports/ # Model 1: age and sex adjusted, Model 2: Model 1 + smoking
status, alcohol status, low income, history of diabetes, hypertension, dyslipidemia and chronic kidney disease
adjusted. Table 3. Subgroup analyses according to the age and sex group and risk for all-cause fracture. IR incidence
rate, aHR adjusted hazard ratio, PY person-years. # Model 1: age and sex adjusted, Model 2: Model 1 + smoking
status, alcohol status, low income, history of diabetes, hypertension, dyslipidemia and chronic kidney disease
adjusted. fracture risk. Fourth, exercising ≥ 1000 MET-min/week, namely moderate-to-vigorous exercise, was significantly
associated with a reduced risk of fractures than mild-intensity physical activity. In our study, the cumulative incidence of fracture in post-stroke survivors was 8.22% during a median of
4.13 years, which is slightly lower than that in the multicenter stroke registry of Korea, which reported a 13%
cumulative incidence at 4 years24. While mild-to-moderate stroke patients had the highest cumulative incidence
in that study, those who were either independent or infirm enough to be bedridden had a lower incidence of
fractures. As our study population mostly consisted of independent to mild stroke patients who were able and
willing to visit ambulatory health examinations provided by the government after stroke, our population may
have a healthier lifestyle, which may explain the lower incidence of fractures of any kind compared to the multi-
center prospective stroke registry study. The incidence of fractures was almost 1.6-fold higher in our stroke cohort
than in the general Korean population without a previous history of stroke. Overall, the incidence of fractures
in the Korean stroke population was slightly higher than that in the Caucasian population. US and Canadian
studies have shown the 2-year incidence of fractures in stroke survivors to be 1.7–6.1% in recent studies25,26.t y
Stroke survivors are at an elevated risk of fractures on their paretic sides, most often in the hip or vertebrae. Specifically, ambulatory stroke survivors, such as our study population, often have poor balance, lower bone mass,
and disuse osteoporosis in the paretic arm or legs27,28. Therefore, fragile fractures are common in stroke survivors. Scientific Reports | (2023) 13:16715 | https://doi.org/10.1038/s41598-023-44031-8 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Trends of incidence, mortality, and future projection of spinal fractures in Korea using nationwide claims data. J. ,
g
J
,
(
)
18. Kim, T. Y. et al. Trends of incidence, mortality, and future projection of spinal fractures in Korea using nationwide claims data. J. Korean Med. Sci. 31, 801–805. https://doi.org/10.3346/jkms.2016.31.5.801 (2016).f i. 31, 801–805. https://doi.org/10.3346/jkms.2016.31.5.801 (2016) p
g
j
19. Jang, S. Y. et al. What is the difference in the risk of suicide death between spine fracture in patients older than 65 years and matched
controls? A large-database study from South Korea. Clin. Orthop. Relat. Res. 478, 2422–2430. https://doi.org/10.1097/corr.00000
00000001414 (2020).h 0. Lee, K. B. et al. The epidemiology of fracture in patients with acute ischemic stroke in Korea. J. Korean Med. Sci. 34, e164. https://
doi.org/10.3346/jkms.2019.34.e164 (2019). 21. Park, D. H. et al. Long-term proton pump inhibitor use and risk of osteoporosis and hip fractures: A nationwide population-based
and multicenter cohort study using a common data model. J. Gastroenterol. Hepatol. 37, 1534–1543. https://doi.org/10.1111/jgh.
15879 (2022). (
)
22. Gu, K. M. et al. Acute exacerbation of COPD increases the risk of hip fractures: A nested case-control study from the Korea Nat
Health Insurance Service. Korean J. Intern. Med. 37, 631–638. https://doi.org/10.3904/kjim.2021.152 (2022). 23. Ahn, S. H. et al. Osteoporosis and osteoporotic fracture fact sheet in Korea. J. Bone Metab. 27, 281–290. https://doi.org/10.11005/
jbm.2020.27.4.281 (2020).h j
24. Lee, K. B. et al. The epidemiology of fracture in patients with acute ischemic stroke in Korea. J Kor. Med. Sci. 34, 1 (2019). l
l
k f f
ft
k
l
f
h
k
l
h
d 5. Kapral, M. K. et al. Risk of fractures after stroke: Results from the Ontario Stroke Registry. Neurology. 88, 57–64. https://doi.org
10.1212/WNL.0000000000003457 (2017).t 26. Whitson, H. E. et al. Adding injury to insult: fracture risk after stroke in veterans. J. Am. Geriatr. Soc. 54, 1082–1088. https://doi.
org/10.1111/j.1532-5415.2006.00769.x (2006).i g
j
7. Hyndman, D. & Ashburn, A. People with stroke living in the community: Attention deficits, balance, ADL ability and falls. Disabil
Rehabil. 25, 817–822. https://doi.org/10.1080/0963828031000122221 (2003). g
28. Mackintosh, S. F., Hill, K. D., Dodd, K. J., Goldie, P. A. & Culham, E. G. Balance score and a history of falls in hospital predict
recurrent falls in the 6 months following stroke rehabilitation. Arch. Phys. Med. Rehabil. 87, 1583–1589. https://doi.org/10.1016/j.
apmr.2006.09.004 (2006).fi p
9. Karlsson, M. Author contributions D.Y.C. and M.L. wrote the main manuscript. K.H. performed the statistical analysis. J.H.L. and B.Y.C. reviewed
he main manuscript. K.H.Y. supervised this study. All authors reviewed the manuscript. www.nature.com/scientificreports/ H., Gonyea, W. J. & Stray-Gundersen, J. Contrasts in muscle and myofibers of elite male and female
bodybuilders. J. Appl. Physiol. 67, 24–31. https://doi.org/10.1152/jappl.1989.67.1.24 (1989).i y
J
pp
y
,
p
g
j pp
(
)
38. Giladi, M., Milgrom, C., Simkin, A. & Danon, Y. Stress fractures: Identifiable risk factors. Am. J. Sports Med. 19, 647–652. https://
doi.org/10.1177/036354659101900617 (1991). y
pp
y
g
j
38. Giladi, M., Milgrom, C., Simkin, A. & Danon, Y. Stress fractures: Identifiable risk factors. Am. J. Sports Med. 19, 647–652. htt
doi.org/10.1177/036354659101900617 (1991). References https://doi.org/10.1161/STR.00000
00000000022 (2014). https://doi.org/10.1038/s41598-023-44031-8 Scientific Reports | (2023) 13:16715 | www.nature.com/scientificreports/ Has exercise an antifracture efficacy in women?. Scand. J. Med. Sci. Sports. 14, 2–15. https://doi.org/10.1111/j.1600-
0838.2003.00322.x (2004).t (
)
0. Dean, C. M. & Shepherd, R. B. Task-related training improves performance of seated reaching tasks after stroke. A randomized
controlled trial. Stroke. 28, 722–728. https://doi.org/10.1161/01.str.28.4.722 (1997). g
31. Duncan, P. et al. A randomized, controlled pilot study of a home-based exercise program for individuals with mild and mod
stroke. Stroke. 29, 2055–2060. https://doi.org/10.1161/01.str.29.10.2055 (1998).i p
g
2. Macko, R. F. et al. Treadmill exercise rehabilitation improves ambulatory function and cardiovascular fitness in patients with
h
i
t
k
A
d
i
d
t
ll d t i l St k
36 2206 2211 htt
//d i
/10 1161/01 STR 0000181076 91805 89 (2005) g
32. Macko, R. F. et al. Treadmill exercise rehabilitation improves ambulatory function and cardiovascular fitness in patients with
chronic stroke: A randomized, controlled trial. Stroke. 36, 2206–2211. https://doi.org/10.1161/01.STR.0000181076.91805.89 (2005).l 32. Macko, R. F. et al. Treadmill exercise rehabilitation improves ambulatory function and cardiovascular fitness in patients with
chronic stroke: A randomized, controlled trial. Stroke. 36, 2206–2211. https://doi.org/10.1161/01.STR.0000181076.91805.89 (2005). 33. Marigold, D. S. et al. Exercise leads to faster postural reflexes, improved balance and mobility, and fewer falls in older persons with p
g
3. Marigold, D. S. et al. Exercise leads to faster postural reflexes, improved balance and mobility, and fewer falls in older persons with
chronic stroke. J. Am. Geriatr. Soc. 53, 416–423. https://doi.org/10.1111/j.1532-5415.2005.53158.x (2005). p
g
j
34. Vearrier, L. A., Langan, J., Shumway-Cook, A. & Woollacott, M. An intensive massed practice approach to retraining bal
post-stroke. Gait. Posture. 22, 154–163. https://doi.org/10.1016/j.gaitpost.2004.09.001 (2005). 35. Lee, Y. K., Yoon, B. H. & Koo, K. H. Epidemiology of osteoporosis and osteoporotic fractures in South Korea. Endocrinol. Metab. (Seoul). 28, 90–93. https://doi.org/10.3803/EnM.2013.28.2.90 (2013).ft 35. Lee, Y. K., Yoon, B. H. & Koo, K. H. Epidemiology of osteoporosis and osteoporotic fractures in South Korea. Endocrinol. M
(Seoul). 28, 90–93. https://doi.org/10.3803/EnM.2013.28.2.90 (2013).ft p
g
36. Kim, J. S., Lee, K. B., Roh, H., Ahn, M. Y. & Hwang, H. W. Gender differences in the functional recovery after acute stroke. J. Neurol. 6, 183–188. https://doi.org/10.3988/jcn.2010.6.4.183 (2010).i p
g
j
7. Alway, S. E., Grumbt, W. H., Gonyea, W. J. & Stray-Gundersen, J. Contrasts in muscle and myofibers of elite male and female
bodybuilders. J. Appl. Physiol. 67, 24–31. https://doi.org/10.1152/jappl.1989.67.1.24 (1989).i p
g
j
37. Alway, S. E., Grumbt, W. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 9. Eng, J. J., Pang, M. Y. & Ashe, M. C. Balance, falls, and bone health: role of exercise in reducing fracture risk after stroke. J. Rehabil. Res. Dev. 45, 297–313. https://doi.org/10.1682/jrrd.2007.01.0014 (2008). p
g
j
10. Shin, D. W., Cho, J., Park, J. H. & Cho, B. National General Health Screening Program in Korea: History, current status, and fu
direction. Precis. Future Med. 6, 9–31. https://doi.org/10.23838/pfm.2021.00135 (2022).fi p
g
p
1. Cha, M. J. et al. Effectiveness and safety of non-vitamin K antagonist oral anticoagulants in asian patients with atrial fibrillation
Stroke. 48, 3040–3048. https://doi.org/10.1161/STROKEAHA.117.018773 (2017).i f
y
g
8, 3040–3048. https://doi.org/10.1161/STROKEAHA.117.018773 p
g
2. Kang, S. H. et al. Risk of ischemic stroke in patients with non-valvular atrial fibrillation not receiving oral anticoagulants—Korean
nationwide population-based study. Circ. J. 81, 1158–1164. https://doi.org/10.1253/circj.CJ-16-1267 (2017). 3. Lee, S. R. et al. Non-vitamin K antagonist oral anticoagulants in asian patients with supranormal renal function. Stroke. 50
1480–1489. https://doi.org/10.1161/STROKEAHA.118.024264 (2019).i p
g
4. Lee, S. R. et al. Optimal rivaroxaban dose in Asian patients with atrial fibrillation and normal or mildly impaired renal function
Stroke. 50, 1140–1148. https://doi.org/10.1161/STROKEAHA.118.024210 (2019). p
g
(
)
5. Oh, J. Y., Yang, Y. J., Kim, B. S. & Kang, J. H. Validity and reliability of Korean version of international physical activity question-
naire (IPAQ) short form. J. Korean Acad. Fam. Med. 28, 532–541 (2007). 6. Jeong, S. W. et al. Mortality reduction with physical activity in patients with and without cardiovascular disease. Eur. Heart J. 40
3547–3555. https://doi.org/10.1093/eurheartj/ehz564 (2019). 3547–3555. https://doi.org/10.1093/eurheartj/ehz564 (2019). p
g
j
7. Choi, E.-K. Cardiovascular research using the Korean National health information database. Korean Circ. J. 50, 754–772 (2020). 8 Ki
T Y t l T
d
f i
id
t lit
d f t
j
ti
f
i
l f
t
i K
i
ti
id
l i
d t
J p
g
j
17. Choi, E.-K. Cardiovascular research using the Korean National health information database. Korean Circ. J. 50, 754–772 (2020). 18. Kim, T. Y. et al. Trends of incidence, mortality, and future projection of spinal fractures in Korea using nationwide claims data. J. Korean Med. Sci. 31, 801–805. https://doi.org/10.3346/jkms.2016.31.5.801 (2016).f p
g
j
Choi, E.-K. Cardiovascular research using the Korean National health information database. Korean Circ. J. 50, 754–772 (2020). Kim, T. Y. et al. Fundingh Funding
This research was supported by Basic Science Research Program through the National Research Foundation of
Korea (NRF) funded by the Ministry of Education (2022R1F1A1076131) and by the Bio&Medical Technology
Development Program of the National Research Foundation (NRF) funded by the Korean government (MSIT)
(No. RS-2023-00223501). Competing interests h 7
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Intimate partner violence among ever-married Afghan women: patterns, associations and attitudinal acceptance
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Abstract Intimate partner violence (IPV) is one of the most prevalent forms of violence that women suffer globally. Women in
Afghanistan have been exposed to high levels of IPV which coincided with high levels of conflict during more than four
decades. We cross-sectionally examined the Afghanistan Demographic and Health Survey responses of 21,234 ever-married
Afghan women. We first performed the frequency distribution analysis to determine the prevalence of IPV and the basic
socio-demographic characteristics of the participants. Subsequently we examined the relationship between the independent
and dependent variables followed by the bivariate and survey versions of logistic regression analyses. We report odds ratios
in order to depict the strength and direction of the associations between the IPV and selected independent variables. P-values
less than 0.05 were considered statistically significant. The analyses showed that 55.54% of Afghan women experienced
some form of physical, emotional, or sexual violence by their intimate partners during the recall period partners. The most
common form of IPV found was physical violence (50.52%). Factors such as being exposed to inter-parental violence
(respondent woman’s father physically abused her mother) (adjusted OR=3.69, CI=3.31–4.10) and respondent’s acceptance
of IPV (aOR=1.85, 1.51–2.26) were associated with increased exposure to IPV. Having a spouse with at least a primary
education (aOR=0.76, CI=0.64–0.91) or a respondent with at least a primary education (aOR=0.82, CI=0.68–0.98) was
associated with lower exposure to reported IPV. The lifetime experience of IPV occurs to a high extent among Afghan
women, and several socio-demographic factors have predisposing attributes. IPV policy formulation and strategizing may
benefit from considering these factors. Keywords Domestic violence · Women’s health · Sexual violence · Attitudinal acceptance · Inter-parental violence Keywords Domestic violence · Women’s health · Sexual violence · Attitudinal acceptance · Inter Intimate partner violence among ever-married Afghan women:
patterns, associations and attitudinal acceptance Rehana Shinwari1 · Michael Lowery Wilson1 · Olumide Abiodun2
· Masood Ali Shaikh2 Received: 3 March 2021 / Accepted: 9 May 2021
© The Author(s) 2021
/ Published online: 13 July 2021 Michael Lowery Wilson
michael.wilson@uni-heidelberg.de https://doi.org/10.1007/s00737-021-01143-2
Archives of Women’s Mental Health (2022) 25:95–105 https://doi.org/10.1007/s00737-021-01143-2
Archives of Women’s Mental Health (2022) 25:95–105 ORIGINAL ARTICLE Olumide Abiodun
abiodun.olumide@ictuniversity.org 2
Injury Epidemiology and Prevention (IEP) Research Group,
Turku Brain Injury Centre, Turku University Hospital
and University of Turku, Turku, Finland Introduction and/or psychological violence — which are known to
disproportionately affect women. Of the 87,000 homicidal
deaths of women in 2017, more than one-third (34%) had
been murdered by their current or former intimate partner. Asia (20,000) followed by Africa (19,000) are the most
affected world regions by reported deaths resulting from
domestic violence (Crime on Drugs UNO 2018). There is a
significant gendered gradient in the prevalence of intimate
partner homicide, with 82% of the victims being female
(UNODC 2019). A WHO study investigating violence in
10 countries estimated that between 15 and 71% of women
affected by IPV disclosed physical or sexual violence by a
male intimate partner at some point in their lives (Garcia-
Moreno et al. 2006). A more recent study in 46 low- and
middle-income countries showed that the prevalence of IPV
varies across countries from a prevalence rate of about 5 to
40% (Coll et al. 2020). The most common forms of reported
physical violence include pushing, slapping, punching, and
hitting with an object (Fikree et al. 2005). Intimate partner violence (IPV) is a global problem
of significant public health importance which includes
a constellation of harmful behaviors being perpetrated
within intimate partnerships — including physical, sexual, Olumide Abiodun
abiodun.olumide@ictuniversity.org 1
Heidelberg Institute of Global Health (HIGH), University
of Heidelberg, Heidelberg, Germany 2
Injury Epidemiology and Prevention (IEP) Research Group,
Turku Brain Injury Centre, Turku University Hospital
and University of Turku, Turku, Finland 2
Injury Epidemiology and Prevention (IEP) Research Group,
Turku Brain Injury Centre, Turku University Hospital
and University of Turku, Turku, Finland 96 R. Shinwari et al. and reproductive health and reproductive health rights as
per the 1994 International Conference on Population and
Development and the 2005 Beijing World Conference on
Women. Violence against women is thought to be prevalent
in all societies, but IPV is particularly common in conflict
settings (Gibbs et al. 2018; Jewkes et al. 2018) and associ-
ated with economic instability, and gender norms (Menj´ıvar
and Salcido 2002). A study of 935 married Afghan women
found a 35% one-year prevalence of emotional and/or phys-
ical violence (Gibbs et al. 2018). A 2012 survey of 4,700
Afghan households across 16 provinces showed that almost
90% of women had experienced at least one form of vio-
lence, while 62% had faced several (Humanitarian Response
2012). Methodology Our study is a secondary analysis of the Afghanistan
Demographic Health Survey (AfDHS) 2015. The detailed
methodology for the AfDHS was developed through a
collaboration between the Central Statistics Organization of
the Republic of Afghanistan and the United States Agency
for International Development and is available elsewhere
https://microdata.worldbank.org/index.php/catalog/2786. An analysis of Demographic Health Survey (DHS)
data from 10 countries showed that apart from the
partner’s alcohol consumption and inter-parental violence,
other risk factors were consistently associated with IPV. However, some factors including women’s age at marriage,
occupation, and attitude toward IPV were significantly
associated in about six out of the ten countries. The
other significant risk factors were the womens’ and their
partners’ ages, the women’s levels of education, household
wealth, and place of residence (Hindin et al. 2008). In
Europe, low-income, young age (18–24 years), and a
history of childhood physical or sexual violence were
associated with current physical/sexual violence among
women. Psychological violence was mostly experienced by
women with childhood abuse experience and among those
with a migrant background. Also, women living in countries
with high unemployment rates and areas with excessive
alcohol consumption are more likely to experience IPV
(Sanz-Barbero et al. 2018). Introduction Afghan women are, exposed to high levels of abuse,
which has possibly been exacerbated by severe levels of
conflict for more than 40 years (Humanitarian Response
2012). Additionally, Afghanistan experiences some of the
worst forms of gender inequity and was ranked 154th out of
157 nations (UNDP 2015). A social-ecological model-based characterization of
the causes and risk factors for IPV by the WHO
identified four levels of influence: individual, interpersonal,
community, and societal factors. At the individual level,
age, education, childhood abuse, inter-parental violence,
substance
abuse,
justifying behavior, and
personality
disorders were associated with increased perpetration,
victimization, or both in a relationship. Interpersonal
risk factors for IPV included conflict, male dominance,
financial burdens, polygamous unions, and a women’s
higher educational status relative to their male partners. At the community and societal levels, IPV is occurs
within a complex social fabric knitted by gender inequity,
societal norms, women’s socioeconomic status, poverty,
armed conflict, as well as weak legal and community
authorizations (Organization et al. 2012). However, the
evidence at these latter two levels remains weak (Heise et al. 1994). IPV is associated with general, mental, and reproductive
health outcomes. It is associated with an increase in the
occurrence of depression, suicidal thoughts, and post-
traumatic stress disorder (PTSD) (Gibbs et al. 2018). A
WHO study of its 194 Member States showed an association
between IPV and newborn deaths (Oza et al. 2015). IPV
influences both maternal and child health. Women who
report higher severity of spousal violence are at greater
risk of having low birth weight (LBW) or premature babies
(Alhusen et al. 2014). Exposure to IPV during pregnancy
has long term negative implications for unborn children. Women who are anemic due to IPV, give birth to anemic
children who in turn are susceptible to chronic diseases in
adulthood (Organization 2019). In the presence of a paucity of recent representative
studies on IPV in Afghanistan, the current study will
provide information on the epidemiology of IPV among
Afghan women and will help to adjust priorities for
additional research aimed at enabling women to live a better
and healthier life. The study aims to examine the patterns
and demographic associations of IPV among ever-married
Afghan women. Dependent variable Our study’s outcome variable is the presence of any type
of physical, psychological/emotional, or sexual intimate
partner violence among ever-married women aged 15–
49 years in Afghanistan. In the questionnaire of the
domestic violence module, ever-married women were asked
questions about physical, emotional, sexual violence, and
controlling behavior. The emotional violence questions
asked information regarding (1) if the respondent were
humiliated in front of others, (2) threatened to hurt or harm
her or someone she cared about, and (3) if insulted or The fifth Sustainable Development Goal (SDG) empha-
sizes gender equality and calls for universal access to sexual Intimate partner violence among ever-married Afghan women: Patterns, associations... 97 decisions was re-coded as “capacity” and if she did not in
none of the decisions was computed as “no capacity”. made her feel bad about herself. While sexual violence
questions involved information about (1) if the respondent
was physically forced to have sexual intercourse with her
husband/ partner when she did not desire, (2) physically
forced her to perform any other sexual acts when she did not
want to, and (3) threatened to perform sexual acts when she
did not desire. Education level For the DHS survey, a separate ques-
tionnaire was developed for ever-married women. In the
questionnaire, they were asked three questions about their
education. First, if they have ever attended school following
by a second question of the type (Madrassa) of school they
attended. If the response to the first question was yes, then
they were asked about their highest level of education. The
dataset had categorized the education level into 4 categories
of no education (1), primary education (2), secondary edu-
cation (3), and higher education (4). We used the same in
our study. No education was used as a reference category
and all other categories were compared against it. The physical violence included questions regarding if the
respondent women were ever (1) pushed or shook, or had
something thrown at them, (2) slapped, (3) kicked, dragged
or beaten up, (4) punched with a fist, or hit with something
hard, (5) burnt or strangled on purpose, (6) threatened or
attacked with a knife, gun, or any other weapon, and (7)
twisted her arm or pulled hairs by their husband/partner. Dependent variable For all the above-mentioned categories of physical violence,
the women who answered; often, sometimes, and yes were
computed into a single variable of “yes” (1) whereas the
women who never had any such experience was calculated
as a “no” (0). Subsequently, a binary outcome for physical
violence was created and re-coded as “yes” if at least one
of the mentioned violent acts were perpetrated against the
respondent and a “no” if none of these acts were committed. After calculating these categories for physical violence, we
created a binary outcome variable and re-coded it as “yes”
if any type of physical, or emotional, or sexual spousal
violence was experienced and “no” if none of these types
of IPV was reported. Thus, this provided us with a final
composite variable referring to any ever-married woman
who has had experienced any type of lifetime violence by
her partner. Spousal educational attainment Similarly, in the survey
questionnaires, women were asked about the educational
attainment of their husbands. The questions included if
their husbands ever attended school and what was the
highest level of school they attended. In the dataset, the
answers were categorized into 5 categories of no education,
primary education, secondary education, higher education,
and don’t know. The respondents who answered “don’t
know” was combined with the category of “no education”. Consequently, we used these four categories for our study
keeping no education group as a reference. Place of residence Place of residence is the setting where
the respondents lived during the survey, which was grouped
into two categories, i.e., urban (1) and rural (2) in the
original questionnaire. We used the same categories for our
analysis. The urban category was taken as a reference. Independent variable our study, If the response to any of these 5 categories was a
“yes” we counted it as a justification of IPV, and a “no” was
coded as hitting by husband is not justified. After comput-
ing each category, we created a binary variable with a “yes”
if the respondent justified violence in at least one of the five
circumstances and a “no” if none of the circumstances was
justified for wife-beating. Women’s occupation In the questionnaire, women were
asked about the type and duration of occupation they
have had which was grouped into 7 groups including
not working, professional, clerical, agriculture, services,
skilled manual, and unskilled manual. For our analysis, we
combined services, clerical, skilled manual, and unskilled
manual into one category whereas kept not working,
professional, and self-employed (agriculture) each in
separate categories. Thus, we used these 4 categories for our
analysis. The category of unemployed women was kept as a
reference against all other job categories. Inter-parental violence Inter-parental violence is another
independent variable that we assumed to influence IPV
prevalence among women. In the questionnaire of the
AfDHS survey, women were asked if their fathers have ever
beaten their mothers. The answer was categorized into three
groups of yes, no, and don’t know. We combined the “don’t
know” and “no” categories resulting in a binary variable. Independent variable We selected the independent variables for our study based
on the previous literature review and basic assumptions on
the factors that influence intimate partner violence against
women. These variables include decision-making capacity,
women’s education, her partner’s education, place of
residence, age, wealth index, women’s occupation, number
of living children, region, partner’s alcohol consumption,
attitudinal acceptance, and inter-parental violence. Age For the variable of age, respondents were asked about
their age on their last birthday. In the survey, the variable
of age was divided into 7 age groups with a difference of 5
years in each. For our study, we used the same age groups
which are as follows: 15–19, 20–24, 25–29, 30–34, 35–39,
40–44, and 45–49. The age group of 15–19 was used as a
reference group against all the other age groups. Decision-making capacity To assess women’s participation
in decision-making, we used questions regarding three types
of household decisions: the respondent’s health care; major
household purchases; and visits to family or relatives. The
answers given were in 5 groups, wife alone; wife and
husband together; husband alone; someone else; and other. These categories were generated into a new binary variable
as “alone = 1” if the wife decides alone or together with her
husband otherwise “not alone = 0” was re-coded. Finally,
if the women participated in any of the three households’ Wealth The variable for the wealth index was created by
asking a few questions to the respondents in the household
questionnaire section. The questionnaire included questions
about the ownership of goods, agricultural land, and any
livestock and household characteristics. The respondents
were asked if they had certain goods at home such as
radio, TV, mobile or landline phone, computer, refrigerator,
furniture, sewing machine, and generator. Besides, housing
characteristics included questions regarding access to
electricity, source of drinking water, number of sleeping 98 R. Shinwari et al. rooms, type of toilet, type of material used in housing
structure, and type of cooking fuel. The variable of wealth
index was already categorized in the dataset of the DHS
survey thus we used the same categories from scale 1 to 5 for
our study: 1 = Poorest, 2 = Poorer, 3 = Middle, 4 = Richer,
5 = Richest. Group 1 (poorest) was chosen as the reference
group. Data analysis and presentation We used STATA software version 16.1 to analyze our data. The dataset used was only limited to the ever-married
women aged 15–49 years old. The number of living children For our study, we used the
variable “number of living children” to an ever-married
woman that was grouped into 17 parts in the DHS dataset
from 0 to 16 children. We categorized it into five groups as
follows: no children (0), 1–2 children (1), 3–4 children (2),
and 5–16 children (3). We took the group of women with no
children as a reference for our analysis. The variables required for this analysis were created in a
subset and were described earlier. We have presented a short
description of the variables and their categories in Table 1. A frequency distribution analysis was performed to
describe the women’s distribution according to their
background and characteristics. Subsequently, a survey
version of the test was performed to examine the frequency
of types of IPV against women by predictor variables. Logistic regression analysis was performed to explore
the association between our explanatory variables and the
outcome variable. Initially, the bivariate logistic regression
analysis was conducted for each explanatory variable
independently. The insignificant variables were removed
before running a multiple logistic regression analysis. Afterward, we performed a multiple logistic regression
analysis. Statistical significance was set to (p<0.05) and a
95% confidence interval was considered in the regression
analysis. Region Afghanistan is divided into 34 provinces and each
province is further subdivided into 458 districts. Based on
the location of these provinces, they are regrouped into
geographical regions. The participants were asked about the
province they belonged to. For our study, we categorized
these provinces into 7 regions according to the UN division
of regions in Afghanistan including Central region (1),
Eastern region (2), North Eastern region (3), Western region
(4), South Eastern region (5), North Eastern region (6), and
South Western region (7). The Central region was used as a
reference group. Alcohol consumption In the questionnaire, ever-married
women were asked if their former or current husbands
consumed alcohol and if yes, how often did they get drunk. In the dataset, the variable is already grouped into “yes” if
they did drink and “no” if they did not. We used the same
categories for our study as well. Indicates the number of living children. No children (1); 1–2 children (2); 2–4 children
(3); 5–16 children (4) Provinces re-coded into seven regions
based on the United Nation’s division of
regions in Afghanistan. Central (1); Eastern (2); North Eastern (3);
Western (4); South Western (5); North Western
(6); South Western (7) IPV not justified under any circumstances (0);
IPV justified at least under any one or more
circumstances (1) This variable shows the accepting attitude
of women toward IPV under given five
circumstances. It was categorized into
a binary variable of “yes” If IPV was
justified in at least one of the 5 situations
and “no” if in none of the situations. No/Don’t know (0); Yes (1) No/Don’t know (0); Yes (1) Urban (1); Rural (2) Women’s place of residence in terms of
urban and rural status. Poorest (1); Poorer (2); Middle (3); Richer (4);
Richest (5) Household wealth of the respondent orig-
inally categorized into 5 groups. Displays the occupation of women and
re-coded into 4 groups Not
working
(1);
services/clerical/skilled
manual/unskilled
manual
(2);
Profes-
sional/technical (3); Self-employed/agriculture
(4) Not
working
(1);
services/clerical/skilled
manual/unskilled
manual
(2);
Profes-
sional/technical (3); Self-employed/agriculture
(4) Indicates the number of living children. Includes any type of IPV (physical, emotional,
and/or sexual) Displays the level of woman’s educational
attainment that is categorized into 4
groups. No Education (1); Primary (2); Secondary (3);
Higher (4) No Education/Don’t know (1); Primary Level
(2); Secondary Level (3); Higher Level (4) Displays the level of partner’s educational
attainment that is categorized into 4
groups. 15–19 (1); 20–24 (2); 25–29 (3); 30–34 (4);
35–39 (5); 40–44 (6); 45–49 (7) 15–19 (1); 20–24 (2); 25–29 (3); 30–34 (4);
35–39 (5); 40–44 (6); 45–49 (7) Age was originally coded into 7 groups,
and the same was kept for this analysis. Urban (1); Rural (2) Descriptive statistics Table 2 presents the cumulative proportions of our outcome
and explanatory variables. Attitudinal acceptance of IPV In the questionnaire, respon-
dents were asked if their husbands are justified hit-
ting/beating them under the five given circumstances: (1) if
she goes out without telling her husband, (2) if she neglects
the children, (3) if she argues with him, (4) if she refuses
to have sex with him, and (5) if she burns the food. For The majority (21.2%) of respondents belonged to the age
group of 25–29 years followed by the age group 20–24
(20%), while only 6.2% were in their teenage years. Table 1
shows that almost 85% of the women had no education,
while 7% had a secondary level of schooling. Whereas for
the partners, 41.68% had some form of education, out of Intimate partner violence among ever-married Afghan women: Patterns, associations... 99 Table 1 Description of the variables used in the study with coding, Afghanistan, DHS (20
Variable name
Definition
Outcome variable
Intimate partner violence (IVP)
Includes any type of IPV (physical, emotional,
and/or sexual)
Predictor variables
Women’s educational Attainment
Displays the level of woman’s educational
attainment that is categorized into 4
groups. Partner’s educational attainment
Displays the level of partner’s educational
attainment that is categorized into 4
groups. Age
Age was originally coded into 7 groups,
and the same was kept for this analysis. Place of residence
Women’s place of residence in terms of
urban and rural status. Wealth index
Household wealth of the respondent orig-
inally categorized into 5 groups. Occupation
Displays the occupation of women and
re-coded into 4 groups
Number of children
Indicates the number of living children. Region
Provinces re-coded into seven regions
based on the United Nation’s division of
regions in Afghanistan. Attitudinal acceptance of IPV
This variable shows the accepting attitude
of women toward IPV under given five
circumstances. It was categorized into
a binary variable of “yes” If IPV was
justified in at least one of the 5 situations
and “no” if in none of the situations. Inter-parental violence
Respondent ever witnessed her father beat
her mother. Women’s decision-making capacity
Women’s participation in decision-making
on
three
areas
of
decision-making:
woman’s
healthcare
seeking,
major
household purchases, and visits to relatives. No/Don’t know (0); Yes (1) 100 violence act reported were being “slapped” (30.3%),
“pushed or shaken” (27.3%), “arm-twisted” (19%), and
“punched” (15%) by a partner/husband. The least common
forms of physical violence were “being threatened with
weapons” and “burnt on purpose” with an equal proportion
(2.3%). There were provincial variations in the prevalence
of lifetime IPV experience among ever-married Afghan
women. The highest prevalence provinces were Herat, Ghor,
Kandahar, Logar, Paktia, and Baghlan (64.6% to 92.2%). The prevalence among women in Farah, Faryab, Sare-
pul, Parwan, Laghman, Nangarhar, Nooristan, and Takhar
ranged between 50 and 64.6%. Women who experienced
the least rates (6.4–25.4%) were from Nimrod, Helmand,
Urozgan, Bamyan, Samangan, Panjsher, Badakhshan, and
Khost provinces. Overall. the South Western region
contributed the highest proportion of any form of IPV
(12.6%) followed by the Central (9.6%) and Western (8.5%)
regions. The Eastern region had the lowest contribution
(4%). Table 2 Cumulative proportion of factors in ever married women aged
15–49 interviewed for domestic violence module in Afghanistan, DHS
2015
Variable
Percentage
Physical violence
50.52
Sexual violence
7.43
Emotional violence
37.36
Any type of Violence
55.54
Decision-making capacity
64.79
Women’s educational attainment
No education
83.56
Primary
7.80
Secondary
6.77
Higher
1.87
Spousal educational attainment
No education/Don’t know
58.32
Primary
13.53
Secondary
21.36
Higher
6.79
Residence (Rural)
77.79
Age groups
15–19 years
6.22
20–24 years
20.8
25–29 years
21.22
30–34 years
14.80
35–39 years
15.16
40–44 years
10.24
45–49 years
11.78
Wealth index
Poorest
20.38
Poorer
21.01
Middle
20.4
Richer
19.86
Richest
18.35
Occupation
Not working
86.85
Professional/Technical/Managerial
6.32
Agricultural/Self-employed
2.39
Clerical/Services/Unskilled & Skilled laborer
4.44
Living children
No children
10.00
1–2 children
24.61
3–4 children
26.18
5–16 children
39.21
Attitudinal acceptance of IPV
80.56
Father ever beat mother
38.51 Table 2 Cumulative proportion of factors in ever married women aged
15–49 interviewed for domestic violence module in Afghanistan, DHS
2015 About two-thirds (64.79%) of the women had no
decision-making power. The majority (80.56%) of the
respondents justified wife-beating for one or more reasons. The most agreed reason for IPV justification was “if the
wife goes out without telling her husband” (67%) followed
by “if she argues with her partner” (59.4%). Whereas about
48% and 33% of the women justified spousal violence if
wife neglects children and “if the wife refuses to have sex
with husband”, respectively. The least mentioned reason
was “if the wife burns food” (18%). No/Don’t know (0); Yes (1) Respondent ever witnessed her father beat
her mother. Did not participate in any of the three decisions
(0); If participated in one or more of the three
decisions (1) Did not participate in any of the three decisions
(0); If participated in one or more of the three
decisions (1) Women’s participation in decision-making
on
three
areas
of
decision-making:
woman’s
healthcare
seeking,
major
household purchases, and visits to relatives. which 21% completed higher education. More than 75%
of the participants were living in rural settings. Concerning
the wealth index, women belonging to the poorest, poorer,
and middle groups were almost equally distributed (about
21%) followed by the richer group with about 19.86%,
and the richest with 18.35%. About 87% of the women
were unemployed. Of the 13% employed women, 6% were
professional workers, while only 2.4% were self-employed
or worked in agriculture. Most (39.2%) of the respondent
women had more than 5 living children. Only 10% of
women had no children at all. Only 39 (0.18%) respondents
had partners who consumed alcohol, therefore, the variable
was excluded from our study. All the rates referred to the lifetime IPV experience
of the studied women. The prevalence of some form
of physical, emotional, or sexual violence among ever-
married women in their lifetime was 55.54%. The most
common form of spousal violence experienced was
physical violence (50.52%) followed by emotional violence
(37.4%), and sexual violence (7.43%). Out of a total of
21,234 respondents, 28.55% reported both physical and
emotional violence, 8.11% reported physical and sexual
violence whereas around 6.25% of women experienced
both emotional and sexual violence However, 1,355 women
(6.29%) reported all three types of physical, emotional,
and sexual violence. The commonest types of physical which 21% completed higher education. More than 75%
of the participants were living in rural settings. Concerning
the wealth index, women belonging to the poorest, poorer,
and middle groups were almost equally distributed (about
21%) followed by the richer group with about 19.86%,
and the richest with 18.35%. About 87% of the women
were unemployed. Of the 13% employed women, 6% were
professional workers, while only 2.4% were self-employed
or worked in agriculture. Most (39.2%) of the respondent
women had more than 5 living children. Only 10% of
women had no children at all. Only 39 (0.18%) respondents
had partners who consumed alcohol, therefore, the variable
was excluded from our study. R. Shinwari et al. No/Don’t know (0); Yes (1) Finally, about 38.5%
of women witnessed inter-parental violence, while the
remaining did not. Bivariate analysis Table 3 shows the crude odds ratios of any type of intimate
partner violence for the non-reference versus reference
categories of all the co-variates. The above Table 3 displays a significant (p<0.001) asso-
ciation of women’s age, education number of children,
attitudinal acceptance of IPV, and experience of inter-
parental violence with the experience of spousal violence. Similarly, rural-dwelling (p=0.002) and the respondents’
husbands/partners’ educational levels (p<0.001) were asso-
ciated with IPV. Table 3 also shows that the richest wealth index shows
a protective effect against IPV (p=0.036), while the other
wealth index categories did not show any significant results
against our outcome variable (p>0.05). Also, women who
worked in agriculture or who were self-employed were
more likely (COR=2.52, 95%CI 1.88–3.38) to experience
IPV compared to unemployed women. IPV was not
associated with the other employment categories (p>0.05). 101 Intimate partner violence among ever-married Afghan women: Patterns, associations... Table 3 Outcomes
of
simple
logistic
regression
models
with
individual variables association with domestic violence among ever
married women aged 15–49 in Afghanistan, DHS 2015
Variable
OR
95% CI
P-value
Decision-making capacity
0.91
0.74–1.11
0.341
Educational attainment
No education
Reference
Primary
0.68
0.57–0.82
<0.001
Secondary
0.53
0.38–0.75
<0.001
Higher
0.36
0.22–0.62
<0.001
Spousal educational attainment
No education/Don’t know
Reference
Primary
0.73
0.61–0.87
<0.001
Secondary
0.74
0.62–0.88
0.001
Higher
0.55
0.42–0.73
<0.001
Residence (Rural)
1.38
1.13–1.68
0.002
Age groups
15–19 years
Reference
20–24 years
1.71
1.34–2.19
<0.001
25–29 years
2.40
1.80–3.20
<0.001
30–34 years
2.21
1.72–2.85
<0.001
35–39 years
2.39
1.88–3.03
<0.001
40–44 years
2.19
1.69–2.84
<0.001
45–49 years
3.19
2.40–4.24
<0.001
Wealth index
Poorest
Reference
Poorer
1.05
0.86–1.28
0.629
Middle
1.20
0.93–1.55
0.165
Richer
1.01
0.80–1.28
0.921
Richest
0.77
0.60–0.98
0.036
Occupation
Not working
Reference
Professional/Technical/Managerial
1.14
0.86–1.52
0.366
Agricultural/Self-employed
2.52
1.88–3.38
<0.001
Clerical/Services/Unskilled
1.32
0.86–2.04
0.204
& Skilled laborer
Living children
No children
Reference
1–2 children
2.44
2.03–2.94
<0.001
3–4 children
2.76
2.21–3.46
<0.001
5–16 children
2.97
2.37–3.70
<0.001
Attitudinal acceptance of IPV
2.16
1.80–2.60
<0.001
Father ever beat mother
3.90
3.49–4.34
<0.001
OR is odds ratios; 95% CI is 95% confidence intervals Multivariate logistic regression analysis Table 3 Outcomes
of
simple
logistic
regression
models
with
individual variables association with domestic violence among ever
married women aged 15–49 in Afghanistan, DHS 2015 In Table 4, we present the results from the multiple
regression analysis to test the individual variables that
were had statistical significance earlier against the outcome
variable. The primary education level of both women (OR=0.82
95%CI=0.68–0.98)
and
their
partners
(OR=0.76
95%CI=0.64–0.91), age groups (p<0.05), number of
children
(p<0.001),
attitudinal
acceptance
(OR=1.85
95%CI=1.51–2.26), and inter-parental violence (OR=3.69
95%CI=3.31–4.10)
showed
a
significant
association
with the experience of IPV among Afghan women. The
goodness-of-fit test revealed that this was a good multi-
variate logistic model for having ever experienced any type
of IPV among ever-married Afghan women aged between
15 to 49 years old ([F: [9, 886] 1.18, p-value: 0.3022). The other variables did not show a statistically significant
relationship. Discussion Afghanistan was found to have a higher prevalence of IPV
compared to other LMICs (Coll et al. 2020). However,
relative to some of the high-income countries, the rates
of emotional and sexual violence in Afghanistan appeared
lower (Costa et al. 2015). For instance, 70.5% of Greek
women experienced emotional violence, while more than
20% of German, Portuguese, and British women reported
spousal sexual violence. However, the rates of physical
violence (23% in Greece) were much lower than among
Afghan women (Costa et al. 2015). However, Iran (78%)
had a higher prevalence of IPV. The high prevalence
of IPV in Afghanistan could be due to the inflexible
traditional views and gender norms prevalent in the country. In Afghanistan, wife-beating is seen as an acceptable act
when women violate the gender norms and resist male
wishes (Echavez et al. 2016). It is believed that Afghan
women might adhere to the rigid traditional gender roles
to protect themselves from partner abuse and reduce the
likelihood of violence against them. Nonetheless, it is
not very clear if these gender roles are used to overlook
violence or if these are practiced to control the violence
(Humanitarian Response 2012). Afghanistan is among the
worst performers within the WPS 2019 Index with regards
to gender equity and women’s empowerment, which is
reflected in the country’s current ranking of 166th out of
167 countries that shows how deep-rooted gender norms
in different countries (GIWPS 2019). It is also possible
that the prevalence of IPV in our study is under-reported
because of the sensitivity attached to gender-based violence
and speaking about women’s problems in the Afghan OR is odds ratios; 95% CI is 95% confidence intervals Unexpectedly, decision-making capacity was not associated
(p=0.341) with the experience of marital violence among
Afghan women. 102 R. Shinwari et al. Discussion Table 4 Outcomes of multivariable analysis of variables associated
with domestic violence among ever married women aged 15–49 in
Afghanistan, DHS 2015
Variable
Adjusted OR
95% CI
P-value
Educational attainment
No education
Reference
Primary
0.82
0.68–0.98
0.032
Secondary
0.81
0.56–1.17
0.265
Higher
0.59
0.33–1.04
0.066
Spousal educational attainment
No education/Don’t know
Reference
Primary
0.76
0.64–0.91
0.003
Secondary
0.92
0.78–1.08
0.306
Higher
0.82
0.60–1.12
0.214
Residence (Rural)
1.13
0.94–1.37
0.192
Age groups
15–19 years
Reference
20–24 years
1.36
1.06–1.7
0.016
25–29 years
1.65
1.25–2.18
<0.000
30–34 years
1.49
1.13–1.97
0.005
35–39 years
1.60
1.19–2.15
0.002
40–44 years
1.49
1.07–2.08
0.018
45–49 years
2.13
1.57–2.90
<0.001
Living children
No children
Reference
1–2 children
2.23
1.82–2.73
<0.001
3–4 children
2.23
1.66–3.00
<0.001
5–16 children
2.25
1.64–3.08
<0.001
Attitudinal acceptance of IPV
1.85
1.51–2.26
<0.001
Father ever beat mother
3.69
3.31–4.10
<0.001
OR is Odds Ratios, 95% CI is 95% confidence intervals
Statistically significant at p-value of <0.01 level Our study finding of primary education alone being
protective against IPV negated the hypothesis that the
likelihood of spousal violence should reduce with the
increasing level of education of the couple. There seems
to be a contextual difference in the relationship between
IPV and education. For instance, the Demographic Health
Surveys in Uganda and Malawi found that higher levels
of education are associated with increased risk of IP
(Peterman et al. 2020). However, other studies (Akmatov
et al. 2008; Weitzman 2018) have shown that women’s
education is protective from IPV, while a lower level of
education increased the risk (Sabri et al. 2014). There may
also be an attenuated effect with regard to education, as
the great majority of women were in the low education
category. The insufficient variability in the data in this
regard possibly played a role in being unable to detect
statistically significant differences. Table 4 Outcomes of multivariable analysis of variables associated
with domestic violence among ever married women aged 15–49 in
Afghanistan, DHS 2015 The multivariate analysis confirmed the association
between the woman’s age and her experience of partner
violence and several studies have approved this relationship
(Tuyishime 2015; Bott et al. 2012; Sanz-Barbero et al. 2018). The direction of the relationship between age and
IPV varies among countries. While some studies found that
younger women were more likely to experience spousal
abuse compared to older (Bott et al. 2012; Sanz-Barbero
et al. Discussion 2018), others, like the current study, suggest the
opposite (Hindin et al. 2008). Interestingly, a study of
Afghan women showed no relationship between age and
IPV within the preceding 12 months of data collection
(Gibbs et al. 2018). Younger Afghan women may be less
resistant to male dominance and more fearful of divorce and
the associated stigma so that they are more tolerant to abuse
and less likely to report them. In the current study, the risk of IPV seems to increase
with the number of children the woman has. Our data agree
with a Cambodian study that found a higher odds of physical
violence in women with five or more children (Yount and
Carrera 2006). Other studies from different settings agree
that the risk of IPV increases with the number of children
women have (Sabri et al. 2014; Ali et al. 2011; Hussain et al. 2017; Putra et al. 2019). There are many possible reasons
for higher rates of violence among women with a greater
number of children. First, spouses with more children have
a greater amount of responsibilities in the process of their
upbringing that may put extra pressure on them thus leading
to disagreements and violence. This may also limit women’s
options for leaving a toxic relationship (Sabri et al. 2014). Second, women with an increased number of living children
may have lesser chances of having a professional life,
working outside, and have an income, due to increased
responsibility. This may lead them to be dependent on
their husbands financially thus increasing their chances of
experiencing violent behavior (Putra et al. 2019). Third, context (Echavez et al. 2016). It is most probable that
the low prevalence of sexual IPV in our study is the
result of under reporting. Many women do not consider
forced sex as violence and, therefore, under report it
(Metheny and Stephenson 2019; Abirafeh 2009; Zakar
et al. 2012). Comparison with previous findings among
Afghan women, which showed the prevalence of 52%
and 74% for the lifetime experience of spousal physical
and emotional violence respectively in 2008 (Humanitarian
Response 2012), suggests a reducing trend in IPV in the
country over time. However, this may also result from the
differences between the characteristics of the respondents. The provincial variation in IPV prevalence in Afghanistan
could be due to the distribution of contextual characteristics
like ethnicity, gender beliefs, and educational attainment. context (Echavez et al. 2016). Strength and limitations An attitude that justifies IPV is a significant predictor
of spousal abuse and our data corroborates this assertion,
which has been reported by other authors from varied
settings (Amir-ud Din et al. 2018; Putra et al. 2019; Solanke
2018; Jesmin and Jesmin 2015; Tran et al. 2016). Women
who accept wife-beating under any circumstance are more
likely to experience it. A previous multi-country study
found that Afghanistan had the highest acceptance rate
for spousal violence (90%) among 39 LMICs (Tran et al. 2016). The current study found an acceptance rate of 80.5%. A majority of Nigerians also appear to justify spousal
abuse (Solanke 2018). Afghans have a rigid perception of
gender roles (Humanitarian Response 2012), therefore, the
women approve of the idea of them being in a subordinate
social position, resulting in the justification of attitudes
toward violence (Jesmin and Jesmin 2015). It has also been
suggested that women who justify partner violence have had
learned it as normal behavior resulting from the experience
of inter-parental violence (Yount and Carrera 2006; Wood
2001; Vung and Krantz 2009) or personal experience of
violence or abuse in childhood (Vung and Krantz 2009). Besides, in settings where violence against women is
widespread, women tend to accept interventions to resolve
the issues. These justifying behaviors may be transferred
to the upcoming generations (Jesmin and Jesmin 2015). The fear of stigmatization may also explain the justifying
behaviors. In conservative societies, divorce and separation
are not respectable options for spousal crisis resolution. To avoid the stigma, many women rather suffer spousal
violence silently and refrain from resisting it (Jesmin and
Jesmin 2015). The main strengths of our study were the large sample size
(21, 234) and the generalizability of the data. However,
there were some limitations. One of the limitations of our
study is that the women might have under-reported their
experience of IPV due to the sensitivity of the topic in
the country, especially sexual violence. We measured the
lifetime experience of IPV among ever-married Afghan
women thus, the responses on IPV which happened many
years prior may have been subject to recall bias. Males were
excluded from the questions in IPV. Increasing attention
is being given to the importance of having also intimate
partner responses in questionnaires concerning the subject
of IPV. Strength and limitations Data could not be collected from the province of
Zabul due to security concerns, thus this study remains
silent on the responses from women living in Zabul. The
data are cross-sectional, thus causality cannot be inferred
from these results. Discussion It is most probable that
the low prevalence of sexual IPV in our study is the
result of under reporting. Many women do not consider
forced sex as violence and, therefore, under report it
(Metheny and Stephenson 2019; Abirafeh 2009; Zakar
et al. 2012). Comparison with previous findings among
Afghan women, which showed the prevalence of 52%
and 74% for the lifetime experience of spousal physical
and emotional violence respectively in 2008 (Humanitarian
Response 2012), suggests a reducing trend in IPV in the
country over time. However, this may also result from the
differences between the characteristics of the respondents. The provincial variation in IPV prevalence in Afghanistan
could be due to the distribution of contextual characteristics
like ethnicity, gender beliefs, and educational attainment. Intimate partner violence among ever-married Afghan women: Patterns, associations... 103 children’s neglect might also lead to women’s violence. When a woman has an increased number of children, she
might not be able to pay the required attention to each one
of them and take good care of them, which could lead her
partner to show aggressive behavior toward her. overall tolerant behavior of women due to male dominance
and gender disparities play a critical role in IPV (Ali and
Gavino 2008; Jejeebhoy and Sathar 2001). Funding Open Access funding enabled and organized by Projekt
DEAL. References Abirafeh L (2009) Gender and International Aid in Afghanistan. McFarland Incorporated Publishers, USA Gibbs A, Corboz J, Jewkes R, Gibbs A, Corboz J, Jewkes R
(2018) Factors associated with recent intimate partner violence
experience amongst currently married women in Afghanistan and
health impacts of ipv: a cross sectional study. BMC Publ Health
18(1):593. https://doi.org/10.1186/s12889-018-5507-5 Akmatov MK, Mikolajczyk RT, Labeeb S, Dhaher E, Khan MM
(2008) Factors associated with wife beating in Egypt: Analysis
of two surveys (1995 and 2005). BMC Womens Health 8:15. https://doi.org/10.1186/1472-6874-8-15 Alhusen JL, Bullock L, Sharps P, Schminkey D, Comstock E,
Campbell J (2014) Intimate partner violence during pregnancy
and adverse neonatal outcomes in low-income women. J Womens
Health 23(11):920–926. https://doi.org/10.1089/jwh.2014.4862 p
g
GIWPS (2019) Women, peace and security index. https://reliefweb. int/sites/reliefweb.int/files/resources/WPS-Index-2019-Report. pdf. Accessed 10 June 2020 Heise LL, Raikes A, Watts CH, Zwi AB, Heise LL, Raikes A, Watts
CH, Zwi AB (1994) Violence against women: A neglected public
health issue in less developed countries. Soc Sci Med 39(9):1165–
1179. https://doi.org/10.1016/0277-9536(94)90349-2 Ali PA, Gavino MIB (2008) Violence against women in Pakistan: A
framework for analysis. J Pak Med Assoc 58(4):198–203. https://
pubmed.ncbi.nlm.nih.gov/18655430 Ali TS, Asad N, Mogren I, Krantz G (2011) Intimate partner violence
in urban Pakistan: prevalence, frequency, and risk factors. Int J
Womens Health 3:105. https://doi.org/10.2147/IJWH.S17016 Hindin MJ, Kishor S, Ansara DL (2008) Intimate partner vio-
lence
among
couples
in
10
DHS
countries:
Predictors
and
health
outcomes. https://dhsprogram.com/publications/
publication-as18-analytical-studies.cfm. Accessed 10 June 2020 Aslam SK, Zaheer S, Shafique K (2015) Is spousal violence being
“Transmitted” through Victims? Findings from the Pakistan
demographic and health survey 2012-13. PLoS ONE vol 10(6). https://doi.org/10.1371/journal.pone.0129790 Humanitarian Response (2012) Domestic violence in Afghanistan. https://www.humanitarianresponse.info/sites/www.humanitarian
response.info/files/documents/files/Living%20with%20Violence
%20-%20A%20National%20Report%20on%20Domestic%20Vio-
lence%20in%20Afghanistan%2C%20Global%20Rights%2C%20
2008.pdf. Accessed 10 June 2020 Bancroft L, Silverman JG, Ritchie D (2012) The batterer as parent:
Addressing the impact of domestic violence on family dynamics. ResearchGate pp 1–325. https://doi.org/10.4135/9781452240480 Bensley L, Van Eenwyk J, Simmons KW, Bensley L, Van Eenwyk
J, Simmons KW (2003) Childhood family violence history and
women’s risk for intimate partner violence and poor health. Am
J Prev Med 25(1):38–44. https:/doi.org/10.1016/s0749-3797(03)
00094-1 Hussain S, Usman M, Sabir M, Zakar R, Usman A (2017) Prevalence
of spousal violence and associated risk factors: Facts from
Pakistan demographics and health survey 2012-13. J Fam Viol
32(7):711–719. https://doi.org/10.1007/s10896-017-9915-6 (7):711–719. https://doi.org/10.1007/s10896-017-9915- Islam TM, Tareque MdI, Tiedt AD, Hoque N (2014) The intergener-
ational transmission of intimate partner violence in Bangladesh. Glob Health Action 7(1):23591. Conclusions The lifetime experience of IPV is highly prevalent
among Afghan women. The commonest form of violence
was physical violence. Sociodemographic factors such
as education, age, number of living children, attitudinal
acceptance of IPV, and inter-parental violence were
significantly associated with spousal abuse. Whereas
many factors such as women’s decision-making capacity,
occupation, place of residence, and wealth index did not
show any significant association. Afghanistan governmental
and non-governmental agencies need to step up efforts
aimed at educating Afghans against IPV and challenging
the norms that underline the phenomenon. Interventions
aimed at controlling spousal violence should be targeted at
multiple strata, including individual and community levels. Afghanistan must formulate and implement policies that
promote gender equity and mainstream the strategy into
all aspects of public and private life. The capacity of the
health care setting to identify victims and mount an effective
response to IPV should be enhanced. There is a need for
further research to explain the mechanisms between the risk
factors and IPV among Afghans. Our findings that witnessing inter-parental violence
increases the likelihood of IPV among women were in line
with some of the previous studies, which indicates that the
women who observed violence between their parents are
more inclined to approve it (Solanke 2018; Aslam et al. 2015; Bensley et al. 2003; Vung and Krantz 2009; Islam
et al. 2014; Chernet and Cherie 2020). Parents and the
family are the primary socialization agents for a child. Children of aggressive families tend to display violent
behavior as adults (Kalmuss 1984). The witnessing of inter-
parental violence by children may affect their behavior
and quality of life. The case is particularly worse for girl
children (Kitzmann et al. 2003). The likelihood of spousal
violence experience may be heightened if the girls are made
to believe that the mothers are responsible for the inter-
parental violence (Bancroft et al. 2012). In essence, the 104 R. Shinwari et al. https://doi.org/10.1177/0886260518787809 Crime on Drugs UNO (2018) Global study on homicide: Gender-
related killing of women and girls. UNODC, United Nations
Office on Drugs and Crime Echavez CR, Mosawi S, Pilongo LWR (2016) The other side of gender
inequality: Men and masculinities in Afghanistan. ResearchGate. https://www.researchgate.net/publication/291350543-The-Other-
Side-of-Gender-Inequality-Men-and-Masculinities-in-Afghanistan Gender-Inequality-Men-and-Masculinities-in-Afghanistan Fikree FF, Razzak JA, Durocher J (2005) Attitudes of Pakistani men to
domestic violence: a study from Karachi, Pakistan. J Men’s Health
Gender 2(1):49–58. https://doi.org/10.1016/j.jmhg.2005.01.004 Garcia-Moreno C, Jansen HA, Ellsberg M, Heise L, Watts CH (2006)
Prevalence of intimate partner violence: findings from the WHO
multi-country study on women’s health and domestic violence. Lancet 368(9543):1260–1269. https://doi.org/10.1016/S0140-6736
(06)69523-8 Declarations Chernet AG, Cherie KT (2020) Prevalence of intimate partner violence
against women and associated factors in Ethiopia. BMC Womens
Health 20(1):1–7. https://doi.org/10.1186/s12905-020-0892-1 Since we performed secondary data analysis of the AfDHS 2015
data, our study did not require any ethical approval. The ICF
Institutional Review Board (IRB) and the Ministry of Public Health
of Afghanistan approved the survey protocol and the questionnaires
of the AfDHS 2015. They were assured of confidentiality and signed
voluntary informed consents were obtained from the participants
before initiating interviews. The authors declare no competing
interests. Coll CVN, Ewerling F, Garc´ıa-Moreno C, Hellwig F, Barros AJD,
Coll CVN, Ewerling F, Garc´ıa-Moreno C, Hellwig F, Barros
AJD (2020) Intimate partner violence in 46 low-income and
middle-income countries: An appraisal of the most vulnerable
groups of women using national health surveys. BMJ Glob Health
5(1):e002208. https://doi.org/10.1136/bmjgh-2019-002208 1):e002208. https://doi.org/10.1136/bmjgh-2019-002208 Costa D, Soares J, Lindert J, Hatzidimitriadou E, Sundin ¨O, Toth O,
Ioannidi-Kapolo E, Barros H (2015) Intimate partner violence: a
study in men and women from six European countries. Int J Public
Health 60(4):467–478. https://doi.org/10.1007/s00038-015-0663-1 lth 60(4):467–478. https://doi.org/10.1007/s00038-015-0663 Open Access This article is licensed under a Creative Commons
Attribution 4.0 International License, which permits use, sharing,
adaptation, distribution and reproduction in any medium or format, as
long as you give appropriate credit to the original author(s) and the
source, provide a link to the Creative Commons licence, and indicate
if changes were made. The images or other third party material in this
article are included in the article’s Creative Commons licence, unless
indicated otherwise in a credit line to the material. If material is not
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use is not permitted by statutory regulation or exceeds the permitted
use, you will need to obtain permission directly from the copyright
holder. To view a copy of this licence, visit http://creativecommons. org/licenses/by/4.0/. Amir-ud Din R, Fatima S, Aziz S (2018) Is attitudinal acceptance of
violence a risk factor? An analysis of domestic violence against
women in Pakistan. J Interpers Violence pp 0886260518787809. https://doi.org/10.1177/0886260518787809 References https://doi.org/10.3402/gha.v7. 23591 Bott S, Guedes A, Goodwin M, Mendoza JA (2012) Violence against
women in Latin America and the Caribbean: A comparative anal-
ysis of population-based data from 12 countries. ResearchGate. https://www.researchgate.net/publication/258874187-Violence-
Against-Women-in-Latin-America-and-the-Caribbean-A-Com-
parative-Analysis-of-Population-Based-Data-from-12-Countries Jejeebhoy SJ, Sathar ZA (2001) Women’s autonomy in India and
Pakistan: The influence of religion and region. Popul Dev Rev
27(4):687–712. https://doi.org/10.1111/j.1728-4457.2001.00687.x Intimate partner violence among ever-married Afghan women: Patterns, associations... 105 and associated factors in Europe. J Epidemiol Commun Health
72(7):611–616. https://doi.org/10.1136/jech-2017-209701 Jesmin SS, Jesmin SS (2015) Married women’s justification of
intimate partner violence in Bangladesh: Examining community
norm and individual-level risk factors. Violence Vict 30(6):984–
1003. https://doi.org/10.1891/0886-6708.VV-D-14-00066 Solanke BL (2018) Does exposure to interparental violence increase
women’s risk of intimate partner violence? Evidence from Nigeria
demographic and health survey. BMC Int Health Hum Rights
18(1):1–13. https://doi.org/10.1186/s12914-018-0143-9 Jewkes R, Corboz J, Gibbs A, Jewkes R, Corboz J, Gibbs A (2018)
Trauma exposure and IPV experienced by Afghan women: Anal-
ysis of the baseline of a randomised controlled trial. PLoS ONE
13(10):e0201974. https://doi.org/10.1371/journal.pone.0201974 Tran TD, Nguyen H, Fisher J, Tran TD, Nguyen H, Fisher J
(2016) Attitudes towards intimate partner violence against women
among women and men in 39 low- and middle-income countries. PLoS ONE 11(11):e0167438. https://doi.org/10.1371/journal.pone. 0167438 Kalmuss D (1984) The intergenerational transmission of marital
aggression. J Marriage Fam 46(1):11–19. https://www.jstor.org/
stable/351858 Tuyishime E (2015) Modeling factors associated with intimate part-
ner violence against women in intimate partnership in Rwanda. http://www.jkuat.ac.ke/campuses/kigali/publications/tuyishime-
elysee-2015-modeling-factors-associated-with-intimate-partner-vio-
lence-against-women-in-intimate-partnership-in-rwanda. Accessed
11 June 2020 Kitzmann KM, Gaylord NK, Holt AR, Kenny ED (2003) Child
witnesses to domestic violence: A meta-analytic review. J Consult
Clin Psychol 71(2):339–352. https://doi.org/10.1037/0022-006x. 71.2.339 Menj´ıvar C, Salcido O (2002) Immigrant women and domestic
violence: Common experiences in different countries. Gend Soc
16(6):898–920. https://doi.org/10.1177/089124302237894 UNDP (2015) Gender inequality index (GII) | human devel-
opment reports. http://hdr.undp.org/en/content/gender-inequality-
index-gii. Accessed 18 July 2020 Metheny N, Stephenson R (2019) Help seeking behavior among
women who report intimate partner violence in Afghanistan: An
analysis of the 2015 Afghanistan demographic and health survey. J
Fam Viol 34(2):69–79. https://doi.org/10.1007/s10896-018-0024-y UNODC (2019) Global study on homicide. https://www.unodc. org/documents/data-and-analysis/gsh/Booklet5.pdf. Accessed 10
June 2020 Organization WH (2019) Mental health aspects of women’s repro-
ductive health: A global review of the literature. https://apps.who. int/iris/bitstream/handle/10665/43846/9789241563567-eng.pdf? sequence=1. Accessed 10 June 2020 Vung ND, Krantz G (2009) Childhood experiences of interparental vio-
lence as a risk factor for intimate partner violence: a population-
based study from northern Vietnam. References J Epidemiol Community
Health 63(9):708–714. https://doi.org/10.1136/jech.2008.076968 sequence=1. Accessed 10 June 2020 Organization WH et al (2012) Understanding and addressing violence
against women. Intimate partner violence. Tech. rep., World
Health Organization Weitzman A (2018) Does increasing women’s education reduce their
risk of intimate partner violence? Evidence from an education pol-
icy reform. Criminology 56(3):574–607. https://doi.org/10.1111/
1745-9125.12181 Oza S, Lawn JE, Hogan DR, Mathers C, Cousens SN, Oza S, Lawn
JE, Hogan DR, Mathers C, Cousens SN (2015) Neonatal cause-
of-death estimates for the early and late neonatal periods for
194 countries: 2000-2013. Bull World Health Organ 93(1):19–28. https://doi.org/10.2471/BLT.14.139790 Wood JT (2001) The normalization of violence in heterosexual
romantic relationships: women’s narratives of love and violence. J
Soc Pers Relat 18(2):239–261. https://doi.org/10.1177/02654075
01182005 Peterman A, Behrman J, Palermo T (2020) paa2015. https://paa2015. princeton.edu/abstracts/151336. Accessed 14 July 2020 Yount KM, Carrera JS (2006) Domestic violence against married
women in Cambodia. Soc Forces - Soc forces 85(1):355–387. https://doi.org/10.1353/sof.2006.0151 Putra IGNE, Pradnyani PE, Parwangsa NWPL (2019) Vulnerability to
domestic physical violence among married women in Indonesia. J
Health Res. https://doi.org/10.1108/JHR-06-2018-0018 Zakar R, Zakar MZ, Faist T, Kraemer A (2012) Intimate partner
violence against women and its related immigration stressors
in Pakistani immigrant families in Germany. SpringerPlus 1:5. https://doi.org/10.1186/2193-1801-1-5 Sabri B, Renner LM, Stockman JK, Mittal M, Decker MR (2014) Risk
factors for severe intimate partner violence and violence-related
injuries among women in India. Women Health 54(4):281–300. https://doi.org/10.1080/03630242.2014.896445 Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations. Sanz-Barbero B, L´opez Pereira P, Barrio G, Vives-Cases C (2018)
Intimate partner violence against young women: prevalence
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pH metric study of complexes of some metal ions with salicylaldehyde-4-phenyl-3-thiosemicarbazone
|
Zenodo (CERN European Organization for Nuclear Research)
| 1,975
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cc-by
| 1,972
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* For Correspond&nce J. INDIAN OHE}I. SOC. VOL. LII, DECEMBER 197o I 500 om -l (benzene ring). The UV spectrum of the
compound B in ethanol shows absorption maxima at
247. 257. and 322 nm whieh i"' Yery similar to that of
oitropten3. That compound B contains an epoxide
ring is eYident from the fact that it gave a. diol (JJ). C.5H 180 5, m.p. 124°, on treatmE-nt with aqueous
oxalic acid (·1 %). The diol consumed
out~ molt•
of periodic acid. The lR spectrum of the diol in
Nujol mull ::Jhows hands at 3510 em 1 an(~ 3400 cm-1
(gly<'olio OH group::~). 1725 om-1 (laetone carhonyl). 1610 cm-1
and 1500 cm-1
(aromatic ring}. The Y. N. BHATT, K. K. PATEL. K. J. SHAH, R. s. PATlilL"'
l)epartment of Ohomiiltry, Sam·a.shtra Univ~rsity
Bhavnagar LIGANDS ~uch as thiOI!emioarhazonos are
~<tudied
to a limitAd extent pH-metricallr
Here sa.lit'yla-
ldehyde-4-phenyl-3-thio~emica.rbazon~ (SPT) deriva-
tive i~ stuclioo with metals like V02+, Cu2-t, Co~~+,
Ni:t+. The formation constants foJ· their metals w&e
calculated by Calvin and WilRO~l pH t.itration techni-
que a.'! modified hy Irving and Rosl3otti. The atabi-
Iitv < Qnl'ltant~
&"P
cl.et£-rmined in 50% aqueous
dioxane medium. (11) Acknowledgement Tb.e authors are indebted to Prof. S. M. Sil'oa.r,
Director of BOJ>e InHliitute for his interest in thi& work. They are also grateful to D.liS. A. P. B. Sinha and
K. G. Das of National Chemical Laboratory, Poona. l'ndia, for the 1\-r:MR and ma.~s 'Spectra of the com·
pounds. One of us (A. B.) is indebt!'-tl to C.S.I.R. ·
for financial RRI'!i~tance. References l. P. K. SANYAL and P. K. HosE, Srierlce and Otdtuf'l',
1969, 35. 332. (
g}
HCO
3
(I)
(11)
::NMR spectrum of the diol (CD013, 60MHz} ~thowH
a pair of doublets centered at 6.22 8(IH, d, J = 9.5Hz)
and 7.628 (JH, d, J = 9.5Hz) for the 3-H alld 4-H
respectively. The two aromatic prouons namely
5-H and 6-H app0ar as another p,air of doublet~
eenwrctl at 6.8M (lH, rl, J = 8.5Hz) and 7.348
OR. d. J = 8.5Hz), OM of the peaks of the latter
oV61'laps with that of OHCI3. The t;h.roo p!oton"' of
t·h.e mP.th.oxvl group ( -OCHs) and lihe six proton"l of
the two met'hyl gJ.'oups attached t-o one of the carbinol
carbon atom [-C(OH)-(CH3hl appNv a.ss harp singlets
at 3.983 (3H) an~ 1.31$ (6H) .respectively. Th(' pea.k
at 2.626 (2H) wPich di::~appears on D20 trflatme-.nt lS
due to the two proton"! of the ~:tlcoholic hydroxyl
groups (-C-OH). The carhiuyl prot.Qn (-CHOH}
appaa.rs a•/ a broad multiplet <;entel·ed at 3.648 (IH). 'l'he two benzylic proton"! appea..r as ~ typical multi-
plot centered at 3.038. Th.o above data show t.h.o.t
th., diol h~ the struetute (II). Direct crompa..ri11on
with an a.u.thllntic sample has not been p~liil>le but
there m little doubt that compound B is idf•nt.i(;al with
a.ur&ptE'n4 (I, Lit. rt>t,ord~ m.p. 98°). HCO
3
(I) ·>
D. P. OHAXR.ABORTY A.nd H. K. CHOWDHUBY, Tetrahedron
lett., 1967, 36, ~471. :l. D. P. CHAKRABORTY and S. K. 0HAKRABORTI, Trans. Bose Ru. lmt., 1961, 24, 15. 4. Dictionary of Organic C01ngunds, Eyre & Spottiswoode
Publishers r.td, E. & F. N. Spon Ud., Vol. I, 1964,
p. 293. J. INDIAN OHE}I. SOC. VOL. LII, DECEMBER 197o J. INDIAN OHE}I. SOC. VOL. LII, DECEMBER 197o pH rn.etric study of complexes of some
metal ions with salicylaldehyde·4·
phenyl·3-thiosemicarbazone
Y. N. BHATT, K. K. PATEL. K. J. SHAH, R. s. PATlilL"'
l)epartment of Ohomiiltry, Sam·a.shtra Univ~rsity
Bhavnagar
JllantUJCript received 29 March 1975; revi.Bed 24 ,July 1975;
accepted 14 Augtut 1975 (I) Results and Discussions The practical proton-ligand stabifiy;y consr,ant for
uhe ligand was calculated whh the help of nA valueM
at different B values (pH meter readings in 50%
a.queou.s dioxan-medium). nA is caleula.ted by th.P. method of lrvin.g & Rossotti3 as adoptf'd by Jahalpur-
Wala et al•. Plot of Jog n.d/1-n...t aga.insli B is done
T:<> obtain a best straight line. From this straight
hne the values of th£ practical ~tability cOJll'!tam
was obtained ,using the relation ]i'ol' log K1, it is obsel'V'(>(! that stability uou~t11.11h1
t\l'e in the- order Cu2t >Co2+-> \·02r >Ni2*-. Thus, Jrving William's rule is not violatC'd e:x:eep(;
for Ni2-+ which fm m~o~ le-s~ lita.blE' complex. log PK1B = B+log
~.A-
1-nA Metal titration.s : wa.~ not much difference between log K 1 and log K2• wa.~ not much difference between log K 1 and log K2•
The average. sta.hHity oonstants a1•e givE>n in Tablo 1. A mixture of 2 ml of O.IM HN03+5 ml of I.OM
k.N03+o mJ of 0.01 ligand+ I ml of O.OlM metal
nitrate solution (in case of cobalt 0.5 m1 solution waR
taken) +20 ml ofdioxane+17 ml ofwator was tit.ra-
ted with 0.2M NaOH solution. The average. sta.hHity oonstants a1•e givE>n in Tablo 1. Table 1
}[eta I
JogK1
logK2
log /J2
Cu2 •
10.28
('o2-'-
9.84
9.46
19.30
V02 •
9.44
N12+
$.94 Experimental in whicli( 1~n/JL wa1-
plottPd against (n-2)HL
d OI
'
' od
1
(1-=..n)
an
E>rup s metl
waH a so used as tht>Jf' (n-2)HL
d OI
'
' od
1
(1-=..n)
an
E>rup s metl
waH a so used as tht>Jf' (n-2)HL
d OI
'
' od
1
(1-=..n)
an
E>rup s metl
waH a so used as tht>Jf' Experimental Tb.o
li~and .;a lie yla.ld.ehyO.e-4· phcnyl-3-t hio::~emi
ca.rbozont> was synthesizoo and cryatallised to get ana-
lytically pure (m.p. 188°)1. ThE' ~tolution of SPT was
prepared in dioxane ('BDli, AnalaR ,re.de) which was
further rul'ifit.'Cl by standard method of Vogel2. Metal
nitrates 1lf!M wer(• of AnalaR grade which were esti-
mated by standard gravimetrin and Yolumetric
methods. Phillips pH mot('r wa8 USt:ld for pH
mca.suremeutR. pH muter was c:,alihrat(•d with huffer
'!olutions having pH~ 33 an(l 9.1 at 25°±1°. All tit.rations wen• carried out in a 100 ml beaker
kt~pt in water thermostat maintained at 25°±1(;. T!lirty five to forty 6vt\ roadingH were taken in ea.clt
oa~e. Eanh tJitra.tion waR rtlpea'OOcl. to get reproducible
results. The metal ligarut J'atio w~ maintained l :5. 1'h,c titration eurves are giwn in lfig. 1. Blank titraticms: A mixture of 2ml of Q1M
HN03, 5 rol of l.OM KN03, 25 mi dioxanf' and 18 mJ
of water (to make tht> tnt.al of 50 ml in a.IJ t.irra.tions} 1214 OTES NOTES purwa.la et al4. The va.lues of fi a.ro plotted against
pL. The fOJmatJOn em ves a1e representffi 111 F111. 2. I 'Ya~hs titl'ato(t with 0.2M NaOH until the pH J'if'f"'S to
a OUt 12. I
~ -8
0 >
C 2+ N :tt-
pl FOR
0
t
I
_
__,82
'{0
100
. ~ 0
J
" ;:Jt
l·'t ~
,.,
n
ll. 0)'1
.... t·O
G
J:<'ig. 2. Formation curves fot• SPT systemR. 12.·0
110
10·0
6
H
Cu
.5·0
Ill
4·0
3C
0·6
0·7
0·8
0·
. ·0
1-1
VOLUME OF ALKAL-I ('ml) ---!>
\
Fig. 1. Titration curve11 for SPT systems. J:<'ig. 2. Formation curves fot• SPT systemR. \
Fig. 1. Titration curve11 for SPT systems. Tlu• value~:~ of log K1 wer£' calculated by half iutegi'al
ID(>th<)(l. But for calt•ula.tion of loll K 2 least squru·t•
whicli( 1~n/JL Liga·nd titrations: A mixy;ure of 2 ml of O.IM HN03,
~ ml of l.OM KN03, 5 ml of O.OIM Hgand solution
In dioxane, 20 rnl of dioxane and 18 ml of wa.tt•r
was titrated with 0.2M NaOH solution. method? Referoaees J. I. D. SH.A.li: and J. P. TRtVBDI, J. Indian Ohem. Soc.,
1963, 40, 889. ,
,
2. A. I. VoGEL, "A Text Book of Practical Organic Chemis-
try,., Longmans, 1961, p. 177. All attempts to isolate cinnamic acid were unsuc. cessful. Ea.rlil".r, a striking example had been cited
where identical condhions were employed'. 3. H. IRVING and H. RossoTTI, J. Ohem. Soc.,
4
K E
K E 3. H. IRVING and H. RossoTTI, J. Ohem. Soc., 1954, 2904. 4. K. E. J.ABALPURWALA. K. E. VDNXATACHALA¥ and :u. B. KABA»I, J. l'TIM'g. Nuclear Ohem., 1964, 266, 1927. 1011. A probable explanation is afforded as follows :
Since the reaction is base-oa.talysed, the ca.rbonion
formed from N-substituwd succinimide is secondary
as compared to the formation of primary carbanion
fl'om acetic anh.ydride. Thf>refore the seoonda.ry
oa.rbanion is relatively le~R stab](> and more reactive,
hence the product. 5. H. IltVOl'G and H. RossoTTI, J. Ohem. Soc., 1953, 3397. 6
l
i
di 6. H. OLE.RUl>, "Ja.rnakloriderna.s Kompl exitate (diss.),
Lindstetnitta wnitJersities-Bokhawlel, Lund, 1944. A few benzylidme derivadves of
N-substituted succinimides
D'OosTA RosABIO AND V. V. NADK.A:RNY*
~adkarny Sacasa Research Laboratory,
Ohemistry Department,
St. Xavier's College, Bomba.y-1. llafH#scnpt received 23 Aprill975, rooi8ed 28 .fftly 1975,
accepted 12 At-UJust 1975. ~llafH#scnpt received 23 Aprill975, rooi8ed 28 .fftly 1975,
accepted 12 At-UJust 1975. 1
N the Perkin's1 Reaction,
a..roma.tic
aldehydes
condense with acin anhydrides possessing two
hydrogt>n
atoms on an a-carbon atom, in the
presence of sodium acetate to give fl-arylaorylic acidl4. Th
i
h
b
difi d
d
i M 1
N the Perkin's1 Reaction,
a..roma.tic
aldehydes
condense with acin anhydrides possessing two
hydrogt>n
atoms on an a-carbon atom, in the
presence of sodium acetate to give fl-arylaorylic acidl4. The reaction has been modified and connensatioM
}LaVe boon effected using a.Jdeb,ydes ann oa.rboxy}i<•
acids pO&Jessing a-hydrogen atoms. De Ta.r2 u.soo
this prooooure in the conden.c;a.tion of ll-nitrohenza.Jde-
llyde with phenyla.of>tic a.cid. with triE>thylamine as 1N *(All the melting points are taken in a t-oncentrated H2S04
bath a.nd may need further correction). *(All the melting points are taken in a t-oncentrated H2S04
bath a.nd may need further correction). @t]:::ti-(Q) ~~-©Yp:~"'"'
f
I
Ar
loY
11
Ill
a= -phr-ny1. b = -p-cblorophenyJ,
<' = -rutphthyl a= -phr-ny1. b = -p-cblorophenyJ,
<' = -rutphthyl Acknowledgement The a11t.lwr~ ul'e thankful to Dr. K. A. Thakot,
Profes~or and Head, Depattmtmt of Chemistry,
Saurashtra Univw:sity, Bhu.vnagac, fol' pro,,iding The proton ligand sta.bHity consta.nt obtained i".l
7.94 X 109. For metal ligand litabHity oonstants fi
and pL were calculated by the method of .Jabal- 1215 J. INDIAN CHEM. SOC., VOL. LII, DECEMBER 1975 laboratory fa.cilitjE'l:l; to C.S.I.R., New Delhi and
Gujarat State for the award of sohola.rships tO Y.N.B. and K.K.P., respectively. cinimidea were synthesized according to the method
as given in Wilds. cinimidea were synthesized according to the method
as given in Wilds. TheN-substituted imides, as obtained above were
condensed with benzaldehyde, acetic anhydride and
fused sodium acetatf> for 4-5 h,r under the conditions
of Perkin's reaction to afford compounds of the type
II. A few benzylidme derivadves of
N-substituted succinimides Similar condensations were offeoted using a.nisalde-
hyde to yield compounns of the type nr. ~adkarny Sacasa Research Laboratory,
Ohemistry Department, TABLE. DERIVATIVES OJo' THE TYPE II* AND m* TABLE. DERIVATIVES OJo' THE TYPE II* AND m*
C
d
M l f
l
0 TABLE. DERIVATIVES OJo' THE TYPE II* AND m*
Compound
Mol. formula
m.p. 0 0
II a. C24H 1102N
125°-6 .. Tib
Ca.HteO:aNCl
160°-1°
He
C:aaRtt~OllN
170°-1°
lila
C26H:atO"N
186°
IIIb
C26H:roO"-NOI
165°-6°
me
C3oH230"'N
166 .. -7°
*(All th
lti
i t
t k
i
t
t
t d H S0 St. Xavier's College, Bomba.y-1. D'OosTA RosABIO AND V. V. NADK.A:RNY* The nm-ivatives gave e~ted nitrogen analysis. Experimental procedure a ba.sa. The most significant applications
are :
condensation of cyclic sulphur oompoundf.l such &.<I
rhodaninea (containing active methylene groups)
wit.h aromatic ald('hydes and the famous 'Erlenmeyer's
azalaetona synthet~is. A mi.xtm·o of N-aubstitut<•d i>Uccinimide (0.5 g) and
aromatic aldehyde (0.6 g), acetic anhydride (2.5 ml)
and fused sodium acetate (0.1 g), is refl:uxed at 170°-
1900 and ma.intaiJ1ed at that temperature for 4-5 hr. Tho reaction fl.ask is then connooted to steam distil·
la.tiou apparatus and excess of aldehyde is steam
di111tilled. The resultant mixture, on oooling, affords
long needles of the product which on crystallization N-Substituted succinimidfla contain active methy~
lene groups and the aim of the pl'et;ant inve,;tigation
ia the extension of Perkins reaction to thesa imides
to form
bt>.nozylidine
d~rivatives. Therf>fOf('. N-
phehyl-. N·p-chloropht»1}1~, and N~na.pllthyl-. :mc- 1216
|
https://openalex.org/W4321017677
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https://www.researchsquare.com/article/rs-2521438/latest.pdf
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Atomically visualizing cyclic self-refreshing of gas-oxide interfaces
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Atomically visualizing cyclic self-refreshing of gas-
oxide interfaces Page 1/20
oxide interfaces
Xianhu Sun
Lawrence Berkeley National Laboratory
https://orcid.org/0000-0002-9509-2950
Dongxiang Wu
Binghamton University
Wenhui Zhu
Binghamton University
Yaguang Zhu
Binghamton University
Wissam Saidi
University of Pittsburgh
https://orcid.org/0000-0001-6714-4832
Jorge Boscoboinik
Brookhaven National Laboratory
https://orcid.org/0000-0002-5090-7079
Judith Yang
University of Pittsburgh
Renu Sharma
National Institute of Standards and Technology
https://orcid.org/0000-0001-7521-8790
Guangwen Zhou
Binghamton University
https://orcid.org/0000-0002-9243-293X
Article
Keywords:
Posted Date: February 16th, 2023
DOI: https://doi.org/10.21203/rs.3.rs-2521438/v1
License:
This work is licensed under a Creative Commons Attribution 4.0 International
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Read Full License
Additional Declarations: There is NO Competing Interest. Article License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Additional Declarations: There is NO Competing Interest. Page 1/20 Page 1/20 Abstract Surface-induced breaking of translation symmetry of a crystalline oxide results in various types of
coordinately unsaturated cations/anions and surface restructuring 1-11, yet identifying the stability,
functionality and activity of the coordinated unsaturated sites of gas-oxide interfaces remains
challenging owing to their dynamic behaviors in reacting gas and temperature environments and issues
with current characterization tools. Through direct in-situ transmission electron microscopy observations
and atomistic modeling, here we report cyclic self-refresh between oxygen-rich and oxygen-deficient
surface reconstructions of CuO in H2 that are chemically active and inactive for H2O formation,
respectively. After a period of chemical inactivity, the oxygen-deficient surface re-oxidizes back to the
oxygen-rich termination due to the outward diffusion of lattice oxygen from the subsurface. This cyclic
surface refresh is intrinsically induced by the disparity in chemical activity of undercoordinated surface
atoms in modulating H2-induced loss of lattice oxygen at the surface and subsequent oxygen
replenishment from the subsurface, which results in spatiotemporally separated redox reaction steps at
the oxide surface. The atomistic mechanism has significant implications in manipulating the surface
reactivity of oxides by tuning this separation of the redox steps at oxide surfaces. Full Text Figure 1a
illustrates a high-resolution TEM (HRTEM) image of the as-formed CuO from the oxidation of Cu, showing
a step-terrace morphology composed of
and
microfacets with the overall surface
(
−
2 02)
(
−
1 10)
[
] followed by switching to H2 gas flow (0.5 Pa) that induces the oxide reduction reaction. Figure 1a
illustrates a high-resolution TEM (HRTEM) image of the as-formed CuO from the oxidation of Cu, showing
a step-terrace morphology composed of
and
microfacets with the overall surface
(
−
2 02)
(
−
1 10)
[
−
] orientation along the
direction. The inset in Fig. 1a is a diffractogram of the HRTEM image,
[
−
3 12] orientation along the
direction. The inset in Fig. 1a is a diffractogram of the HRTEM image,
[
−
3 12] which can be indexed as monoclinic CuO oriented along the
zone axis. Figure 1b is an enlarged
[
−
1
−
1
−
1] g
[
−
1
−
1
−
1] zone axis. By comparing the experimental and simulated HRTEM images in Fig. 1b, we can conclude that
the Cu-O columns show bright image contrast whereas the pure Cu columns have significantly low image
contrast, as indicated by the atomic model overlaid on the simulated HRTEM image. Our in-situ TEM
images indicate that the CuO lattice is relatively stable at 300 ℃ in vacuum for at least 5 min, and the
continuous electron beam irradiation does not induce noticeable oxide reduction, as confirmed from the
negligible changes in the lattice contrast and the step-terrace surface morphology (Extended Data Fig. 1
and Supplementary Movie 1). [
] Figure 1c-f presents a time sequence of HRTEM images (Supplementary Movie 2) showing the CuO
surface evolution at 300°C after switching to 0.5 Pa of H2 gas flow. The H2 exposure results in the oxide
reduction from the surface, and the initially stepped surface becomes atomically flat in some local areas,
as marked by the white dashed box in Fig. 1c. Compared to stoichiometric CuO, this atomically flat
surface region is slightly O-deficient with a surface composition of Cu8O7, as determined from a
combination of DFT modeling and HRTEM image simulations (Fig. 1g-j). This trend of the surface
flattening continues, and another surface configuration (white dashed box in Fig. 1d) of the flattened
region becomes visible, which is significantly O-deficient (Cu8O2), (Fig. 1h, j, k). Full Text Coordinated unsaturated sites determine the surface reactivity for a wide range of interfacial phenomena
and are crucial to many materials applications such as in catalysis, fuel reactions, and corrosion
protection. Even with the advent of in-situ capabilities of surface science tools, directly probing the
activity and dynamics of such coordinated unsaturated atoms at oxide surfaces is challenging because
the insulating (or wide-band-gap) nature of bulk oxides hampers ion and electron spectroscopic
measurements. By contrast, the development of environmental transmission electron microscopy (ETEM)
allows direct imaging of gas-solid interfacial reactions by introducing a reactive gas to the sample area
while simultaneously probing atomic structural evolution in the outermost surface layer and sub-surface
layers of the oxides 7,12−16. Here we report direct ETEM imaging of cyclic gas-oxide interfacial
restructuring induced by the disparity in chemical activity of undercoordinated surface atoms of active
oxides that are used widely as catalysts and catalyst supports. Such dynamic gas-solid interfacial
restructuring shown here cannot be observed outside of reaction conditions and underscores the need of
in-situ microscopy observations to probe the intrinsic kinetics of the gas-solid reactions by
spatiotemporally resolving local reaction dynamics. Cupric oxide (CuO) reduction by hydrogen (H2) is chosen as a model reaction because of its wide
relevance to various catalytic processes including the water-gas-shift reaction 17, methanol synthesis and
oxidation 18,19, and oxidative dehydrogenation of alcohols 20, wherein CuO is an active catalyst/support
and undergoes the reduction reaction because H2 is involved either as a reactant or a product present in
the surrounding 17–21. Our in-situ TEM experiments involve two steps, starting with the in-situ formation
of CuO inside the environmental TEM by oxidizing metallic Cu at 300°C in 0.5 Pa of oxygen (O2) pressure, Page 2/20 Page 2/20 Page 2/20 followed by switching to H2 gas flow (0.5 Pa) that induces the oxide reduction reaction. Figure 1a
illustrates a high-resolution TEM (HRTEM) image of the as-formed CuO from the oxidation of Cu, showing
a step-terrace morphology composed of
and
microfacets with the overall surface
orientation along the
direction The inset in Fig 1a is a diffractogram of the HRTEM image
(
−
2 02)
(
−
1 10)
[
−
3 12] followed by switching to H2 gas flow (0.5 Pa) that induces the oxide reduction reaction. Full Text Once the entire surface
becomes flattened, the surface configurations are subject to cyclic interconversions between the Cu8O7
and Cu8O2 reconstructions while constantly maintaining the flat surface morphology and the
surface orientation (Fig. 1e, f and Extended Data Fig. 2). No other stable surface restructuring is observed
besides the Cu8O7 and Cu8O2 reconstructions. [
−
3 12] Figure 1g is an enlarged HRTEM image of the Cu8O7 surface configuration as marked with the yellow box
in Fig. 1e. Microscopically, the oxide reduction is induced by the loss of lattice O from the surface. During
the H2 exposure, hydrogen reacts with lattice O at the CuO surface to form H2O molecules that desorb
from the surface at elevated temperatures. Accompanying the surface desorption of H2O molecules is the
concomitant formation of O vacancies at the oxide surface. DFT is employed to examine the energetics Page 3/20 Page 3/20 (
−
3 12) of O vacancy formation at different surface sites of bulk terminated CuO
consisting of
,
(
−
3 12)
Osurf
2
f
f of O vacancy formation at different surface sites of bulk terminated CuO
consisting of
,
and
(Fig. 1i). Figure 1j shows the minimum-energy surface configuration upon the surface
desorption of one of the most vulnerable O atoms (as marked by the red arrow in Fig. 1i) that are
coordinated with two Cu atoms and possess the lowest vacancy formation energy. This process thereby
results in the slight O deficiency (Cu8O7) consisting of
,
and
, in which the
displacements of the remaining Cu and O atoms within the three topmost Cu-O layers convert the other
at the top left corner to
. The inset in Fig. 1g is a simulated HRTEM image (Extended Data
Fig. 3) based on the DFT-relaxed Cu8O7 surface model in Fig. 1j, which matches well with all the key
feature of the experimental HRTEM image, including dimmed and blurry image contrast of the atom
columns owing to the O deficiency and resulting distortions of the atom positions, mostly in the two
topmost layers. Figure 1h corresponds to an enlarged HRTEM view of the Cu8O2 surface of the red box
region marked in Fig. 1f, where the surface becomes more O-deficient due to the continued reduction by
H2. As shown in Fig. 1j, the O atoms marked by the red arrows are coordinates with three Cu atoms and
have the similar vacancy formation energies. Full Text Their desorption from the surface not only results in the
significant O deficiency (Cu8O2 consisting of
and
) but also induces large displacements
(as indicated by the blue arrows) of the remaining Cu and O atoms in the three topmost Cu-O layers. The
inset in Fig. 1h is a simulated HRTEM image (Extended Data Fig. 4) based on the DFT-relaxed structure of
the Cu8O2 model (Fig. 1k), which matches well with the experimental HRTEM image, including the relative
intensity of the Cu-O (bright) and Cu (dim) columns and the relatively large hollow space resulting from
the reconfiguration of the Cu and O atoms in the three topmost Cu-O layers. (3 12)
O
f
2
Osurf
3
Cusurf
3
Osurf
3
Cusurf
3
Cusurf
2
Osurf
2
Osurf
3
Osurf
3
Cusurf
2 and
(Fig. 1i). Figure 1j shows the minimum-energy surface configuration upon the surface
desorption of one of the most vulnerable O atoms (as marked by the red arrow in Fig. 1i) that are
coordinated with two Cu atoms and possess the lowest vacancy formation energy. This process thereby
results in the slight O deficiency (Cu8O7) consisting of
,
and
, in which the
displacements of the remaining Cu and O atoms within the three topmost Cu-O layers convert the other
at the top left corner to
. The inset in Fig. 1g is a simulated HRTEM image (Extended Data
Fig. 3) based on the DFT-relaxed Cu8O7 surface model in Fig. 1j, which matches well with all the key
feature of the experimental HRTEM image, including dimmed and blurry image contrast of the atom
columns owing to the O deficiency and resulting distortions of the atom positions, mostly in the two
topmost layers. Figure 1h corresponds to an enlarged HRTEM view of the Cu8O2 surface of the red box
region marked in Fig. 1f, where the surface becomes more O-deficient due to the continued reduction by
H2. As shown in Fig. 1j, the O atoms marked by the red arrows are coordinates with three Cu atoms and
have the similar vacancy formation energies. Their desorption from the surface not only results in the
significant O deficiency (Cu8O2 consisting of
and
) but also induces large displacements
(as indicated by the blue arrows) of the remaining Cu and O atoms in the three topmost Cu-O layers. The
inset in Fig. 1h is a simulated HRTEM image (Extended Data Fig. Full Text Figure 2c illustrates the Cu8O7 → Cu8O2 conversion via surface pitting (marked
by the yellow arrow) at the Cu8O7 terrace that results in the nucleation of two new steps of three atomic
layer height (3×d
) and their lateral receding motion (Supplementary Movie 5). Figure 2d shows
the Cu8O7 → Cu8O2 conversion via the receding flow of a monoatomic step (1×d
along the surface (Supplementary Movie 6), where the step flow rate is measured as 0.412±0.002 nm s−1. (
−
3 12)
(
−
3 12)
(
−
3 12) ≈1.31\AA ) Figure 2b-d presents in-situ HRTEM observations of the Cu8O7 → Cu8O2 conversions and demonstrates
the step-flow mechanism for the surface transformation. Figure 2b corresponds to a surface consisting
of an upper Cu8O7 terrace and a lower Cu8O2 terrace separated by a bilayer atomic step (2×d
). The upper Cu8O7 terrace gradually transforms to the lower Cu8O2 terrace via the receding motion of the
bilayer step (marked by the white arrow) with the measured step flow rate of 0.221 ± 0.001 nm s− 1
(Supplementary Movie 4) Figure 2c illustrates the Cu8O7 →Cu8O2 conversion via surface pitting (marked
(
−
3 12) Figure 2b-d presents in-situ HRTEM observations of the Cu8O7 → Cu8O2 conversions and demonstrates
the step-flow mechanism for the surface transformation. Figure 2b corresponds to a surface consisting
of an upper Cu8O7 terrace and a lower Cu8O2 terrace separated by a bilayer atomic step (2×d
). (
−
3 12) along the surface (Supplementary Movie 6), where the step flow rate is measured as 0.412±0.002 nm s−1. Figure 2e-f demonstrates that the Cu8O2 → Cu8O2 conversion also occurs via the receding motion of
surface steps. Figure 2e corresponds to the surface consisting of two Cu8O2 terraces separated by a
monoatomic step (Supplementary Movie 7) whereas Fig. 2f shows the surface composed of two Cu8O2
terraces separated by a double-layer step (Supplementary Movie 8). In both cases, surface refresh (Cu8O2
→ Cu8O2) happens through the retraction motion of the atomic steps toward the upper terrace side, where
the step-flow rates are measured as 0.262 ± 0.002 nm s− 1 and 0.137 ± 0.001 nm s− 1 for the monoatomic
and double-layer steps, respectively. The observed retraction motion of surface steps is induced by the
oxide decomposition along the step, where the lattice O is lost due to the H2-induced H2O formation. Full Text 4) based on the DFT-relaxed structure of
the Cu8O2 model (Fig. 1k), which matches well with the experimental HRTEM image, including the relative
intensity of the Cu-O (bright) and Cu (dim) columns and the relatively large hollow space resulting from
the reconfiguration of the Cu and O atoms in the three topmost Cu-O layers. (
)
Osurf
3
Cusurf
3
Osurf
3
Cusurf
3
Cusurf
2
Osurf
2
Osurf
3
Osurf
3
Cusurf
2 Figure 2 illustrates time-lapse HRTEM images of the interconversion between the Cu8O7 and Cu8O2
surface reconstructions and a direct comparison of their chemical reactivity governing the oxide
reduction in the H2 gas flow. Overall, their interconversion can be grouped into three categories: i) Cu8O2
→ Cu8O7 (Fig. 2a), ii) Cu8O7 → Cu8O2 (Fig. 2b-d), and Cu8O2 → Cu8O2 (Fig. 2e-f). Figure 2a corresponds
to the snapshots of the Cu8O2 → Cu8O7 conversion (Supplementary Movie 3), where the surface has the
Cu8O2 reconstruction at the beginning of the observation. It takes an incubation time of ≈ 7 s to initiate
the Cu8O2 → Cu8O7 conversion via the nucleation and growth of a Cu8O7 segment out of the atomically
flat Cu8O2 surface. As marked by the white double arrow in Fig. 2a, the Cu8O7 segment has nearly the
same surface height as the parent Cu8O2 surface. Since the Cu8O7 is O-richer than the Cu8O2, the
nucleation and growth of the Cu8O7 segment is induced by the outward diffusion of O from the
subsurface to replenish the lost lattice O at the surface. The observed Cu8O2 → Cu8O7 conversion also
demonstrates the inertness of the Cu8O2 surface towards further H2, inhibiting H2O formation and lattice
O loss. Therefore, the inactivity of the Cu8O2 allows time for the subsurface O to refill O vacancies at the
surface for recovering the Cu8O7. Page 4/20 Figure 2b-d presents in-situ HRTEM observations of the Cu8O7 → Cu8O2 conversions and demonstrates
the step-flow mechanism for the surface transformation. Figure 2b corresponds to a surface consisting
of an upper Cu8O7 terrace and a lower Cu8O2 terrace separated by a bilayer atomic step (2×d
). The upper Cu8O7 terrace gradually transforms to the lower Cu8O2 terrace via the receding motion of the
bilayer step (marked by the white arrow) with the measured step flow rate of 0.221 ± 0.001 nm s− 1
(Supplementary Movie 4). Full Text At
the same time, Cu atoms released from the receding step diffuse to the inner surface region of the oxide
and aggregate into a Cu thin layer, as indicated by the Moiré fringe contrast of the TEM image formed as
a result of overlapping of the Cu with the CuO in Extended Data Fig. 5). Figure 2e-f demonstrates that the Cu8O2 → Cu8O2 conversion also occurs via the receding motion of
surface steps. Figure 2e corresponds to the surface consisting of two Cu8O2 terraces separated by a
monoatomic step (Supplementary Movie 7) whereas Fig. 2f shows the surface composed of two Cu8O2
terraces separated by a double-layer step (Supplementary Movie 8). In both cases, surface refresh (Cu8O2
→Cu8O2) happens through the retraction motion of the atomic steps toward the upper terrace side, where The Cu8O7 → Cu8O2 conversion requires the loss of lattice O in the Cu8O7 terrace. By contrast, the Cu8O2
→ Cu8O7 conversion require addition of O that could be provided by the outward diffusion of lattice O
from the subsurface region of the CuO without further lattice O loss from the surface. Their requirement
difference for the interconversion of the two surface reconstructions indicates that the Cu8O7 terminated
terrace is more receptive for H2 that results in H2O formation than the Cu8O2 terminated terrace. This is
also confirmed by the faster step flow rates during the Cu8O7 → Cu8O2 conversion (0.412 ± 0.002 nm/s
and 0.221 ± 0.001 nm/s for monoatomic and double-layer steps, respectively) than that during the Cu8O2
→ Cu8O2 conversion (0.262 ± 0.002 nm/s and 0.137 ± 0.001 nm/s for the monoatomic and double-layer
steps, respectively). The difference in the surface reactivity toward H2 is further confirmed by the
observed surface pitting at the Cu8O7 terrace (Fig. 2c), as a result of the oxide lattice collapse induced by
the lattice O loss. By contrast, the Cu8O2 terminated terraces are more stable and do not show any
surface pitting (except for the step flow) because of its lower reactivity toward H2 adsorption. This is The Cu8O7 → Cu8O2 conversion requires the loss of lattice O in the Cu8O7 terrace. By contrast, the Cu8O2
→ Cu8O7 conversion require addition of O that could be provided by the outward diffusion of lattice O
from the subsurface region of the CuO without further lattice O loss from the surface. Full Text Their requirement
difference for the interconversion of the two surface reconstructions indicates that the Cu8O7 terminated
terrace is more receptive for H2 that results in H2O formation than the Cu8O2 terminated terrace. This is
also confirmed by the faster step flow rates during the Cu8O7 → Cu8O2 conversion (0.412 ± 0.002 nm/s
and 0.221 ± 0.001 nm/s for monoatomic and double-layer steps, respectively) than that during the Cu8O2
→ Cu8O2 conversion (0.262 ± 0.002 nm/s and 0.137 ± 0.001 nm/s for the monoatomic and double-layer
steps, respectively). The difference in the surface reactivity toward H2 is further confirmed by the
observed surface pitting at the Cu8O7 terrace (Fig. 2c), as a result of the oxide lattice collapse induced by
the lattice O loss. By contrast, the Cu8O2 terminated terraces are more stable and do not show any
surface pitting (except for the step flow) because of its lower reactivity toward H2 adsorption. This is Page 5/20 Page 5/20 evidenced from the above in-situ TEM observation that the Cu8O2 surface cannot be reduced easily,
instead, it is re-oxidized back to the Cu8O7 with the supply of O from the subsurface when surface steps
are absent on the Cu8O2 surface in Fig. 2a. It is worth noting that the in situ TEM observations are made
with the same dose rate of the electron beam, so the difference in the reducibility between the Cu8O7 and
Cu8O2 reconstructions is intrinsic rather than an artifact caused by the electron beam irradiation during
the TEM observation. In addition, comparative experiments with the beam blanked off suggest
reconstructions and interconversion are independent on the electron beam irradiation (Extended Data
Fig. 6, Supplementary Movie 2). Figure 2g shows the measured oxide reduction rate during the constant H2 gas flow by measuring the
receding motion speed of the atomic step multiplied by the surface height of the step. As expected, the
oxide reduction rate oscillates with periodic fluctuations caused by the cyclic refresh between the Cu8O7
and Cu8O2 surfaces. As labeled in Fig. 2g, the oxide reduction rate depends on the type of the
interconversions, no oxide reduction occurs during the Cu8O2 → Cu8O7 transformation (i.e., red segments
in Fig. 2g, 0 ± 0.003 nm2 s− 1, corresponding to Fig. 2a), whereas the three types of the Cu8O7 → Cu8O2
transformation (light blue segments in Fig. 2g, corresponding to Fig. Full Text 2b-d) show the similar fast reduction
rate (0.061 ± 0.007 nm2 s− 1), and the two types of the Cu8O2 → Cu8O2 transformation have a slower
reduction rate (0.033 ± 0.004 nm2 s− 1) (purple segments, corresponding to Fig. 2e, f). To further elucidate the reaction pathway between H2 and the CuO surface, ambient-pressure X-ray
photoelectron spectroscopy (AP-XPS) is employed to monitor the evolution of reaction intermediates. Same as the in-situ TEM experiments, the AP-XPS measurements also start with the oxidation of clean Cu
into CuO in O2, followed by switching to H2 gas flow. As shown in the lower panel of Fig. 3a, the O 1s
obtained from the as-oxidized Cu in O2 shows a relatively symmetrical line shape with the peak located at
the binding energy of 529.7 eV, which corresponds to lattice O in CuO. Upon switching from O2 to H2, the
O 1s spectrum (upper panel of Fig. 3a) is broadened with the presence of a large shoulder at the higher
binding energy of 531.4 eV. The shoulder peak matches well with the binding energy of hydroxyl (OH)
species 22. The presence of OH species provides a clear indication of the dissociative H2 adsorption; that
is, H2 → 2H and H + O → OH, where O is lattice O at the CuO surface and OH is a reaction intermediate
before its transformation to H2O. The surface coverage of the OH species depends on the surface density
of lattice O, and it is therefore reasonable to expect a stronger OH peak intensity for the Cu8O7 than that
for Cu8O2. To test the dependence of the OH coverage on the CuO surface reconstructions, we then
introduce O2 to the XPS chamber while maintaining the constant H2 gas flow at 0.13 Pa. As shown in
Fig. 3b, the peak intensity of the OH component increases with increasing O2 partial pressure during the
O2 + H2 co-dosing. This indicates that H2 induced loss of lattice O can be partly replenished with the
supply of O2 gas to favor the O-rich surface such as Cu8O7 over Cu8O2. Page 6/20 To shed light on the mechanism of the interconversion between Cu8O7 and Cu8O2, DFT calculations are
performed to examine the relative stability and activity of the two surface reconstructions toward
dissociative H2 adsorption and the resulting H2O formation. Full Text At the beginning of the reaction, one H2
molecule is placed slightly (0.02 ± 0.01 nm) above the top of the
of the Cu8O7 surface (Fig. 3c). Upon structure relaxation, the H2 molecule dissociates spontaneously into two H atoms that bond with
the adjacent
to form two OH groups with a decrease of the system energy by 0.72 eV (Fig. 3d). The
OH groups are stable instead of forming water molecules before the surface is fully covered by one
monolayer of OH (Fig. 3e). Then, it is favorable to adsorb an additional H atom on the top of existing OH
to form a H2O molecule with a system energy drop by 1.13 eV (Fig. 3f). The desorption of H2O molecules
at elevated temperatures results in the loss of lattice O, which destabilizes the surface and leads to the
lattice collapse of the surface layer. This corroborates well with our in-situ TEM observations, showing the
surface pitting (Fig. 2c) and the surface decay by the receding motion of the atomic steps (Fig. 2b-d). The
H2-induced surface O loss from the Cu8O7 can be partly replenished with the supply of O from the
adjacent subsurface region, which results in the significant O deficiency in the subsurface. However, the
rate of the O replenishment from the subsurface cannot keep up with the faster rate of O loss at the
Cu8O7 surface in the reducing atmosphere. This results in the lattice collapse of the Cu8O7 surface and
drives the reduction of the Cu8O7 to Cu8O2 via the nucleation and growth of the Cu8O2 upon the surface
pitting of the Cu8O7 layer (Fig. 2c). Osurf
3
Osurf
3 Similarly, our DFT calculations show that H2 molecules at the Cu8O2 surface also spontaneously
dissociate into atomic H and form OH groups at
(Fig. 3g, h). However, after all the lattice O at the
surface is converted to OH, additional H adsorption by the OH for H2O formation is energetically
Osurf
3 Similarly, our DFT calculations show that H2 molecules at the Cu8O2 surface also spontaneously
dissociate into atomic H and form OH groups at
(Fig. 3g, h). However, after all the lattice O at the
surface is converted to OH, additional H adsorption by the OH for H2O formation is energetically
unfavorable. Instead, the additional H bounces off from the OH site to adjacent Cu with an increase of the
system energy by 0.13 eV (Fig. Full Text 3i, j). This indicates a lower reducibility of the Cu8O2 surface compared to
the Cu8O7, the latter of which favors the H2O formation after reaching one monolayer of the OH coverage. The lower reducibility of the Cu8O2 surface can be attributed to its significant O deficiency that lowers the
coordination number of surface Cu from
in Cu8O7 (Fig. 3c) to
in Cu8O2 (Fig. 3g). As a
result, the lattice O at the Cu8O2 surface is coordinated by more metallic-like
(Extended Data
Fig. 7) that results in stronger Pauli repulsion toward further H adsorption. The OH-saturated Cu8O2
surface can even be re-oxidized back to the Cu8O7 with the supply of lattice O from the subsurface
(Fig. 2a), which takes a relatively long incubation time ≈ 7 s to initiate the Cu8O2 → Cu8O7 conversion
because of the large O diffusion barrier (1.51 eV) through the Cu8O2 layer (Extended Data Fig. 8). This
large diffusion barrier can be related to the relatively hollow configuration of the Cu8O2 (Fig. 1k) that
results in the longer diffusion distance of the O atoms compared to that in the Cu8O7 (Fig. 1j). Osurf
3
Cusurf
3
Cusurf
2
Cusurf
2 surface is converted to OH, additional H adsorption by the OH for H2O formation is energetically
unfavorable. Instead, the additional H bounces off from the OH site to adjacent Cu with an increase of the
system energy by 0.13 eV (Fig. 3i, j). This indicates a lower reducibility of the Cu8O2 surface compared to
the Cu8O7, the latter of which favors the H2O formation after reaching one monolayer of the OH coverage. The lower reducibility of the Cu8O2 surface can be attributed to its significant O deficiency that lowers the
coordination number of surface Cu from
in Cu8O7 (Fig. 3c) to
in Cu8O2 (Fig. 3g). As a
result, the lattice O at the Cu8O2 surface is coordinated by more metallic-like
(Extended Data
Fig. 7) that results in stronger Pauli repulsion toward further H adsorption. The OH-saturated Cu8O2
surface can even be re oxidized back to the Cu O with the supply of lattice O from the subsurface
Cusurf
3
Cusurf
2
Cusurf
2 unfavorable. Instead, the additional H bounces off from the OH site to adjacent Cu with an increase of the
system energy by 0.13 eV (Fig. 3i, j). Full Text This indicates a lower reducibility of the Cu8O2 surface compared to
the Cu8O7, the latter of which favors the H2O formation after reaching one monolayer of the OH coverage. The lower reducibility of the Cu8O2 surface can be attributed to its significant O deficiency that lowers the
coordination number of surface Cu from
in Cu8O7 (Fig. 3c) to
in Cu8O2 (Fig. 3g). As a
result, the lattice O at the Cu8O2 surface is coordinated by more metallic-like
(Extended Data
Cusurf
3
Cusurf
2
Cusurf
2 Fig. 7) that results in stronger Pauli repulsion toward further H adsorption. The OH-saturated Cu8O2
surface can even be re-oxidized back to the Cu8O7 with the supply of lattice O from the subsurface
(Fig. 2a), which takes a relatively long incubation time ≈ 7 s to initiate the Cu8O2 → Cu8O7 conversion
because of the large O diffusion barrier (1.51 eV) through the Cu8O2 layer (Extended Data Fig. 8). This
large diffusion barrier can be related to the relatively hollow configuration of the Cu8O2 (Fig. 1k) that
results in the longer diffusion distance of the O atoms compared to that in the Cu8O7 (Fig. 1j). We now address the question of why the Cu8O7 → Cu8O2 and Cu8O2 → Cu8O2 transformations occur
preferentially via the receding motion of surface steps. O atoms along step edges have less coordinated
Cu atoms and thus less Pauli repulsion for capturing H (Extended Data Fig. 7c, d), which is conducive to Page 7/20 Page 7/20 the OH and H2O formation along the step edges. In addition, our DFT calculations also indicate that O
vacancies produced from the H2O formation and desorption in the terrace area tend to segregate to the
step edge via surface diffusion (Extended Data Fig. 9), thereby further destabilizing the step-edge Cu
atoms and contributing to the observed receding motion of surface steps via step-edge detachment of
under-coordinated Cu atoms. DFT calculations also indicate that differences exist in the energetics of the
vacancy formation between the Cu8O7 and Cu8O2 surfaces. For the Cu8O7, the O vacancy formation
energy along the step edge is 2.1 eV, which is slightly smaller than that (2.2 eV) for the terrace region
(Extended Data Fig. 10a). By contrast, the Cu8O2 surface has the O vacancy formation energies of 2.4 eV
along the step edge and 2.8 eV at the terrace (Extended Data Fig. 10b), respectively. Full Text Continued H2O formation leads to the accumulation
of O vacancies and induces surface pitting via local lattice collapse of the Cu8O7 layer, where the bottom
of the surface pits has the Cu8O2 reconstruction (Fig. 4d, e). The Cu8O7→Cu8O2 conversion can occur via
the growth of a monoatomic-deep pit with the receding motion of the two resulting monoatomic steps
(Fig. 4d, e), thereby completing a cycle by reverting the surface back to the Cu8O2 (Fig. 4e, a). Multiple
locations of the surface pitting with the depths of one or two atomic layers can also occur in the Cu8O7
layer (Fig. 4f), and the Cu8O7→Cu8O2 conversion via the lateral growth of these surface pits results in the
creation of Cu8O2-terminated terraces separated by a monoatomic step (Fig. 4g, h). This switches the
step-flow Cu8O7→Cu8O2 transformation to the step-flow Cu8O2→Cu8O2 surface refresh that also leads to
the completion of the cycle by reverting the surface to the Cu8O2 (Fig. 4h, a). Due to the intrinsic
difference of the chemical activity of the Cu8O7 and Cu8O2 surfaces, the cyclic switching between these
two surface reconstructions induced by dynamic interplay between the H2-induced loss of lattice O in the
topmost surface and slower O supply from the subsurface manifests as the oscillatory kinetics of the 8
7
y
2
y
dissociative H2 adsorption. Surface desorption of H2O molecules results in the formation of O vacancies
in both the surface and subsurface regions (Fig. 4c). Continued H2O formation leads to the accumulation
of O vacancies and induces surface pitting via local lattice collapse of the Cu8O7 layer, where the bottom
of the surface pits has the Cu8O2 reconstruction (Fig. 4d, e). The Cu8O7→Cu8O2 conversion can occur via
the growth of a monoatomic-deep pit with the receding motion of the two resulting monoatomic steps
(Fig. 4d, e), thereby completing a cycle by reverting the surface back to the Cu8O2 (Fig. 4e, a). Multiple
locations of the surface pitting with the depths of one or two atomic layers can also occur in the Cu8O7
layer (Fig. 4f), and the Cu8O7→Cu8O2 conversion via the lateral growth of these surface pits results in the
creation of Cu8O2-terminated terraces separated by a monoatomic step (Fig. 4g, h). This switches the
step-flow Cu8O7→Cu8O2 transformation to the step-flow Cu8O2→Cu8O2 surface refresh that also leads to
the completion of the cycle by reverting the surface to the Cu8O2 (Fig. 4h, a). Full Text Such differences in
the O vacancy formation explain the lower reducibility of the Cu8O2 and the slower step-flow rate of the
surface refresh (Cu8O2 → Cu8O2) compared to that for the surface transformation (Cu8O7 → Cu8O2). For
both the Cu8O7 and Cu8O2, step edges have lower O vacancy formation energies than terraces, which lead
to the receding motion of surface steps and the cyclic oscillation in the step-flow decay of the surface
layers of the oxide (Fig. 2g). In-situ TEM observations described above and DFT results are mutually consistent and deliver strong
evidence that the O-deficient Cu8O2 surface has lower reducibility than the O-rich Cu8O7. Figure 4
schematically summarizes the cyclic interconversions of the two reconstructions resulting from the
spatiotemporally separated reduction and oxidation reaction steps at the oxide surface. The Cu8O2
surface is saturated with the OH coverage and the lack of chemical activity toward further H2 adsorption
allows its oxidation to the Cu8O7 via the Cu8O2 + O (lattice) → Cu8O7 conversion by uptaking lattice O
right below in the subsurface (Fig. 4a, b). This conversion is slow as evidenced by a relatively long
incubation time because of the large diffusion barrier (1.51 eV) of the subsurface O to the Cu8O2 layer. The formation of the Cu8O7 makes the surface chemically active toward the H2O formation by In-situ TEM observations described above and DFT results are mutually consistent and deliver strong
evidence that the O-deficient Cu8O2 surface has lower reducibility than the O-rich Cu8O7. Figure 4
schematically summarizes the cyclic interconversions of the two reconstructions resulting from the
spatiotemporally separated reduction and oxidation reaction steps at the oxide surface. The Cu8O2
surface is saturated with the OH coverage and the lack of chemical activity toward further H2 adsorption
allows its oxidation to the Cu8O7 via the Cu8O2 + O (lattice) → Cu8O7 conversion by uptaking lattice O
right below in the subsurface (Fig. 4a, b). This conversion is slow as evidenced by a relatively long
incubation time because of the large diffusion barrier (1.51 eV) of the subsurface O to the Cu8O2 layer. The formation of the Cu8O7 makes the surface chemically active toward the H2O formation by
dissociative H2 adsorption. Surface desorption of H2O molecules results in the formation of O vacancies
in both the surface and subsurface regions (Fig. 4c). Full Text Due to the intrinsic
difference of the chemical activity of the Cu8O7 and Cu8O2 surfaces, the cyclic switching between these
two surface reconstructions induced by dynamic interplay between the H2-induced loss of lattice O in the
topmost surface and slower O supply from the subsurface manifests as the oscillatory kinetics of the Page 8/20 Page 8/20 oxide reduction (Fig. 2g). This oscillatory kinetics is governed by the spatiotemporally separated redox
reactions of the Cu8O7 reduction by H2 and Cu8O2 oxidation via the subsurface lattice O. Reducibility is key for the use of oxide catalysts in chemical transformations such as catalytic oxidation
reactions and oxidative dehydrogenation processes following the Mars-van Krevelen (MvK) mechanism
23. In this mechanism, the oxide itself serves as a source of O to form oxygenated compounds that
desorb from the surface. As shown from our in-situ TEM observations in Fig. 2 and DFT modeling in
Fig. 3, the coordinatively unsaturated
exerts more repulsion force than
for dissociative H2
adsorption at
, which not only drives cyclic interconversions of the surface reconstructions (Cu8O7
vs. Cu8O2) but also results in a period of chemical inactivity before the
is re-converted to
by the outward diffusion of lattice O in the subsurface. These results demonstrate that the microscopic
oscillatory kinetics of the oxide reduction comprises spatiotemporally separated steps of the oxide
oxidation and reduction even under a steady reaction condition. However, macroscopically, the oscillatory
kinetics cannot be detected readily. This is mainly due to facet-dependent reaction kinetics on different
facets of catalysts, and the ensemble average of countless microscopic kinetics washes out the
uncorrelated oscillations at different surface areas and facets, thereby giving rise to a superimposed
macroscopic kinetics with few chances to display oscillatory manner. Cusurf
2
Cusurf
3
Osurf
3
Cusurf
2
Cusurf
3 Oscillations in chemical reactions operate far from equilibrium on the way towards equilibrium. Therefore, oscillations in a chemical reaction are driven by the decrease in free energy of the reacting
system. This decrease is what drives all chemical reactions, but not all chemical reactions display the
oscillatory kinetics. There must be some feature peculiar to the oscillatory reactions that allows them to
periodically change the reaction rate under stationary conditions. In the field of heterogeneous catalysis,
this feature is tied to the periodic changes of the surface state of the catalyst. Full Text The origin of the
oscillations is attributed to exothermic-/endothermic-reaction-induced temperature change, coverage-
dependent adsorption, autocatalytic free site generation on the surface, oxide formation and reduction,
and inhibitory effect of the product adsorption, etc. 24–30. Departing from these known mechanisms that
are derived largely from carbon monoxide oxidation 28–32 and nitric oxide reduction on platinum group
metals 33–36, our in-situ TEM imaging and analysis shown above demonstrate that the disparity in the
surface activity between under-coordinated O and Cu exerts an intrinsic control of the microscopically
oscillatory redox kinetics of the CuO surface. The atomistic insights from the H2 oxidation over CuO may
be directly applicable to a wide range of oxide-catalyzed oxidation reactions for controlling the
transformation kinetics. This is because the protypes of the elemental processes governing the surface
reactivity exhibit the similarities, including the involvement of lattice O following the MvK mechanism and
disparity in the chemical activity among different types of undercoordinated surface atoms in mediating
the spatiotemporal separation between the catalyst oxidation and reduction. In summary, we have identified the atomistic origin of the oscillatory kinetics of CuO reduction in H2. Using in-situ TEM imaging, we observe cyclic self-interconversions between the Cu8O7 and Cu8O2 surface Page 9/20 reconstructions in constant H2 gas flow. The O-rich Cu8O7 is chemically active toward H2O formation and
undergoes self-transformation to the O-deficient Cu8O2. The O-deficient Cu8O2 reconstruction results in a
period of chemical inactivity before its re-oxidation back to the Cu8O7 with the supply of lattice O in the
subsurface. This self-oscillatory kinetics of the oxide reduction is induced by the disparity in the chemical
activity of undercoordinated surface atoms in modulating the rates between H2-induced loss of lattice O
at the surface and O replenishment from the subsurface. The atomistic mechanism is closely relevant to
a wide range of oxide catalyzed reactions and has significant implications in manipulating the surface
reactivity of oxides by manipulating the spatiotemporally separated redox reaction steps at the oxide
surface. Data availability: All data generated or analyzed during this study are included in this published article (and its
supplementary information files). Acknowledgement: We acknowledge Drs. Meng Li and Stephen D. House (University of Pittsburgh) for
helpful discussions. This work was supported by the U.S. Department of Energy, Office of Basic Energy
Sciences, Division of Materials Sciences and Engineering under Award No. DE-SC0001135. This research
used resources of the Center for Functional Nanomaterials, the 23-ID-2 beamline at the National
Synchrotron Light Source II, and the Scientific Data and Computing Center, a component of the
Computational Science Initiative, at Brookhaven National Laboratory, which is supported by the US
Department of Energy, Office of Basic Energy Sciences, under Contract No. DE-SC0012704. This work
also used the computational resources from the Extreme Science and Engineering Discovery Environment
(XSEDE) through allocation TG-DMR110009, which is supported by National Science Foundation grant
number OCI-1053575, and CRC resources at the University of Pittsburgh. Author contributions G.Z. conceived the experiments and supervised the project. W.Z., X.S., and Y.Z. performed the
experiments. D.W. and W.S. conducted DFT calculations. J.B., R.S, and J.Y. provided experimental
instruments and guidance. X.S. and G.Z. analyzed data and wrote the paper. All the authors commented
on the manuscript. Competing interests Authors declare no competing interests. Additional information Additional information Supplementary information The online version contains supplementary material available at
https://doi.org/******. Page 10/20
Correspondence and requests for materials should beaddressed to G.Z. Correspondence and requests for materials should beaddressed to G.Z. Peer review information Nature Materials thanks the anonymous reviewers for their contributions to the
peer review of his work. Reprints and permissions information is available online at http://www.nature materials.com/reprints. R f Reprints and permissions information is available online at http://www.nature materials.com/reprints. References 1. Campbell, C. T. & Peden, C. H. Oxygen vacancies and catalysis on ceria surfaces. Science 309, 713-
714, (2005). 2. Al-Abadleh, H. A. & Grassian, V. H. Oxide surfaces as environmental interfaces. Surf. Sci. Rep. 52, 63-
161, (2003). 3. Shibata, N., Goto, A., Choi, S.-Y., Mizoguchi, T., Findlay, S. D., Yamamoto, T. & Ikuhara, Y. Direct
imaging of reconstructed atoms on TiO2 (110) surfaces. Science 322, 570-573, (2008). 4. Ganduglia-Pirovano, M. V., Hofmann, A. & Sauer, J. Oxygen vacancies in transition metal and rare
earth oxides: current state of understanding and remaining challenges. Surf. Sci. Rep. 62, 219-270,
(2007). 5. Batzill, M. & Diebold, U. The surface and materials science of tin oxide. Prog. Surf. Sci. 79, 47-154,
(2005). 6. Parkinson, G. S., Novotny, Z., Jacobson, P., Schmid, M. & Diebold, U. Room temperature water splitting
at the surface of magnetite. J. Am. Chem. Soc. 133, 12650-12655, (2011). 7. Yuan, W. T., Zhu, B., Li, X.-Y., Hansen, T. W., Ou, Y., Fang, K., Yang, H., Zhang, Z., Wagner, J. B., Gao, Y. &
Wang, Y. Visualizing H2O molecules reacting at TiO2 active sites with transmission electron
microscopy. Science 367, 428-430, (2020). 8. Parkinson, G. S. Iron oxide surfaces. Surf. Sci. Rep. 71, 272-365, (2016). 9. Rousseau, R., Glezakou, V.-A. & Selloni, A. Theoretical insights into the surface physics and chemistry
of redox-active oxides. Nat. Rev. Mater. 5, 460-475, (2020). 9. Rousseau, R., Glezakou, V.-A. & Selloni, A. Theoretical insights into the surface physics and chemistry
of redox-active oxides. Nat. Rev. Mater. 5, 460-475, (2020). 10. Sun, K., Kohyama, M., Tanaka, S. & Takeda, S. Reaction mechanism of the low-temperature water–
gas shift reaction on Au/TiO2 catalysts. J. Phys. Chem. C 121, 12178-12187, (2017). 10. Sun, K., Kohyama, M., Tanaka, S. & Takeda, S. Reaction mechanism of the low-temperature water–
gas shift reaction on Au/TiO2 catalysts. J. Phys. Chem. C 121, 12178-12187, (2017). 11. Polo-Garzon, F., Bao, Z., Zhang, X., Huang, W. & Wu, Z. Surface reconstructions of metal oxides and
the consequences on catalytic chemistry. ACS Catal. 9, 5692-5707, (2019). 12. Fujita, T., Guan, P., McKenna, K., Lang, X., Hirata, A., Zhang, L., Tokunaga, T., Arai, S., Yamamoto, Y.,
Tanaka, N., Ishikawa, Y., Asao, N., Yamamoto, Y., Erlebacher, J. & Chen, M. Atomic origins of the high
catalytic activity of nanoporous gold. Nat. Mater. 11, 775-780, (2012). 13. Hansen, T. W. & Wagner, J. B. References Catalysts under controlled atmospheres in the transmission electron
microscope. ACS Catal. 4, 1673-1685, (2014). 13. Hansen, T. W. & Wagner, J. B. Catalysts under controlled atmospheres in the transmission electron
microscope. ACS Catal. 4, 1673-1685, (2014). 14. Ross, F. M. & Minor, A. M. in Springer Handbook of Microscopy (eds Peter W. Hawkes & John C. H. Spence) 101-189 (Springer International Publishing, 2019). Page 11/20 15. Chao, H.-Y., Jiang, H., Ospina-Acevedo, F., Balbuena, P. B., Kauppinen, E. I., Cumings, J. & Sharma, R. A
structure and activity relationship for single-walled carbon nanotube growth confirmed by in situ
observations and modeling. Nanoscale 12, 21923-21931, (2020). 16. Yoshida, H., Kuwauchi, Y., Jinschek, J. R., Sun, K., Tanaka, S., Kohyama, M., Shimada, S., Haruta, M. &
Takeda, S. Visualizing gas molecules interacting with supported nanoparticulate catalysts at
reaction conditions. Science 335, 317-319, (2012). 17. Newsome, D. S. The water-gas shift reaction. Catal. Rev. Sci. Eng. 21, 2 18. Klier, K. Methanol synthesis. Adv. Catal. 31, 243-313, (1982). 18. Klier, K. Methanol synthesis. Adv. Catal. 31, 243-313, (1982). 19. Rodriguez, J. A., Kim, J. Y., Hanson, J. C., Pérez, M. & Frenkel, A. I. Reduction of CuO in H2: in situ time-
resolved XRD studies. Catal. Lett. 85, 247-254, (2003). 20. Poreddy, R., Engelbrekt, C. & Riisager, A. Copper oxide as efficient catalyst for oxidative
dehydrogenation of alcohols with air. Catal. Sci. Tech. 5, 2467-2477, (2015). 21. Svintsitskiy, D. A., Kardash, T. Y., Stonkus, O. A., Slavinskaya, E. M., Stadnichenko, A. I., Koscheev, S. V.,
Chupakhin, A. P. & Boronin, A. I. In situ XRD, XPS, TEM, and TPR study of highly active in CO
oxidation CuO nanopowders. J. Phys. Chem. C 117, 14588-14599, (2013). 22. Zhang, Q., Huang, L., Kang, S., Yin, C., Ma, Z., Cui, L. & Wang, Y. CuO/Cu2O nanowire arrays grafted by
reduced graphene oxide: synthesis, characterization, and application in photocatalytic reduction of
CO2. RSC Adv. 7, 43642-43647, (2017). 23. Mars, P. & van Krevelen, D. W. Oxidations carried out by means of vanadium oxide catalysts. Chem. Eng. Sci. 3, 41-59, (1954). 24. Eckert, E., HlavÁČEk, V. & Marek, M. Catalytic oxidation of CO on CuO/Al2O3. ii. measurement and
description of hysteresis and oscillations in a laboratory catalytic recycle reactor. Chem. Eng. Commun. 1, 95-102, (1973). 25. Vendelbo, S. B., Elkjaer, C. F., Falsig, H., Puspitasari, I., Dona, P., Mele, L., Morana, B., Nelissen, B. References & Imbihl, R. Interaction of NO and H2 with Pt(100). Appl. Surf. Sci. 48-49, 130-134,
(1991). 35. Lombardo, S. J., Fink, T. & Imbihl, R. Simulations of the NO+NH3 and NO+H2 reactions on Pt(100):
Steady state and oscillatory kinetics. J. Chem. Phys. 98, 5526-5539, (1993). 35. Lombardo, S. J., Fink, T. & Imbihl, R. Simulations of the NO+NH3 and NO+H2 reactions on Pt(100):
Steady state and oscillatory kinetics. J. Chem. Phys. 98, 5526-5539, (1993). 36. Suchorski, Y., Datler, M., Bespalov, I., Zeininger, J., Stoger-Pollach, M., Bernardi, J., Gronbeck, H. &
Rupprechter, G. Visualizing catalyst heterogeneity by a multifrequential oscillating reaction. Nat. Commun. 9, 600, (2018). References J.,
van Rijn, R., Creemer, J. F., Kooyman, P. J. & Helveg, S. Visualization of oscillatory behaviour of Pt
nanoparticles catalysing CO oxidation. Nat. Mater. 13, 884-890, (2014). 26. Miller, B. K. & Crozier, P. A. Linking changes in reaction kinetics and atomic-level surface structures on
a supported Ru catalyst for CO oxidation. ACS Catal. 11, 1456-1463, (2021). 26. Miller, B. K. & Crozier, P. A. Linking changes in reaction kinetics and atomic-level surface structures on
a supported Ru catalyst for CO oxidation. ACS Catal. 11, 1456-1463, (2021). 27. Suchorski, Y., Zeininger, J., Buhr, S., Raab, M., Stöger-Pollach, M., Bernardi, J., Grönbeck, H. &
Rupprechter, G. Resolving multifrequential oscillations and nanoscale interfacet communication in
single-particle catalysis. Science 372, 1314-1318, (2021). 28. Wolff, J., Papathanasiou, A. G., Kevrekidis, I. G., Rotermund, H. H. & Ertl, G. Spatiotemporal
addressing of surface activity. Science 294, 134-137, (2001). 28. Wolff, J., Papathanasiou, A. G., Kevrekidis, I. G., Rotermund, H. H. & Ertl, G. Spatiotemporal
addressing of surface activity. Science 294, 134-137, (2001). 29. Rotermund, H. H., Engel, W., Kordesch, M. & Ertl, G. Imaging of spatio-temporal pattern evolution
during carbon monoxide oxidation on platinum. Nature 343, 355-357, (1990). 29. Rotermund, H. H., Engel, W., Kordesch, M. & Ertl, G. Imaging of spatio-temporal pattern evolution
during carbon monoxide oxidation on platinum. Nature 343, 355-357, (1990). 30. Nolte, P., Stierle, A., Jin-Phillipp, N. Y., Kasper, N., Schulli, T. U. & Dosch, H. Shape changes of
supported Rh nanoparticles during oxidation and reduction cycles. Science 321, 1654-1658, (2008). Page 12/20 Page 12/20 31. Kuzovkov, V. N., Kortlüke, O. & von Niessen, W. Kinetic oscillations in the catalytic CO oxidation on Pt
single crystal surfaces: Theory and simulation. J. Chem. Phys. 108, 5571-5580, (1998). 32. Engel, T. & Ertl, G. Elementary steps in the catalytic oxidation of carbon monoxide on platinum
metals. Adv. Catal. 28, 1-78, (1979). 33. lmbihl, R. & Ertl, G. Oscillatory kinetics in heterogeneous catalysis. Chem. Rev. 95, 697-733, (1995). 33. lmbihl, R. & Ertl, G. Oscillatory kinetics in heterogeneous catalysis. Chem. Rev. 95, 697-733, (1995). 34. Madden, H. H. & Imbihl, R. Interaction of NO and H2 with Pt(100). Appl. Surf. Sci. 48-49, 130-134, 33. lmbihl, R. & Ertl, G. Oscillatory kinetics in heterogeneous catalysis. Chem. Rev. 95, 697-733, (1995). 34. Madden, H. H. & Imbihl, R. Interaction of NO and H2 with Pt(100). Appl. Surf. Sci. 48-49, 130-134,
(1991). 34. Madden, H. H. Methods We also performed “comparison experiments” by blanking and un- Page 13/20 Page 13/20 blanking the electron beam and confirmed that the electron beam effect had a negligible effect on the
formation of two surface terminations and their interconversion. Section 2. Ambient-pressure X-ray photoelectron spectroscopy (AP-XPS). AP-XPS experiments were
performed at the IOS beamline of the National Synchrotron Light Source II (NSLS-II), Brookhaven National
Laboratory. The AP-XPS station is equipped with an analysis chamber with the base pressure lower than
6.7 × 10− 7 Pa, a hemispherical analyzer, and an Ar-ion sputtering gun. The AP-XPS system has several
differential pumping stages between the analysis chamber and the hemispherical analyzer which allows
maintaining ultrahigh vacuum (UHV) conditions (lower than 1.3 × 10− 5 Pa) in the analyzer when the
pressure in the analysis chamber is several hundreds of Pascals. Photoemitted electrons leave the high-
pressure chamber through a small aperture in a conical piece into the differentially pumped transfer
lenses system toward the electron energy analyzer. XPS spectra can be acquired in this system at
pressures up to ≈ 600 Pa. The photon energy range of the beamline is from 250 eV to 2000 eV. Same as
the ETEM experiments, Cu foils (99.9%) were used in the AP-XPS experiment with the similar treatment by
rinsing in deionized water and ultrasonication in acetone. After loading into the AP-XPS chamber, the Cu
foil was further cleaned by cycles of ion sputtering and annealing to remove any native oxide. The
surface cleanliness was confirmed by XPS measurements of the Cu 2p and O 1s peaks. The cleaned Cu
was then oxidized to form a CuO layer at 400 °C by flowing O2 at the pressure of ≈ 133 Pa within the AP-
XPS chamber. Upon completing the oxidation, the sample was cooled down to 100 °C in the O2
atmosphere. The analysis chamber was then evacuated, followed by flowing H2 gas to the chamber at
0.13 Pa. For O2/H2 co-dosing experiments, the H2 gas flow rate (and thus H2 partial pressure) was kept
constant while the O2 gas flow (and its partial pressure) was gradually increased to O2/H2 = 0, 1, and 3. Methods Section 1. In Situ environmental TEM (ETEM) experiments. Our in-situ TEM experiments started from the
preparation of pristine CuO by Cu oxidation in O2, followed by switching the gas flow to H2. Both
processes were conducted in a dedicated ETEM equipped with an objective-lens aberration corrector and
a gas manifold that enabled control of the gas flow rate and partial pressure of various gases in the
specimen area. Cu foils (99.9% purity) were used in the in-situ ETEM experiments. The Cu foils were first
thoroughly rinsed in deionized water followed by ultrasonication in acetone for 10 min. The Cu foils were
then treated by plasma cleaning before loading into the TEM column. The Cu foils were further cleaned
inside the TEM by heating to 400 °C in a H2 gas flow at the pressure of ≈ 100 Pa to remove any native
oxide. The cleaned Cu was directly oxidized at 400 °C to form a thick CuO layer inside the TEM by flowing
O2 gas at the pressure of ≈ 0.5 Pa in the specimen area of the TEM column. After the oxidation step, the
specimen was then cooled down to 300 °C in the same O2 atmosphere. The TEM column was then
evacuated and the CuO was confirmed to be stable at 300 °C and in vacuum under the electron beam
irradiation. Afterwards, H2 was then introduced into the specimen region of the TEM column at ≈ 0.53 Pa
with a H2 gas flow rate of ≈ 0.01 SCCM (cubic centimeters per minute), and the specimen temperature
was maintained at 300°C during the H2 gas flow. This two-step process of in-place CuO preparation by
oxidation and subsequent observations of the oxide reduction in H2 has the advantage of minimizing
potential sample contamination. In-situ TEM observations were performed at an accelerating voltage of 300 keV and the electron beam
current of ≈ 1.1 × 105 e‧nm− 2·s− 1. In-situ movies were obtained by recording the TEM images with a
frame rate of 5 frames per second. The image frames were aligned to compensate any thermal drift. To
rule out any effects from the electron beam irradiation on the in-situ TEM results, the e-beam effect was
carefully minimized by adjusting the imaging condition in one area and then moving to a neighboring,
fresh area for HRTEM imaging. Methods It
is worth mentioning that, compared to chemical syntheses of oxide nanostructures, the CuO formed from
the thermal oxidation of Cu in our ETEM and AP-XPS experiments is of both high crystallinity due to the
high temperature and high purity for the elimination of any other chemical intermediaries. Section 3. DFT modeling. DFT calculations were performed using the Vienna Ab initio Simulation
Package (VASP) 37. Perdew, Burke, and Ernzerhof (PBE) generalized gradient approximation (GGA) 38 and
projector augmented-wave (PAW) 39 potential were performed to describe the electron–electron exchange
and core-electron potential separately. We employed DFT + U in our DFT calculations since the
conventional DFT functions cannot describe the strong correlation effect among the partially filled Cu 3d
states in CuO 40. According to the previous study, the values of U and J were selected as 7 eV and 0 eV for
CuO, respectively 41. The plane-wave cutoff energy was set to be 400 eV for all the calculations, and spin-
polarized calculations were performed since CuO has an antiferromagnetic ground state. The Brillouin-
zone integration was performed using (8 ⋅ 8 ⋅ 8) K-point meshes based on Monkhorst-Pack grids. We used
t 2
p
vacancy formation energy, respectively. All the atomic structures were visualized using the Visualization
for Electronic and Structure Analysis (VESTA). vacancy formation energy, respectively. All the atomic structures were visualized using the Visualization
for Electronic and Structure Analysis (VESTA). Page 14/20 Page 14/20 Page 14/20 Section 4. HRTEM simulations. DFT-relaxed structure models of Cu8O7 (Fig. 1j) and Cu8O2 (Fig. 1k) were
used as input files for HRTEM image simulations. The HRTEM image simulations were performed using
the multi-slice method in Simula TEM software with the parameters carefully matched to the
experimental conditions (accelerating voltage: 300 keV, the spherical aberration: 0.001 mm, defocus: -5 ~
65 nm, thickness: 15 ~ 40 nm, defocus spread: 4.5 nm, slice thickness: 0.9 nm. The frozen phonon model
was applied to reduce the elastic scattering and increase the background intensity. Section 5. Quantification of the oxide reduction kinetics. The oxide reduction kinetics is measured from in
situ HRTEM image sequences by frame-by-frame tracking the projection area of the oxide and lateral
length of the surface reconstructions. We employ the selection brush tool in ImageJ software to count the
number of pixels, followed by converting into length and projection area. Methods The pixel size for TEM imaging
is 0.02174 nm × 0.02174 nm, i.e., the length of 46 pixels is equal to 1 nm. The errors mainly result from
both multiple measurements (due to the small uncertainty in the interface location of the two surface
reconstructions) and numerical approximation to three decimal places. For example, the uncertainty in
the pixel size results in a measurement error of 0.02174 nm for the migrating distance of the surface step
of the Cu8O7 → Cu8O2 conversion in Fig. 2d. Dividing this measurement error by the total transformation
time (12 s) in Fig. 2d leads to an average measurement error (0.02174nm/12s = 0.00181 ~ 0.002 nm/s)
of the receding motion rate of the surface step. The temporal evolution of the projection area of the oxide
is measured from the in-situ TEM imaging, which is then used to determine the oxide reduction rate by
differentiating the projection area with time. Similarly, the errors of the oxide reduction rate are from both
multiple measurements of the projection area and numerical approximation to three decimal places. References: 37 Kresse, G. & Furthmüller, J. Efficiency of ab-initio total energy calculations for metals and
semiconductors using a plane-wave basis set. Comput. Mater. Sci. 6, 15–50, (1996). 37 Kresse, G. & Furthmüller, J. Efficiency of ab-initio total energy calculations for metals and
semiconductors using a plane-wave basis set. Comput. Mater. Sci. 6, 15–50, (1996). 38 Perdew, J. P., Burke, K. & Ernzerhof, M. Generalized gradient approximation made simple. Phys. Rev. Lett. 77, 3865, (1996). 38 Perdew, J. P., Burke, K. & Ernzerhof, M. Generalized gradient approximation made simple. Phys. Rev. Lett. 77, 3865, (1996). 39 Kresse, G. & Joubert, D. From ultrasoft pseudopotentials to the projector augmented-wave method. Phys. Rev. B 59, 1758, (1999). 39 Kresse, G. & Joubert, D. From ultrasoft pseudopotentials to the projector augmented-wave method. Phys. Rev. B 59, 1758, (1999). 40 Wang, L., Maxisch, T. & Ceder, G. Oxidation energies of transition metal oxides within the GGA + U
framework. Phys. Rev. B 73, 195107, (2006). 40 Wang, L., Maxisch, T. & Ceder, G. Oxidation energies of transition metal oxides within the GGA + U
framework. Phys. Rev. B 73, 195107, (2006). 41 Maimaiti, Y., Nolan, M. & Elliott, S. D. Reduction mechanisms of the CuO (111) surface through surface
oxygen vacancy formation and hydrogen adsorption. Phys. Chem. Chem. Phys. 16, 3036–3046, (2014). Figures Page 15/20 Figure 1 See image above for figure legend. Page 16/20 Page 16/20 Figure 2
In-situ HRTEM visualization of the cyclic interconversions between the Cu8O7 and Cu8O2 surface
reconstructions of CuO at 300 °C and 0.5 Pa of H2 gas flow. a, Cu8O2→Cu8O7 conversion at the
atomically flat Cu8O2 terminated surface (Supplementary Movie 3). b, Cu8O7 → Cu8O2 conversion by the
receding motion of a double-layer step (Supplementary Movie 4). c, Cu8O7 → Cu8O2 conversion by the
receding motion of a three-layer step (Supplementary Movie 5). d, Cu8O7 → Cu8O2 conversion by the
receding motion of a monoatomic step (Supplementary Movie 6). e, Cu8O2 → Cu8O2 by the retraction Figure 2
In-situ HRTEM visualization of the cyclic interconversions between the Cu8O7 and Cu8O2 surface
reconstructions of CuO at 300 °C and 0.5 Pa of H2 gas flow. a, Cu8O2→Cu8O7 conversion at the
atomically flat Cu8O2 terminated surface (Supplementary Movie 3). b, Cu8O7 → Cu8O2 conversion by the
receding motion of a double-layer step (Supplementary Movie 4) c Cu8O7 →Cu8O2 conversion by the In-situ HRTEM visualization of the cyclic interconversions between the Cu8O7 and Cu8O2 surface
reconstructions of CuO at 300 °C and 0.5 Pa of H2 gas flow. a, Cu8O2→Cu8O7 conversion at the
atomically flat Cu8O2 terminated surface (Supplementary Movie 3). b, Cu8O7 → Cu8O2 conversion by the
receding motion of a double-layer step (Supplementary Movie 4). c, Cu8O7 → Cu8O2 conversion by the
receding motion of a three-layer step (Supplementary Movie 5). d, Cu8O7 → Cu8O2 conversion by the
receding motion of a monoatomic step (Supplementary Movie 6). e, Cu8O2 → Cu8O2 by the retraction
motion of a monoatomic step (Supplementary Movie 7). f, Cu8O2 → Cu8O2 by the retraction motion of a Page 17/20 Page 17/20 Page 17/20 double-layer step (Supplementary Movie 8). g, Shrinkage rates of the oxide projection area as a function
of the H2 exposure time (Supplementary Movie 9), revealing the cyclic oscillation of the oxide reduction
rates, where the error bars represent standard deviation uncertainties based on multiple measurements. The letter symbols correspond to the different types of the interconversions shown in a-f. Figure 3
AP-XPS measurements and DFT calculations of hydrogen adsorption. a, Photoelectron spectra of O 1s Figure 3
AP-XPS measurements and DFT calculations of hydrogen adsorption. a, Photoelectron spectra of O 1s
obtained from the CuO surface at T=350 °C and pO2≈133 Pa (lower panel). Figure 1 Switching to 0.13 Pa of the
H2 gas flow at T=100 °C results in the OH peak in addition to the lattice O in CuO (upper panel). b,
Evolution of the O 1s spectra during the CuO exposure to the mixtures of H2 and O2, showing the Figure 3 AP-XPS measurements and DFT calculations of hydrogen adsorption. a, Photoelectron spectra of O 1s
obtained from the CuO surface at T=350 °C and pO2≈133 Pa (lower panel). Switching to 0.13 Pa of the
H2 gas flow at T=100 °C results in the OH peak in addition to the lattice O in CuO (upper panel). b,
Evolution of the O 1s spectra during the CuO exposure to the mixtures of H2 and O2, showing the Page 18/20 Page 18/20 increasingly stronger peak intensity of the OH component with increasing the O2 partial pressure. c-f
Evolution of the minimum-energy structures of the Cu8O7 showing the dissociative H2 adsorption leading
to the H2O formation after reaching one monolayer of the OH coverage. Additional H forms an H2O
molecule marked by the black arrow. g-j, Minimum-energy structures of the Cu8O2 showing the
dissociative H2 adsorption that self-saturates at the one monolayer of the OH coverage. Yellow, red, and
sky (and blue) balls represent Cu, O, and H atoms, respectively. increasingly stronger peak intensity of the OH component with increasing the O2 partial pressure. c-f
Evolution of the minimum-energy structures of the Cu8O7 showing the dissociative H2 adsorption leading
to the H2O formation after reaching one monolayer of the OH coverage. Additional H forms an H2O
molecule marked by the black arrow. g-j, Minimum-energy structures of the Cu8O2 showing the
dissociative H2 adsorption that self-saturates at the one monolayer of the OH coverage. Yellow, red, and
sky (and blue) balls represent Cu, O, and H atoms, respectively. Figure 4
Pictorial illustration of cyclic conversions of the surface reconstructions of CuO in H2. a, b The low
reducibility of the Cu8O2 surface promotes its oxidation to the Cu8O7 by absorbing lattice O in the
subsurface. c, The formation and surface desorption of H2O molecules from the highly reducible Cu8O7
surface results in a high concentration of O vacancies in the surface and subsurface region of the oxide. Figure 1 d, e, Accumulative H2 adsorption and H2O formation results in the step-flow Cu8O7→Cu8O2 conversion via
the nucleation and growth of a monoatomic-deep surface pit in the Cu8O7 layer, leading to a complete
cycle of the surface conversion. f-h, Step-flow Cu8O7→Cu8O2 conversion via the nucleation and growth of
one and two atomic layer deep pits results in the encountering of the Cu8O2-terminated terraces
separated by a monoatomic step, and the step-flow Cu8O2→Cu8O2 refresh also leads to a complete cycle Figure 4 Pictorial illustration of cyclic conversions of the surface reconstructions of CuO in H2. a, b The low
reducibility of the Cu8O2 surface promotes its oxidation to the Cu8O7 by absorbing lattice O in the
subsurface. c, The formation and surface desorption of H2O molecules from the highly reducible Cu8O7
surface results in a high concentration of O vacancies in the surface and subsurface region of the oxide. d, e, Accumulative H2 adsorption and H2O formation results in the step-flow Cu8O7→Cu8O2 conversion via
the nucleation and growth of a monoatomic-deep surface pit in the Cu8O7 layer, leading to a complete
cycle of the surface conversion. f-h, Step-flow Cu8O7→Cu8O2 conversion via the nucleation and growth of
one and two atomic layer deep pits results in the encountering of the Cu8O2-terminated terraces
separated by a monoatomic step, and the step-flow Cu8O2→Cu8O2 refresh also leads to a complete cycle Pictorial illustration of cyclic conversions of the surface reconstructions of Pictorial illustration of cyclic conversions of the surface reconstructions of CuO in H2. a, b The low
reducibility of the Cu8O2 surface promotes its oxidation to the Cu8O7 by absorbing lattice O in the
subsurface. c, The formation and surface desorption of H2O molecules from the highly reducible Cu8O7
surface results in a high concentration of O vacancies in the surface and subsurface region of the oxide. d, e, Accumulative H2 adsorption and H2O formation results in the step-flow Cu8O7→Cu8O2 conversion via
the nucleation and growth of a monoatomic-deep surface pit in the Cu8O7 layer, leading to a complete
cycle of the surface conversion. f-h, Step-flow Cu8O7→Cu8O2 conversion via the nucleation and growth of
one and two atomic layer deep pits results in the encountering of the Cu8O2-terminated terraces
separated by a monoatomic step, and the step-flow Cu8O2→Cu8O2 refresh also leads to a complete cycle yclic conversions of the surface reconstructions of CuO in H2. a, b The low Page 19/20 Page 19/20 Page 19/20 of the surface conversions. Included at the bottom of each panel are the corresponding experimentally
observed surface reconstructions in Fig. 2. Red and cyan balls represent O and H atoms, respectively. of the surface conversions. Included at the bottom of each panel are the corresponding experimentally
observed surface reconstructions in Fig. 2. Red and cyan balls represent O and H atoms, respectively. Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Movie1.avi
Movie2.avi
Movie3.avi
Movie4.avi
Movie5.avi
Movie6.avi
Movie7.avi
Movie8.avi
Movie9.avi
EXTENDEDDATAFIGURELEGENDS.docx Movie1.avi
Movie2.avi
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J. Indian Chern. Soc.,
Vol. 78, October-December 2001, pp. 743-755
The reactions of phenols with a,,B-unsaturated aromatic acids in presence of
polyphosphoric acid : synthetic and mechanistic studies
P. L. Majumder*, S. Chatterjee (nee Lahiri) and N. Mukhoti
Department ofChemistry, University College of Science, 92, Acharya Prafulla Chandra Road,
Kolkata-700 009, India
Manuscript received 30 August 2001
The reactions of cinnamic acid with phenol itself, catechol, hydroquinone, pyrogallol and 2-naphthol in presence PPA were
studied and that with resorcinol was reinvestigatcd. With phenol itself and hydroquinone were obtained 3,4-dihydro-4phenylcoumarin (I r) and 6-hydroxy-3,4-dihydro-4-phenylcoumarin (Is), respectively, as the sole products. Catechol and pyrogallol, on the other hand, afforded, besides the corresponding 3,4-dihydrocoumarins, viz. 8-hydroxy-3,4-dihydro-4phenylcoumarin (It) and 7,8-dihydroxy-3,4-dihydro-4-phenylcoumarin (lu), as the major products, 5,6-dihydroxy-3phenylindanone (Sb) and the chalcone 1-(2',3',4'-tl"ihydroxy)-3-phenylpropan-2-en-1-one (3d), respectively, as minor products. But contrary to earlier observation, resorcinol was found to give 7-hydroxy-3,4-dihydro-4-phenylcoumarin (lv) instead
of 7-hydroxyflavanone (4e). 2-Naphthol, on the other hand, afforded the chalcone derivative l-f2'-hydroxynaphthyiJ-3phenylpropan-2-ene-1-one (7) as the exclusive product. The yields of the major products in the above reactions were 5067% except that with phenol itself yielding I r in 40% yield. The structures of all the products 1 r, 1s, 1 t, 1 u, 1v, 3d, Sb and
7 were established from their various spectral (IR, 1Hand 13 C NMR and mass) data. Evidence for plausible mechanisms of
formation of the above products was provided.
The reactions of phenols with a,,B-unsaturated aromatic acids
cinnamic acid, 2,4-, 3,4- and 3,5-dimethylphenols were
in presence of different condensing agents evoked
found to give the 3,4-dihydro-4-phenylcoumarins Ia (56%),
considerable interest both from synthetic and mechanistic
I d ( 17%) and 1h (20% ), respectively. The same reaction of
points of view during the last few decades. The early work
these phenols with 4-methoxycinnamic acid in presence of
in this area includes the synthesis of a fairly large number of PPA also afforded the 3,4-dihydro-4-phenylcoumarins I b
3,4-dihydro-4-phenylcoumarins by reactions of phenols with
(57%), le (21 %) and 1i (24%), respectively. In the reactions
cinnamic acids and those of cinnamates or the corresponding
between 3,4- and 3,5-dimethylphenols with 4-methoxynitriles with various condensing agents, viz. AICI 3, ZnC1 2,
cinnamic acid were also obtained traces of 6,7-dimethylBCI 3, H2S0 4 , HCI orpolyphosphoric acid (PPA) 1- 8 . Among
coumarin (2a) and 5,7-dimethylcoumarin (2b ), respectively.
These authors, however, observed that the reactions of these
the various condensing agents, PPA was found to be most
phenols with 3-methoxycinnamic acid were not as selective
suitable. Subsequent studies 9- 13 on this reaction with
as in the cases with 2- and 4-methoxycinnamic acids. Thus,
different phenols and cinnamic acids and those on various
phenyl cinnamates in presence ofPPA have yielded, besides
with 3-methoxycinnamic acid, 2,4-, 3,4- and 3,5several new 3,4-dihydro-4-phenylcoumarins, a few
dimethylphenols were found to give 3-(3'-methoxyphenyl)2", 4" -dimethylpropenoate (24%) and 3,4-dihydrocoumarin
chalcones, flavanones and indanones. In course of these
lc (17%), the chalcone 3a (32%) and the 3,4-dihydrostudies it was observed 9- 13 that the course of these reactions
coumarins 1f(21 %) and lg (1 0%), the flavanone 4a (17%)
and hence the nature of the products obtained was found to
and the 3,4-dihydrocoumarin lj (25%), respectively.
depend on various factors, viz. nature of the phenols,
positions and the nature of the substituents in the phenyl
Chenault and Dupin 10 also studied the reactions of sevring of the cinnamic acid and cinnamates, stoichiometry of eral arylcinnamates in presence of PPA at 80° for 1.5 h in
the reactants, composition of PPA, temperature, the reaction
order to examine the effects of substituents on the aryl ring
period, solvent, etc. Thus, Chenault and Dupin 9 studied the
on the reactivity of such cinnamates. Thus, while they failed
reactions of2,4-, 3,4- and 3,5-dimethylphenols with 2-, 3to obtain any product with phenyl-, 4-nitrophenyl- and 4and 4-methoxycinnamic acids in presence of PPA at a
acetylphenylcinnamates, 4-chloro-3,5-dimethylphenyl-, 3,5temperature close to 50° for 24 h with a view to examining
di methy I phenyl-, 3 ,5-d ich lorophenyl-, 2,4-dimethy !the influence of the methoxy groups at different positions in
phenyl-, 2-methoxyphenyl-, 3,5-methoxyphenyl-, and 3,4,5the phenyl rings of the cinnamic acid. With 2-methoxytrimethoxyphenylcinnamates were shown to give the 3,4743
J. Indian Chern. Soc., Vol. 78, October-December 2001
dihydrocoumarins lk (60%), II (90%), lm (40%), In, the
indanone Sa (22%), the flavanone 4b (24%); and a mixture
of 3,4-dihydrocoumarin to and the flavanone 4c, respectively.
of PPA at 50-70° for 2 h. The same reaction when carried
out at 85-90° and I 00° for 25 min afforded 6 • 11 4e in 12.7%
and 20%, respectively. Similar condensation of resorcinol
monomethyl ether with cinnamic acid at 50-70° for 2 h led 13
2a: R1=H. R'=R 1=Me
2b: R 1=R'=Me. R'=H
Ia:
lb:
lc:
ld:
le:
If:
lg:
lh:
R 1=R'=R 6 =R 7 =H. R 2=R 4 =Me. R'=OMe
R'=R'=R'=R6 =H. R'=R'=Me. R 7=0Me
R'=R ..=R'=R. 7=H. R 2=R'=Me. R6=0Me
R'=R'=R 6 =R 7=H. R'=R'=Me, R'=OMe
R'=R'=R'=R'=H. R 2 =R'=Me, R 7=0Me
R'=R 4=R'=R'=H. R 2=R'=Me, R6=0Me
R'=R'=Me, R1=R'=R'=R'=H. R6=0Me
R'=R'=Me. R'=R'=R'=R'=H. R'=OMe
li: R 1=R 1=Me. R'=R 4=R1=R'=H, R 7=0Me
lj: R'=R 1=Me. R'=R 4=R'=R 7=H. R6 =0Me
lk: R'=R'=Me. R'=CI. R'=R'=R 6=R 7 =H
II: R'=R 1 =Me, R'=R'=R'=R'=R 7=H
lm: R'=R'=CI, R'=R'=R'=R'=R'=H
In: R'=R'=R'=R'=R'=H. R2=R'=Me
lo: R'=R 2=R 3=0Me, R'=R'=R'=R'=H
lp: R'=R'=R'=R'=R 6=H. R'=OH, R7=0Me
lq: R'=R 2=R 4 =R'=R'=H. R'=R'=OMc
lr: R'=R'=R'=R 4 =R'=R'=R 7 =H
Is: R 1=R 3=R'=R'=R 6 =R 7=H. R'=OH
It: R'=R'=R 1 =R'=R'=R'=H. R'=OH
lu: R'=R'=R'=R'=R'=H. R'=R'=OH
h: R'=R'=R 4 =R'=R'=R 7 =H, R'=OH
lw: R1=R'=R'=R'=R'=R'=H. R 7=0Mc
lx: R'=R'=R'=R'=H. R'=R'=OH, R7 =0Me
Ri
3a:
3b:
3c:
3d:
3e:
0
R 1=0H, R 2=R'=H, R 3=R'=Me. R"=OMe
R 1=0H, R2=R 4=R'=R'=H, R'=OMe
R 1=0Me, R 2=R'=R'=R'=H. R1 =011
R 1=R'=R 1=0H, R 4 =R'=R 6 =H
R 1=R'=R'=R1=0Me, R'=R'=H
R 1=R 1 =Mc. R'=H. R4=0Mc
R'=R 3=0Me, R'=R'=H
R '=R'=R'=OMc. R'=H
R'=R1=0H. R'=R'=H
R'=OH, R'=R'=R'=II
4r: R'=R'=R 1=R'=H
4a:
4b:
4c:
4d:
4e:
..
·o
Sa: R 1=0H. R'=OMe
Sh: R 1=R'=OH
Talapatra eta!. 12 synthesized pinocembrin ( 4d) in 50%
yield by condensation of phloroglucinol with cinnamic acid
in presence of PPA along with a minor product 6 ( 14%).
They also obtained 13 7-hydroxytlavanone ( 4e) in 60% yield
by the reaction of resorcinol with cinnamic acid in presence
0
,.
6
7
744
to an isomeric mixture of the chalcones 3b ( 12%) and 3c.
The chalcone 3b had also been obtained by Reichel and
Proksch 8 by the same reaction in 11% yield. However, when
the same reaction was carried out 13 with resorcinol and 4methoxycinnamic acid in presence of PPA in xylene at 70°
for I h, neither the corresponding chalcone nor the llavanone
could be isolated, and, instead, the 3,4-dihydrocoumarin lp
was obtained in 56% yield. The same reaction of 4methoxycinnamic acid with resorcinol monomethyl ether
afforded 13 the 3,4-dihydrocoumarin I q in 44% yield.
The results of the above investigations thus clearly demonstrate that the course of these reactions is highly sensitive
to the various factors stated earlier. This has prompted us to
Majumder e( a/. :The reactions of phenols with a,,B-unsaturated aromatic acids in presence of polyphosphoric acid
study this reaction with some phenols which have not been
used earlier and reinvestigate the reaction with some phenols
already reacted with cinnamic acids in presence ofPPA and
also to carry out further investigations for arriving at a plausible mechanism of formation of the products in each case.
The results of these studies are described in this paper.
Results and Discussion
We have studied this reaction with phenol itself, hydroquinone, catechol, pyrogallol and 2-naphthol and
reinvestigated that with resorcinol using cinnamic acid in
presence ofPPA at boiling water-bath temperature (95-100°)
for 2 h. Phenyl cinnamate and phenyl4'-methoxycinnamate
were separately treated with pyrogallol in presence of PPA
under the above reaction condition as part of our studies on
the mechanism of this reaction.
1740-1750cm-l [Jr, Jtand tv: vmax 1740cm- 1; Is and
lu: l-Jnax 1750 cm- 1]. TheIR spectra of 3d and 7, on the
other hand, showed bands at 1640 and 1645 cm- 1, respectively, which were attributed to conjugated keto carbonyl
functions involved in intramolecular hydrogen bonding with
phenolic hydroxyl functions. The chelated phenolic hydroxyl
functions in 3d and 7, as expected, showed absorptions at
vmax 3200 and 3400 cm- 1, respectively. TheIR spectrum
of Sb exhibited strong band at 1785 cm- 1, indicating the
presence of a five-membered keto carbonyl function of the
indanone moiety.
More compelling evidence for the assigned structures of
the above compounds was provided by their 1H NMR spectral data (Table 2 for compounds 1r-x). Thus, the presence
of a 3,4-dihydro-4-substituted (by phenyl group) coumarin
moiety in each of I r, Is, It, I u and 1v was indicated by the
Phenol itself and hydroquinone when separately treated
appearance of a one-proton apparent triplet at iS 4.19-4.40
with cinnamic acid in presence ofPPA afforded 3,4-dihydro[Jr: 54.35; Is: 4.40; It: 4.29; tu: 4.38; tv: 4.19] and a
4-phenylcoumarin ( t r) and 6·hydroxy-3 ,4-dihydro-4two-proton doublet of AB qua11et at 52.96-3.19 [Jr: c5
phenylcoumarin (Is), respectively, as the sole products.
3.19; Is: 3.03, It: 3.02; tu: 3.05; tv: 2.96] in the specUnder the same reaction condition catechol and pyrogallol,
trum of each of these compounds, which were attributed to
on the other hand, gave, besides the corresponding 3,41-1-4 and 1-1 2 -3 of their respective 3,4-dihydrocoumarin moidihydro-4-phenylcoumarins l t and t u as the major prodety. The observed multiplicity of these signals was rationalized by the generation of a chiral centre at C-4 in each of
ucts, the indanone derivative Sb and the chaclone 3d, rethese compounds giving rise to an ABX system. The alterspectively, as minor products. But contrary to earlier obsernative isomeric flavanone formulations of these compounds
vations6~13, resorcinol under the above reaction condition
having
their H-2 and 1-1 2 -3 also constituting similar ABX
yielded 7-hydroxy-3,4-dihydro-4-phenylcoumarin ( 1v) as the
system
was ruled out by the fact that H-2 of the flavanone
sole product instead of 7-hydroxyflavanone ( 4e) obtained
derivatives were reported 9 •10 · 11 • 13 to resonate at a much
by Talapatra et al. 13 in the same reaction under slightly difdownfield position (at ca c5 5.5) compared to H-4 of the
ferent reaction conditions. 2-Naphthol, on the other hand,
3,4-dihydro-4-phenylcoumarins,
although the chemical shifts
afforded the chalcone derivative 7 as the exclusive product.
of
H
z-3
of
both
types
of
compounds
are quite comparable.
The yields of the products in the above reactions are shown
The
chemical
shift
of
the
proton
at
54.19
of 1v obtained by
in Table I. Except that fort r ( 40%), the yields of the major
us
in
the
reaction
ofresorcinol
and
cinnamic
acid in presproducts with other phenols were found to vary from 50 to
ence of PPA corresponds to that of 1-1-4 of a 3,4-dihydro-467%.
phenylcoumarin rather than that of 1-1-2 (b' 5.46) of 7hydroxyflavanone ( 4e) found by Talapatra et a!. 13 in the
Table 1. Yields or products obtained from reactions of different
same reaction at 50-70°. The presence of one phenolic hyphenols with cinnamic acid in presence ofl'PA
droxyl function in each of 1s. It and 1v [Is : c5 8.3; It :
Phenol
Yield(%)
l'roduct(s)
6.96;
Jv: 6.0], that for two such groups in 1 u (b' 8.07 and
Phenol
lr
40
8.15) and none in 1 r, coupled with the chemical shifts and
Hydroquinone
Is
65
splitting
patterns of the aromatic protons of all the above
Catechol
62 (It), 25 (Sb)
It, Sb
five
compounds
are also consistent with their assigned strucPyrogallol
lu,Jd
67 (lu), 18 (3d)
tures.
Resorcinol
64
2-Naphthol
'"
7
50
The structures of I r, Is. 1t. t u. ! v, 3d, Sb and 7 were
firmly established from the following spectral evidence. The
presence of an unconjugated 0-lactone function in each of
lr, Is, It, lu and lv was indicated by strong IR bands at
The 11-1 NMR spectrum of5b showed signals at £52.77
(2H, d ABq, 1 1 I 6 Hz and 12 6.0 Hz) and 4.49 (I H, dd. 1 1
6.0 Hz and 12 3 Hz), which were attributed to llr2 and H3, respectively, of its indanone moiety. The spectrum ofthe
compound also showed signals for two phenolic hydroxyl
protons ( 6 6.40, 2H, br s ), two aromatic protons at () 6.63
745
J. Indian Chern. Soc., Vol. 78, October-December 2001
Table 2. 11-1 NMR spectral data• oflr, Is, II, lu, lv, lw and 1x
i5 ppm (multiplicity, J in Hz)
Protons
Jr
Is
_It
Hr3
3.19
(d, ABq,
Jl 15.0,
J2 6.2)
4.35 (app.
t, J 6.2)
3 03
(d, ABq.
J 1 15.0,
J2 6 0)
4.40 (app.
t, J 6.0)
3.02
(d, ABq,
Jl 15.0,
J 2 6.0)
4.29 (app.
t. J 6.0)
7.07-7.38
(complex
multiplet)
[H-5- H-8,
1-1-2'- H-6']
6.49 (d, J
24) [H-5]
J, 8.5, J2
H-4
Aromatic
protons
(H-5- 1-1-8,
H-2'-H-6')
6.97 (dd,
4.38 (app
t, J 6'0)
lv
2.96
(d, ABq,
J, 15.0,
J2 6 0)
4.19 (app.
t, J 6.0)
lw
2.97
(d, Al3q,
J, 15.0,
J2 6.0)
4.29 (app.
t, J 6 5)
661 (d, J
8.1) [1-1-5]
7.73 (d, J
9.0) [H-5]
7.08-7.39
(m) [H-5H-8J
6.36 (d, J
8.1) [H-6]
J, 9.0,
6.75(d,J
J2 2 5)
9 0) [H-3',
H-5']
lu
3 05
(d, ABq.
J, 15.0,
J2 6.0)
2 5) [H-5]
6.78 (dd,
J, 8.7, J2
7.16 (app.
[H-6}
t)
24) [H-7]
6.94 (d, J
8 7) [H-8]
6.44 (dd,
01-1
8.3 (br s)
7.29-7.35
(m) [1-1-2'H-6')
6.96 (br s)
7.23-7.28
(m) [H-2'1-1-6')
6.0 (br s)
OMe
*Spectra in CDC\ 3 .
and 7.11 (each IH, s) assigned for H-4 and H-7, respectively, and five aromatic protons at 8 7.24 of the phenyl
group at C-3.
The 1H NMR spectrum of 3d and 7, on the other hand,
showed signals at 87.53 and 7.84 (each IH, d, J 15.6 Hz)
and 86.61 and 7.83 (each JH, d, J 15.0 Hz), respectively,
which were attributed to H-2 and H-3 of their respective
chalcone formulations. The presence of a chelated phenolic
hydroxyl proton in each of 3d and 7 was indicated by the
signals at 8 13.4 and 13 .0, respectively, in the spectra of the
compounds. The two unchelated phenolic hydroxyl protons
of 3d appeared at 8 5.59 and 5.93 (each I H, br s). The
spectrum of 3d also showed signals for seven aromatic protons. Two of these aromatic protons appeared at 86.52 and
7.40 (each d, J 9 Hz), which cones ponded to H-5' and H6', respectively. The five protons of the phenyl group at C3 of 3d resonated at 87.34 (3H, m) and 7.60 (2H, m). The
above protons in 7 similarly appeared at 8 7.35 (3H, m)
and 7.53 (2H, m). The spectrum of 7 also showed signals at
6'7.40 (d J9 Hz) and 7.23 (dd, 1 1 9Hz and 12 3Hz), which
746
[H-6]
6.36 (d, J
2.5) [H-8 J
8.07 (br s)
8.15(brs)
6.61 (d, J
8.0) [H-5],
6.36 (d. J
8 0) [1-1-6]
6.49 (dd,
[H-7]
J, 8.0, J2
2.4)
7.25-7.30
(m) [H-2'H-6']
7.23-7.28
(m) [H-2'H-6']
1x
3.0
(d, ABq,
J, 16.0,
J2 6.2)
4.40 (app.
t, J 6 2)
7.04 (d, J
9.0) [11-2',
H-6']
3.90 (s)
6.84 (d, J
9.0) [H-3',
H-5'1
7.06 (d, J
9.0) [H-2',
H-6'1
6.0 (br s)
[2xAr-OH]
3. 92 (s)
were attributed to H-4' and H-3', respectively, the further
splitting of the signal for H-3' presumably being due to long
range coupling with the chelated phenolic hydroxyl proton.
The four-proton complex multiplet at around 87.75 in the
spectrum of 7 corresponded to its H-5', H-6', H-7' and H8'. Thus, the structures of 3d and 7 are consistent with their
above 1H NMR spectral data.
The structures of lr, Is, It, lu, lv, 3d, Sb and 7 were
also supported by their characteristic mass spectral fragmentations. The mass spectra of lr, Is, It, lu, lv, 3d, Sb
and 7 showed molecular ion peaks at mlz 224, 240, 240,
256, 240, 256, 240 and 274, respectively, confirming their
respective molecular formulae. The spectra of 1r, Is, l t, I u
and I v were characterized by the appearance of intense peaks
at mlz 147 and 146, 163 and 162, 163 and 162, 179 and
178, and 163 and 162, respective!), which c. , . •punded to
the ion-fragments a and b, c and d, e and f, g and h, and i
and j, respectively (Scheme l'). The ion-fragments a, c, e, g
and i were generated by a benzylic cleavage at C-4 of their
respective molecular ions. Loss of a II' from C-3 of the
Majumder eta!. :The reactions of phenols with a,fi-unsaturated aromatic acids in presence ofpolyphosphoric acid
or 4nl
l;.
R'XX)'-
~
R:)yllo~O
R2
0
Ri
Ri
lr:
Is:
It:
lu:
h :
R'=R'=R'=H (m/z 224)
R'=R'=H. R'=OH (rn/z 240)
R'=OH. R'=R'=H (m/z 240)
R'=R'=OH. R-'=H (m/z 256)
R'=R '=H. R'=OH (m/z 240)
a: R'=R'=R'=H (m/z 147)_
c: R'=R'=H. R'=OH im/z 163)
e: R'=OH. R 2=R'=H (rnlz 163)
g: R'=R'=OH. R'=H (m/z 179)
i: R'=R-'=H. R 1=0H (m/z 163)
h: R 1=R 1=R '=H (m/z 1461
d: R'=R'=H. R-'=OH (mil 16::>)
r: R'=OH. R'=R'=H irnlz 162)
h: R 1=R'=OH. R'=H irnlz 1781
j: R'=R'=H. R'=OH (m/z 162)
1-CO
1-CO
R~
R2~ 0
HO~+
Rl
HO~
a':R'=R 1=R'=H{m/zll9)
0
c': R'=R'=H. R'=OH (m/z 135)
k: m/z 163
e': R'=OH, R 1=R·'=H (m/z 135)
g': R'=R'=OH. R'=H (mlz 151)
i': R'=R'=H, R 1=0H (m/z 135)
HOJJ=n l
HO
+.
-CO
II-
~
HOYIT----n l
HO~
b': R'=R'=R'=H(rnlz 118)
d': R 1=R 1 =H. R'=OH (rnlz 134
R'=OH. R'=R'=H (m/z 134
h': R 1=R 1=0H. R'=H (m/z 15G
j': R'=R-'=H. R 1=0H (m/z 134
r·:
+·
0
I: m/z 162
m: rnlz 134
+
[3d]
+'
HO'n
1 1 1P-+~!"
rO
r
~OH
+
HO~c=o
m/z274
OH
vu
o:m/zl71
m: m/z 153
CH,
n: rnlz 104
q: m/z 142
I': m/z 170
Scheme I. Mass spectral fragmentations oflr-, Is, lt. lu, lv, Jd, Sb and 7.
above ion-fragments gave rise to the ion-fragments b, d, f,
h and j as shown in Scheme l. The mass spectra of I r, Is,
1t, 1u and 1v were further characterized by the appearance
of intense peaks at mlz l I 9 and 118, I 35 and I 34, I 35 and
134, 150, 135 and 134, respectively. These peaks corresponded to the ion-fragments a' and b', c' and d', e' and f,
h', and i' and j', respectively (Scheme I). While the ionfragments a', c', e' and i' were assumed to be formed by the
loss of CO from the ion-fragments a, c, e and i, respectively, the ion-fragments b', d', f', h' and j' must have originated by similar loss ofCO from the ion-fragments b, d, f.
h and j, respectively. It is interesting to note that the mass
spectrum of I u is devoid ofthe'peak at mh: I 51 corresponding to the ion-fragment g'. This is presumably because of
the fact that the loss of a H' from the ion-fragment g (m/z
179) to form the ion-fragment h (m/z 178) is much more
faster than the loss of CO from g. The loss of CO from h
directly gives rise to the peak at mlz 150 (ion-fragment h')
in the mass spectrum of I u. An alternative route to the genesis of the ion-fragments b', d', rand j' may be the loss of
H' from the ion-fragments a', c', e' and i', respectively. The
mass spectrum ofthe indanone Sb also showed intense peaks
at mlz I 63, 162 and 134 which were attributed to the ionfragments k, I and m, respectively, foi·med by the loss of a
phenyl radical by the benzylic cleavage at C-3 followed by
sequential loss of a 1-l' and CO (Scheme I). The mass spectral fragmentations of 3d and 7 are typical of a chalcone
derivative. Thus, cleavage of the C- 1- C-2 bond of 3d and
7 afforded intense peaks at mlz 153 (ion-fragment m) and
171 (ion-fragment o ), respectively. The same cleavage fol747
JICS-32
J.lndian Chem. Soc., Vol. 78, October-December2001
lowed by uptake of a H" generated the moderately intense
peak at mlz 104 (ion-fragment n) in the mass spectra of
both 3d and 7. The mass spectrum of7 is similarly characterized by the appearance of intense peaks at mlz 170 (ionfragment p) and 142 (ion-fragment q), which were assumed
to be formed by sequential loss of a H" and a molecule of
CO from the ion-fragment o (mlz 171) (Scheme 1).
The structures of the above compounds were finally confirmed by their 13 c NMR spectral data (Tables 3 and 4).
The degree of proto nation of the carbon atoms of each compound was determined by DEPT experiments and the assignments of the carbon chemical shifts were made by comparison with the c)c values of structurally similar compounds.
Thus, the carbon chemical shifts of I r were virtually identical with the repo11ed 14 c)c values of the compound which
were distinctly different from those of the isomeric flavanone
4f. The most diagnostic 13 C NMR spectral features of the
3,4-dihydro-4-phenylcoumarin and the isomeric flavanone
Table 3. 13 C NMR spectral data of I r. Is, It, I u, lv, .. f and Sb
Chemical shifts in Jppm
I r*
Is**
It**
I u••
lv*
c
C-2
167.5
165.9
167.0
167 8
165.5
C-1
C-3
36.9
37.1
37.0
37.9
36.8
c
4f*
Sb*
C-2
79.5
47.0
44.5
44.1
118.0
188.0
C-4
40.6
41.2
41.3
41.2
40.3
C-3
C-4a
II 1.8
111.3
II 0.9
104.0
104.3
C-3a
C-5
117.0
116.6
119.4
118.8
118.8
C-4
191.6
C-6
124. 5
154.6
125.1
112.0
111.7
C-4a
114.2
C-7
127.5
115.3
116.5
144.0
153.9
C-5
129.6
C-8
129.0
129.5
153.3
142.9
109.8
C-6
121.5
147 7
C-8a
151.6
145.7
146.0
146.7
151.5
C-7
136.0
1216
C-1'
140.2
141.9
140.9
142.9
140.9
C-7a
C-2'
127.4
128.0
128.1
128.4
126.8
C-8
118.0
115.2
155.9
142 6
C-3'
129.1
129.5
129.5
129.7
129.1
C-8a
150.2
C-4'
128.7
127.9
127.9
128.1
127.5
C-1'
138.6
142.5
C-5'
129.1
129.5
129.5
129.7
129.1
C-2'
126.0
127.6
C-6'
127.4
128.0
128.1
128.4
126.8
C-3'
128.7
129.0
C-4'
128.0
127.2
C-5'
128.7
129.0 •
C-6'
126.0
127.6
= 5cocl + 76.9 ppm.
3
with OrMs = J(d6-acetone) + 29.6
*Spectra were run in CDCJ:3 and chemical shills measured with OrMs
**Sp..:ctra were run in d 6 -acetone and chemical shifts measured
ppm.
Table 4. 13 C NMR spectral data ofJd, Je and 7
Chemical shifts in 5 ppm
Chemical shifts in 8 ppm
c
3d
7
Je
c
C-1
193.3
192.0
192.6
C-8'
123.3
C-2
119.0
117.1
117.3
C-9'
134.1"
C-3
144.8
147.1
147.1
C-10'
C-1'
116.2
116.0
108.4
C-1"
135.1
134.7"
135.0
C-2'
154.2
153.5
159.9
C-2"
128.0
128.2
128.1
C-3'
136.1
109.5
96.3
C-3"
128.8
128.7
128.6
C-4'
157.1
117.1
162.4
C-4"
129.9
130.5
130.0
C-5'
108.1
129.6
135.1
C-5"
128.8
128.7
128.6
C-6'
127.3
126.2
154.7
C-6"
128.0
128.2
128.1
C-7'
127.5
Spectra were run in CDC~ and chemical shifts measured with 8rMs = 6coc1 3 + 76.9 ppm.
avalues are interchangeable.
748
Jd
7
3e
134.7"
Majumder et al. :The reactions of phenols with a,,B-unsaturated aromatic acids in presence ofpolyphosphoric acid
oc
isomeric flavanone derivatives resonated at a much lower
field at cab~ 79.0 ( 4f: ac 79.5). The sp 3-methylene carbons
(C-3) Of3,4-dihydro-4-phenylcoumarins lr, Is, It, lu and
lv (lr: ac 36.9; Is: 37.1; It: 37.0; lu: 37.9; lv: 36.8)
also showed downfield shifts of ca 7.5 ppm compared with
those of the C-3 of the isomeric flavanone derivatives ( 4f :
44.5). The chemical shifts of the aromatic carbons of the
above 3,4-dihydro-4-phenylcoumarins are also in conformity with the proposed structures of the compounds.
values of
derivatives are the observed differences in the
their sp 3 -methine (C-4 of 3,4-dihydro-4-phenylcoumarins
and C-2 of the isomeric flavanones) and their sp 3-methylene carbons (C-3 of both types of compounds) as well as
those of their carbonyl carbons. Thus, while the carbonyl
carbons of the 3,4-dihydro-4-phenylcoumarins appeared at
ca
167.0 (lr: ac 167.5; Is: 165.9; It: 167.0; lu:
167.8; 1v : 166.5), the corresponding carbonyl carbons of
the isomeric flavanone derivatives resonated at ca
191192 (4f: ac 191.6). Again, while the sp 3-methine carbons
(C-4) ofthe 3,4-dihydro-4-phenylcoumarins appeared at ca
ac 40-41 (lr: ac 40.6; Is: 41.2; It: 41.3; lu: 41.2; tv:
40.3), the corresponding sp 3 -methine carbons (C-2) of the
oc
oc
oc
"~~'
The structure of the indanone derivative Sb was also
confirmed by its 13 c NMR spectral data (Table 3). Thus,
the carbonyl carbon of Sb appeared at ac 188.0 and the sp 3
methine (C-3) and methylene (C-2) carbons of the compound
~OMe
PPA
H0~-
Me
0
2.4-. 3.4- and 3.5·
2·. 3- and 4-~rethyoxy
d1me1hylphcnols
cinnami~.:
acids
OMe
l
,.
H 20
:~OMo
M~e
I
0
l
lOMe at
lOMe at C-31
Cyclization
H
c.z· orC-4"1
-
(i)-HIll
( ii)H 10
Me
M~O»OMe
Me
Mw-
1l
M~e
I
0
Me
Me
~,~· ~ :~·
,;?
I
"Wo ' 0
f"'y'OMe
0
0
I
-
Me
::,._
OM<
GloMe
I
PPA
Scheme 2. Plausible mechanisms of the reactions of phenols and cinnamic acids and those of aryl cinnamatcs in presence of PPA proposed by
Chenault and Dupin9.l O
749
J. Indian Chern. Soc., Vol. 78, October-December 2001
clization of the chalcone derivatives would finally give the
corresponding tlavanones. The formation of an indanone
derivative was also assumed to involve similar Fries rearrangement at the position para to the phenoxy oxygen atom
followed by cyclization.
resonated at Jc 44.1 and 47.0, respectively. The aromatic
carbons of Sb resonated at the expected positions.
The structures of the chalcone derivatives 3d and 7 were
also further confi1med by their 13 c NMR spectral data (Table
4 ). The virtually identical Jc values of C-1, C-2 and C-3 of
3d and 7 with those of the corresponding carbon atoms of
the chalcone derivative 3c 15 (3d : Jc 193.3 (C-1 ), 119.0 (C2), 144.8(C-3); 7: Jc 192.0(C-l), 117.1 (C-2), 147.1 (C3); 3e: Jc 192.6 (C-1), 117.3 (C-2); 147.1 (C-3)] firmly
established the chalcone formulations of 3d and 7. The r5c
values of the aromatic carbons of 3d and 7 are also in conformity with their proposed structures.
If the reaction with aryl esters of 2- and 4-methoxy·
cinnamic acids proceeds exclusively through intramolecular Friedei-Crafts cyclization, it should not give any crossover products when the reaction is carried out with a
phenylcinnamate in presence of another phenol. With a view
to verifying this contention we have carried out the reaction
with (i) phenylcinnamate and (ii) phenyl 4'-methoxycinnamate, both in presence of pyrogallol under the reaction condition used by us. In each case, the products were
found to be a mixture of 3,4-dihydro-4-phenylcoumarins
obtained from phenol and pyrogallol separately. Thus, with
phenyl cinnamate and pyrogallol the products were found
to be 3,4-dihydro-4-phenylcoumarins 1rand 1u along with
traces of 3d (Scheme 3 ). Similarly, the reaction of phenyl
4'-methoxycinnamate with pyrogallol afforded 3,4-dihydro4' -methoxyphenylcoumarins 1w and lx (Scheme 3 ). The
structures of 1wand 1 x were established mainly from their
1H NMR spectral data (Table 2). It is interesting to note
that in both the reactions no flavanone derivative was obtained. The above observations clearly indicate that thereactions of aryl esters of cinnamic acids proceed mostly, if
not exclusively, through an intermolecular mechanism involving the cleavage of the aryl cinnamates to the acylium
ions and the phenols. The acylium ions appear to be the
obligatory intermediates. A Michael type 1,4-addition of
the phenols to the acylium ions followed by cyclization finally gives the 3,4-dihydro-4-phenylcoumarins (Scheme 4).
Although considerable synthetic work has been carried
put on the reactions of phenols with a,/}- unsaturated aromatic acids in presence of PPA, the exact mechanisms of
these reactions are still obscure. Chenault and Dupin 9· 10
were the first to propose plausible mechanisms for these
reactions. These authors assumed the formation of aryl
cinnamates as the obligatory intermediates in these reactions, and depending on the nature of the substituents in the
phenyl rings of the phenols and the cinnamic acid, PPA was
assumed to form coordination complex either with the ester
carbonyl (with aryl esters of 2 - and 4-methoxycinnamic
acids) or with the phenoxy oxygen atom (with the aryl esters of3-methoxycinnamic acid) of the cinnamates (Scheme
2). In the former case an intramolecular Friedel-Crafts cyclization followed by hydrolysis would give 3,4-dihydro-4phenylcoumarins, while in the latter case heterolytic cleavage ofthe cinnamates would form acylium ions, which, by
an intermolecular Fries rearrangement followed by hydrolysis, would give the chalcone derivatives (Scheme 2). Cy-
3d
([races)
PPA. 90-IOO"C, 2 hr
lr
ago
#
<?I
·:::,....
OMe
lu
OMe
OMe
OH
H~OH
"''
PPA. 90-IOO"C, 2 hr
c&
:::,....
+
I
0
lw
0
H
OH
1x
Scheme 3. Formation of lr. lu. 1w and lx in the reactions of phenylcinnamate and phenyl 4'-methoxycinnamate separately with pyrogallol in
presence of PPA.
750
Majumder eta/. : The reactions of phenols with a,_,8-unsaturated aromatic acids in presence of polyphosphoric acid
are reminiscent of the additions of Grignard reagents to
a,_,8-unsaturated carbonyl compounds. The formation of the
chalcones or tlavanones would be favored with bulkier
phenols which are unable to react in the l ,4-mode due to
steric interaction with the phenyl groups of the acylium ions,
while phenols of lower bulk would favor the formation of
3,4-dihydro-4-phenylcoumarins involving addition mostly
through 1,4-mode. The additions of phenols of intermediate bulk seem to be nonregioselective forming mixture of
The formation of the tlavanones may be assumed to proceed through a Michael type 1,2-addition of the phenols to
the acylium ions to give the chalcones which on cyclization
finally give the tlavanones (Scheme 4). The indanones may
also be assumed to be formed through a I ,2-addition ofthe
appropriate phenols to the acylium ions, the attack of the
phenols taking place through a site ofthe phenols ditTerent
from that giving the chalcones or tlavanones. Such Michael
type additions of phenols to the intermediate acylium ions
-
-
PPA
PPA
1.4-addition
1.2-addition
1
1
Rl
RJ
R3
R5
R5
R6
R6
R7
R7
l
Rl
Rl
0
R3
[3d: RI=R2=0H
R3=R4=R5=R6=R7=HJ
R3
l
R~
lr: R I.R2=R3=R•=R3:R6.R7=H
CO
h: RI:R2=R . .Rl:R6=R7=H.R-1.QH
§-H
II: R I.QH. R2•R·'•R4:RloR6.R7•H
lu: RI=R2=0H, R)=R4=R-~=R6=R7=H
h: RI:Rl=R4=Rl:R6.R7=H. R2.0H
~
lw: R 1=R2=RJ=R"'=R$2 R6=H.R7:o:OMc
lx: R 1=R2=0H. Rl:R4:Rl=R6=H. R7=0M•
j
HO'()--(Ph
HO~
Ph
l
0
Sb
PPA
-
+
·~)
fx· ~
addition
'
Ph
7
Scheme 4 (contd.)
751
J. Indian Chem. Soc., Vol. 78, October-December 200 I
H
M~OH
I .
+
+
M
2.4·, J ,4· and
3.5-dimethy !phenols
2.4-. 3.4· and
3.5-dime!hy !phenols
OMe
l
PPA
+
x'
l
~~-~
X
(oMe
OMe
•
y
y'
:~e
0
(.+H
Me
0
l
:~e
I~~~+
0
OMc
?'
l
OMc
M
M
0
-
~'
M~e
M
+H+
1-Q
OH
OMe
OMe
--
l
Me
0-H
M
l'
OMc
"W"
I
lt
Me
0
2b
OM.
M
OMe
M x x : r oo
•
Me
6,._
I
h
-6
M
·
2a
Scheme
~-
Proposed plausible mechanisms of the reactions of phenol, catechol, hydroquinonc, pyrogallol, 2-naphtlwl mel other phenols with
cinnamic acids and those of different arylcinnamates in presence of I'PA.
both 3,4-dihydro-4-phenylcoumarins and chalcones or tlavanones, the ratio oft he products being again dependent on
the bulk of the phenols and temperature of the reaction. This
was corroborated by the fact that 2-naphthol gave exclusively the chalcone derivative 7. Pyrogallol gave the 3,4752
dihydrocoumarin I u as the major product, while the chalcone
derivative was obtained as a minor product. The reaction of
resorcinol with cinnamic acid at 50-70° gave the flavanone 13
as the sole product, \vhile the same reaction at 95--100° afforded exclusively the 3,4-dihydrocoumarin lv. Similarly,
Majumder eta!. : The reactions of phenols with a,,B-unsaturated aromatic acids in presence of polyphosphoric acid
added with stirring at room temperature. The reaction mixthe reactions of 3,4, 3,5- and 2,4-dimethylphenols with 39
ture was then heated on a boiling water-bath (95-1 00°) for
methoxycinnamic acid afforded mixtures of 3,4-dihydro2 h. It was then cooled to room temperature, poured into
coumarins and chalcones or flavanones and cinnamate (only
in case of2,4-dimethylphenol) as stated earlier, formed by
crushed ice and left for 2 h. The liberated solid in each case
both I ,4- and I ,2-additions of the pheno Is to the correspondwas extracted with Et 2 0, washed with H 20, dried and the
ing acylium ions. The fact that the reaction of2,4-, 3,4- and
solvent removed. The crude products obtained by the reac3,5-dimethylphenols with 2- and 4-methoxycinnamic acids
tion of 1 g each of phenol. hydroquinone, catechol, pyroafforded exclusively the 3,4-dihydro-4-phenylcoumarins and
gallol, resorcinol and 2-naphthol with 1.5, 1.33, 1.33, 1.18,
no chalcones or flavanones, may be rationalized by assum1.33 and 1.04 g of cinnamic acid, respectively, in presence
ing the participation of the more stable resonating forns x'
ofpolyphosphoric acid (20 ml) in each case under the reacandy' rather than the less stable forms x andy, respectively,
tion conditions stated above, were separately chromaof the 2- and 4-methoxycinnamyl ions, which can only form
tographed.
the 3,4-dihydrocoumarin derivatives as shown in Scheme
Reaction of phenol with cinnamic acid : The petroi4. The elimination of the anisyl group from the 3,4EtOAc (80 : I) eluate in the chromatography of the crude
dihydrocoumarin I i involving coordination ofPPA with the
reaction product obtained by the reaction of phenol and cinoxygen of the 4' -methoxyl group as proposed by Chenault
namic acid afforded I r (0.89 g), crystallized from petroland Dupin 9 appears to take place by protonation at C-1' of
EtOAc, m.p. 95°; vmax 1740 (5-lactone C=O), 1600, 1565,
I i as shown in Scheme 4. Under the reaction conditions
1490, 1440, 830, 810, 750, 660 and 610 cm- 1 (aromatic
(95-100°) used by us, formation of phenyl cinnamates may
nucleus); mlz (rei. int.) 224 (M+, 100), 147 (45), 146 (70),
not necessarily be an obligatory step in the reactions of phe119 (40) and 118 (46).
nol, hydroquinone, catechol, resorcinol, pyrogallol and 2Reaction of hydroquinone with cinnamic acid : Chronaphthol with cinnamic acid in presence of PPA. Cinnamic
matography
of the crude reaction product obtained from the
acid at the above high temperature may directly be converted
ofhydroquinone
and cinnamic acid gave Is (1.42
reaction
to the corresponding acylium ion which may undergo either
g)
in
the
petrol-EtOAc
(50:
1), crystallized from the same
a Michael type 1,4- or 1,2-addition with the above phenols
solvent
mixture,
m.p.
125°
(Found : C, 74.96; H, 4.98.
depending upon their nature to give different products as
C15H1203 requires: C, 75.0; H, 5.04%); vmax 3450 (OH),
shown in Scheme 4. The phenyl cinnamates, if at all formed
1750 (5-lactone C=O), 1615, 1580, 1500, 835, 775, 720,
initially, are possibly converted to the acylium ions under
665 and 625 cm- 1 (aromatic nucleus); mlz (rei. int.) 240
the high temperature used.
(M+, 46), 163 (62), 162 (100), 135 (32), 134 (48) and 106
(37).
Experimental
M.ps. are uncorrected. Silica gel (1 00-200 mesh) were
used for column chromatography and silica gel G for TLC.
JR spectra (KBr) were run on a Perkin-Elmer spectrophotometer (Model 782). 1H NMR spectra (300 MHz) were
recorded on a Bruker Supercon FT NMR instrument using
TMS as the internal standard and 13 C NMR spectra (75
MHz) in the same instrument. Mass spectra were recorded
with a direct inlet system at 70 eV and the figures in the
parentheses attached to each mlz value represent relative
intensities of the peaks. All analytical samples were routinely dried over P 20 5 for 24 h in vacuo and were tested for
purity by TLC and MS. Anhydrous Na 2S0 4 was used for
drying organic solvents. Petrol used had b.p. 60-80°.
General method for the reactions ofphenols with cinnamic acid in presence of PPA :
To the freshly prepared polyphosphoric acid [prepared
by vigorously stirring a mixture of 12.5 g P 20 5 and 20 ml
orthophosphoric acid (d = 1.75 g/ml) at 100° for 2 h] a
mixture of the appropriate phenol and cinnamic acid was
Reaction of catechol and cinnamic acid : The crude
reaction products obtained by the reaction of catechol and
cinnamic acid in presence of PPA were chromatographed.
The petroi-EtOAc (50: I) eluate afforded It (1.35 g), crystallized from petrol-EtOAc, m.p. II 0° (Found : C, 74.98;
H, 4.92. C 15 H 12 0 3 requires : C, 75.0; H, 5.04%); vmax
3420(0H), 1740(5-lactoneC=O), 1620,1590,1465,1410,
870, 840, 690 and 625 cm- 1 (aromatic nucleus); m!z (rei.
int.)240(M+,48), 163(63), 162(100), 135(32), 134(48),
107 (47), 106 (37).
Further elution of the column with petroi-EtOAc
(20 : I) gave 5b (0.55 g), crystallized from the same solvent mixture, m.p. 122° (Found : C, 74.92; H, 4.95.
ci5HI203 requires: C, 75.0; H, 5.04%); Vmax 3425 (OH),
1785 (5-membered cyclic conjugated C=O), 1635, 1620,
1490, 875, 815, 800, 790, 670 and 640 cm- 1 (aromatic
nucleus); m/z (rei. int.) 240 (M+, 100), 163 (55), 162 (69),
134 (3 I), I 06 (35) and 78 (25).
Reaction of pyrogallol with cinnamic acid : The mix753
J. Indian Chern. Soc., Vol. 78, October-December 2001
ture of products obtained by the reaction of pyrogallol with
cinnamic acid in presence of PPA was chromatographed.
The petrol-EtOAc (30 : I) eluate gave I u ( 1.36 g), crystallized from petrol-EtOAc, m.p. \72° (Found : C, 70.29; H,
4.69. C 15 H 12 0 4 requires: C, 70.3; H, 4.7%); vmax 3420
(OH), 1750 (6-lactone C=O), 1620, 1520, 1480, 890, 775,
730, 620 and 610 cm- 1 (aromatic nucleus); mlz (rei. int.)
256 (M+, 100), 179 (48), 178 (65), 150 (50), 122 (32), 94
(27) and 66 (20).
Further washing the column with petroi-EtOAc ( 10 : I)
afforded 3d (0.37 g), crystallized from petroi-EtOAc, m.p.
152° (Found: C, 70.28; H, 4.68. C 15 H 12 0 4 requires: C,
70.3; H, 4. 7%); vmax 3440 (unchelated Ar-01-1), 3200 ( chelatedAr-OH), !640(conjugatedchelatedC=O), 1610,1575,
1515, \435, 860,790,770,745,680 and 6\5 cm- 1 (aromatic nucleus); m!z (rei. int.) 256 (M+, 100). 153 (84), 124
(53), I 04 (33), 96 (35) and 63 (24).
Reaction of resorcinol with cinnamic acid : The reaction product obtained by the reaction of resorcinol with cinnamic acid in presence of PPA under the reaction condition
stated above was chromatographed. The petroi-EtOAc
(50: I) eluate gave lv (1.40 g), crystallized from petroiEtOAc, m.p. I 25° (Found : C, 74.95; H, 4.94. C 15 H 12 0 3
requires: C, 75.0; H, 5.04%); vmax 3400 (OH), 1740 (c)lactone C=O), 1610, 1575, 1440, 880, 830, 735, 660 and
620 cm- 1 (aromatic nucleus); ml::: (rei. int.) 240 (M+, I 00),
163 (53), 162 (69), 135 (26), 134 (32), 106 (33) and 78
(24).
Reaction of 2-naphthol with cinnamic acid : The residue obtained by the reaction of 2-naphthol with cinnamic
acid in presence of PPA was chromatographed. The petrolEtOAc (50: I) eluate afforded 7 (0.95 g), crystallized from
the same solvent mixture, m.p. 178° (Found: C, 83.18; H,
5.10. c19HI402 requires: C, 83.2; H, 5.15%); Vmax 3340
( chelated Ar-OH), 1645 (conjugated chelated C=O), 1610,
1550, 1520, 1440, 1430, 850, 820, 790, 680 and 625
cm- 1 (aromatic nucleus); mlz (rei. int.) 274 (M+, I 00), 171
(48), 170 (56), 142 (42)and 104 (26).
Preparation ofphenyl cinnamate and 4 '-methoxyphenyl
cinnamate: Phenyl cinnamate and 4' -methoxyphenyl
cinnamate were prepared by treating a solution of phenol
(0.1 mol) and pyridine (0.1 mol) in dry benzene (100 ml)
separately with cinnamoyl chloride (0.1 mol) and 4methoxycinnamoyl chloride (0.1 mol), respectively, in dry
~enzene (30 ml). The mixtures were separately heated for I
r'l on a boiling water-bath. After filtration and evaporation
of the solvent, the cinnamates were obtained in solid state
(ca 99%): phenylcinnamate: 56.46 (I H. d,.! 15Hz, H-2),
7.41-7.56 (IOH, m, H-2'-H-6' and H-2"-H-6"), 7.80 (I H,
754
d,.!! 5Hz, H-3); 4' -methoxyphenylcinnamate: 53.95 (3H,
s, Ar-OMe), 6.88 (2H, d,.! 8.7 Hz, H-3' and H-5'), 7.28
(51-1, m, H-2"-H-6"), 7.38 (IH, d, .! 15 Hz, H-2), 7.55
(21-1, d, .! 8.7 Hz, H-2' and H-6') and 7.80 (I H, d, J 15 Hz,
I-1-3).
Reaction ofphenyl cinnamate with pyrogallol in presence of PPA :A mixture of phenyl cinnamate (I g), pyrogallol (0.5 g) and PPA (20 ml) was heated on a boiling
water-bath for 2 h. The crude reaction products after usual
work-up were chromatographed. The petroi-EtOAc (80: I)
eluate afforded I r (0.15 g). Further elution of the column
with petroi-EtOAc (30 : I) gave I u (0.4 7 g).
Reaction of 4 '-methoxyphenyl cinnamate with pyrogallol in presence ofPPA: A mixture of 4'-methoxyphenyl
cinnamate (I g) and pyrogallol (0.55 g) was treated with
PPA (20 ml). The mixture was heated on a boiling waterbath for 2 h. The products after usual work-up were
chromatographed. The petrol-EtOAc (50 : I) eluate gave
I w (0.2 g), crystallized from the same solvent mixture, m.p.
115°. Further washing the column with petrol-EtOAc
( 10 : 1) afforded I x (0.46 g), crystal! ized from petrol-EtOAc,
m.p. 122°.
Acknowledgement
The authors are thankful to Prof. W. Kraus, University
of Hohenheim, Stuttgart, Germany, for the mass spectra.
The work was suppot1ed by C.S.I.R. and U.G.C., New Delhi.
References
.J.D. Simpson and H. Stephen.J. Chem. Soc., 1956, 1382.
2.
A. K. Dasgupta and M.S. Paul,J. indian Chem. Soc .. 1970, 47,
1017.
3.
A. K. Dasgupta, K. R. Das and A. Dasgupta,/ndian J. Chem.,
1972,10.32.
4
T Manimaran, T K. Thiruvengadam and V. T. Ramakrishnan.
Synthesis, I 975, II, 739.
5.
N. Hasebe, J. Chem. Soc. Jpn., Pure Chem. Sect. (Nippon
Kagaku Zam), 1961, 82, 1728.
6.
N. Hasebe, J. Chem. Soc. Jpn., Pure Chem. Sect. (Nippon
Kagaku Zani), 1962, 83, 96.
7.
F. H. Marquardt, Helv. Chim . ...leta, 1965, 48, 1476.
8.
1.. Reichel and G. Proksch, Justus Lie bigs Ann. Chem., 1971,
745, 59.
9.
J. Chenault and J. F. E. Dupin, Heterocycles. 1983,20,437.
I 0.
J. Chenault and J. F. E. Dupin, Helerocycles, I 983, 20, 240 I.
II.
T. Matsui, Bull. Facul(v Eng. Miva=aki Univ., 1984, 30. 141
(Chem. Abs/1:. 1985, 102, 95441).
12.
B. Talapatra, T. Deb and S. K. Talapatra,/ndian J. Chem., Sect.
E. 1985, 24. 561.
Majumder eta!. :The reactions of phenols with a,jl-unsaturated aromatic acids in presence ofpolyphosphoric acid
13.
B. Talapatra, T Deb and S. K. Talapalra,/ndwnJ. Chem., Sect.
B, 1986. 25, 112.
14.
P. Joseph-Wathan, .J. Mares, M. C. Hernandez and J. N. Shoolery,
J. Magn. Reson.. 1974. 16, 44 7.
15.
A. Patra, G. Ghosh, 1'. K. Sengupta and S. Nalh. Magn Reson.
Chem., 1987, 25, 734.
755
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Ueber die beeinflussung der wirkung narcotischer medicamente durch antipyretica
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XXXIV.
Aus dem med.-chem, und pharmakol. Institut der Universitat Bern
(Director: Prof. Dr. E m i l Btirgi).
Ueber die Beeinflussung der
Wirkung narcotischer Medicamente durch Antipyretica.
Von
Sophie Lomonosoff aus Kijew.
Biirgi und seine Schiller haben nachgewiesen, dass bei der gleichzeitigen Verwendung zweier verschiedener Narcotica die einzeinen narcotischen Effecte sich nicht nur addiren, sondern in ungeahnter Weise versts
Weitere Untersuchungen zeigten dann, dass eine solche Vermehrung der Narcosewirkung nur erfolgt, wenn die Medieamente verschiedenen Arzneigruppen, denen verschiedene Zellreptoren, allgemeiner gesagt Angriffspunkte, entsprechen, entstammen. Zwei Narcotica, die z. B.
beide zu den Nareotiea der Fettreihe gehSren, addiren sieh entweder
bloss, oder zeigen dann doch eine sehr geringe Verst~trkung ihrer Einzelwirkung. Es schien nun yon besonderem Interesse, nachzuforschen, ob
die Arzneimittel der Antipyreticagruppe die Wirkungen eigentlicher Nareotiea zu verst~rken im Stande seien und in welchem Grade. Die Antipyretica gelten ja als eine Art yon Narcotica, die, wenn wir vom Chinin
absehen, vermSge ihrer st~rkeren Verwandtschaft zu gewissen Theilen dos
Centralnervensystems in ers[er Linie das Wgrmeregulationseentrum beruhigen und die W~trmeabgabe vermehren, unter Umst~nden aber aueh
eine Allgemeinnarcose hervorrufen kSnnen und wegen dieser Eigensehaft
als Antineuralgica ausgedehnte Yerwendung finden. Ob ihnen als Narcotiea die gleichen Angriffspunkte zukommen wie anderen eigentlichen
Narcotica, k a n n a priori nieht sieher entschieden werden. O v e r t o n
maeht allerdings wahrseheiniich, dass sie wie die Narcotica der Fettreihe
in Folge ihrer LipoidlSslichkeit narcotisch wirken.
Wir haben mit einer Untersuchung des L a c t o p h e n i n s begonnen
und lassen hier die Resultate folgen:
Versuch 1. Ein 1850 g schweres Kaninchen erhielt Nachmittags 3 Uhr 0,01 g
Morph. hydr. pro Kilogramm KSrpergewicht. Nach 15 Min. sitzt das Thier ruhig
im Kiifig~ nimmt keine Nahrung zu sich: reagirt abet auf Stiche und li~sst sich nicht
auf den giicken oder auf die Seite legen. Dieser Zustand dauert 2 Stunden. Nachher
nimmt das Thier wieder 1%hrung zu sich und bewegt sieh im K~ifig.
Ueber d. Beeinllussung d. Wirkung narcotischer Medicamente durch Antipyretica. 567
Versuch 2. Parallelversuch zu 1, ~hnliche Erscheinungen.
Versuch 3. 0,015 Morph. hydr. pro Kilogramm KSrpergewicht~ ~hnliches Verhalten wie 1 und 2.
Versuch 4. 0,02 Morph. hydr. Die Reaction auf Stiche nach 15 Min. gering,
abet nie ganz aufgehoben. Keine Annahme abnormer Stellungen.
Versuch 5. Parallelversuch zu 4. Aehnliche Erscheinungen.
Versuch 6. Kaninchen, 1940 g schwer, bekam !) Uhr 30 Min. Vormittags 07025
Morphium pro Kilogramm K6rpergewicht. 9 Uhr 40 Min. liegt das Thier platt auf
dem Bauch, reagirt aber auf Stiehe und liisst sich nicht auf den giicken legen. 9 Uhr
50 Min liegt es in derselben Lage, reagirt nicht auf Stiche und liisst sich ab und zu
eine abnorme Stellung geben. Nach 2 Stunden fSmgt es an sich zu erholen.
Diese crsten Versuehe mit Morphium allein dienten dazu, die minimal
narcotisirende Morphiumdosis festzustellen. Naeh den Untersuehungen
von H a u e k o l d
und L i n d e m a n n
betrug sic 0,02 Morphium pro Kilogramm K6rpergewieht.
Wit fanden sic etwas h6her (0,025). Der Unterschied zwisehen den Ergebnissen der genannten Autoren und den meinigen
ist dadurch bedingt, dass wir nut ganz ausgesprochene S y m p t o m e als
eigentlieh nareotisehe ansahen.
Ueber diesen P u n k t haben sich B i i r g i
und S a r a d s e h i a n
wiederholt des genaueren geSussert, so dass wir bier
auf eine Beschreibung dcr Methodik verzichtcn ki3nnen. Auch 0,025
Morpbium hydroehloricum pro Kilogramm K6rpergcwieht batten keino
eigentliche Narcose ergeben.
Versuch 7. Kaninchen, 1810 g schwer, bekam 11 Uhr 5 Min. Vormittags 0,02
Morphium pro Kilogramm Kih'pergewicht subcutan und 11 Ubr 15 Min. eine Pravatzsche Spritze yon abfiltrirter 2proc. bactopheninlSsung (nieht pro I{ilogramm K~irpergewicht). 11 Uhr 50 Min. liisst sich das Thier giickenlage gefallen~ reagirt nicht
mehr auf Stechen. 12 Uhr 5 Min. Auf Stechen mit der Nadel steht es auf, ebenso
bei Prfifung der Reflexe, li~sst sich aber wieder hinlegen. 12 Uhr 10 Min. lgsst es
sich nicht mehr auf den R/icken legen und bewegt sich im Kafig.
Versuch 8. Kaninchen, 2150 g schwer, bekam 4 Uhr 10 Min. 0,02 Morphium
pro Kilogramm Kgrpergewicht subcutan und 4 Uhr 20 Min. eine Pravaz'sche Spritze
derselben Lactopheninl/isung wie in Versuch 7 subcutan. 5 Uhr 5 Min. lgsst sich das
Thier am den giieken legen. Bei Priifung auf Stiche springt es auf, l[isst sich abet
wieder hinlegen. 5 Uhr 55 Min. l~isst es sich nicht mehr niederlegen. Gegen 7 Uhr
erholt es sich vollstS~ndig.
Versueh 9. Kaninchen, 1645 g schwer, bekam um 5 Uhr nachmittags 5 ecru
derselben LaetopheninlSsung subcutan. 5 Uhr 45 Min. wird das Thier etwas ruhiger.
6 Uhr 30 Min. bewegt es sieh im KS~fig und nimmt Nahrung. Andere Symptome waren
nicht aufgetreten.
Versuch 10. Kaninchen, 1179 g schwe 5 bekam i0 ccm derselben Lactophenin15sung subeutan. Verhielt sich wie 9.
Versuch 11. Kaninchen, 1487 g schwer, bekam 10 Uhr 25 Min. Vormittags
0,02 Morphium pro Kilogramm Kiirpergewieht subeutan und 10 Uhr 35 Min. eine halbe
Pravaz'sehe Spritze derselben 2proc. LactopheninlSsung subcutan. Nach 50 Min.
wird das Thier ruhiger, reagirt schwaeh auf Stiche, 15~sst sich keine abnorme Stellung
geben. Gegen 12 Uhr erholt es sich vollst~indig.
Versuch 12. Kaninchen, 1765 g schwer~ bekam 4 Uhr 13 Min. Nachmittags
0,01 Morphium pro Kilogramm K~irpergewicht subcutan, und 4 Uhr 23 Min. zwei
Pravaz'sche Spritzen derselben lloroc. LactopheninKisung subcutan. 4 Uhr 35 Min.
liegt das Thier ausgestreekt auf dem Baueh~ reagirt sehwaeh auf Stieh% l~sst sieh
aber nieht auf den g(ieken legen; yon 5 Uhr 15 Min. an vgllige Erholung.
568
Sophie Lomonosoff,
In dicscn Versuehen wurde theils Morphium und
Laetophenin
allein, theils win'den beidc Substanzen zusammen gegeben. Wir hat'ten
durch einen heisscn wg.ssrigeu Auszug eine concentrirte Lactophenin15sung, die wir vor dem Gebraueh kalt werdcn ]lessen, hergestellt. 1 g
dieser LSsung enthielt u,gef/ihr 0,002 Lactophenin. 10 ccm = 0,02 Lactophenin ergaben noch keine vollstSndige Narcose, und wit haben deshalb
verzichtet, die minimal narcotisirende Dosis fiir das Lactophenin iiberhaupt zu ermitteln. Wir sahen aber (Versuch 7 und 8), dass eine Spritze
= 0,002 im Stande ist, 0,02 Morphium zu einer narcotisirenden Dosis
zu machen, ja aueh schon dis halbe Menge zeigte einen deutliehen Einfluss. Dagegen erzielten wir mit 0,01 Morphium und 2 ccm der Laetopheninl5sung nur eine halbe Narcose. Wir haben diese Versuche nieht
welter ausgedehnt, well uns daran gelegen war, mii, subcutanen Injectionen zu arbeitcn und das Lactophcnin sich hierf(ir wenig eignete. Wir
gingen daher zu einer Untersuchung der Combination Morphium-Antipyrin fiber.
Versuch 13. Kaninchen, 1320 g schwer, bekam 3 Uhr 25 Min. Nachmittags
0,02 Morphium pro Kilogramm KSrpergewicht subcutan und 3 tihr 35 Min. 0,2 Antipyrin pro Kilogramm K~rpergewicht subcutan. 3 Uhr 45 Min. liegt das Thier platt
gestreckt auf dem Bauch, liisst sich noch nicht auf den l~iicken legen und ist sehr
empfindlich gegen Stiche. 4 Ui~r 5 Min. liisst es sich auf den Riicken legen. 4 Uhr
25 Min. schon nicht mehr: sitzt abel ruhig da und nimmt keine Nahrung. Gegen 5 Uhr
erholt es sich vollstSndig.
Versuch 14. Kaninchen, 1820 g schwer, bekam 4 Uhr Nachmittags 0,02 Morphium pro Kilogramm I(Srpergewioht und 4 Uhr 10 Mfn. 0,1 Antipyrin pro Kilogramm
KSrpergewicht, beides subcutan. 4 Uhr 25 Min. liegt es ausgestreckt auf dem Bauch
und bleibt so mehr als 2 Stunden liegen. Die Reaction auf Stiche ist herabgesetzt.
Nach 2 Stunden tritt Erholung ein.
Yersuch 15. Kaninchen, 2100 g schwer, bekam 4 Uhr 15 Min. Nachmittags
0,02 Morphium pro Kilogramm KSrpergewicht und 4 Uhr 25 Min. 0,05 Antipyrin pro
Kilogramm KSrpergewicht, beides subeutan. 4 Uhr 55 *lin. sitzt das Tier ruhig da,
liisst sich nicht auf den Riieken legen. Die Reaction auf Stiehe ist normal. 7 Uhr
Abends gleiches u
Versuch 16. l{aninchen, 2100 g schwer, bekam 5 Uhr 40 Min. Naehmittags
0,01 Morphium pro Kilogramm K5rpergewieht und 5 Uhr 50 Min. 0,2 Antipyrin pro
Kilogramm KSrpergewicht, beides subeutan. 6 Uhr 15 Min l~sst sich das Tier auf den
Rfieken legen. Scbmerzempfindung herabgesetzt. 6 Uhr 45 Min. stebt es auf und ist
nicht mehr hinzulegen.
Versuch 17. Kaninchen, 2560 g schwer, bekam 5 Uhr 45 Min. 0,01 Morphium
pro Kilogramm KSrpergewicht und 5 Uhr 55 Min. 0,1 Antipyrin pro Kilogramm KSrpergewicht, beides subcutan. 6 Uhr 15 Min. liisst sich das Tier auf den Rtieken legen,
springt aber bald wieder auf. Schmerzempfindung herabgesetzt. Von 6 Uhr 45 Min.
an allmRhliche Erholung.
u
18. Kaninchen, 2480 g schwer, bekam 10 Uhr 10 Min. Vormittags
0,01 Morphium pro Kilogramm t(Srpergewicht und 10 Uhr 20 Min. 0,05 Antipyrin pro
Kilogramm KSrpergewicht, beides subcutan. 10 Uhr 30 Min. sitzt das Tier ruhig im
KSfig, nimmt keine Nahrung zu sich. Narcotische Symptome dauern etwa 5--10 Min.
Versuch 19. 0,01 Morphium und (10 Min. sp~iter) 0,025 Antipyrin. Aehnliche
Erscheinungen wie in Versuch 1S.
Versuch 20. Ein 1710 g schweres Kaninohen erhiilt 4 Uhr Nachmittags 1,0 Anti-
Ueber d. Beeinflussung d. Wirkung narcotiseher Medicamente dutch Antipyretica. 569
pyrin pro Kilogramm KSrpergewicht subcutan. 4 Uhr 15 Min. starkc Dyspnoc, niemals
eigentliche Narcose.
Versuch 21. 0,01 Morphium und (10 Min. spiiter)0,0125 Antipyrin, nach 30 Min.
freiwillige Bauchlage, deutliche Bernhigung~ abet keine ausgcsprochene Narcose.
Versuch 22. '2,0 Antipyrin pro Kilogramm KSrpergewicht, nach einer Viertelstunde ErstickungskrS.mpfe. lCeaction auf Stiehe erhalten. Nach 45 Min. Exitus.
Versuch :23. 0,005 Morph. hydrochl, und naeh 10 Min. 0~2 Antipyrin. Nach
15 Min. schwache Dyspno% die allmShlich zunimmt und fast so stark wird win in
Versueh 20 (1,0 Antipyrin aliein), t(eine Nareose. Nach 3 Stunden vollst~indigc Erholung.
Versueh 24. 0~005 Morphium und nach 10 Min. 0~25 Antipyrin. Das Tier wird
ruhiger, hat aber starke Athemnoth. lCeagirt auf Stiche. Die Athemnoth wird heftiger,
dennoch vollstSmdige Erholung.
Versuch 25. 0,005 Morphium und 10 Min. spS~ter0,3 Antipyrin. Nach weiteren
10 Min. starke Dyspnoe. Naeh 1 Std. 10 Min. wird R~ckenlage angenommen. Schmerzempfindung nieht vSllig aufgehoben. Das Thief liegt da, ohne sich zu bewegen. 10Min.
sp~iter springt es auf und erholt sich yon da an allm~hlich ganz.
Versuch 26. 0,005 Morphium und (10 Min. spSter) 0,4 Antipyrin. Nach 15 Min.
Dyspnoe. Nach 40 Min. Annahme der tlfickenlage. Schmerzempfindung stark herabgesetzt, aber nicht aufgehoben. 11/4 Stunde nach der Injection springt das Thier
spontan auf~ erholt sich yon da an ailm~ihlich.
Versuch 27. 0,005 Morphium und (10 Min. sp~iter) 0,5 Antipyrin. Dyspnoe
nach 10 Min. Nach 30 Min. beginnende Narcose win in Versuch 2(;. Dauer etwa
2 Stunden. VollstSndige ]/~etablirung.
Versuch 28. 070025 Morphium und 0,3 Antipyrin. Schwacho Dyspnoe nach
25 Min. Einziges Symptom~ das etwa 1 Stunde anh~lt.
Versuch 29. 0,0025 Morphium und 0,4 Antipyrin. Erscheinungon ungef/ihr
win in Versuch 28.
Versuch 30. 0,0025 Morphium und 0~5 Antipyrin. Erseheinungen win in Versuch 28 und 29.
Versuch 31. 0,0025 Morphium und 1,0 Antipyrin. Starke Dyspnoe nach 10Min,
gleiehzeitig Narcoseerscheinungen. Das Kaninchen lSsst sich auf die Seite legen.
Schmerzempfindung herabgesetzt. Nach 35 Min. Convulsione% die rasch vor/ibergehen. Sensibilit~it herabgesetzt. Die Kriimpfe wiederholen sieh noeh einige Male~
werden aber schw~cher. Nach 4 Stunden vSllige Erholung.
Versuch 32. 0,0025 Morphium und 0,6 Antipyrin. Dyspnoe nach 15 Minuten.
Geringe nareotische Symptome, sonst nichts Besonderes.
Versuch 33. 0,0025 Morphimn und 0,8 Antipyrin. Aehnliche Erscheinungen.
Unruhiger Schlaf, der etwa 1 Stunde anhS.lt. Herabsetzuug der Schmerzempfindung.
Dyspnoe.
Aus diesen Versuchen geht in crs(~er Linic horror, dass man mit
Antipyrin allcin eine Nareose nicht erzielen konnte. 2,0 Antipyrin (Versuch '22) wirkten durch L/ihmung des Athmungs- und Vasomotorencentrums
tgdtlich und 1,0 Antipyrin erzeugte nine starkc Dyspnoe, die mehr als
2 Stunden dauertc, aber keinc Narcose. Ging man yon einer glcichbleibenden Morphiumdosis yon 0,02 aus, so erhielt man dutch Hinzufiigung yon 0,1 Antipyrin gerade noeh narcotische Erseheinungen. Betrug
die Morphiumdosis 0,01, so war 0 , 1 - - 0 , 2 Antipyrin nSthig, um ganz
schwache Narcose zu erzeugenl). Geringere Quantitaten riefen wohl auch
I) Die Schmerzempfindung war in beiden FS~llen stark herabgesetzt, aber ninht
ganz aufgehoben.
570
SophieLomonosoff,
noch BetKubungszust~nde hervor, niemals aber eigentliche Narcosen. Wir
gingen dann mit der Morphiumdosis noch welter herunter, zuerst auf
0,005, dann auf 0,0025, und erzielten mit der ersten Quantit/it noch
eine schwache Narcose, wenn wir ihr 0,3 Antipyrin zufSgten, mit der
zweiten bei einer Combination mit wenigstens 0:6 Antipyrin.
Morphium-Pyramidon-Versuche.
V e r s u c h 34. Kaninchen, 2445 g schwer, bekam 3 Uhr 25 Min. 0,005 Morphium
und um 3 Uh,' 35 Min. 0,2 Pyramidon pro Kilogramm KSrpergewicht, beides subcutan.
Um 4 Uhr liegt das Thief ausgestreckt auf dem Baueh, hat starka Dyspnoe~ auf don
Riicken l~sst as sich nicht logan. Um 4 Uhr 5 Min. bekommt das Thief heftige clonische Kr~impfe, die rasch voriibergehan, aber bald wiedarkahren und dann wieder
voriibergehen. Dabai bestoht starker Speichelfiuss und Zg,hneknirschan und w~hrand
der Krgmpfe iiberschl~gt sich das Thier nach rfickw~irts. In den Intervallen zwischen
dan Kr~mpfan~ walcha etwa 5 Min. dauarn, li~sst sich das Thier auf den Riickan legen.
Die Kr~mpfe dauern zwei Tage an. Am Enda des zweitan Tagas beruhigt sich das
Thier. Wiihrend dieser Zait verlor as 200 g an Gawiaht. Wiihrand des Krampfes
war die Reaction auf Stiche aufgehoben, Reflexe blieben unveriindert.
V e r s u c h 35. Kaninchen: 2180 g sehwer~ bekam um 9 Uhr 25 Min. Vormittags
0,005 g Morphium und um 9 Uhr 35 Min. 0~1 g Pyramidon pro Kilogramm KSrpergewicht, beides subcutan. 9 Uhr 40 Min. bekommt das Thier starke Dyspno% welche
2 Stundan dauert, dann abet voriibergeht; as tritt vollst~ndiga Erholung ein.
V e r s u c h 36. Kaninchen~ 1S10 g schwer, bekam 9 Uhr 40 Min. Vormittags
0~2 g Pyramidon pro Kilogramm KSrpergewicht subeutan. Um 10 Uhr 15 Min. bekam
das Thief starke Dyspnoa und 15 Min. darauf Zittern am ganzan KSrper. Gegen
12 Uhr tritt vollst~ndiga Erholung ein.
V e r s u e h 37. Kaninchen, 1990 g sehwer: bekam 3 Uhr 25 Min. Nachmittags
0:005 g Morphium und 3 Uhr 35 Min. 0:2 g Pyramidon. Beides pro Kilogramm KSrpergewicht subcutan, l0 Min. darauf tritt starka Dyspnoe und urn'4 Uhr treten
gleiche Kriimpfe wie im Versueh 34 auf. Die Erholung trat schon am folgenden
Tage auf.
Versuch 38. Kaninchen~ 1450 g schwer: bakam 4 Uhr 25 Min. Naehmittags
0,4 g Pyramidon pro Kilogramm KSrpergewicht subeutan. Um 4 Uhr 45 Min. bekam
es dieselbcn Kdimpfe wie in den Versuchen No. 36 und 39. Um 4 Uhr 55 Min.
kommen zu den friiheren Kr~mpfan noch Erstickungskr~mpfa hinzu. Das Thier schreit
laut auf, worauf der Tod erfolgt.
Versuche 38 und 36 haben uns gezeigt, dass wit auch mit dem
Pyramidon: wenn wit es fiir sich allein geben: keine Narcose erzeugen
kSnnen. 0,4 g wirken tSdtlich, 0,2 g stark toxisch, aber nicht narcotisirend. Das Pyramidon hat bei Kaninchen untcr Anderem eine starke
Dyspnoe und Kriimpfe erzcugende Wirkung. Wit waren nicht im Stande,
bci einer gleichbleibenden Dosis yon 0,005 Morphium durch Zusatz yon
Pyramidon Narcose zu erzeugen und haben deshalb diese Versuchsreihe
abgeschlossen.
Versuche mit Urethan-Antipyrin und Urethan-Pyramidon.
Auf eine detaillirte Wiedergabe dieser Versuche will ich verzichten,
um nicht zu ermtiden.
Aus de~! frtiheren Versuchsreihen wussten wir, dass 1:0 g7 besser
Ueber d. Booinflussungd. Wirkungnarcotischer Medicamentedurch Antipyretica. 571
gesagt 0,9 g Urethan als die minimal-narcotisirendc Dosis fiir diese Substanz anzusehen ist.
Wit gaben zuerst 0,5 g Urethan und 0,2 g Pyramidon und crhieltcn
cine schwaehe abet siehere Nareose. Doeh war der Schlaf so unruhig,
dass wit yon nun an niemals mehr Pyramidon flit unserc Versuehe verwendetcn. In einer weitercn Vcrsuehsrcihe (Versuch 41--44) gaben wit dann
zu einer gleiehbleibcnden Antipyrinmenge von 0,2 g Urethan in absteigenden Quantitiiten. Geringfiigige narcotisehc Symptome erhielten wit
aueh noah bei einer Zugabe yon 0,2 g Urethan, doeh handelte es sieh
um keine wirkliehe Narcose mehr. Wir erg/inzten diese Versuche noah
dureh weitere Experimente (Versuehe 46, 47, 49, 50 und 52). Aber
obwohl uns 0,2 g Antipyrin auch noah mit 0,4, 0,3 und 0,2 g Urethan
zusammen nareotische Symptome gab, sahen wir doeh, dass die minimalnareotisirende Menge des Gemisches erst bei 0,2 g Antipyrin + 0,5 g
Urethan zu suchen ist. Bei 0,2 g Antipyrin -t- 0,2 g Urethan entstanden,
wie schon gesagt, noah einige schwaehe Hypnosesymptome (minimal-nareotisirende Dosis naeh der friiheren Auffassung), verminderte man dann
die Urethanmenge noah mehr, so war iiberhaupt nichts Besonderes mehr
wahrnehmbar. Ebenso crlosehen die Nareosesymptome, wenn man zu
ciner gleichbleibenden Dosis yon 0,5 g Urethan wcniger als 0,2 g Antipyrin
zufiigte. Vielleieht war noah bci 0,5 g Urethan und 0,15 g Antipyrin etwas
weniges zu bemerken. Wit haben nun in dieser Vcrsuehsreihe aueh noah
die Frage genauer untersueht, ob eventuell ein Unterschied in der Wirkung eintritt, wenn man bald das eine, bald das andere der zwei eombinirten Medicamcnte zuerst injieiric. Das Antipyrin flit sieh allein gegeben, wirkt raseher toxisch als das Urethan nareotisirend.
Es sehien
daher a priori angezeigt, zuerst das Urethan und dann das Antipyrin zu
geben. Wir injieirten im Allgemeinen das Antipyrin zehn Minuten nach
dem Urethan und glaubten, dass auf diese Weise die Haupteffeete der
beiden Arzneien zusammenfallen wiirden.
Wit haben dann abet aueh das hntipyrin vor dam Urcthan gegeben
und die Injectionen dabei in verschiedenen Zeitr/iumen aufeinander folgen
lassen. Schliesslich haben wir aueh, wenn wir das Urcthan zuerst gaben,
naehgcsehen, ob evcntuell zur Erzie]ung grSsscrer Nareosen ein andercs
als das genannte Intervall yon 10 Minuten gews
warden musste, um
den hSchsten Effect zu erzielen. Wit haben dabei die folgenden Res u l t a t e erhalten:
Vcrsuch
No.
41
60
61
62
Urethan Intervall Antipyrin
Narcose.
g
Min.
g
Eintritt nach:
Dauer:
0,5
10
0,2
5 Min.
3 Stun& 10 Min.
0,5
10
0,2
10 .
3
.
10 ,,
0,5
20
0,~
30 ,
2
,, 40 ,
0,5
30
0,2
20
3
-aber schwacheWirkung
Antipyrin
Urethan
46
0,2
10
0,5
I0 ,,
2 Stund. 10 Min.
53 und 58
0,2
20
0,5
35 ,,
30 " keine Nareose
54
0,2
30
0,5
59
,0,2
~o
0,5
35 ,
-- Stund. 55 IVlin.
.
572
Sophie l~omonosoff,
Wir sehen aus dieser Versuchsanordnung, dass es nicht sehr viel
ausmacht, ob man das Antipyrin odcr das Urethan zuerst giebt. Wir
konnten nut feststellen, dass wenn man alas Antipyrin zuerst giebt und
Urethan dann erst naeh 30 Minuten fo]gen litsst, die Wirkung sehr abgeschw/icht ist oder sogar fehlt. Es ist ja klar, dass mall die Intervalle nieht beliebig verlSngern kann, und da das Antipyrin sehneller
wirkt, als das Urethan, die lntervalle dann, wenn das Antipyrin zuerst
gegeben wird, kfirzer sein miissen, als bei einem Beginn mit Urethan.
lch babe auch (Versuch 64 und 65) untersucht, wie die M o r p h i u m A n t i p y r i n - C o m b i n a t i o n wirkt, wenn das Antipyrin nieht wie in den
friiheren Versuchen nach dem Morphium, sondern vor demselben gegeben
wird. Wenn wit 0,02 Morphium und 10 Minuten nachher Antipyrin gegeben hatten, so war 10 Minufen nach der zweiten Injection Benommenheir, 30 Minuten nachher eine Narcose yon 20 Minuten Dauer eingetreten.
Gaben wit dagegen zuerst Antipyrin und 10 Minuten nachher Morphium,
so entstand iiberhaupt keine Narcose. Aehnliches erhielten wir bei den
Dosen 0,2 Antipyrin und 0,01 Morphium. Diese Thatsaehen erkli/ren
sigh leicht, wenn man annimmt, dass die Wirkung des Antipyrins viel
rascher eintritt und viel raseher vergeht, als dig des Morphiums.
Wenn wir, gestiitzt auf die vorliegenden Versuche, beurtheilen wollen,
ob und in welehem Grade die Antipyretica narcotische Wirkungen andGrer
Arzneien zu verst~irken im Stande sind, so miissen wir jede yon unseren
Versuchsreihen noch einmal in Kiirze gesondert betracbten. Wir haben
allerdings yon keinem einzelnen Antipyreticum eine eigentliche minimal
narcotisirende Menge finden kSnnen und die Ausrechnung ist dadurch bedeutend erschwert. Wit sahen, dass 0,02 Lactophenin ffir sich allein
noch nicht narcotisiren, wohl aber wurden 0,02 Morphium ----- 4/5 x durch
0,002 Lactophenin narcotisch. Eine Dosis von 0,01 Morphium = -~/~
bedurfte zu ihrer Compleftirung 0,1 Antipyrin; 0,005 Morphium = 1/5 x
0,3 und 0,0025 Morphium-----1/1 o x 0,6 Antipyrin. 0,2 Urethan -+- 0,2
Antipyrin wirkten kaum narcotisch; 0,5 Urethan = ~/~ x -t- 0,2 Antipyrin entsprachen der minimal-narcotisirenden Menge.
Zu bemerken ist, dass wir eigentlieh mit diesen Combinationen niemals sehr vollst/indige, schSne Narcosen erhalten haben. Offenbar liegt
in der sonstigen Wirkung der Antipyretica ein die Allgemeinnarcose
stSrendes Moment. Aus diesem Grunde ist es auch besonders schwer,
zu entscheiden, ob die Combinationen yon antipyretischen Medicamenten
mit sogenannten eigentlichen Narcoticis zu Potenzirungen der Wirkungen
fiihren. Noch sehwieriger wird diese Frage dureh den Umstand, dass
sich fiir dig Antipyretica, wenn sic fiir sich allein gegeben werden, eine
minimal-narcotisirende Dosis iiberhaupt night ermitteln 15~sst. Geringe
Dosen machen sehwaehe narcotische Symptome, hShere erzeugen ganz
andere Vergiftungserscheinungen, die immer mehr in den Vordergrund
treten und damit allmiihlich das Narcosebild verwischen. S i c h e r k a n n
man mit Hilfe der C o m b i n a t i o n den N a r c o t i c a c h a r a k t e r der
von uns u n t e r s u c h t e n A n t i p y r e t i c a , der aus a n d e r e n Griinden
schon b e h a u p t e t w o r d e n ist, beweisen. Es ist also keine Frage,
dass sich dis narcotische Kraft dGr Antipyretica zu derjenigen eines
Ueber d. Beeinflussung d.Wirkung narcotischer Medicamente durch Antipyretica.
573
anderen, eines eigentlichen Narcotieums hinzuaddirt, weniger sicher, ob
dabei auch einc Potenzirung der Einzeleffecte zu Stande kommt. Wir
haben immerhin gcsehen, dass schon ziemlich kleine Dosen von Antipyrin
und von Lactophenin kleine, an und fiir sich unwirksame Mengen cigentlicher Narcotica wirksam machen kSnnen. Am auffallcndsten schien uns
diese Verst/irkung bei der Combinaton des Morphiums mit einem Antipyreticum. Da sich abet ein mathematischer Ausdruck fSr diese Verst/i.rkung nicht finden l/isst, weil die minimal-narcotisirende Dosis fiir das
Antipyreticum nicht festgestellt werden kann, lassen sich vorlgufig keine
bestimmte Angaben darfiber, ob einfache Addition oder Potcnzirung vorliegt, machen. Jedenfalls ist die Potenzirung, wenn sic iiberhaupt vorhanden ist, nicht so bedeutend wie in anderen Fgllen. So haben wit
z. B. flit das Scopolamin beim Kaninchen auch keine minimal-narcotisirende Menge angeben kSnnen, da das Medicament, fiir sich allein gegeben, bei diesem Thiere nahezu unwirksam ist; aber die Verstgrkung
bei seiner Vereinigung mit einem anderen Narcoticum war sine so starke,
dass die Potenzirung daraus mit absoluter Sicherheit resultirte. Bei der
Combination Morphium-Urethan war die Potenzirung weniger stark aber
deutlich und, da fiir beide Mittel minimal-narcotisirende Mengen festgestcllt werden kSnnen, auch sicher beweisbar. Nach der Ansehauung
Biirgi's sollten in unseren Versuchsreihen namentlich bei Combination
von Morphium mit antipyretischen Arzneien Potenzirungen zu beobachten
sein, w~hrend bei Vereinigung von Narcoticis der Fettreihe mit den
gleichen Mitteln eventuell in Folge der Identit5t des Zellreceptors eine
abnorme Verst/irkung vielleicht nicht zu Stande kommen kSnnte. Meine
Resultate scheinen diese aus den bisherigen Thatsachen abgeleitete Anffassung im Allgemeinen zu bewahrheiten, doch sind sit immerhin nicht
eindeutig und zahlreich genug und es wird jedenfalls nothwendig sein,
sis noch zu vermehren. Wit werden zu diesem Zweck in erster Linie
auch die Antipyretica, die man per os geben muss - - leider sind das
die meisten --, mit in den Bereich unserer Untersuchungen ziehen m~issen,
vor Allem auch das Lactophenin, das von allen antipyretischen Arzneien
die starkste narcotische Kraft besitzen soll und das wir nur in sehr
kleinen Dosen anwenden konnten, da wit von einer Verabreichung per os
absehen wollten, lnteressant wird es dann schliesslieh sein, zu sehen,
ob die eigentlichen Narcotica andererseits auch die temperaturherabsetzenden Eigenschaften der Antipyretica in unerwartet hohem Grade zu
vermehren im Stande sind oder nicht.
Fiir die Praxis lgsst sich aus meincn Resultaten bis dahin nut der
Schluss ziehen, dass in einer Combination zwischen einem Antipyreticum
und einem eigentlichen Narcoticum die dem ersteren innewohnende narcotische Kraft sich wirklich geltend macht. Ucber die GrSsse der eintretenden Verstgrkung kSnnen wir uns noch nicht mit Bestimmtheit aussprechen.
Versuch No.
1.
2.
3.
4.
Morphium
0,01
0,01
0,015
0,02
Lactophenin
-----
Dauer der Narcose
Keine Narcose.
Heine Narcose.
Kcine Narcose.
Halbe Narcose.
574
SophieLomonosoff,
Versuch No.
5.
6.
7.
Morphium
0,02
0,025
0,02
8.
9.
[0.
11.
0,02
--0,02
Versueh No.
13.
[4.
15.
16.
[7.
[8.
19.
20.
21.
9.9..
23.
24.
9.5.
26.
27.
9.8.
29.
30.
31.
32.
33.
Morphium
0,02
0,02
0,02
0,01
0,01
0,01
0,01
Lactophenin
-
-
-1 Pravaz'sche Spritze Iproc.
LactopheninlSsung
1 Pravaz'sche Spritze derselben LSsung
5 ecru dcrselben LSsung
10 eem derselben LSsung
1/2 Pravaz'sc.he Spritze derselben Liisung
Dauer der Narcose
Halbe Narcose.
Halbe Narcose.
20 Min.
50 Min.
Keine Narcose.
Keine Narcose.
lIalbe Narcose.
0,01
-0,005
0,005
0,005
0,005
0,005
0,0025
0,0025
0,0025
0,0025
0,0025
0,0025
Antipyrin
0,2
0,1
0,05
0,2
0,1
0,05
0,025
1,0
0,0125
2,0
0,2
0,25
0,3
0,4
0,5
0,3
0,4
0,5
1,0
0,6
0,8
Dauer dcr Narcosc
20 Minuten.
Benommenheit, abet keine Narcose.
Keine Narcose.
l0 Minuten.
30 Minuten.
Keine N~rcose.
geine N~rcose.
Keine Nareose.
Benommenheit, aber keine Nareose.
Ted naeh 1/2 Stunde
Keine Nareose.
Keine Nareose.
10 Minuten.
30 Minutcn.
1 Stunde 30 Minuten.
Kcine Narcose.
t(cine Narcose.
geine Narcose.
-45 Minuten.
1 Stunde.
Versuch No.
63.
64.
Antipyrin
0,2
0,2
Morphium
0,02
0,01
Dauer der Narcosc
Keine Nareose.
Keine Narcose.
Versueh No.
34.
35.
36.
37.
38.
Morphium
0,005
0,005
Pyramidon
0,2
0,1
0,2
0,2
0,4
Dauer der Nareose
Keine Nareose, Kriimpfe w~hrend 2 Tage.
Keine Narcose, Betiiubung w~hrend 2 Std.
Keine Narcose, nur Aufregung.
Dasselbe wie in Versueh 34.
Nach 30 Minuten Ted.
Versueh No.
40.
Urethan
0,5
u
No.
41.
42.
43.
44.
45.
47.
48.
50.
51.
56.
60.
61.
69..
Versueh No.
46.
49.
52.
53.
-
-
-
-
0,005
--
Urethan
0,5
0,4
0,3
0,2
0,5
0,1
0,5
0,15
0,5
0,5
0,5
0,5
0,5
Pyramiden
0,2
Daucr der Narcose
35 Minuten (sehwaeh).
[ntervall
10 Min.
l0 Min.
10 Min.
10 Min.
10 Min.
10 Min.
10 Min.
10 Min.
[0 Min.
10 Min.
10 Min.
20 Min.
30 Min.
Antipyrin
0,2
0,2
0,2
0,2
0,15
0,2
0,1
0,9.
0,05
0,09.5
0,9.
0,9.
0,9.
Dauer der Narcose
3 Std. 10 5Iin.
2 Std. Keine reehte Nareose.
35 Min. Keine reehte Nareose.
25 Min. geine rechte Nareose.
2 Std. 10 Min. Keine reehte Nareose.
Keine Narcose.
Keinr Nareose.
Keine Nareose.
Keine Nareose.
Keine Nareose.
3 Stunden 10 Minuten.
2 Stunden 40 Minuten.
3 Stunden.
Antipyrin Intervall
0,9.
10 Min.
0,9.
10 Min.
0,9.
10 Min.
0,9.
20 Min.
Urethan
0,5
0,4
0,3
0,5
Dauer der Nareose
9. Stunden 10 Minuten.
? 2 Stunden.
? 1 Stunde 20 Minuten.
2 Stunden 35 Minuten.
Ueber d. Beeinflussung d. Wirkung narcotischer Medicamente durch Antipyretica. 575
Versuch No. Antipyrin Intervall
54.
0,2
30 Min.
55.
0,2
10 ~,f~n.
57.
0,2
10 Min.
58.
0,2
20 Min.
59.
0,2
30 Min.
Urethan
0,5
0,25
0,5
0,5
0,5
Dauer der Narcose
Kcine Narcose.
Kcinc Narcose.
3 Stunden.
2 Stunden 30 Minuten.
55 Minuten.
Literaturiibersicht.
1. Bfirgi, E., Die Wirkung von Narcoticacombinationen. Deutsche med. Wochenschr.
1910. No. l u. 2.
2. D e r s e l b e , Allgemeine Bemerkungen etc. Diese Zeitschrift.
3. H a u c k o l d , E., Ueber die Beeinfiussung yon Narcoticis durch Scopolamin. Diese
Zeitschrift. Bd. 7.
4. K at z e a eI s o n ~ Ueber die Wirkung gleichzeitig gegebener Narcotica der Fettreihe.
Diese Zeitschrift.
5. L i n d e m a n n , F., Versuehe fiber die Morphium-Urethannarcosc. Diese Zeitschrift.
Bd. 7.
6. O v e r t o n , E., Studien fiber die Narcose. Jena 1901. Fischeffs Verlag.
7. S a r a d s c h i a n , Ueber die Wirkung gleichzeitig gegebener Narcotica der Fettreiho
bei intravenSser Injection. Diese Zeitschrift.
8. v. Z e e l e n , Ueber die Wirkung combinirter Opiumalkaloide. Diese Zeitschrift.
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https://openalex.org/W3183565977
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https://ieeexplore.ieee.org/ielx7/4609443/9314330/09497702.pdf
|
English
| null |
Graph Convolutional Networks-Based Super-Resolution Land Cover Mapping
|
IEEE journal of selected topics in applied earth observations and remote sensing
| 2,021
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cc-by
| 13,493
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Graph Convolutional Networks-Based
Super-Resolution Land Cover Mapping g Ge, Senior Member, IEEE, Feng Ling
, Jin Chen, Yuehong Chen
, and Yuanxin Jia Xining Zhang
, Yong Ge, Senior Member, IEEE, Feng Ling
, Jin Chen, Yuehong Chen Abstract—Super-resolution mapping (SRM) is an effective tech-
nology to solve the problem of mixed pixels because it can be used
to generate fine-resolution land cover maps from coarse-resolution
remote sensing images. Current methods based on deep neural
networks have been successfully applied to SRM, as they can
learn complex spatial patterns from training data. However, they
lack the ability to learn structural information between adjacent
land cover classes, which is vital in the reconstruction of spatial
distribution. In this article, an SRM method based on graph con-
volutional networks (GCNs), named SRMGCN, is proposed to
improve SRM results by capturing structure information on the
graph. In SRMGCN, a supervised inductive learning strategy
with mini-graphs as input is considered, which is an extension of
the GCN framework. Furthermore, two operations are designed
in terms of adjacency matrix construction and an information
propagation rule to help reconstruct detailed information of ge-
ographical objects. Experiments on three datasets with different
spatial resolutions demonstrate the qualitative and quantitative
superiority of SRMGCN over three other popular SRM methods. pixels are common in low-, medium-, and even high-resolution
remote sensing images. Hard classification simply assigns the
most likely land cover class to each mixed pixel; however, this
process may inevitably bring tremendous information loss [5]. Soft classification provides membership grades for each class
which can also be considered as the area proportions of different
land cover classes within coarse-resolution pixels in the form of
fraction images [5]–[7]; however, it is unable to determine the
correct position of land cover classes inside the mixed pixel. Therefore, super-resolution mapping (SRM) technology was
developed to address this problem [6]–[9]. p
p
[ ] [ ]
In the past 20 years, various SRM technologies have been
developed. These technologies can be roughly classified into
two groups, according to the model used to describe the spatial
pattern of land cover classes. One group uses explicit prior
models to analyze the spatial distribution of land cover classes
through clear formulations. The maximal spatial dependence
model based on the first law of geography [10] is used most fun-
damentally and commonly [8]. Manuscript received April 3, 2021; revised June 20, 2021; accepted July 22,
2021. Date of publication July 27, 2021; date of current version August 12,
2021. This work was supported by the National Natural Science Foundation for
Distinguished Young Scholars of China under Grant 41725006. (Corresponding
authors: Yong Ge; Jin Chen.) 7667 7667 IEEE JOURNAL OF SELECTED TOPICS IN APPLIED EARTH OBSERVATIONS AND REMOTE SENSING, VOL. 14, 2021 Graph Convolutional Networks-Based
Super-Resolution Land Cover Mapping The spatial position of each class
in mixed pixels is determined by maximizing the spatial depen-
dence at the subpixel scale, the subpixel/pixel scale, the object
scale [11], or multiple scales [12]. Typical algorithms include
the spatial attraction model (SAM) [13], the vectorial boundary
model (VBSPM) [14], the maximum a posteriori (MAP) based
methods [15], etc. In recent years, some optimization algorithms
have been developed to acquire optimal SRM results. Ma et
al. [16] proposed multiobjective subpixel land-cover mapping
frameworktoresolvetheregularizationparameterdetermination
problem in the MAP-based SPM methods; based on this, Song et
al. [17] introduced multiple shifted images to the multiobjective
model to produce more accurate mapping results. However,
the aforementioned methods need to take the fraction images
generated by soft classification as input, and cannot fully exploit
the spectral information of the original image. Therefore, some
SRM approaches incorporating the original spectral information
and the concept of spatial dependence have been proposed
[18]. Although maximal spatial dependence is a widely used
basic assumption of SRM, the spatial heterogeneity distribution
characteristics of geographical objects are also universal [19];
this makes the maximal spatial dependence model unsuitable for
some situations, especially for intricate and fragmented patterns
in an extreme situation [20]. Index
Terms—Deep
neural
networks
(DNNs),
graph
convolutional
networks
(GCNs),
land
cover,
subpixel,
super-resolution mapping (SRM). This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ Xining Zhang and Yong Ge are with the State Key Laboratory of Resources
and Environmental Information System, Institute of Geographic Sciences and
Natural Resources Research, Chinese Academy of Sciences, Beijing 100101,
China, and also with the University of Chinese Academy of Sciences, Beijing
100049, China (e-mail: zhangxn@lreis.ac.cn; gey@lreis.ac.cn). Manuscript received April 3, 2021; revised June 20, 2021; accepted July 22,
2021. Date of publication July 27, 2021; date of current version August 12,
2021. This work was supported by the National Natural Science Foundation for
Distinguished Young Scholars of China under Grant 41725006. (Corresponding
authors: Yong Ge; Jin Chen.)
Xining Zhang and Yong Ge are with the State Key Laboratory of Resources
and Environmental Information System, Institute of Geographic Sciences and
Natural Resources Research, Chinese Academy of Sciences, Beijing 100101,
China, and also with the University of Chinese Academy of Sciences, Beijing
100049, China (e-mail: zhangxn@lreis.ac.cn; gey@lreis.ac.cn).
Feng Ling is with the Key Laboratory of Monitoring and Estimate for
Environment and Disaster of Hubei Province, Innovation Academy for Precision
Measurement Science and Technology, Chinese Academy of Sciences, Wuhan
430077, China (e-mail: lingf@whigg.ac.cn).
Jin Chen is with the State Key Laboratory of Earth Surface Processes and
Resource Ecology, Beijing Normal University, Beijing 100875, China (e-mail:
chenjin@bnu.edu.cn).
Yuehong Chen is with the College of Hydrology and Water Resources, Hohai
University, Nanjing 210098, China (e-mail: chenyh@lreis.ac.cn).
Yuanxin Jia is with the Academy of Forest Inventory and Planning, Na-
tional Forestry and Grassland Administration, Beijing 100714, China (e-mail:
jiayx@lreis.ac.cn).
Digital Object Identifier 10.1109/JSTARS.2021.3100400 Feng Ling is with the Key Laboratory of Monitoring and Estimate for
Environment and Disaster of Hubei Province, Innovation Academy for Precision
Measurement Science and Technology, Chinese Academy of Sciences, Wuhan
430077, China (e-mail: lingf@whigg.ac.cn). Yuehong Chen is with the College of Hydrology and Water Resources, Hohai
University, Nanjing 210098, China (e-mail: chenyh@lreis.ac.cn). Jin Chen is with the State Key Laboratory of Earth Surface Processes and
Resource Ecology, Beijing Normal University, Beijing 100875, China (e-mail:
chenjin@bnu.edu.cn). A. Basic Theory of SRM The averaging filter in this process
can be regarded as an error free soft classifier that does not
requiretraining.DuringtheSRMprocess,eachcoarsepixelisdi-
vided into small subpixels and each subpixel is regarded as pure
so that only one of the classes is assigned to it. As a result, a finer
resolution representation of the land cover map can be attained. In recent years, owing to good performance in flexibly extract-
ing structural feature representations from complicated graph
data, graph convolutional networks (GCNs) [33] have attracted
more attention. Although GCNs initially focused on applica-
tions in which the data were represented typically in the form
of graphs, including social networks, traffic networks, citation
networks, and so on, the important roles of GCNs in the field
of image processing have been explored and proven lately. The
researchonimageprocessingrevealedthestrongperformanceof
GCNs in image recognition [34], [35], image semantic segmen-
tation [36], and hyperspectral image classification [37]–[40],
even if images have long been treated as regular grids in Eu-
clidean space rather than graph structure data. It was found that
in remote sensing image classification, local spatial structure
information between adjacent land covers can be encoded by
GCNs to flexibly preserve the class boundaries [37], which can
also provide new perspectives for better addressing the SRM
problem. To be specific, let C be the number of land cover classes and
suppose that fraction images with spatial resolution r have been
estimated by a soft classifier. Assuming fraction images cover
n pixels, then the coarse fraction images can be represented
as X = {xi,c | i = 1, 2, . . . , n; c = 1, 2, . . . , C}. Given a scale
factor S, a fine spatial-resolution land cover map with spatial
resolution R (R = r/S) can be obtained through SRM by de-
composing each pixel into S × S subpixels. The fine land cover map can be represented as Y =
{yj,c | j = 1, 2, . . . , N; c = 1, 2, . . . , C}
and N = n × S2,
where yj,c ∈{0, 1} is defined in (1); this means that each
subpixel is allocated a value 1 or 0 for each class. Meanwhile,
each subpixel should be allocated only one land cover class,
meaning that the condition C
c=1 yj,c = 1 should be satisfied
for all j = 1, 2, . . . A. Basic Theory of SRM The aim of SRM is to determine the appropriate spatial distri-
bution of land cover classes within coarse mixed pixels by taking
the fraction images as input [7], [8]. In the real situation, the frac-
tion images are obtained through soft classification. However,
the process of soft classification inevitably contains some errors
and uncertainties. Therefore, the experimental part of this article
uses synthetic fraction images to verify the effect of the proposed
SRM method without soft classification errors. For a given
fine-resolution land cover map, synthetic coarse fraction images
can be simulated by averaging the pixel values of the original
fine-resolution map contained within each coarse pixel with a
certain scale factor [24], [27]. The averaging filter in this process
can be regarded as an error free soft classifier that does not
requiretraining.DuringtheSRMprocess,eachcoarsepixelisdi-
vided into small subpixels and each subpixel is regarded as pure
so that only one of the classes is assigned to it. As a result, a finer
resolution representation of the land cover map can be attained. To be specific, let C be the number of land cover classes and
suppose that fraction images with spatial resolution r have been
estimated by a soft classifier. Assuming fraction images cover
n pixels, then the coarse fraction images can be represented
as X = {xi,c | i = 1, 2, . . . , n; c = 1, 2, . . . , C}. Given a scale
factor S, a fine spatial-resolution land cover map with spatial
resolution R (R = r/S) can be obtained through SRM by de-
composing each pixel into S × S subpixels. The aim of SRM is to determine the appropriate spatial distri-
bution of land cover classes within coarse mixed pixels by taking
the fraction images as input [7], [8]. In the real situation, the frac-
tion images are obtained through soft classification. However,
the process of soft classification inevitably contains some errors
and uncertainties. Therefore, the experimental part of this article
uses synthetic fraction images to verify the effect of the proposed
SRM method without soft classification errors. For a given
fine-resolution land cover map, synthetic coarse fraction images
can be simulated by averaging the pixel values of the original
fine-resolution map contained within each coarse pixel with a
certain scale factor [24], [27]. A. Basic Theory of SRM , N to ensure the uniqueness of subpixel class In this article, an SRM approach based on GCN is proposed,
called SRMGCN. This approach considers an enhanced spatial
neighborhood relation by building graph structures based on
spatial distance and class-fractional similarity; hence, the inher-
ent relationship between adjacent land covers can be captured
by utilizing the learned model. To the best of our knowledge,
this is the first time that GCN has been applied to solving
SRM problems. To exhaustively exploit the input features and
reconstruct detailed information of geographical objects, we
have improved the GCN [33] in two aspects. First, in the
construction of the adjacency matrix, dynamic edge weight ad-
justment is realized by adding a layer-wise trainable parameter. yj,c =
1, subpixel j belongs to class c
0, otherwise
. (1) yj,c =
1, subpixel j belongs to class c
0, otherwise
. (1) (1) IEEE JOURNAL OF SELECTED TOPICS IN APPLIED EARTH OBSERVATIONS AND REMOTE SENSING, VOL. 14, 2021 Both of them are dedicated
to describing the relationship between fractions in the local
window and the spatial distribution of subpixels in the central
coarse pixel, but they have difficulty learning elusive non-linear
hidden representation. Recently, methods based on deep neural
networks (DNNs), such as convolution neural networks (CNNs)
[24]–[26] and generative adversarial networks [27] for SRM
have gradually emerged. Some SRM methods based on DNNs
comprise two steps: fraction image super-resolution and land
cover allocation for subpixels [28], [29], and the successful
experience of DNNs in image super-resolution can be used as
references for the first step of SRM. He et al. [30] summarized
a unified deep learning framework for SRM, into which the
majority of the state-of-the-art networks could be easily em-
bedded. DNNs can extract complicated feature representations
from low to high levels by composing multiple simple but
nonlinear modules [31], which have considerable advantages in
modeling the complex relationships between coarse-resolution
images and fine-resolution land cover categorical maps [24],
[32]. Although compared with other approaches, DNNs-based
methods have shown superiority in SRM, it is still a challenge to
recover the details of SRM results with existing learning modes. Moreover, most current SRM methods based on DNNs choose
to design deeper and more complex network structures with
many parameters, ignoring the potential structural relationship
between adjacent land cover classes; this hinders the represen-
tation capability of networks. Second, in the rule of information propagation, we introduce
an input feature transformation matrix into each layer to fuse
the relatively shallow features. On top of this, we extend the
original GCN proposed for semisupervised node classification
to a generalizable classifier with a supervised inductive learning
strategy in which the mini-graphs are constructed to train the
GCN. In this way, GCN can be used to solve node classification
where unlabeled data are inaccessible during the training stage. The remainder of this article is organized as follows. Sec-
tion II elaborates on the proposed SRMGCN. Section III presents
experimental results of three datasets with different spatial res-
olutions using the proposed method in comparison with three
popular SRM methods. Sections IV and V state discussions and
conclusions, respectively. II. METHODOLOGY IEEE JOURNAL OF SELECTED TOPICS IN APPLIED EARTH OBSERVATIONS AND REMOTE SENSING, VOL. 14, 2021 7668 [21] and back-propagation neural networks [22], [23] have been
proposed to achieve this goal. Both of them are dedicated
to describing the relationship between fractions in the local
window and the spatial distribution of subpixels in the central
coarse pixel, but they have difficulty learning elusive non-linear
hidden representation. Recently, methods based on deep neural
networks (DNNs), such as convolution neural networks (CNNs)
[24]–[26] and generative adversarial networks [27] for SRM
have gradually emerged. Some SRM methods based on DNNs
comprise two steps: fraction image super-resolution and land
cover allocation for subpixels [28], [29], and the successful
experience of DNNs in image super-resolution can be used as
references for the first step of SRM. He et al. [30] summarized
a unified deep learning framework for SRM, into which the
majority of the state-of-the-art networks could be easily em-
bedded. DNNs can extract complicated feature representations
from low to high levels by composing multiple simple but
nonlinear modules [31], which have considerable advantages in
modeling the complex relationships between coarse-resolution
images and fine-resolution land cover categorical maps [24],
[32]. Although compared with other approaches, DNNs-based
methods have shown superiority in SRM, it is still a challenge to
recover the details of SRM results with existing learning modes. Moreover, most current SRM methods based on DNNs choose
to design deeper and more complex network structures with
many parameters, ignoring the potential structural relationship
between adjacent land cover classes; this hinders the represen-
tation capability of networks. Second, in the rule of information propagation, we introduce
an input feature transformation matrix into each layer to fuse
the relatively shallow features. On top of this, we extend the
original GCN proposed for semisupervised node classification
to a generalizable classifier with a supervised inductive learning
strategy in which the mini-graphs are constructed to train the
GCN. In this way, GCN can be used to solve node classification
where unlabeled data are inaccessible during the training stage. The remainder of this article is organized as follows. Sec-
tion II elaborates on the proposed SRMGCN. Section III presents
experimental results of three datasets with different spatial res-
olutions using the proposed method in comparison with three
popular SRM methods. Sections IV and V state discussions and
conclusions, respectively. [21] and back-propagation neural networks [22], [23] have been
proposed to achieve this goal. I. INTRODUCTION R
EMOTEsensingimagesareconsideredthemostimportant
input to produce land cover maps. However, mixed pixels,
which refer to pixels that contain more than one land cover class,
constituteasignificantobstacletoaccurateclassification[1]–[3]. Due to the complex distribution patterns of geographical objects
andthelimitationofthesensor’sspatialresolution[1],[4],mixed R Yuehong Chen is with the College of Hydrology and Water Resources, Hohai
University, Nanjing 210098, China (e-mail: chenyh@lreis.ac.cn). The other group uses learning-based methods to address SRM
issues without explicit prior models, but with implicit relation-
ships between coarse-resolution images and fine-resolution land
cover maps learned from sample pairs. Support vector regression Yuanxin Jia is with the Academy of Forest Inventory and Planning, Na-
tional Forestry and Grassland Administration, Beijing 100714, China (e-mail:
jiayx@lreis.ac.cn). Yuanxin Jia is with the Academy of Forest Inventory and Planning, Na-
tional Forestry and Grassland Administration, Beijing 100714, China (e-mail:
jiayx@lreis.ac.cn). Yuanxin Jia is with the Academy of Forest Inventory and Planning, Na-
tional Forestry and Grassland Administration, Beijing 100714, China (e-mail:
jiayx@lreis.ac.cn). Digital Object Identifier 10.1109/JSTARS.2021.3100400 C. The Proposed SRM Method C. The Proposed SRM Method The framework is presented in Fig. 1. When input fraction
images are given, they are preprocessed by interpolating and
splitting to generate mini-graphs. In the training stage, the pro-
posed GCN is designed to train labeled data regarding subpixels
as nodes by minimizing a certain loss function. In the testing
stage, unlabeled data are fed into the trained model to produce fi-
nal fine-resolution land cover maps through two class-allocation
algorithms. The three critical aspects are detailed below. To acquire the hidden representation of nodes, spectral con-
volutions on a graph are defined by multiplying a graph sig-
nal with C input channels x ∈RN×C with a spectral filter
gθ(Λ) = diag(θ) in the Fourier domain, as (2) expresses gθ ⋆x = Ugθ (Λ) U T x
(2) (2) gθ ⋆x = Ugθ (Λ) U T x 1) Mini-graphs Generation: In studies to date, the applica-
tions of GCN in remote sensing image classification are mostly
based on feeding all samples in one big graph into the network
[37]–[39]; two main problems arise from this. First, the massive
nodes and edges of all samples will slow calculation and run
out of memory. Second, it is impossible to build a generalizable
classifier to predict unknown input through inductive learning. where U is the matrix of eigenvectors of the symmetric
normalized Laplacian matrix L = IN −D−1/2 AD−1/2 =
UΛU T . Here, IN is an identity matrix. D is a diagonal degree
matrixofthegraphwithelementDii =
j Aij .Λisadiagonal
matrix whose elements are the eigenvalues of L. In (2), U T x
represents the graph Fourier transform of x. gθ(Λ) is considered
a function of the eigenvalues of L. p
p
g
g
To solve these problems, a training strategy based on mini-
graphs is adopted in this article. As shown in Fig. 2, on the
one hand, input coarse fraction images are first interpolated to
nominal fineresolutionimages withascalefactor of S. Here, this
operation is realized by the nearest neighbor interpolation. Then
the nominal fine resolution images are used to extract a series
of patches by a certain size of sliding window with a suitable
sampling stride. For each patch, a mini-graph is constructed
with each subpixel in the patch taken as a node; the graph node
features are initialized with class fractions of subpixels after
interpolation, which is locally expressed in Fig. 2 (a). C. The Proposed SRM Method Each mini-
graph is a spatial distance-based structure graph in which each
node as center node connects to its first-order neighborhoods and
Fig. 2 (b) shows additional details. On the other hand, the ref-
erence map is extracted from the original labeled classification
map using the same size of sliding window and sampling stride;
it then forms a sample pair with the corresponding mini-graph. In
this way, the SRM is transformed into a graph node classification
problem at subpixel scale. Subsequently, these sample pairs
based on mini-graphs are used for training and predicting in
batches. To reduce the computational costs and realize a local con-
nection [33], [41], [42], Hammond et al. [43] established an
approximating formula about the spectral filter gθ(Λ) with
Chebyshev polynomials Tk(·) up to the order Kth to circumvent
computing the eigenvectors of the Laplacian matrix L. Then the
convolution of a graph signal x with a spectral filter gθ′ can be
defined as gθ′ ⋆x ≈
K
k=0
θk
′Tk
˜L
x
(3) (3) where ˜L = 2/(λmax)L −IN with λmax being the largest eigen-
value of L. θk′ ∈RK is a vector of Chebyshev coefficients. This
formulation is K-localized since it only depends on the nodes
in the Kth-order neighborhoods of the central node. Considering that a linear function of spectral convolutions on
a graph can improve modeling capacity on a number of domains,
Kipf and Welling [33] limited K = 1 to simplify this model and
further approximated λmax of L by 2. By doing so, (3) can be
rewritten as gθ′∗x ≈θ
IN + D−1
2 AD−1
2
x
(4) (4) 2) Proposed SRM Network: The proposed SRM model
(SRMGCN) adopts the GCN as the backbone. The proposed
model improves the construction of the adjacency matrix and
the rule of information propagation in each graph convolutional
layer to obtain node attribute information in a more detailed and
comprehensive manner. by allowing the free parameters satisfy θ = θ0′ = −θ1′ to
address overfitting and minimize the number of operations
per layer. Kipf and Welling further introduced a renormaliza-
tion trick and applied it to the convolution matrix to enhance
stability during network training: IN + D−1/2AD−1/2 →
˜
D
−1/2 ˜
A ˜
D
−1/2, with ˜
A = A + IN and ˜
Dij =
j ˜
Aij . ZHANG et al.: GRAPH CONVOLUTIONAL NETWORKS-BASED SUPER-RESOLUTION LAND COVER MAPPING 7669 parameters and the bias vector, respectively, included by the lth
layer. a layer-wise propagation rule. The aggregation of feature in-
formation is conducted in each graph convolutional layer to
extract high-level features from neighbors to their center nodes. Formally, let G = (V, E) be an undirected graph with N nodes
vi ∈V, edges (vi, vj) ∈E. An adjacency matrix A (weighted
or unweighted) of G indicates whether each pair of nodes is
connected and contains the structure information of the graph. B. Definition of GCN Based on spectral theory, GCN [33] is a multilayer neural
network architecture that directly operates on arbitrary graph
structure data and aims to generate node representations via IEEE JOURNAL OF SELECTED TOPICS IN APPLIED EARTH OBSERVATIONS AND REMOTE SENSING, VOL. 14, 2021 7670 Fig. 1. Framework of our method. Fig. 2. Generating progress of the mini-graph and the sample pair. In the middle is an enlarged schematic view of the lower right corner of the mini-gra
constructed based on the patch. (a) Graph node features derived from class fractions of subpixels. (b) First-order neighborhood diagram in the mini-graph a
every node denotes a subpixel node. The orange node is an inner node on the graph with eight yellow first-order neighborhoods. The blue node is a corner node
the graph with three yellow first-order neighborhoods. The green node is a border node on the graph with five yellow first-order neighborhoods. The arrows po
to the direction of information aggregation, that is, to their center nodes. Fig. 1. Framework of our method. Fig. 2. Generating progress of the mini-graph and the sample pair. In the middle is an enlarged schematic view of the lower right corner of the mini-graph
constructed based on the patch. (a) Graph node features derived from class fractions of subpixels. (b) First-order neighborhood diagram in the mini-graph and
every node denotes a subpixel node. The orange node is an inner node on the graph with eight yellow first-order neighborhoods. The blue node is a corner node on
the graph with three yellow first-order neighborhoods. The green node is a border node on the graph with five yellow first-order neighborhoods. The arrows point
to the direction of information aggregation, that is, to their center nodes. Fig. 2. Generating progress of the mini-graph and the sample pair. In the middle is an enlarged schematic view of the lower right corner of the mini-graph
constructed based on the patch. (a) Graph node features derived from class fractions of subpixels. (b) First-order neighborhood diagram in the mini-graph and
every node denotes a subpixel node. The orange node is an inner node on the graph with eight yellow first-order neighborhoods. The blue node is a corner node on
the graph with three yellow first-order neighborhoods. The green node is a border node on the graph with five yellow first-order neighborhoods. The arrows point
to the direction of information aggregation, that is, to their center nodes. on the weighted A, an additional trainable edge weighted param-
eter is added in each layer so that the correlation between land
cover information represented by adjacent subpixels defined
by elements of A can be dynamically adjusted via layer-wise
training. C. The Proposed SRM Method The
˜
A means an adjacency matrix with added self-loops. Therefore,
a layer-wise propagation rule in GCN is as follows: In the first place, the GCN is dependent on the adjacency
matrix A to propagate information between nodes; hence, how
to build A is critical for the GCN [44]. Here, we select the
similarity distance-based weighted A. Each element of it, also
known as the weight of the edge between each pair of nodes, can
be computed by using the heat kernel function [40]. However,
in a graph-based SRM problem with subpixels as nodes, the
similarity between nodes will change after aggregation of layer-
wise information. There is a high computational cost associated
with assigning each edge a dynamic adjustment parameter or
recalculating the similarity distances of each layer. Thus, based H(l) = σ
˜
D
−1
2 ˜
A ˜
D
−1
2 H(l−1)W (l) + b(l)
(5) (5) where H(l)(l ≥1) denotes the output in the lth graph convo-
lutional layer; σ(·) represents a nonlinear activation function,
such as the rectified linear unit (ReLU) and Softmax used in this
article; W (l) and b(l) denote the trainable weight matrix of filter IEEE JOURNAL OF SELECTED TOPICS IN APPLIED EARTH OBSERVATIONS AND REMOTE SENSING, VOL. 14, 2021 IEEE JOURNAL OF SELECTED TOPICS IN APPLIED EARTH OBSERVATIONS AND REMOTE SENSING, VOL. 14, 2021 Each element of A(l) in the lth layer be measured as transformation matrix W (l)
2
of transformation parameters to
the above propagation rule to further improve the process of
feature representation. Now, we have the following layer-wise
propagation rule in this article: H(l) A(l)
ij =
θ(l)e−γ∥xi−xj∥2, if vi ∈N (vj) or vi ∈N (vj)
0, otherwise
(6 = σ
˜
D
(l)−1
2 ˜
A
(l) ˜
D
(l)−1
2 H(l−1)W (l)
1 + H(l−1)W (l)
2 + b(l)
(7) (6)
(7) where xi and xj denote the feature vectors associated with the
nodes vi and vj. N(vi) (or N(vj)) is the set of neighbors of vi
(or vj). θ(l) is the trainable parameter in the lth layer with an
initial value of 1, and each element of A is multiplied by this
parameter to get the A(l). As for the parameter γ, we empirically
set 0.2 referring to [37] and [39]. where W (l)
1
is equivalent to W (l) in (5) and W (l)
2
has the
same dimensions with W (l)
1 . ˜
A
(l) and ˜
D
(l) vary with A(l)
in each layer. ˜
D
(l)−1
2 ˜
A
(l) ˜
D
(l)−1
2 H(l−1)W (l)
1
represents the
features after information aggregation; H(l−1)W (l)
2 is the trans-
formation of input features which represents relatively shallow
features. In the second place, the propagation rule expressed in (5)
can only propagate the aggregated features, but cannot directly
fuse the input features (in other words, the relatively shallow
information) of each layer. Actually, the input features are ex-
pected to help supplement the boundary and contour information
of geographical objects, so we add a trainable input feature In the SRMGCN network, a shallow GCN with four convo-
lutional layers is used to train and predict the input datasets,
where the first three graph convolutional layers are followed
by the ReLU function and the last graph convolutional layer ZHANG et al.: GRAPH CONVOLUTIONAL NETWORKS-BASED SUPER-RESOLUTION LAND COVER MAPPING 7671 Fig. 3. Diagram of propagation of feature information. The superscripts l (l = 1, 2, . . .) of H denote the output of the lth layer. The superscripts l (l = 1, 2, . . .)
of W1 and W2 denote trained variables of the lth layer. The superscripts l (l = 1, 2, . . III. EXPERIMENT AND ANALYSIS where yG is the node indices in the training set and Y denotes
the label matrix. Toeliminatetheuncertaintyof soft classification, experiments
were performed on three different resolution datasets with syn-
thetic coarse fraction images to evaluate the proposed SRMGCN
method. The synthetic images were created by degrading the
actual fine land cover map to coarse fraction images by applying
an averaging filter with a scale factor of 3. The three original land
cover maps all represented the real distribution of geographical
objects on the earth’s surface and were, respectively, used as
reference images to assess the effectiveness of mapping results. A traditional hard classifier and three existing SRM methods
with relatively remarkable performances, VBSPM [14], SAM
[13], and SRMCNN [25], were compared with the proposed
SRMGCN method. 3) Class-Allocation Algorithm: The allocation of classes for
subpixels after the soft attribute estimation values are obtained at
the subpixel scale is also a significant issue that directly affects
the result of SRM [45]. In this article, we adopt two algorithms,
direct hardening (DH) [46], [47] and linear optimization tech-
niques (LOTs) [48], to complete class allocation. Like the traditional hard classification, DH assigns each sub-
pixel to the class with the maximum soft attribute value [47]. Its formula is shown in (9), where pj,c and pj,c′ denote the soft
attribute value of class c and c′ respectively. This approach is
simple and efficient, but it easily creates overly smooth mapping
results because it does not take constraints from class fractions
into account IEEE JOURNAL OF SELECTED TOPICS IN APPLIED EARTH OBSERVATIONS AND REMOTE SENSING, VOL. 14, 2021 .) of A are calculated from the trained variables θ(l) in the lth layer. Fig. 3. Diagram of propagation of feature information. The superscripts l (l = 1, 2, . . .) of H denote the output of the lth layer. The superscripts l (l = 1, 2, . . .)
of W1 and W2 denote trained variables of the lth layer. The superscripts l (l = 1, 2, . . .) of A are calculated from the trained variables θ(l) in the lth layer. branch-and-boundalgorithm[49]isemployedforthispurposein
this article. Although LOT is a little time-consuming compared
with DH due to many iterations, the experiments in related
studies have shown that it can usually produce pleasing mapping
results [45], [50]. Note that this method is only applied to mixed
pixels, whereas subpixels of pure pixels are still allocated land
cover classes by the DH method to save time is followed by the Softmax function. In general, the proposed
network takes an initial node feature matrix X and an adjacency
matrix A as inputs and produces node features Z ∈RN×C by
stacking several graph convolutional layers of the form of (7),
where C is the output dimension (i.e., the number of land cover
classes in our cases). Finally, a C-dimensional feature vector
for every node is produced, which represents a soft attribute
estimation value of each subpixel, that is, the probability of each
subpixel belonging to each class. For clarity, Fig. 3 demonstrates
the propagation of feature information when stacking the first
two graph convolutional layers. Maximize F =
S×S
j=1
C
c=1
yj,c × pj,c
(10)
subject to
⎧
⎪
⎪
⎨
⎪
⎪
⎩
C
c=1
yj,c = 1
S×S
j=1
yj,c = round
xi,c|j × S2. (11) Maximize F =
S×S
j=1
C
c=1
yj,c × pj,c
(10) (10) For the SRM task, we adopt the cross-entropy loss function
to evaluate the difference between the network output and the
labels in the training set, which is (11) loss = −
g∈yG
C
c = 1
Y gc ln Zgc
(8) (8) C. Results From the perspective of visual evaluation and quantitative ac-
curacy assessment, the effectiveness of the proposed SRMGCN
and aforementioned baseline methods on the test set of the three
datasets are evaluated. The per-class accuracy, overall accuracy
(OA), average accuracy (AA), and kappa coefficient (Kappa) are
employed as the metrics for the latter. g
The third dataset is a set of 20 chips very-high-resolution
images named Zurich Summer (https://sites.google.com/site/
michelevolpiresearch/data/zurich-dataset), which were cap-
tured by the QuickBird satellite in August 2002 over the city
of Zurich, Switzerland [51]. The spatial resolution of the corre-
sponding ground truth masks considered as reference images is
0.61 m as pansharpened panchromatic images, and the average
size of the masks is 1182 × 1051 pixels. We used four chips
(No. 1, 5, 6 and 13) to generate mini-graphs for testing and the
rest to generate mini-graphs for training. Nine different urban
and periurban classes were manually annotated in ground truth
masks: roads; buildings; trees; grass; bare soil; water; railways;
swimming pools; and others (Background). p y
1) Results on the GlobeLand30 Dataset: A visual compar-
ison of SRM results generated by different methods on
the GlobeLand30 dataset is shown in Fig. 4(c)–(g). The
reference map and hard classification map are also pro-
vided in Fig. 4(a) and (b). The classification maps of
VBSPM and SAM result in salt-and-pepper noise on the
edge of the objects, especially linear objects. Contrarily,
SRMCNN, SRMGCN(DH), and SRMGCN(LOT) are able
to yield smoother visual effects. Additionally, it is also
notable that the linear water body is discontinuous in
the SRMCNN result. In contrast, since SRMGCN(DH)
and SRMGCN(LOT) take effort to construct and learn
the spatial relationship between nodes, the continuity and
integrity of these elongated features are well preserved. Note that, in the SRMGCN(DH) result, some subpixels of
“Shrubland” dotted on the “grass land” are misclassified
into “grass land” because the DH class-allocation algo-
rithm brings overly smooth results to some extent. 1) Results on the GlobeLand30 Dataset: A visual compar-
ison of SRM results generated by different methods on
the GlobeLand30 dataset is shown in Fig. 4(c)–(g). The
reference map and hard classification map are also pro-
vided in Fig. 4(a) and (b). The classification maps of
VBSPM and SAM result in salt-and-pepper noise on the
edge of the objects, especially linear objects. Contrarily,
SRMCNN, SRMGCN(DH), and SRMGCN(LOT) are able
to yield smoother visual effects. C. Results Additionally, it is also
notable that the linear water body is discontinuous in
the SRMCNN result. In contrast, since SRMGCN(DH)
and SRMGCN(LOT) take effort to construct and learn
the spatial relationship between nodes, the continuity and
integrity of these elongated features are well preserved. Note that, in the SRMGCN(DH) result, some subpixels of
“Shrubland” dotted on the “grass land” are misclassified
into “grass land” because the DH class-allocation algo-
rithm brings overly smooth results to some extent. Some key information about these three datasets is given in
Table I. Note that the proportion of pure pixels in the last column
refers to the ratio of the number of pixels containing only one
land cover class to the total number of coarse resolution pixels
in fraction images. TABLE I
KEY INFORMATION ABOUT DATASETS TABLE II
SIZE OF PATCHES AND NUMBER OF MINI-GRAPHS TABLE I
KEY INFORMATION ABOUT DATASETS Cultivated Land, Forest, Grass Land, Shrubland, Wetland, Water
Body, Artificial Surfaces, and Bareland. Cultivated Land, Forest, Grass Land, Shrubland, Wetland, Water
Body, Artificial Surfaces, and Bareland. The second dataset is the example dataset of EOPatches for
Slovenia 2019 (http://eo-learn.sentinel-hub.com). This dataset
contains 25 groups of Sentinel-2 time series images and the cor-
responding 10-m resolution land use and land cover maps with
500 × 500 pixels. In our experiments, 20 land cover maps were
used to generate training set and 5 were used to generate testing
set. The land cover classes of this dataset are summarized into
eight classes: Cultivated Land, Forest, Grassland, Shrubland,
Water, Wetland, Artificial Surface, and Background. A. Datasets yj,c =
1, if pj,c = max (pj,c′) and 1 ≤c, c′ ≤C
0, otherwise. (9) (9) The first dataset was extracted from GlobeLand30 V2020,
the 30-m resolution global land cover data product containing
966 tiles developed by China. This data could be obtained
from the Global Land Cover Data Product Service website of
the National Geomatics Center of China (www.globeland30. org; DOI: 10.11769). We selected one of the tiles marked
N49_30_2020LC030 for experiments. In this tile comprising
19796 × 18988 pixels, we randomly extracted 80 chips with 540
× 540 pixels, 65 of which were used to generate mini-graphs
for training, and the rest were used to generate mini-graphs for
testing. The land cover map of the study area, which is regarded
asthereferenceimage,includeseightprimarylandcoverclasses: LOT introduces an objective function for maximizing the sum
of soft attribute values of all subpixels in each coarse resolution
pixel. This objective function expressed in (10) needs to be sub-
jected to constraints from class uniqueness and class fractions
given in (11) [45], [48], where xi,c|j denotes the class fraction
value of class C in pixel i corresponding to the given subpixel LOT introduces an objective function for maximizing the sum
of soft attribute values of all subpixels in each coarse resolution
pixel. This objective function expressed in (10) needs to be sub-
jected to constraints from class uniqueness and class fractions
given in (11) [45], [48], where xi,c|j denotes the class fraction
value of class C in pixel i corresponding to the given subpixel
j. Both the function to maximize and the constraints are linear
equations,sothetheoreticallyoptimalsolution(i.e.,optimalland
cover classes of subpixels) of the above mathematical model
can be find by LOT. Several algorithms can be applied, and the j. Both the function to maximize and the constraints are linear
equations,sothetheoreticallyoptimalsolution(i.e.,optimalland
cover classes of subpixels) of the above mathematical model
can be find by LOT. Several algorithms can be applied, and the IEEE JOURNAL OF SELECTED TOPICS IN APPLIED EARTH OBSERVATIONS AND REMOTE SENSING, VOL. 14, 2021 7672 TABLE II
SIZE OF PATCHES AND NUMBER OF MINI-GRAPHS TABLE I
KEY INFORMATION ABOUT DATASETS B. Experimental Settings TableIIIquantitativelyreportstheassessmentresultsobtained
by different methods on the GlobeLand30 dataset. Since the
input fraction images produced by degrading reference images
are error free, the values of the assessment indexes of the
five SRM results are all relatively high. Consistent with the
above visual evaluation, the OA, AA, and Kappa values of
the DNNs-based SRM methods, SRMCNN, SRMGCN(DH), and
SRMGCN(LOT), are higher than those of VBSPM and SAM. Our GCN-based methods perform better than the SRMCNN
because elongated features are fragmentary using SRMCNN. In
general, SRMGCN(LOT) with LOT class-allocation algorithm
achieves the optimal performances in AA and most specific
class accuracies, especially “Shrubland,” whose geo-objects are
spotted, and SRMGCN(DH) provides the highest values in OA
and Kappa. According to Section II-C, a series of mini-graphs of each
dataset were generated and split into a training set, a validation
set, and a test set to test our model. The training sets of the three
datasets involve samples of all classes. The size of the patch used
to construct the mini-graph and the number of mini-graphs of
the three datasets are given in Table II. In our experiments, the numbers of input and output channels
of other layers are set to 64; the exception is that the number
of input channels of the first layer and output channels of the
last layer are equal to the number of land cover classes. The
proposed network was completed via the Python-3.6 platform
[52] and PyTorch-1.4.0 framework [53]. Training was run on an
NVIDIA Quadro RTX 5000 GPU with 16 GB GPU memory. We selected the Adam algorithm [54] to optimize the network
with a learning rate equal to 0.005. In the process of network
training, the number of epochs and batch size were set to 200
and 8, respectively. 2) Results on the Slovenia Dataset: The reference map,
hard classification result, and five SRM results on the
Slovenia dataset are displayed in Fig. 5. It is obvious 2) Results on the Slovenia Dataset: The reference map,
hard classification result, and five SRM results on the
Slovenia dataset are displayed in Fig. 5. It is obvious ZHANG et al.: GRAPH CONVOLUTIONAL NETWORKS-BASED SUPER-RESOLUTION LAND COVER MAPPING 7673 Fig. 4. Example of classification maps with 496 × 496 pixels from the test set of the GlobeLand30 dataset. (a) Reference map. (b) Hard classification map. (c)–(g) SRM results of VBSPM, SAM, SRMCNN, SRMGCN(DH), and SRMGCN(LOT), respectively. B. Experimental Settings The second and third rows are zoomed-in area 1 and
zoomed-in area 2 of the first row, respectively. Fig. 4. Example of classification maps with 496 × 496 pixels from the test set of the GlobeLand30 dataset. (a) Reference map. (b) Hard classification map. (c)–(g) SRM results of VBSPM, SAM, SRMCNN, SRMGCN(DH), and SRMGCN(LOT), respectively. The second and third rows are zoomed-in area 1 and
zoomed-in area 2 of the first row, respectively. TABLE III
PER-CLASS ACCURACY (%), OA (%), AA (%), AND KAPPA COEFFICIENT ON THE GLOBELAND30 DATASET TABLE III PER CLASS ACCURACY (%), OA (%), AA (%), AND KAPPA COEFFICIENT ON THE GLOBELAND30 DATASET
Note: The best one is shown in bold. SRMGCN(LOT) are better reconstructed because of the
increased constraints from class fractions by utilizing the
LOT algorithm. that the traditional hard classification can only provide an
extremely rough description of the distribution pattern of
geographicalobjects,whereastheotherfiveSRMmethods
can portray this spatial distribution in more detail. In
addition, it can be seen clearly from the comparison of two
critical zoomed-in areas that the slender linear artificial
surface objects are disconnected and even almost vanish
in the fine classification maps produced by VBSPM and
SRMCNN.AlthoughSAMretainsmoredetails,itisunable
to determine the correct spatial position of each class but
creates discrete messy small patches. Due to building the
spatial contextual structure via graph, SRMGCN(DH) and
SRMGCN(LOT) generate results that are more similar to
the reference maps than other methods with the correct and
complete linear information retained in the regions that are
difficult to recover. Also, as expected, details produced by that the traditional hard classification can only provide an
extremely rough description of the distribution pattern of
geographicalobjects,whereastheotherfiveSRMmethods
can portray this spatial distribution in more detail. In
addition, it can be seen clearly from the comparison of two
critical zoomed-in areas that the slender linear artificial
surface objects are disconnected and even almost vanish
in the fine classification maps produced by VBSPM and
SRMCNN.AlthoughSAMretainsmoredetails,itisunable
to determine the correct spatial position of each class but
creates discrete messy small patches. Due to building the
spatial contextual structure via graph, SRMGCN(DH) and
SRMGCN(LOT) generate results that are more similar to
the reference maps than other methods with the correct and
complete linear information retained in the regions that are
difficult to recover. Also, as expected, details produced by The accuracy assessments of the five methods on the Slovenia
dataset bear out the aforementioned findings. B. Experimental Settings Similar to the
GlobeLand30 dataset, the results in Table IV indicate that the
OA, AA, and Kappa of VBSPM and SAM are at least 2%
lower than those gained by the SRM methods based on deep
learning, i.e., SRMCNN, SRMGCN(DH), and SRMGCN(LOT). Moreover, the highest values of OA, AA, and Kappa are captured
by our proposed two GCN-based methods, i.e., SRMGCN(DH)
and SRMGCN(LOT), which validates the strength of the graph
structural features in SRM technology. As for the way of class
allocation, the LOT results are not that different from the DH
results, whereas the visual effect of the former is superior to that
of the latter, as mentioned above. IEEE JOURNAL OF SELECTED TOPICS IN APPLIED EARTH OBSERVATIONS AND REMOTE SENSING, VOL. 14, 2021 7674 Fig. 5. Example of classification maps with 390 × 390 pixels from the test set of the Slovenia dataset. (a) Reference map. (b) Hard classification map. (c)–(g)
SRM results of VBSPM, SAM, SRMCNN, SRMGCN(DH), and SRMGCN(LOT), respectively. The second and third rows are zoomed-in area 1 and zoomed-in
area 2 of the first row, respectively. Fig. 5. Example of classification maps with 390 × 390 pixels from the test set of the Slovenia dataset. (a) Reference map. (b) Hard classification map. (c)–(g)
SRM results of VBSPM, SAM, SRMCNN, SRMGCN(DH), and SRMGCN(LOT), respectively. The second and third rows are zoomed-in area 1 and zoomed-in
area 2 of the first row, respectively. TABLE IV
PER-CLASS ACCURACY (%), OA (%), AA (%), AND KAPPA COEFFICIENT ON THE SLOVENIA DATASET TABLE IV
PER-CLASS ACCURACY (%), OA (%), AA (%), AND KAPPA COEFFICIENT ON THE SLOVENIA DATASET
Note: The best one is shown in bold. TABLE IV 3) Results on the Zurich Summer Dataset: Fig. 6 illustrates
the SRM results of five different methods on the Zurich
summer dataset, where some enlarged areas of the first
row are further provided in the last two rows for better
visual inspection. Although the results of the five meth-
ods show no significant differences in this dataset from
the overall perspective, the local views still indicate that
SRMCNN, SRMGCN(DH), and SRMGCN(LOT) are excel-
lent at suppressing speckle noise and blurred boundaries
compared with VBSPM and SAM. For instance, “build-
ings,” “roads,” and “trees” all have clearer boundaries in
the results generated by these three SRM methods based
on DNNs, particularly when there are small protrusions in
the boundaries. B. Experimental Settings On
this foundation, approaches 2 and 3, respectively, only use the
proposed adjacency matrix construction operation and informa-
tion aggregation operation. The last graph convolution approach
is our proposed SRMGCN(DH), which contains both of the above
operations. except that the per-class accuracies of “roads,” “buildings,” and
“water” are modestly lower than the results of SRMGCN(DH). B. Experimental Settings The quantitative evaluation results of the OA, AA, and Kappa
on the Zurich Summer dataset are given in Table V. It is apparent
that the performance of all methods is better than that of the Glo-
beLand30 and Slovenia datasets. This can be attributed mainly
to the higher proportion of pure pixels (see the last column
of Table I) and simpler spatial patterns of the Zurich Summer
dataset. Consistent with visual perception, the statistical results
of the evaluation indexes of all methods are similar; however,
slightsuperioritiescanstillbeobservedwhenSRMGCN(DH) and
SRMGCN(LOT) are compared with SRMCNN and other com-
petitors in terms of all experimental assessment metrics, which
highlights the potential of the proposed GCN-based method
in SRM analysis. It is also worth noting that SRMGCN(LOT)
secures maximum values with respect to almost all statistics ZHANG et al.: GRAPH CONVOLUTIONAL NETWORKS-BASED SUPER-RESOLUTION LAND COVER MAPPING 7675 Fig. 6. Example of classification maps with 1362 × 1293 pixels from the test set of the Zurich Summer dataset. (a) Reference map. (b) Hard classification map. (c)–(g) SRM results of VBSPM, SAM, SRMCNN, SRMGCN(DH), and SRMGCN(LOT), respectively. The second and third rows are zoomed-in area 1 and
zoomed-in area 2 of the first row, respectively. Fig. 6. Example of classification maps with 1362 × 1293 pixels from the test set of the Zurich Summer dataset. (a) Reference map. (b) Hard classification map. (c)–(g) SRM results of VBSPM, SAM, SRMCNN, SRMGCN(DH), and SRMGCN(LOT), respectively. The second and third rows are zoomed-in area 1 and
zoomed-in area 2 of the first row, respectively. TABLE V
PER-CLASS ACCURACY (%), OA (%), AA (%), AND KAPPA COEFFICIENT ON THE ZURICH SUMMER DATASET TABLE V
PER-CLASS ACCURACY (%), OA (%), AA (%), AND KAPPA COEFFICIENT ON THE ZURICH SUMMER DATASET Note: The best one is shown in bold. experiments except for graph convolution operations of convo-
lutional layers. At a scale factor of 3, the visual comparison of the
SRM results using four different graph convolution approaches
on another test area of the Slovenia dataset are exhibited in
Fig. 7(c)–(f), and their varied accuracy assessment results are
given in Table VI. Approach 1 uses the original convolutional
layers with the similarity distance-based weighted adjacency
matrix, but without the trainable edge weighted parameter based
on it and the trainable input feature transformation matrix. A. Usefulness of the Proposed Graph Convolutional Layer As mentioned in the introduction and Section II-C, we are
committed to improving the original graph convolution opera-
tion in the construction of the adjacency matrix and the infor-
mation propagation rule. Here, we conducted an ablation study
on the Slovenia dataset to illustrate the usefulness of these two
operations. The DH algorithm was employed for all approaches
to determine the optimal land-cover labels of subpixels, and the
network structures were kept identical to the abovementioned IEEE JOURNAL OF SELECTED TOPICS IN APPLIED EARTH OBSERVATIONS AND REMOTE SENSING, VOL. 14, 2021 7676 Fig. 7. Example of classification maps with 440 × 440 pixels of the Slovenia dataset achieved by different graph convolution approaches. (a) Reference map. (b)
Hard classification map. (c)–(e) SRM results using the graph convolution of approaches 1 to 3, respectively. (f) SRM result of SRMGCN(DH). The second and
third rows are zoomed-in area 1 and zoomed-in area 2 of the first row, respectively. Fig. 7. Example of classification maps with 440 × 440 pixels of the Slovenia dataset achieved by different graph convolution approaches. (a) Reference map. (b)
Hard classification map. (c)–(e) SRM results using the graph convolution of approaches 1 to 3, respectively. (f) SRM result of SRMGCN(DH). The second and
third rows are zoomed-in area 1 and zoomed-in area 2 of the first row, respectively. B. Comparison of SRM Results on Three Datasets Our experiments have obtained the SRM results using differ-
ent methods on three datasets with different spatial resolutions. Since the overall assessment results of OA, AA, and Kappa
in Tables III–V are affected by the proportion of pure pixels
in the fraction images with different spatial resolutions, we
summarized the statistical results of removing pure pixels on the
three datasets, as given in Table VII. In Table VII, adjusted OA,
adjusted AA, and adjusted kappa denote the OA, AA, and Kappa
coefficients after excluding pure pixels, respectively. From the
resultant table, we find that the SRM results of all methods on the
three datasets have a certain degree of reduction after removing
the pure pixels; the Zurich Summer dataset has the smallest
reduction. Meanwhile, the gap in the quantitative evaluation re-
sults of various methods is further increased, especially between
DNNs-based methods and the traditional methods; the proposed
two methods can result in more accurate spatial location results
of subpixels within mixed pixels. Fig. 8. NP and AI of three datasets. Note that we use the reciprocal of NP, i.e.,
1/NP, to directly reflect the positive correlation with accuracy. which is designed to measure aggregation of spatial patterns. The AI of the highest level of aggregation is equal to 1, and the
AI of the lowest level of aggregation is equal to 0. Fig. 8 indicates that compared with other datasets, the spatial
pattern of the Slovenia dataset is the most fragmented, which
makes the SRM process more difficult because more compli-
cated land covers need to be located; hence, it has the lowest
accuracy. In contrast, the spatial structure of the Zurich summer
dataset is highly aggregated, which can bring more accurate
results with a simpler SRM process. ZHANG et al.: GRAPH CONVOLUTIONAL NETWORKS-BASED SUPER-RESOLUTION LAND COVER MAPPING 7677 TABLE VII
OA (%), AA (%), AND KAPPA COEFFICIENT ON THREE DATASETS AFTER EXCLUDING PURE PIXELS
Note: The best one is shown in bold. E VII
HREE DATASETS AFTER EXCLUDING PURE PIXELS
Fig. 8. NP and AI of three datasets. Note that we use the reciprocal of NP, i.e.,
1/NP, to directly reflect the positive correlation with accuracy. TABLE VII
OA (%), AA (%), AND KAPPA COEFFICIENT ON THREE DATASETS AFTER EXCLUDING PURE PIXELS Note: The best one is shown in bold. Fig. 8. NP and AI of three datasets. Note that we use the reciprocal of NP, i.e.,
1/NP, to directly reflect the positive correlation with accuracy. and approach 3 are relatively close except for several per-class
accuracies, SRMGCN(DH) contributes to the more satisfactory
visual effect, as seen in Fig. 7. The better performance of our
method is attributed to fully considering the spatial relationship
between adjacent nodes representing land covers, while making
full use of relatively shallow shape information. TABLE VI TABLE VI TABLE VI
PER-CLASS ACCURACY (%), OA (%), AA (%), AND KAPPA COEFFICIENT
ACHIEVED BY DIFFERENT GRAPH CONVOLUTION APPROACHES ON THE SLOVENIA DATASET Note: The best one is shown in bold. of “water,” “artificial surface,” and “shrubland.” This further
demonstrates the significance of layer-wise input features in
the SRM process based on GCN. Most notably, SRMGCN(DH)
adopts the proposed approaches of adjacency matrix construc-
tion and information propagation and is capable of restoring
the details to the greatest extent, including boundaries of areal
objects, linear structural features, and fragmented patches. In
Table VI, consistent with visual results, there is a noticeable per-
formance drop in the quantitative results of approaches 1 and 2
compared with other graph convolution approaches that directly
integrate the input features’ transformation information of each
layer. Although the numerical value results of SRMGCN(DH) It can be observed from Fig. 7 that the SRM results obtained
by different graph convolution approaches have significant di-
vergences in the recovery of small-sized patches and elongated
features. By and large, due to the lack of layer-wise input fea-
tures, the resultant classification maps of approaches 1 and 2 are
both visually smooth, such that the details fail to be restructured,
even though the performance of approach 2 is a little better than
that of Approach 1 because of an extra parameter for each layer
to regulate the edge weights (e.g., the partial linear features
of “artificial surface” are captured in Fig. 7(d–1), but not in
(c–1)). Contrarily, the results of approach 3 and SRMGCN(DH)
retain more details, especially the narrow linear information TABLE VIII
OA (%), AA (%), AND KAPPA COEFFICIENT OF DIFFERENT SCALE FACTORS ON THE GLOBELAND30 DATASET Note: The best one is shown in bold. TABLE IX
PARAMETERS OF SRMGCN AND SRMCNN MODELS ON THE SLOVENIA DATASET Note: The best one is shown in bold TABLE IX TABLE IX
PARAMETERS OF SRMGCN AND SRMCNN MODELS ON THE SLOVENIA DATASET be more easily seen by observing the quantitative results from
Tables III and VIII. The possible reason is that the spatial
locations of subpixels within mixed pixels increased with the
increase of scale factor which makes the SRM process more
complicated [45]. only four convolutional layers operating in the graph. In ad-
dition, SRMCNN requires approximately 18 times the model
parameters of our methods. This indicates that the proposed
method only needs significantly fewer parameters to better learn
the complex nonlinear relationship between the coarse fraction
images and fine resolution land cover maps; this also reveals the
effectiveness of the structure information defined on the graph
in SRM. In the meantime, the smaller number of parameters also
makes it possible to learn with smaller samples. D. Further Comparison between SRMCNN and SRMGCN To further understand the CNN-based and GCN-based SRM
methods used in this article, a further comparison is made
with respect to model structure and number of parameters. Taking the Slovenia dataset as an example, where the number
of output channels in this dataset is 8, the statistical results of
the model structure and parameters of each trainable variable
about SRMGCN are given in Table IX, and the total number of
parameters of SRMCNN at a scale factor of 3 and SRMGCN are
also reported in it. C. Impact of Different Scale Factors on the SRM Results 90 × 190 pixels of different scale factors of the GlobeLand30 dataset. (a) Hard classification map. (b)–(f) SRM results of
CN(DH), and SRMGCN(LOT), respectively. TABLE VIII
OA (%), AA (%), AND KAPPA COEFFICIENT OF DIFFERENT SCALE FACTORS ON THE GLOBELAND30 DATASET C. Impact of Different Scale Factors on the SRM Results Moreover, the accuracy results of the five methods on the
GlobeLand30 dataset after pure pixels are removed are slightly
higher than those of the Slovenia dataset. On the Zurich sum-
mer dataset, the results of all methods increase significantly,
which is similar to the conclusions obtained from comparing
Tables III–V. Under the same scale factor, the reason for these
results may be related to the spatial distribution characteristics
of the geographical objects in different datasets. To clarify this
further, we calculated the values of two landscape indices on
the test set of the three datasets to represent the spatial pattern
characteristics; the results are illustrated in Fig. 8. The number
of patches (NP) per 10 000 grids can be used to describe the
heterogeneity of the entire landscape, and its value has a good
positive correlation with the degree of fragmentation of the
landscape. Generally speaking, the larger the NP, the higher the
degree of fragmentation. The aggregation index (AI) [55] is a
ratio of actual shared edges to maximal possible shared edges, The influence of different scale factors on the SRM results is
also worthy of investigation. Therefore, the SRM performances
with different scale factors were analyzed by studying on the
GlobeLand30 dataset. Based on the experiments with a scale
factor of 3 in Section III, two scale factors S = 4 and S = 5
wereaddedandtestedontheGlobeLand30datasetforevaluating
the accuracy of the five SRM methods. Fig. 9 and Table VIII
compare the visual performance and quantitative assessments,
respectively. The results shown in Fig. 9(b)–(f) indicate that speckle noise
and blurred boundary are significantly improved by three DNNs-
based methods, especially at the scale factor of 5. In addition, the
results of SRMGCN are closer to the reference image, because
the mapping results of SRMCNN look slightly smoother, which
makes some details disappear. Moreover, the mapping accuracy
gradually decreased with an increased scale factor, which can IEEE JOURNAL OF SELECTED TOPICS IN APPLIED EARTH OBSERVATIONS AND REMOTE SENSING, VOL. 14, 2021 7678 Fig. 9. Classification maps with 190 × 190 pixels of different scale factors of the GlobeLand30 dataset. (a) Hard classification map. (b)–(f) SRM results of
VBSPM, SAM, SRMCNN, SRMGCN(DH), and SRMGCN(LOT), respectively. Fig. 9. Classification maps with 190 × 190 pixels of different scale factors of the GlobeLand30 dataset. (a) Hard classification map. (b)–(f) SRM results of
VBSPM, SAM, SRMCNN, SRMGCN(DH), and SRMGCN(LOT), respectively. V. CONCLUSION [17] M. Song, Y. Zhong, A. Ma, X. Xu, and L. Zhang, “Multiobjective subpixel
mapping with multiple shifted hyperspectral images,” IEEE Trans. Geosci. Remote Sens., vol. 58, no. 11, pp. 8176–8191, May. 2020. Inspired by the successful applications of GCN in graph
structure data, a GCN-based SRM method with simple architec-
ture, named SRMGCN, is proposed in this article. As opposed
to the original graph convolution operation, we designed two
operations in terms of adjacency matrix construction and the
information propagation rule to improve the performance of
GCN in SRM. Meanwhile, to build a generalizable classifier,
we propose a supervised inductive learning strategy of GCN
with the mini-graphs as input. The experimental results of three
datasets with different spatial resolutions demonstrate that the
SRMGCN is capable of yielding better performance in both
visual and quantitative assessments. It is particularly conducive
to restructuring small patches, and linear features draw support
from the graph structure information. In future studies, the
integration of other available information, such as object-based
features and auxiliary data, with SRMGCN is recommended
for more accurate SRM analysis. Simultaneously, other means
of graph construction for remote sensing images are worth
exploring to further improve the restorability of GCN-based
SRM methods. [18] X. Xu et al., “A new spectral-spatial sub-pixel mapping model for remotely
sensed hyperspectral imagery,” IEEE Trans. Geosci. Remote Sens., vol. 56,
no. 11, pp. 6763–6778, Nov. 2018. [19] Y. Ge et al., “Principles and methods of scaling geospatial earth science
data,” Earth Sci. Rev., vol. 197, Jul. 2019, Art. no. 102897. [20] F. Ling et al., “Learning-Based superresolution land cover mapping,” IEEE
Trans. Geosci. Remote Sens., vol. 54, no. 7, pp. 3794–3810, Mar. 2016. [21] Y. Zhang, Y. Du, F. Ling, S. Fang, and X. Li, “Example-based super-
resolution land cover mapping using support vector regression,” IEEE J. Sel. Topics Appl. Earth Observ. Remote Sens., vol. 7, no. 4, pp. 1271–1283,
Apr. 2014. p
[22] L. Wang, Y. Zhang, and J. Li. “BP neural network based subpixel mapping
method,” in Proc. Int. Conf. Intell. Comput., 2006, pp. 755–760. [23] L. Zhang, K. Wu, Y. Zhong, and P. Li, “A new sub-pixel mapping
algorithm based on a BP neural network with an observation model,”
Neurocomputing, vol. 71, no. 10–12, pp. 2046–2054. Jun. 2008. [24] F. Ling and G. M. Foody, “Super-resolution land cover mapping by deep
learning,” Remote Sens. Lett., vol. 10, no. 6, pp. 598–606, Jun. 2019. ZHANG et al.: GRAPH CONVOLUTIONAL NETWORKS-BASED SUPER-RESOLUTION LAND COVER MAPPING 7679 relations that help improve the effect of SRM. Therefore, other
graph construction methods based on remote sensing images
are worth exploring. Inspired by the advantages of object-based
image analysis in processing remote sensing images [11], [56],
using objects as graph nodes is a promising way to address
the above problems and is more conducive to maximizing the
advantages of GCN. [3] Y. Ge, “Sub-pixel land-cover mapping with improved fraction images upon
multiple-point simulation,” Int. J. Appl. Earth Observ. Geoinf., vol. 22,
no. 1, pp. 115–126, Jun. 2013. [4] N. Keshava and J. F. Mustard, “Spectral unmixing,” IEEE Signal Proc. Mag., vol. 19, no. 1, pp. 44–57, Feb. 2002. [5] D. Lu and Q. Weng, “A survey of image classification methods and
techniques for improving classification performance,” Int. J. Remote Sens.,
vol. 28, no. 5, pp. 823–870, Mar. 2007. pp
[6] Y. Ge, S. Li, and V. C. Lakhan, “Development and testing of a subpixel
mapping algorithm,” IEEE Trans. Geosci. Remote Sens., vol. 47, no. 7,
pp. 2155–2164, Jul. 2009. Second, to simply compare the performances of the SRM
algorithms, the synthetic coarse fraction images are used as input
in this article, and the results produced by our experiments are
the most ideal. However, the errors caused by soft classification
algorithms are inescapable. For the sake of avoiding uncertainty
propagation from soft classification to SPM, several DNNs-
based SRM methods taking coarse remote sensing multiband
images as input rather than fraction images have achieved the
joint optimization of soft classification and SRM [30], [32]. So,
similar approaches are expected to be further examined for the
GCN-based methods. [7] P. M. Atkinson, “Issues of uncertainty in super-resolution mapping and
their implications for the design of an inter-comparison study,” Int. J. Remote Sens., vol. 30, no. 20, pp. 5293–5308, Oct. 2009. [8] P. M. Atkinson, “Mapping sub-pixel boundaries from remotely sensed
images,” in Innovations in GIS, vol. 4, Z. Kemp, Ed., London, U.K.: Taylor
and Francis, 1997, pp. 166–180. [9] Q. Wang, C. Zhang, X. Tong, and P. M. Atkinson, “General solution to
reduce the point spread function effect in subpixel mapping,” Remote Sens. Environ., vol. 251, pp. 112054, Dec. 2020. pp
[10] W. R. Tobler, “A computer movie simulating urban growth in the detroit
region,” Econ. Geography, vol. 46, no. 2, pp. 234–240, Jun. 1970. [11] Y. Chen, Y. Ge, G. B. Heuvelink, R. An, and Y. ZHANG et al.: GRAPH CONVOLUTIONAL NETWORKS-BASED SUPER-RESOLUTION LAND COVER MAPPING Chen, “Object-based
superresolution land-cover mapping from remotely sensed imagery,” IEEE
Trans. Geosci. Remote Sens., vol. 56, no. 1, pp. 328–340, Sep. 2017. Finally, in our experiments, SRMGCN performs better than
other baseline methods, but the results still have some short-
comings compared with reference maps. Some fragmented plots
stilldisappear,andtheboundariesofthegeographicalobjectsare
still somewhat smooth. One reason is that only applying fraction
images to extract features at the subpixel scale is insufficient to
tackle the SRM problem [25], [50]. Thus, auxiliary data [50] and
information on other scales [12], [57], such as the object-based
features from coarse remote sensing images (e.g., shape and
texture features), could be considered in the GCN-based SRM
process. [12] Y. Chen, Y. Ge, Y. Chen, Y. Jin, and R. An, “Subpixel land cover mapping
using multiscale spatial dependence,” IEEE Trans. Geosci. Remote Sens.,
vol. 56, no. 9, pp. 5097–5106, Mar. 2018. [13] K. C. Mertens, B. D. Baets, L. P. C. Verbeke, and R. R. D. Wulf,
“A sub-pixel mapping algorithm based on sub-pixel/pixel spatial at-
traction models,” Int. J. Remote Sens., vol. 27, no. 15, pp. 3293–3310,
Aug. 2006. [14] Y. Ge, Y. Chen, S. Li, and Y. Jiang, “Vectorial boundary-based sub-pixel
mappingmethodforremote-sensingimagery,”Int.J.RemoteSens.,vol.35,
no. 5, pp. 1756–1768, Mar. 2014. [15] K. Wu, P. Li, and L. Zhang, “Sub-pixel mapping of remote sensing
image based on MAP model,” in Proc. 4th Int. Conf. Image Graph, 2007,
pp. 742–746. pp
[16] A. Ma, Y. Zhong, D. He, and L. Zhang, “Multiobjective subpixel land-
cover mapping,” IEEE Trans. Geosci. Remote Sens., vol. 56, no. 1,
pp. 422–435, Jan. 2017. E. Limitations and Future Work Although this study has shown the effectiveness of structure
information constructed by SRMGCN, the study has some lim-
itations. First, in terms of encoding an image into a graph, our
experiments use subpixels as nodes and the first-order neighbor-
hoods of each node as edges. As a result, there are many nodes
and edges in the graph; this means a large amount of calculation
is required, and it is inconvenient to extract the long-range spatial Compared with the model structure of SRMCNN in [25],
the proposed SRMGCN has a fairly simple architecture, with [2] P. M. Atkinson, “Downscaling in remote sensing,” Int. J. Appl. Earth
Observ. Geoinf., vol. 22, no. 1, pp. 106–114, Jun. 2013. [1] P. Fisher, “The pixel: A snare and a delusion,” Int. J. Remote Sens., vol. 18,
no. 3, pp. 679–685, Feb. 1997. V. CONCLUSION [25] Y. Jia, Y. Ge, Y. Chen, S. Li, G. Heuvelink, and F. Ling, “Super-resolution
land cover mapping based on the convolutional neural network,” Remote
Sens., vol. 11, no. 15, pp. 1815, Aug. 2019. [26] P. V. Arun, I. Herrmann, K. M. Budhiraju, and A. Karnieli, “Convolutional
network architectures for super-resolution/sub-pixel mapping of drone-
derived images,” Pattern Recognit., vol. 88, pp. 431–446, Apr. 2019. g
g
pp
p
[27] C. Shang, X. Li, G. M. Foody, Y. Du, and F. Ling, “Superresolution land
cover mapping using a generative adversarial network,” IEEE Geosci. Remote Sens. Lett., to be published. IEEE JOURNAL OF SELECTED TOPICS IN APPLIED EARTH OBSERVATIONS AND REMOTE SENSING, VOL. 14, 2021 7680 IEEE JOURNAL OF SELECTED TOPICS IN APPLIED EARTH OBSERVATIONS AND REMOTE SENSING, VOL. 14, 2021 [29] X. Ma, Y. Hong, and Y. Song, “Super resolution land cover mapping of
hyperspectral images using the deep image prior-based approach,” Int. J. Remote Sens., vol. 41, no. 7, pp. 2818–2834, Dec. 2019. [52] T.E.Oliphant,“Pythonforscientificcomputing,”Comput.Sci.Eng.,vol.9,
no. 3, pp. 10–20, Jun. 2007. [53] A. Paszke et al., “Pytorch: An imperative style, high-performance deep
learning library,” in Proc. Adv. Neural Inf. Process. Syst., 2019, pp. 8026–
8037. [30] D. He, Y. Zhong, X. Wang, and L. Zhang, “Deep convolutional neural
network framework for subpixel mapping,” IEEE Trans. Geosci. Remote
Sens., to be published, doi: 10.1109/TGRS.2020.3032475. [54] D. P. Kingma and J. Ba, “Adam: A method for stochastic optimization,”
in Proc. Int. Conf. Learn. Represent., 2014, pp. 1–41. [31] Y. LeCun, Y. Bengio, and G. Hinton, “Deep learning,” Nature, vol. 521,
no. 7553, pp. 436–444, May. 2015. [55] H. He, B. DeZonia, and D. Mladenoff, “An aggregation index (AI) to
quantify spatial patterns of landscapes,” Landscape Ecol., vol. 15, no. 7,
pp. 591–601, Jan. 2000. [32] P. V. Arun, K. M. Buddhiraju, and A. Porwal, “CNN based sub-pixel
mapping for hyperspectral images,” Neurocomputing, vol. 311, pp. 51–64,
May. 2018. [56] T. Blaschke et al., “Geographic object-based image analysis–towards
a new paradigm,” ISPRS J. Photogramm. Remote Sens., vol. 87,
pp. 180–191, Dec. 2014. [33] T. N. Kipf and M. Welling, “Semi-supervised classification with graph
convolutional networks,” in Proc. Int. Conf. Learn. Represent. 2017,
pp. 1–14. [57] P. Wang, G. Zhang, L. Wang, H. Leung, and H. Bi, “Subpixel land
cover mapping based on dual processing paths for hyperspectral image,” [57] P. Wang, G. Zhang, L. Wang, H. Leung, and H. Bi, “Subpixel land
cover mapping based on dual processing paths for hyperspectral image,”
IEEE J. Sel. Topics. Appl. Earth Observ. Remote Sens., vol. 12, no. 6,
pp. 1835–1848, Apr. 2019. [34] M. Fey, J. E. Lenssen, F. Weichert, and H. Müller, “Splinecnn: Fast
geometric deep learning with continuous b-spline kernels,” in Proc. IEEE
Conf. Comput. Vis. Pattern Recognit., 2018, pp. 869–877. cover mapping based on dual processing paths for hyperspectral image,”
IEEE J. Sel. Topics. Appl. Earth Observ. Remote Sens., vol. 12, no. 6,
pp. 1835–1848, Apr. 2019. IEEE J. Sel. Topics. Appl. Earth Observ. Remote Sens., vol. 12, no. 6,
pp. 1835–1848, Apr. 2019. [35] F. Monti, D. Boscaini, J. Masci, E. Rodola, J. IEEE JOURNAL OF SELECTED TOPICS IN APPLIED EARTH OBSERVATIONS AND REMOTE SENSING, VOL. 14, 2021 259–273, Mar. 2019. [43] D. K. Hammond, P. Vandergheynst, and R. Gribonval, “Wavelets on graphs
via spectral graph theory,” Appl. Comput. Harmon. Anal., vol. 30, no. 2,
pp. 129–150, Mar. 2011. pp
[44] Y. Yang and Y. Qi, “Image super-resolution via channel attention and spa-
tial graph convolutional network,” Pattern Recognit, vol. 112, , Dec. 2021,
Art. no. 107798. [45] Q. Wang, W. Shi, and L. Wang, “Allocating classes for soft-then-hard
subpixel mapping algorithms in units of class,” IEEE Trans. Geosci. Remote Sens., vol. 52, no. 5, pp. 2940–2959, Jul. 2013. Feng Ling received the B.S. degree in geology and
the M.S. degree in geophysical prospecting and in-
formation technology from the China University of
Geosciences, Wuhan, China, in 1999 and 2002, re-
spectively, and the Ph.D. degree in system analysis
and integration from the Huazhong University of
Science and Technology, Wuhan, China, in 2006. Feng Ling received the B.S. degree in geology and
the M.S. degree in geophysical prospecting and in-
formation technology from the China University of
Geosciences, Wuhan, China, in 1999 and 2002, re-
spectively, and the Ph.D. degree in system analysis
and integration from the Huazhong University of
Science and Technology, Wuhan, China, in 2006. [46] X. Xu, Y. Zhong, L. Zhang, and H. Zhang, “Sub-pixel mapping based
on a MAP model with multiple shifted hyperspectral imagery,” IEEE J. Sel. Topics Appl. Earth Observ. Remote Sens., vol. 6, no. 2, pp. 580–593,
Apr. 2013. the M.S. degree in geophysical prospecting and in-
formation technology from the China University of
Geosciences, Wuhan, China, in 1999 and 2002, re-
spectively, and the Ph.D. degree in system analysis
and integration from the Huazhong University of
Science and Technology, Wuhan, China, in 2006. He is currently a Professor with the Innovation
Academy for Precision Measurement Science and
Technology, Chinese Academy of Sciences, Wuhan,
China. He has authored more than 80 refereed journal
articles. His research interests include super-resolution mapping and spatial–
temporal fusion of remotely sensed imagery. Dr. Ling was the recipient of the 2016 Boeing Award for Best Scientific
Paper in Image Analysis and Interpretation from the American Society for
Photogrammetry and Remote Sensing as the corresponding author. He has been
supported by several talent programs, including the Hubei Province Natural
Science Fund for Distinguished Young Scholars in 2013 and the Distinguished
Young Scientist Grant of the Chinese Academy of Sciences in 2014. [47] A. J. Tatem, H. G. IEEE JOURNAL OF SELECTED TOPICS IN APPLIED EARTH OBSERVATIONS AND REMOTE SENSING, VOL. 14, 2021 Her current research interests focus on remote sensing image processing and
intelligent super-resolution mapping. [39] S. Wan, C. Gong, P. Zhong, S. Pan, G. Li, and J. Yang, “Hyperspectral
image classification with context-aware dynamic graph convolutional
network,” IEEE Trans. Geosci. Remote Sens., vol. 59, no. 1, pp. 597–612,
May. 2020. [40] D. Hong, L. Gao, J. Yao, B. Zhang, A. Plaza, and J. Chanussot, “Graph
convolutional networks for hyperspectral image classification,” IEEE
Trans. Geosci. Remote Sens, vol. 59, no. 7, pp. 5966–5978, Jul. 2021,
doi: 10.1109/TGRS.2020.3015157. Yong Ge (Member, IEEE) received the Ph.D. degree
in cartography and geographical information system
from the Chinese Academy of Sciences (CAS), Bei-
jing, China, in 2001. She is currently a Professor with the State Key
Laboratory of Resources and Environmental Infor-
mation System, Institute of Geographic Sciences and
Natural Resources Research, CAS. Her research in-
terests broadly focus on the statistical aspects of spa-
tial and spatio-temporal data. She has authored more
than 200 papers in the field of spatial statistics and
related fields. She has been involved in the organization of several international
conferences and workshops. She is currently an Associate Editor of computers
and geosciences and the Editor board member of Spatial Statistics. She is also
the Guest Editor of various special issues of journals. Yong Ge (Member, IEEE) received the Ph.D. degree
in cartography and geographical information system
from the Chinese Academy of Sciences (CAS), Bei-
jing, China, in 2001. [41] M. Defferrard, X. Bresson, and P. Vandergheynst, “Convolutional neural
networks on graphs with fast localized spectral filtering,” in Proc. Adv. Neural Inf. Process. Syst., 2016, pp. 3844–3852. She is currently a Professor with the State Key
Laboratory of Resources and Environmental Infor-
mation System, Institute of Geographic Sciences and
Natural Resources Research, CAS. Her research in-
terests broadly focus on the statistical aspects of spa-
tial and spatio-temporal data. She has authored more
than 200 papers in the field of spatial statistics and
related fields. She has been involved in the organization of several international
conferences and workshops. She is currently an Associate Editor of computers
and geosciences and the Editor board member of Spatial Statistics. She is also
the Guest Editor of various special issues of journals. [42] X. Yan, T. Ai, M. Yang, and H. Yin, “A graph convolutional neural network
for classification of building patterns using spatial vector data,” ISPRS J. Photogramm. Remote Sens., vol. 150, pp. REFERENCES [28] X. Ma, Y. Hong, Y. Song, and Y. Chen, “A super-resolution convolutional-
neural-network-based approach for subpixel mapping of hyperspectral
images,” IEEE J. Sel. Top. Appl. Earth Observ. Remote Sens., vol. 12,
no. 12, pp. 4930–4939, Sep. 2019. [1] P. Fisher, “The pixel: A snare and a delusion,” Int. J. Remote Sens., vol. 18,
no. 3, pp. 679–685, Feb. 1997. [2] P. M. Atkinson, “Downscaling in remote sensing,” Int. J. Appl. Earth
Observ. Geoinf., vol. 22, no. 1, pp. 106–114, Jun. 2013. IEEE JOURNAL OF SELECTED TOPICS IN APPLIED EARTH OBSERVATIONS AND REMOTE SENSING, VOL. 14, 2021 Svoboda, and M. M. Bron-
stein, “Geometric deep learning on graphs and manifolds using mixture
model cnns,” in Proc. IEEE Conf. Comput. Vis. Pattern Recognit., 2017,
pp. 5115–5124. Xining Zhang received the B.Eng. degree in geomat-
ics engineering from the China University of Geo-
sciences, Beijing, China, in 2016, and M.S.E. degree
in photogrammetry and remote sensing from Wuhan
University, Wuhan, China, in 2019. She is currently
working toward the Ph.D. degree in cartography and
geographic information system with the State Key
Laboratory of Resources and Environmental Infor-
mation System, Institute of Geographic Sciences and
Natural Resources Research, Chinese Academy of
Sciences, Beijing, China. Xining Zhang received the B.Eng. degree in geomat-
ics engineering from the China University of Geo-
sciences, Beijing, China, in 2016, and M.S.E. degree
in photogrammetry and remote sensing from Wuhan
University, Wuhan, China, in 2019. She is currently
working toward the Ph.D. degree in cartography and
geographic information system with the State Key
Laboratory of Resources and Environmental Infor-
mation System, Institute of Geographic Sciences and
Natural Resources Research, Chinese Academy of
Sciences, Beijing, China. Her current research interests focus on remote sensing image processing and
intelligent super-resolution mapping. Xining Zhang received the B.Eng. degree in geomat-
ics engineering from the China University of Geo-
sciences, Beijing, China, in 2016, and M.S.E. degree
in photogrammetry and remote sensing from Wuhan
University, Wuhan, China, in 2019. She is currently
working toward the Ph.D. degree in cartography and
geographic information system with the State Key
Laboratory of Resources and Environmental Infor-
mation System, Institute of Geographic Sciences and
Natural Resources Research, Chinese Academy of
Sciences, Beijing, China. Her current research interests focus on remote sensing image processing and
intelligent super-resolution mapping. [36] Y. Lu, Y. Chen, D. Zhao, B. Liu, Z. Lai, and J. Chen, “CNN-G: Convolu-
tional neural network combined with graph for image segmentation with
theoretical analysis,” IEEE Trans. Cogn. Develop. Syst., to be published. doi: 10.1109/TCDS.2020.2998497 [37] S. Wan, C. Gong, P. Zhong, B. Du, L. Zhang, and J. Yang, “Multiscale
dynamic graph convolutional network for hyperspectral image classifica-
tion,” IEEE Trans. Geosci. Remote Sens., vol. 58, no. 5, pp. 3162–3177,
May. 2019. [38] Q. Liu, L. Xiao, J. Yang, and Z. Wei, “CNN-Enhanced graph convolutional
network with Pixel-and superpixel-level feature fusion for hyperspectral
image classification,” IEEE Trans. Geosci. Remote Sens., to be published,
doi: 10.1109/TGRS.2020.3037361. IEEE JOURNAL OF SELECTED TOPICS IN APPLIED EARTH OBSERVATIONS AND REMOTE SENSING, VOL. 14, 2021 Lewis, P. M. Atkinson, and M. S. Nixon, “Increasing
the spatial resolution of agricultural land cover maps using a Hopfield
neural network,” Int. J. Geographical Inf. Sci., vol. 17, no. 7, pp. 647–672,
Oct. 2003. He is currently a Professor with the Innovation
Academy for Precision Measurement Science and
Technology, Chinese Academy of Sciences, Wuhan,
China. He has authored more than 80 refereed journal
articles. His research interests include super-resolution mapping and spatial–
temporal fusion of remotely sensed imagery. He is currently a Professor with the Innovation
Academy for Precision Measurement Science and
Technology, Chinese Academy of Sciences, Wuhan,
China. He has authored more than 80 refereed journal
articles. His research interests include super-resolution mapping and spatial–
temporal fusion of remotely sensed imagery. Dr. Ling was the recipient of the 2016 Boeing Award for Best Scientific
Paper in Image Analysis and Interpretation from the American Society for
Photogrammetry and Remote Sensing as the corresponding author. He has been
supported by several talent programs, including the Hubei Province Natural
Science Fund for Distinguished Young Scholars in 2013 and the Distinguished
Young Scientist Grant of the Chinese Academy of Sciences in 2014. [48] J. Verhoeye and R. D. Wulf, “Land cover mapping at sub-pixel scales using
linear optimization techniques,” Remote Sens. Environ., vol. 79, no. 1,
pp. 96–104, Jan. 2002. [49] E. L. Lawler and D. E. Wood, “Branch-and-bound methods: A survey,”
Oper. Res., vol. 14, pp. 699–719, 1966. [50] Y. Chen, Y. Ge, G. B. Heuvelink, J. Hu, and Y. Jiang, “Hybrid constraints
of pure and mixed pixels for soft-then-hard super-resolution mapping with
multiple shifted images,” IEEE J. Sel. Topics Appl. Earth Observ. Remote
Sens., vol. 8, no. 5, pp. 2040–2052, Apr. 2015. [51] M. Volpi and V. Ferrari, “Semantic segmentation of urban scenes by
learning local class interactions,” in Proc. IEEE Conf. Comput. Vis. Pattern
Recognit. Workshops, 2015, pp. 1–9. 7681 ZHANG et al.: GRAPH CONVOLUTIONAL NETWORKS-BASED SUPER-RESOLUTION LAND COVER MAPPING Jin Chen received the B.A. and M.S. degrees in
geography from Beijing Normal University, Beijing,
China, in 1989 and 1992, respectively, and the Ph.D. degree in civil engineering from Kyushu University,
Fukuoka, Japan, in 2000. Yuanxin Jia received the Ph.D. degree in cartography
and geographical information system from the State
Key Laboratory of Resources and Environmental In-
formation System, Institute of Geographic Sciences
and Natural Resources Research, University of Chi-
nese Academy of Sciences, Beijing, China, in 2020. IEEE JOURNAL OF SELECTED TOPICS IN APPLIED EARTH OBSERVATIONS AND REMOTE SENSING, VOL. 14, 2021 He is currently with the Academy of Forest In-
ventory and Planning, National Forestry and Grass-
land Administration, Beijing, China. His research
interests include natural resource inventorying and
monitoring, remote-sensing image processing, and
g He was a Postdoctoral Researcher with the Uni-
versity of California, Berkeley, CA, USA, from 2000
to 2001, and with the National Institute of Environ-
mental Studies, Tsukuba, Japan, from 2001 to 2004. He is currently a Professor with the Institute of Re-
mote Sensing Science and Engineering, Faculty of
Geographical Science, Beijing Normal University, Beijing, China. His research
interests include remote sensing modeling and vegetation parameter retrieval
through the inversion of remote sensing models. super-resolution mapping. Yuehong Chen received the B.S. degree from Hohai
University, Nanjing, China, in 2010, and the M.Sc. and Ph.D. degrees from the State Key Laboratory
of Resources and Environmental Information Sys-
tem, Institute of Geographic Sciences and Natural
Resources Research, University of Chinese Academy
of Sciences, Beijing, China, in 2013 and 2016, respec-
tively. He is currently an Associate Professor with the
College of Hydrology and Water Resources, Hohai
University, China. His current research interest in-
cludes remote sensing image processing.
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ChatGPT in Organic Chemistry Classrooms: Analyzing the Impacts of Social Environment on Students’ Interest, Critical Thinking and Academic Achievement
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International Journal of Education and Teaching Zone
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ChatGPT in Organic Chemistry Classrooms: Analyzing the Impacts of
Social Environment on Students’ Interest, Critical Thinking and
Academic Achievement Victor Tubosun Babalola1, Suwaiba Said Ahmad2, Halima Sani Tafida3
1Department of Science Education, Yusuf Maitama Sule University, Kano, Nigeria
2Department of Science and Technology Education, Bayero University, Kano, Nigeria
3Department of Science Education, Sule Lamido University, Kafin Hausa, Nigeria Abstract: This study aims to analyze the Impacts of Social
Environment
on
Interest,
Critical
Thinking
and
Academic
Achievement of Students Taught Organic Chemistry Using Artificial
Intelligence ChatGPT. Correlational research design was engaged for
the study. 50-students in SS3 with an average age of 17years, who have
access to android phones and the ChatGPT application from two senior
secondary schools in Gwale Local Government area of Kano State,
Nigeria formed the study sample. The instruments used for data
collection include; the students’ social environment, interest and
critical thinking questionnaire (r=0.96) and Organic Chemistry
Achievement Test (r=0.92). The data were analyzed using mean,
standard deviation and Spearman Rank Order Correlation. The study
revealed a high positive correlation between the social environment
and student’s interest; Social environment and critical thinking; as well
as social environment and students’ achievement in the ChatGPT’s
Organic Chemistry Classroom. The study further found a strong
positive correlation between the students’ interest and critical thinking,
critical thinking and students’ achievement in Organic Chemistry
among students in the ChatGPT condition. It was concluded that social
environment has a strong positive impact on students’ interest, critical
thinking and academic achievement in organic chemistry among
students in the ChatGPT classrooms. The study recommends that, as
the artificial intelligence (AI) ChatGPT is migrating into the chemistry
classrooms, government needs to re-train chemistry teachers, school
librarians and lab-technologists through refreshers courses to improve
their performance in the concepts ______________
Article History:
Received: November 24, 2023
Revised: January 17, 2024
Accepted: January 20, 2024
Published: February 01, 2024 ______________
Article History:
Received: November 24, 2023
Revised: January 17, 2024
Accepted: January 20, 2024
Published: February 01, 2024 _________
Keywords:
ChatGPT,
Educational Intervention,
Learning Outcomes Keywords:
ChatGPT,
Educational Intervention,
Learning Outcomes *Correspondence Author:
lolababa85@ymail.com International Journal of Education and Teaching Zone. Volume 3 (Issue 1): 01-02 (2024)
DOI: https://10.57092/ijetz.v3i1.155 International Journal of Education and Teaching Zone. Volume 3 (Issue 1): 01-02 (2024)
DOI: https://10.57092/ijetz.v3i1.155 INTRODUCTION Science, from the ages, aims at solving global problems by providing explanations
for the working system of the environment through the application of scientific discoveries
in technological ventures (Freedman, 2018; Bradford and Hamer, (2022). By so doing,
many scientific ideas have been helping the chemistry teaching community, including
Chemistry textbook authors, curriculum developers and chemistry teachers to increase
human knowledge and build capacity for technological growth. One of such innovations
in the recent time is the Artificial Intelligence (AI) robot-teachers and the ChatGPT apps. According to Iona University (2023), ChatGPT is a natural language processing tool or International Journal of Education and Teaching Zone | Licence: CC BY-SA 4.0 | Copyright (2024) The (Author)s
Page 60 Page 60 hatGPT in Organic Chemistry Classrooms… | Victor Tubosun Babalola, Suwaiba Said Ahmad, Halima Sani Tafida model trained to produce text that allows users to have human-like conversations with an
AI Chatbot. Specifically, ChatGPT is optimized for dialogue by using reinforcement
learning with human-like feedback that uses demonstrations to guide the learners toward
desired behavior. At the global stage, ChatGPT is gradually finding its way into the
education system and Chemistry teachers in Nigeria needs to be aligned with this
developmental process. This has to be achieved through empirical research backing for its
usability and the quality of impact produced on students’ educational variables. However, the improvement of Chemistry teaching and learning depend largely on
the quality of Science Education as a research field. In that science education through
research of this nature on AI ChatGPT may provide empirical finding for teacher education
institution to train world class Chemistry teachers for Secondary schools, not only in
Nigeria but globally. As described by Babalola, (2023a), science education is the total
package of activities involving the teaching, learning and research into science subjects
including Chemistry. Nevertheless, the teaching of chemistry as a science subject cannot
be said to be efficient, when students’ interest towards Organic Chemistry is low. Students’ interest in Organic Chemistry is a psychological term, referring to likes
and dislikes as well as choices and aspirations that may enhance students’ attention to get
involve and be dedicated to organic concepts and chemistry in general. Harackiewicz,
Smith, and Priniski, (2016), revealed that interest is a powerful reinforcement that promote
learning, guides academic activities and career trajectories which are essential for
academic success. JETZ) | International Journal of Education and Teaching Zone. Volume 3 (Issue 1): 01-02 (2024) INTRODUCTION Interest inspires students to learn Organic Chemistry willingly with
receptive minds which may lead to academic achievement in Chemistry. As reported by
Boesdorfer (2019), the students’ diversity in learning styles, the abstract nature of
chemistry, and the bookish way of teaching chemistry without adequate attention given to
innovation (such as ChatGPT) have been causing decline in students’ interest in Chemistry
including Organic Chemistry concepts. Organic Chemistry can be defined as the branch of Chemistry dealing with the
study of carbon compounds with exception of CO2 gas and –CO32- compounds. Organic
compounds are found in animals and plants that form part of human foods such as
carbohydrate, protein and fruits. Also, Petrol, Diesel and Kerosene used as fuel in homes,
industries and automobiles are organic substances. Organic polymers are used for making
vehicle parts like dashboard, and in health sector for making medical hand gloves and
nylon for surgical operations. Students can only become experts in this area of chemistry
enterprise by gaining admission into tertiary institutions that is well packed with inclusive
skills (Babalola, 2023b) through innovative ways of learning like the ChatGPT. Hence,
lack of interest, critical thinking and underachievement in Organic Chemistry could mean
a reduction in number of future experts in those areas of economy. Sadly, Organic
Chemistry has been contributing to students’ underachievement in Chemistry. Academic achievement in chemistry is the extent to which students realize their
educational goals through examination and continuous assessment scores from tests,
assignments, debates and practical engagements. The problem of academic achievement is
in chemistry is evident in the WAEC Examiner’s report, which shows that out of 2,125
students who sat for WAEC between years 2020 and 2022 in Gwale Secondary schools Page 61 hatGPT in Organic Chemistry Classrooms… | Victor Tubosun Babalola, Suwaiba Said Ahmad, Halima Sani Tafida only 920-students constituting 44.8% passed and 55.2% failed. The problem of poor
achievement in chemistry generally has been militating against students from gaining
admission into tertiary institutions causing inadequate personnel in science related
discipline. As observed by Ali, Babalola and Ibrahim (2023), one of the most annoying
problems of underachievement is that students are carrying their poor knowledge of
chemistry to the tertiary institutions making them to drop-out. Likewise, the scientific
skills and technological knowledge needed for self-reliance and those required for the
nation to move up the ladder of development are under threat among students. INTRODUCTION Unfortunately, lack of interest and Critical-thinking among science students have been
linked to poor academic achievement in the secondary school chemistry. The Bandura,
through his Socio-cognitive theory using Bobo doll experiment assumed that social
environment may help in this situation. Thus; this study hereby examined the impact of
Social environment on these variables among Gwale Secondary school students in Organic
Chemistry taught using ChatGPT. However, It has been found that student’ academic achievement improves
whenever their critical thinking abilities increases (Rokhim, Widarti, & Permatasari,
2023). According to Ennis (2015), the 12-indicators of Critical thinking upon which
students can be assessed includes; comprehension, analyzing, evaluating, and synthesizing
among others. In another words, the cognitive level of bloom taxonomy can be used to
measure critical thinking. However, if students are given adequate opportunities to solve
problems using ChatGPT within the school and home environment, their critical thinking
abilities may develop. Therefore, teachers’ may need to guide and provide their students
with skills of gathering, analyzing and utilizing information from ChatGPT to enable them
solve real life problems. In an effort to solve the problem of low students’ interest, lack of critical thinking
and underachievement in Organic chemistry, Bandura’s theory was considered. Albert
Bandura believes that apart from the teaching resources, such as diagrams, mnemonics and
technology, learners’ academic behaviors may be influenced by the social environment
(Harinie, et al. 2017). As suggested by Nabavi (2014), the persistent change in learners’
behavior as a result of interaction with social environment may improve their academic
achievement. This interaction involves observation and imitation of the Social
environment. Harinie, et al. (2017), found that if children are raised in an entrepreneur
family, there is tendency that they may observe and imitate entrepreneurship skills. Research scholars have viewed Students’ social environment differently, for
instance, Abumchukwu &Okigbo (2023) sees social environment as an online destination
where people come together to create content, share knowledge and learn from one
another. This perspective has been termed social media chemistry groups in this study. Similarly, Nubunga (2020), describe social environment as the combination of social
learning elements like networking, tagging, files sharing and microblogging to create a
safe place to work and learn collaboratively in learning where people gather to share
knowledge. In this study, social environment simply means the human components of the
learners’ academic environment both in and outside the school. International Journal of education and Teaching Zone. Volume 3 ( Issue 1): 01-02 (2024) | (IJETZ METHOD The researchers taught the students Organic Chemistry using the artificial
intelligence ChatGPT for a period of 6-weeks after which the two instruments were
administered. The data was analyzed using Histogram, Mean, Standard deviation and
Spearman Correlation. The range of scores and the decision taken on the hypotheses are;
±0.80 to ± 1.00 (High correlation), ±0.50 to ± 0.79 (Moderate correlation), ±0.00 to ± 0.49
(Low Correlation). METHOD This study analyzed the impact of social environments on students’ interest,
academic achievement and critical thinking among students taught Organic Chemistry
using AI ChatGPT in Gwale Secondary Schools, Kano-Nigeria. Four research questions
and six hypotheses were raised to guide the study. Correlational research design was
adopted for the study. The study population covered 718 science students from eight (8)
secondary schools in the study area. The Cochran’s formula of 1963 was used to determine
the sample size to be 50-students of SS3 with an average age of 17yrs. According to
Babalola (2023c), the Cochran’s formula states that; (n= no/ (1+ (no-1)/385). Nevertheless,
using simple random sampling technique, two intact classes were selected for the study,
the instruments were administered to only 55-students who have access to the ChatGPT. y
The two researcher-made instruments used and their Kuder Richardson formula 20
reliability coefficients (r) include; Social environment, Students’ Interest and Critical-
thinking questionnaire (r = 0.96), and Organic Chemistry Achievement Test (r=0.92). The
achievement test made up of 30-multiple choice WAEC past questions in Organic
Chemistry, while the questionnaire was also made up of 30-items (10-items for each
variable). The questionnaire was made of 4-point scale; Strongly Agreed (SA), Agree (A),
Disagree (D) and Strongly Disagree (SD) rated 4,3,2,1. The instruments were validated by
three Professors in the field of Science Education, Test and Measurement and Linguistics. The researchers taught the students Organic Chemistry using the artificial
intelligence ChatGPT for a period of 6-weeks after which the two instruments were
administered. The data was analyzed using Histogram, Mean, Standard deviation and
Spearman Correlation. The range of scores and the decision taken on the hypotheses are;
±0.80 to ± 1.00 (High correlation), ±0.50 to ± 0.79 (Moderate correlation), ±0.00 to ± 0.49
(Low Correlation). The two researcher-made instruments used and their Kuder Richardson formula 20
reliability coefficients (r) include; Social environment, Students’ Interest and Critical-
thinking questionnaire (r = 0.96), and Organic Chemistry Achievement Test (r=0.92). The
achievement test made up of 30-multiple choice WAEC past questions in Organic
Chemistry, while the questionnaire was also made up of 30-items (10-items for each
variable). The questionnaire was made of 4-point scale; Strongly Agreed (SA), Agree (A),
Disagree (D) and Strongly Disagree (SD) rated 4,3,2,1. The instruments were validated by
three Professors in the field of Science Education, Test and Measurement and Linguistics. INTRODUCTION This suggests that
chemistry teacher, lab-technologist, school librarian, classmates, home-lesson teachers and Page 62 Page 62 hatGPT in Organic Chemistry Classrooms… | Victor Tubosun Babalola, Suwaiba Said Ahmad, Halima Sani Tafida chemistry groups on social media form the bulk of learners’ social environment interested
in this study. Consequently, this article seeks to demonstrate that the quality of chemistry
teaching and learning can be improved using ChatGPT as a resource assisted instruction,
which does not need to take the job from chemistry teachers, school librarians and lab-
technologists. RESULTS AND DISCUSSION Based on the observations and data analysis that have been carried out in this
research, data was obtained from the four research questions created for this research which
were described, stated and answered using a histogram, mean and standard deviation,
where each data obtained was very good. importance or urgency in the education and
teaching process. The question process used is of course about the educational and teaching
environment, both how the forms of behavior are used and how the learning environment
is related to influencing the learning process carried out by students. The learning
environment can be influenced by one of them, the social environment, namely how
students interact both at school and outside the school environment. In this study the
researcher asked a First Research Question, what is the average social environmental rating (IJETZ) | International Journal of Education and Teaching Zone. Volume 3 (Issue 1): 01-02 (2024)
Page 63 Page 63 Page 63 hatGPT in Organic Chemistry Classrooms… | Victor Tubosun Babalola, Suwaiba Said Ahmad, Halima Sani Tafida (SER) of students taught Organic Chemistry in the artificial intelligence ChatGPT
Classroom in Gwale? The data obtained can be seen in Figure 1. Figure1. Histogram of Social Environment Ratings (SER) in ChatGPT Classes Figure1. Histogram of Social Environment Ratings (SER) in ChatGPT Classes Figure1. Histogram of Social Environment Ratings (SER) in ChatGPT Classes As presented in Figure 1, from the research results, the number of students examined
was 50 people, while the maximum social environment rating obtained was 10. This
positively sloping histogram shows a conducive social environment with an average value
of 6. 94 and a standard deviation of 2.351. This means that students' social environment in
the ChatGPT organic chemistry class is conducive. The conducive social environment that
has been formed indicates that the learning process can be carried out well and effectively. Perfect and practical learning conditions or environments are needed to provide
information and knowledge. Then, the researcher continued with the second question,
namely, about what is the average critical thinking score of students taught organic
chemistry in the ChatGPT classroom with artificial intelligence?, where the complete data
that can be collected from this question can be seen in Figure 2. Figure 2. Histogram of Critical Thinking Ratings (CTR) in Organic Chemistry Figure 2. International Journal of education and Teaching Zone. Volume 3 ( Issue 1): 01-02 (2024) | (IJETZ RESULTS AND DISCUSSION Volume 3 (Issue 1): 01-02 (2024) Page 65 Page 65 hatGPT in Organic Chemistry Classrooms… | Victor Tubosun Babalola, Suwaiba Said Ahmad, Halima Sani Tafida The histogram in Figure 4, revealed a positively skewed interest rating of 50-students
with a maximum obtainable rating of 10 in the ChatGPT classroom. The histogram further
revealed the mean interest rating of 6.94 and the standard deviation of 2.736. Since the
mean scores of 6.94>5.00 (50% of the maximum obtainable rating), this implies that
artificial intelligence ChatGPT is an effective technological Application for improving
students’ interest in chemistry. All the six null hypotheses (HO) formulated were tested
using Spearman’s Rank Order Correlation Statistics (rho) of the SPSS version 20 as
follows; Null Hypothesis One (HO1), There is no significant relationship between social
environment Rating and Students’ interest in the ChatGPT’s Organic Chemistry Classes. The relationship between the Social Environment ranking and the Interest ranking in the
ChatGPT Organic Chemistry Class can be seen in Table 1. Table 1: rho Test of relationship between Social Environment ratings and Interest ratings in Organic
Chemistry’s ChatGPT Classrooms. Statistics Variables
SER
SIR
Spearman'
s rho
Social Environment
Ratings(SER)
Correlation Coefficient
1.000
.865
Sig. (2-tailed)
.000
.000
N
50
50
Students’ Interest
Ratings(SIR)
Correlation Coefficient
.865
1.000
Sig. (2-tailed)
.000
. N
50
50 Table 1: rho Test of relationship between Social Environment ratings and Interest ratings in Organic
Chemistry’s ChatGPT Classrooms. Table 1, shows the Spearman’s rho of 0.865. This indicates a High correlation which
is statistically significant at 0.05. (0.001<0.05). Hence, the HO1 is rejected. There is a
significantly high positive correlation between Social environment ratings and students’
interest in Organic chemistry taught with ChatGPT. It can be concluded that social
environment impact students’ interest in Organic Chemistry in the ChatGPT classroom. The null hypothesis one (HO1), was rejected because the result shows a significant
correlation between the student Social environment ratings and students’ interest in organic
chemistry with a Spearman Rank Order correlation (rho = 0.865; P<0.001) which is
significant. The positive direction indicates an increase in students’ Social parameters and
a tremendous increase in student academic interest towards organic chemistry. Conversely,
if there is a decline in quality of student interaction with Social environment parameters
such as the chemistry teacher, laboratory technologist, school librarian, brilliant
classmates, home-lesson teachers, chemistry groups on social media, parents and siblings,
the students may witness low interest in organic chemistry. RESULTS AND DISCUSSION Histogram of Critical Thinking Ratings (CTR) in Organic Chemistry The research data presented in Figure 2 is a histogram graph that measures students'
critical thinking abilities after being taught Organic Chemistry using ChatGPT artificial Page 64 Page 64 hatGPT in Organic Chemistry Classrooms… | Victor Tubosun Babalola, Suwaiba Said Ahmad, Halima Sani Tafida intelligence. As revealed, students' maximum critical thinking score was 10, while the
average score was 6.58, and the standard deviation was 2.572. This shows that students'
critical thinking scores after teaching Organic Chemistry in the ChatGPT class are high,
with an average score of >50% of the maximum score that can be obtained (5/10). Then,
the researchers analyzed Question Three, namely, What is meant by the Organic Chemistry
Achievement Score in the artificial intelligence ChatGPT Classroom at Gwale Secondary
School, Kano? The complete data produced can be seen in Figure 3. Figure 3. Histogram of Organic Chemistry Achievement Scores (OCATS). Figure 3. Histogram of Organic Chemistry Achievement Scores (OCATS). Figure 3 is the histogram of students’ achievement scores in organic chemistry after
they were taught using the artificial intelligence ChatGPT. The histogram shows a bell
shape indicating a normally distributed score of 50 students in a 30-question Organic
Chemistry Achievement test in the ChatGPT classroom. The histogram further revealed
the mean achievement score of 17.18 and the standard deviation of 7.09. Since the mean
achievement scores of 17.18>15.00 (50% of the maximum obtainable marks) in the
achievement test, it can be concluded that ChatGPT is a practical technological Application
for teaching chemistry. Question Four is: What are the mean interest ratings of students
taught Organic Chemistry in the artificial intelligence ChatGPT Classrooms in Gwale
Secondary Schools, Kano? This research question was also answered using Histogram,
Mean, and Standard Deviation of the descriptive statistics, as shown in Figure 4. (IJETZ) | International Journal of Education and Teaching Zone. Volume 3 (Issue 1): 01-02 (2024)
Figure 4. Histogram of Students’ Interest Rating (SIR) in Organic Chemistry
Page 65 Figure 4. Histogram of Students’ Interest Rating (SIR) in Organic Chemistry Figure 4. Histogram of Students’ Interest Rating (SIR) in Organic Chemistry (IJETZ) | International Journal of Education and Teaching Zone. International Journal of education and Teaching Zone. Volume 3 ( Issue 1): 01-02 (2024) | (IJETZ) RESULTS AND DISCUSSION This findings agreed with that
of Ezike (2018) who discovered that conducive classroom environment correlate students’
academic interest and academic achievement. The study also agreed with Igboanugo
(2023), who found a significant impact of teachers’ experience on students’ interest in
chemistry. Null Hypothesis Two (HO2), There is no significant Relationship between
Social Environment Ratings and Critical Thinking ratings in the ChatGPT’s Organic
Chemistry Classrooms. The data obtained regarding the relationship between social
environmental assessment and students' critical thinking abilities in the classroom learning
process can be seen in more detail in Table 2. Page 66 Page 66 ChatGPT in Organic Chemistry Classrooms… | Victor Tubosun Babalola, Suwaiba Said Ahmad, Halima Sani Tafida Table 2. rho Test of Relationship between Social Environment Ratings and Critical Thinking
ratings in Organic Chemistry’s ChatGPT Classrooms. Statistics Variables
SER
OCATS
Spearman's
rho
Social Environment
Rating (SER)
Correlation
Coefficient
1.000
.912
Sig. (2-tailed)
.000
.000
N
50
50
Organic Chemistry
Achievement Test
Scores (OCATS)
Correlation
Coefficient
.912
1.000
Sig. (2-tailed)
.000
. N
50
50 Table 2. rho Test of Relationship between Social Environment Ratings and Critical Thinking
ti
i O
i Ch
i t
’ Ch tGPT Cl The table 2, revealed the Spearman’s rho of 0.912 indicating a high positive
correlation between social environment rating and the Organic Chemistry Achievement
Scores which is statistically significant at 0.05. (0.001<0.05). Hence, the null hypothesis
HO2 is rejected. That is, there is a significantly high positive correlation between Social
environment ratings and students’ Critical thinking rating in Organic chemistry in the
artificial intelligent ChatGPT classrooms. It can be concluded that Social environment
determines students’ achievement in Organic Chemistry in the artificial intelligence
ChatGPT classes. The result of the null hypothesis two (HO2) revealed a significant
correlation between the students’ social environment and Critical thinking in Organic
Chemistry with a Spearman Rank Order Correlation (rho = 0.912; P<0.001) which is
significant. This implies that, there is a strong positive correlation between Social environment
and student Critical thinking in Organic Chemistry. This positive direction implying that,
if there is increase in students Social parameters there will be a laudable increase in student
Critical thinking towards Organic Chemistry. Conversely, if there is a decline in quality of
student Social environment parameters, there will be low Critical thinking ability of
students towards organic chemistry. JETZ) | International Journal of Education and Teaching Zone. Volume 3 (Issue 1): 01-02 (2024) RESULTS AND DISCUSSION This findings correspond with that of Purwanto,
Rahmawati, Rahmayanti, Mardiah and Amalia (2022), who discovered that improvement
in critical thinking skills encourages students’ involvement in the learning process and
provided the required support for using problem-oriented approach in chemistry
classrooms. Null Hypothesis Three (HO3): There is no Significant Relationship between
the Social Environment Ratings and Organic Chemistry Achievement in the ChatGPT’s
Organic Chemistry Classes. The relationship between social environment ranking and
Organic Chemistry Achievement in the ChatGPT Classroom can be seen in Table 3. (IJETZ) | International Journal of Education and Teaching Zone. Volume 3 (Issue 1): 01-02 (2024)
Table 3. rho Test of relationship between the Social environment ratings and Organic
Chemistry Achievement in ChatGPT Classrooms. Statistical tool Variables
SER
CTR
Spearman's
rho
Social Environment
Ratings (SER)
Correlation Coefficient
1.000
.954
Sig. (2-tailed)
.000
.000
N
50
50
Critical Thinking
Rating (CTR)
Correlation Coefficient
.954
1.000
Sig. (2-tailed)
.000
. N
50
50
Page 67 Table 3. rho Test of relationship between the Social environment ratings and Organic
Chemistry Achievement in ChatGPT Classrooms. Statistical tool Variables
SER
CTR
Spearman's
rho
Social Environment
Ratings (SER)
Correlation Coefficient
1.000
.954
Sig. (2-tailed)
.000
.000
N
50
50
Critical Thinking
Rating (CTR)
Correlation Coefficient
.954
1.000
Sig. (2-tailed)
.000
. N
50
50 Table 3. rho Test of relationship between the Social environment ratings and Organic Page 67 hatGPT in Organic Chemistry Classrooms… | Victor Tubosun Babalola, Suwaiba Said Ahmad, Halima Sani Tafida The table 3, shows that the Spearman’s rho of 0.954 indicating a high positive
correlation between social environment rating and students’ critical thinking rating which
is statisically significant at 0.05. (0.001<0.05). Hence, the HO3 is rejected, there is a
significant correlation between the Social environment ratings and Organic chemistry
achievement scores in the ChatGPT’s Organic Chemistry Classes. Result of this Null hypothesis three (HO3) shows a significant relationship between
the students’ social environment and Academic achievement in organic chemistry with a
correlation coefficient (rho = 0.954; P<0.000) which is statistically significant. This
implies that, there is a high positive correlation between the students’ social environment
and students’ academic achievement in organic chemistry. This positive direction signifies
that, if there is increase in students’ social environment there will be a consequential
increase in students’ achievement in Organic Chemistry. International Journal of education and Teaching Zone. Volume 3 ( Issue 1): 01-02 (2024) | (IJETZ RESULTS AND DISCUSSION The relationship between critical thinking assessments and organic chemistry
achievement scores in the ChatGPT class can be seen in Table 5. Table 5: rho Test of significance in relationship between Critical thinking ratings and organic
chemistry achievement scores in ChatGPT Classrooms. Statistical tool Variables
CTR
OCATS
Spearman's rho
Critical Thinking
Ratings(CTR)
Correlation Coefficient
1.000
.911
Sig. (2-tailed)
.000
.000
N
50
50
Organic Chem. Achievement Test
Scores(OCATS)
Correlation Coefficient
.911
1.000
Sig. (2-tailed)
.000
. N
50
50 Table 5: rho Test of significance in relationship between Critical thinking ratings and organic
chemistry achievement scores in ChatGPT Classrooms. The table6 shows the Spearman’s rho of 0.911 indicating a high correlation
between students’ Critical thinking and Academic achievement test scores in organic
chemistry which is statistically significant at 0.05. (0.001<0.05). Hence, the HO5 is
rejected, That is, there is a significant correlation between the students’ Critical thinking
ratings and Organic chemistry achievement scores in the ChatGPT classrooms. It can be
concluded that students’ critical thinking is needed to impact students’ academic
achievement in Organic Chemistry in the artificial intelligent ChatGPT classroom. The result of null hypothesis (HO5) revealed the correlation between students’
critical thinking ratings and academic achievement scores in Organic Chemistry. There is
a significant correlation between the students’ Critical thinking ratings and Organic
chemistry achievement scores in the ChatGPT classrooms (rho = 0.911; P<0.001). It can
be concluded that students’ critical thinking is needed to impact students’ academic
achievement in Organic Chemistry in the artificial intelligent ChatGPT classroom. Null
Hypothesis Six (HO6): There is no significant relationship between students’ interest and
academic achievement scores in the ChatGPT’s Organic Chemistry Classrooms. Significance of the Relationship between Student Interest Ratings and Academic
Achievement Scores in the ChatGPT Organic Chemistry Class. can be seen in Table 6. Table 6. rho Test of Significance in Relationship between Students’ Interest Ratings and
Academic Achievement Scores in Organic Chemistry’s ChatGPT Classes. Statistical tool Variables
SIR
OCATS
Spearman's
rho
Students Interest
Ratings(SIR)
Correlation Coefficient
1.00
0
.782
Sig. (2-tailed)
. .000
N
50
50
Organic Chem. Achievement Test
Scores(OCATS)
Correlation Coefficient
.782
1.000
Sig. (2-tailed)
.000
. N
50
50
N= Number of students; SIR= Students’ Interest Ratings; OCATS=Organic Chem. Ach. Scores Table 6. rho Test of Significance in Relationship between Students’ Interest Ratings an
Academic Achievement Scores in Organic Chemistry’s ChatGPT Classes. RESULTS AND DISCUSSION The this findings agreed with that
of Malik & Rizvi (2018) and Dangara & Madulili (2019) who found that social
environment increases academic achievements in chemistry among students. Null
Hypothesis Four (HO4), There is no Significant Relationship between Students’ Interest
and Critical Thinking Ratings in the ChatGPT’s Organic Chemistry Classes. The
significance in terms of the relationship between student interest ratings and critical
thinking ratings in the ChatGPT Organic Chemistry Classroom can be seen in Table 4. Table 4. rho Test of significance in relationship between students’ interest ratings and
critical thinking ratings in Organic Chemistry’s ChatGPT Classrooms. Statistical tool Variables
SIR
CTR
Spearman's
rho
Students’ interest
Ratings(SIR)
Correlation Coefficient
1.000
.880
Sig. (2-tailed)
.000
.000
N
50
50
Critical Thinking
Ratings(CTR)
Correlation Coefficient
.880
1.000
Sig. (2-tailed)
.000
. N
50
50 Table 4. rho Test of significance in relationship between students’ interest ratings and
critical thinking ratings in Organic Chemistry’s ChatGPT Classrooms. The table5 revealed the Spearman’s correlation (rho) of 0.880 indicating a high
correlation between the students’ interest rating and critical thinking rating which is
statistically significant at 0.05. (0.001<0.05). Hence, the HO4 is rejected, That is, there is
a statistically significant high positive correlation between the Students’ interest ratings
and critical thinking ratings among the students taught organic chemistry in the ChatGPT
classrooms. It can be concluded that students’ interest in Organic Chemistry predicts their
critical thinking in the ChatGPT’s Organic Chemistry Classes. Result of this Null hypothesis four (HO4) shows a significant relationship between
the students’ interest and critical thinking in organic chemistry with a correlation
coefficient (rho = 0.925; P<0.001) which is very much significant. This indicates a strong
positive correlation between students’ interest and students’ critical thinking in organic
chemistry. This indicated that, whenever there is increase in students’ interest, there will
be a consequential increase in students’ critical thinking ability. Conversely, if there is
decrease in student’ interest, the students may experience low critical thinking in organic
chemistry. Null Hypothesis Five (HO5), There is no Significant Relationship between
Critical thinking and Organic Chemistry Achievement Scores in the ChatGPT’s Organic national Journal of education and Teaching Zone. Volume 3 ( Issue 1): 01-02 (2024) | (IJETZ) Page 68 Page 68 hatGPT in Organic Chemistry Classrooms… | Victor Tubosun Babalola, Suwaiba Said Ahmad, Halima Sani Tafida Classes. (IJETZ) | International Journal of Education and Teaching Zone. Volume 3 (Issue 1): 01-02 (2024)
Page 6 RESULTS AND DISCUSSION The table 6, revealed the Spearman’s rho of 0.782 indicating a moderate correlation
between students’ interest rating and academic achievement in Organic Chemistry which
is statistically significant at 0.05. (0.001<0.05). Hence, the HO6 is rejected. That is, there JETZ) | International Journal of Education and Teaching Zone. Volume 3 (Issue 1): 01-02 (2024) Page 69 hatGPT in Organic Chemistry Classrooms… | Victor Tubosun Babalola, Suwaiba Said Ahmad, Halima Sani Tafida is a significant correlation between the students’ interest ratings and Organic chemistry
achievement scores in the artificial intelligent ChatGPT classrooms. It can be concluded
that students’ interest is needed to positively impact academic achievement in Organic
Chemistry in the ChatGPT classrooms. Result of this null hypothesis (HO6) revealed the correlation between the students’
interest ratings and academic achievement in Organic Chemistry. There is a significant
correlation between the students’ interest ratings and Organic chemistry achievement
scores in the artificial intelligent ChatGPT classrooms (rho = 0.782; P<0.001). It can be
concluded that students’ interest is needed to positively impact academic achievement in
Organic Chemistry in the artificial intelligent ChatGPT classrooms. International Journal of education and Teaching Zone. Volume 3 ( Issue 1): 01-02 (2024) | (IJETZ) CONCLUSIONS The significant challenges confronting chemistry learning opportunities in Nigeria
and globally include lack of student interest, low critical thinking, and underachievement,
which were investigated under this study's artificial intelligence ChatGPT Classroom
condition. Showcases that artificial intelligence (AI) robot teachers and ChatGPT are
migrating into chemistry classrooms. The search for the solution to these academic
challenges brought Bandura's Social Learning Theory forward. The theory assumed that
learners' academic behaviours could be improved due to interactions with the social
environment. The impact of the social climate components such as chemistry teachers, lab
technologists, classmates, home lesson teachers, chemistry groups on social media, and
even the school librarians was examined on students' interest, critical thinking and
academic achievement when taught Organic chemistry using the artificial intelligence
ChatGPT. In each case, a strong positive correlation was found between the students' social
environment and the variables above of investigation in Organic chemistry when taught
with artificial intelligence ChatGPT. Artificial intelligence ChatGPT is a plus to chemistry
education in improving students' interest, critical thinking, and academic achievement,
especially when the social environment conditions are favourable. The study
recommended that the Government re-train chemistry teachers and lab technologists on
using ChatGPT through refresher courses such as workshops and Seminars. When using ChatGPT in Organic Chemistry lessons, students should interact
regularly with Social environments such as chemistry teachers, lab technologists, and
school librarians to improve their interest, critical thinking and academic achievement. The
school managers should employ and retain ICT-literate chemistry teachers, lab-
technologists and school librarians who can support ChatGPT usage. Parents should hire
intelligent Science-based home lesson teachers who can operate ChatGPT for their
children to improve academic achievement in Organic chemistry. The school counsellors
should encourage Group discussions and tutorial classes among classmates using ChatGPT
to enhance students’ interest, critical thinking, and academic achievement in Organic
Chemistry. This study believes that if these recommendations are implemented, AI
ChatGPT could be a practical tool for enhancing students’ interest, critical thinking and
academic achievement in Organic Chemistry and Chemistry in General. Page 70 Page 70 ChatGPT in Organic Chemistry Classrooms… | Victor Tubosun Babalola, Suwaiba Said Ahmad, Halima Sani Tafida JETZ) | International Journal of Education and Teaching Zone. Volume 3 (Issue 1): 01-02 (2024) References Amougou, P.M. & Djekou, C.N. (2020). Advocacy for the empowerment of women
through Non- formal education in the locality of Poli in Northern Cameroon,
Research Journal of Educational Studies and Review 6 (1), 23-28. Abumchukwu,A.A. &Okigbo,E.C. (2023). Social Learning Environment as Correlates of
Secondary School Students’ Attitudes in Chemistry, Irish Journal of Educational
Practice, 6(2), 8-15. https://aspjournals.org/Journals/index.php/ijep. Babalola, V.T. (2023a). Effect of mnemonics enhanced tutorial on chemistry education
students' Achievement and mindfulness in a university, Journal of Mathematics
and Science Teacher, 3(1):1-8. https://doi.org/10.29333/mathsciteacher/13073. Babalola,V.T.(2023b). Inclusive Skill Development for Chemistry Education Programme,
UNESA Journal of Chemical Education, 12(2), 102-111. https://doi.org/10.26740/ujced.v12n2.p102-111 Babalola, V.T. (2023c). Size and Sampling Techniques in Chemistry Education
Research: A Meta-Synthesis. Sci Set J of Physics 2(4), 01-07 Bradford, A and Hamer, A. (2022). Science and the scientific method: Definitions and
examples. The scientific method. Live Science publishers. Available at:
https://www.livescience.com/20896-science-scientific-method.html. Boesdorfer, S. B. (2019). Growing teachers and improving chemistry learning: How Best
Practices in chemistry teacher education can enhance chemistry education. Best
Practice in chemistry teacher education. Washington, DC: ACS. Chukwuemeka,O. (2013). Environmental Influence on Academic Performance of
Secondary School Students in Port Harcourt Local Government Area of Rivers
State, Journal of Economics and Sustainable Development, 4(12), 34-38. Dangara, U.Y. & Madulili, C.G. (2019). Evaluation of the Effect of Learning Environment
On Student’s Academic Performance in Nigeria, Chukwuemeka Odumegwu
Ojukwu University, Uli, Anambra State, Nigeria. Ezike,B.U.(2018). Classroom Environment and Academic Interest as Correlates of
Achievement in Senior Secondary School Chemistry in Ibadan South West Local
Government Area, Oyo State, Nigeria Global Journal of Educational Research 17,
61-71. https://dx.doi.org/10.4314/gjedr.v17i1.9 Eilks, I. & Hofstein, A. (2013). Teaching chemistry, Sense Publishers. Pp269-298 Freedman, B. (2018). Science as a Way of Understanding the Natural World,
Environmental Science. https://ecampusontario.pressbooks.pub/environmentalscience/chapter/chapter-2-
copy-paste-from-gitbook/ Harackiewicz, J.M Smith, J.L. and Priniski, S.J. (2016). Interest Matters: The Importance
of Promoting Interest in Education, Policy Insights Behavour Brain Sci., 3(2),
220-227. 10.1177/2372732216655542. Harinie,L. T., Sudiro ,A., Rahayu, M. & Fatchan, A. (2017). Study of the Bandura’s social
Cognitive learning theory for the entrepreneurship learning process. Social
Sciences 6(1): 1-6. http://www.sciencepublishinggroup.com/j/ss International Journal of Education and Teaching Zone. Volume 3 (Issue 1): 01-02 (2024) Page 71 Page 71 ChatGPT in Organic Chemistry Classrooms… | Victor Tubosun Babalola, Suwaiba Said Ahmad, Halima Sani Tafida Ali, H., Babalola, V.T. & Rahinatu, H.I. (2023). Teachers' Perceived Effect of School-
Based Planning (SBP) on Chemistry Achievement in Kano Metropolis Secondary
Schools, Nigeria. Int. Jo. International Journal of education and Teaching Zone. Volume 3 ( Issue 1): 01-02 (2024) | (IJETZ) References of Innovative Research in Edu., Tech. & Soc. Strategies
10(1), 10-22. https://doi.org/10.48028/iiprds/ijiretss.v10.i1.02 Igbaji, C., Miswaru, B. & Sunusi, S.A. (2017). Science Education and Nigeria National
Development Effort: The Missing Link. Int. Jo. of Edu. and Eval., 3(5),46-56. Igboanugo, B.I. (2023). Interactive Effects of Guided Inquiry and Teachers’ Experience
On Chemistry Students’ Interest, Journal of Chemistry, 2013, 1-8. https://doi.org/10.1155/2023/9970946. Kay, D. & Kibble, J. (2016). Learning theories 101: application to everyday teaching and
Scholarship, Advanced Physiological Education 40, 17-25 Malik,R.H. & Rizvi, A.A. (2018). Effect of Classroom Learning Environment on Students'
Academic Achievement in Mathematics at Secondary Level, Bulletin of Education
And Research 40(2), 207-218. Mills,S.(2016). Conceptual Understanding: A Concept Analysis, Qualitative Report,
21(3), 546-557. Nababi, R.T. (2014). Bandura’s social learning theory and social cognitive learning
theory. http:/www.researchgate.net. Nubunga, 1. (2020). Social learning environment and student academic performance in
Federal College of education Jaria Kaduna state, BSU Journal of Science
Mathematics and Computer Education, 1(2), 115-124. Taber, K. S. (2015). Advancing chemistry education as a field, Chemistry Education
Research and Practice, 1(6): 6-8. Tsaparlis, G. (2017). Problems and Solutions in Chemistry Education, Journal of the
Turkish Chemical Society; Chemical Education, 1(1): 1-30. University of California, (2013). What is Science? Berkeley and the Regents of the
University of California. www.understandingscience.org Zhou, M. & Brown, D. (2015). Educational Learning Theories: 2nd Edition. Galileo:
University System of Georgia. Ennis, R. H. (2015). The Nature of Critical Thinking: Outlines of General Critical
Thinking Disposition and Abilities. MIT, Conference Presentation, Cambrigde
University. Roberts, J. D. (1977). Introduction to what Organic Chemistry is all about. Available at:
https://authors.library.caltech.edu/25034/2/BPOCchapter1.pdf. Rokhim, D.A., Widarti, H. R. & Permatasari, A. (2023). The Effectiveness of STAD
Cooperative learning models on critical thinking ability and student learning
Outcomes on Chemical Equilibrium. UNESA Journal of Chemical Education,
12(2), 132-140. Page 72 Page 72
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Post-Stroke Inhibition of Induced NADPH Oxidase Type 4 Prevents Oxidative Stress and Neurodegeneration
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Abstract Ischemic stroke is the second leading cause of death worldwide. Only one moderately effective therapy exists, albeit with
contraindications that exclude 90% of the patients. This medical need contrasts with a high failure rate of more than 1,000
pre-clinical drug candidates for stroke therapies. Thus, there is a need for translatable mechanisms of neuroprotection and
more rigid thresholds of relevance in pre-clinical stroke models. One such candidate mechanism is oxidative stress. However, antioxidant approaches have failed in clinical trials, and the significant sources of oxidative stress in stroke are
unknown. We here identify NADPH oxidase type 4 (NOX4) as a major source of oxidative stress and an effective therapeutic
target in acute stroke. Upon ischemia, NOX4 was induced in human and mouse brain. Mice deficient in NOX4 (Nox42/2) of
either sex, but not those deficient for NOX1 or NOX2, were largely protected from oxidative stress, blood-brain-barrier
leakage, and neuronal apoptosis, after both transient and permanent cerebral ischemia. This effect was independent of age,
as elderly mice were equally protected. Restoration of oxidative stress reversed the stroke-protective phenotype in Nox42/2
mice. Application of the only validated low-molecular-weight pharmacological NADPH oxidase inhibitor, VAS2870, several
hours after ischemia was as protective as deleting NOX4. The extent of neuroprotection was exceptional, resulting in
significantly improved long-term neurological functions and reduced mortality. NOX4 therefore represents a major source
of oxidative stress and novel class of drug target for stroke therapy. Citation: Kleinschnitz C, Grund H, Wingler K, Armitage ME, Jones E, et al. (2010) Post-Stroke Inhibition of Induced NADPH Oxidase Type 4 Prevents Oxidative
Stress and Neurodegeneration. PLoS Biol 8(9): e1000479. doi:10.1371/journal.pbio.1000479
Academic Editor: Malcolm McLeod, University of Edinburgh, United Kingdom Citation: Kleinschnitz C, Grund H, Wingler K, Armitage ME, Jones E, et al. (2010) Post-Stroke Inhibition of Induced NADPH Oxidase Type 4 Prevents Oxidative
Stress and Neurodegeneration. PLoS Biol 8(9): e1000479. doi:10.1371/journal.pbio.1000479 Academic Editor: Malcolm McLeod, University of Edinburgh, United Kingdom Received February 19, 2010; Accepted July 28, 2010; Published September 21, 2010 nschnitz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
tion, and reproduction in any medium, provided the original author and source are credited. Copyright: 2010 Kleinschnitz et al. This is an open-access article distributed under the terms of the Creative Commons Attribut
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract Funding: This work was supported by the NHMRC, Australia, the Deutsche Forschungsgemeinschaft (DFG), Germany (to HHHWS and CK), and by the
Bundesministerium fur Bildung und Forschung within the framework of the NGFN-Plus and the European Commission (EUMODIC). The funders had no role in
study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: HHHWS and KW declare a potential competing interest as shareholder and previous employee, respectively, of Vasopharm GmbH, which
develops NADPH oxidase inhibitors such as VAS2870. All authors declare that they adhere to all PLoS Biology policies on sharing data and materials as detailed in
the PLoS Biology guide for authors. Abbreviations: CISS, constructive interference in steady state; KO, knock out; pMCAO, permanent middle cerebral artery occlu
species; rt-PA, recombinant tissue plasminogen activator; tMCAO, transient middle cerebral artery occlusion; TTC, 2,3,5-triphenyltet
type. onstructive interference in steady state; KO, knock out; pMCAO, permanent middle cerebral artery occlusion; ROS, reactive oxygen
nt tissue plasminogen activator; tMCAO, transient middle cerebral artery occlusion; TTC, 2,3,5-triphenyltetrazolium chloride; WT, wild * E-mail: h.schmidt@farmaco.unimaas.nl (HHHWS); christoph.kleinschnitz@mail.uni-wuerzburg.de (CK) Post-Stroke Inhibition of Induced NADPH Oxidase Type 4
Prevents Oxidative Stress and Neurodegeneration Christoph Kleinschnitz1*, Henrike Grund2, Kirstin Wingler2,3,4,5, Melanie E. Armitage3,5, Emma Jones3,
Manish Mittal2, David Barit6, Tobias Schwarz1, Christian Geis1, Peter Kraft1, Konstanze Barthel7,
Michael K. Schuhmann1,8, Alexander M. Herrmann1,8, Sven G. Meuth1,8, Guido Stoll1, Sabine Meurer3,
Anja Schrewe9, Lore Becker9,10, Vale´rie Gailus-Durner9, Helmut Fuchs9, Thomas Klopstock10, Martin
Hrabe´ de Angelis9,11, Karin Jandeleit-Dahm6, Ajay M. Shah12, Norbert Weissmann2, Harald H. H. W. Schmidt2,3,4,5* 1 Neurologische Klinik und Poliklinik, Universita¨t Wu¨rzburg, Wu¨rzburg, Germany, 2 Rudolf-Buchheim-Institut fu¨r Pharmakologie & Medizinische Klinik, Justus-Liebig-
Universita¨t, Gießen, Germany, 3 Department of Pharmacology and Centre for Vascular Health, Monash University, Melbourne, Australia, 4 Department of Pharmacology
and Toxicology and Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, The Netherlands, 5 National Stroke Research Institute, Florey
Neuroscience Institutes, Melbourne, Australia, 6 Baker IDI Heart and Diabetes Institute, Juvenile Diabetes Research Foundation (JDRF) International Center for Diabetic
Complications Research, Melbourne, Australia, 7 Abteilung Neurologie, Georg-August Universita¨t Go¨ttingen, Go¨ttingen, Germany, 8 Universita¨tsklinik Mu¨nster, Klinik und
Poliklinik fu¨r Neurologie—Entzu¨ndliche Erkrankungen des Nervensystems und Neuroonkologie, Mu¨nster, Germany, 9 Institute of Experimental Genetics, Helmholtz
Zentrum Mu¨nchen, German Research Center for Environmental Health, Mu¨nchen, Germany, 10 Friedrich-Baur-Institut an der Neurologischen Klinik, Klinikum der Ludwig-
Maximilians-Universita¨t Mu¨nchen, Mu¨nchen, Germany, 11 Lehrstuhl fu¨r Experimentelle Genetik, Technische Universita¨t Mu¨nchen, Freising-Weihenstephan, Germany,
12 King’s College London School of Medicine, The James Black Centre, Cardiovascular Division, London, United Kingdom PLoS Biology | www.plosbiology.org NOX4 Is Induced during Ischemic Stroke in Mice and
Humans Because NOX4 mRNA is expressed at higher levels in cerebral
than in peripheral blood vessels [13] and is induced in stroke [14],
we first sought to validate these data not only at the mRNA but
also at the protein level. In all experiments, we followed current
guidelines defining methodological standards for experimental
stroke studies [4,6,7,16,17]. Here we chose a model of acute
ischemic stroke in which mice are subjected to transient middle
cerebral artery occlusion (tMCAO). This disease model is thought
to involve oxidative stress and an induction of Nox4 expression
[18]. Indeed, expression of NOX4 mRNA was significantly higher
12 h and 24 h after tMCAO in the basal ganglia and neocortex of
wild-type mice than in sham-operated controls, in which basal
NOX4 expression was low (Figure 1A). This result was validated
by immunohistochemistry using a specific NOX4 antibody. We
detected a stronger staining in neurons and cerebral blood vessels
in wild-type mice subjected to tMCAO than in sham-operated
controls. Although immunohistochemistry is not quantitative, this
finding suggests higher levels of NOX4 protein (Figure 1B). Importantly, NOX4 staining was also stronger in brain samples
from stroke patients. Although NOX4 was barely detectable in
healthy brain regions, clear positive labeling of NOX4 was seen in
neurons and vascular endothelial cells from the forebrain cortex of
stroke patients. This finding was confirmed by double labeling for
NeuN (a neuronal marker) or von Willebrand factor (an
endothelial marker) and NOX4 in brain tissue (Figure 1B). These
data indicate that NOX4 protein is induced during brain ischemia
in mice, and this observation would be in agreement with a major
functional role for NOX4 in ischemic stroke. Our limited
observations in a small number of human cases provide some
support to the hypothesis that these processes are also important in
human stroke. Although a plethora of drugs for the treatment of acute stroke
are effective in animal models, their translation into clinical
practice has completely failed [3,4]. As a result, many pharma-
ceutical companies have withdrawn from drug discovery in this
area. To overcome this lack of clinically effective neuroprotective
drugs, innovative strategies are urgently needed to identify
pathways that can be targeted with innovative therapies [5]. Higher quality study designs are also required [6,7]. One such high-potential pathway in ischemic stroke may be the
occurrence of oxidative stress, i.e., the increased occurrence of
reactive oxygen species (ROS) above physiological levels. NOX4 Is Induced during Ischemic Stroke in Mice and
Humans Oxida-
tive stress has been suggested for many years to cause tissue
damage and neuronal death. The toxicity of ROS can be further
increased by nitric oxide to produce reactive nitrogen species such
as peroxynitrite (ONOO2), a molecule that causes oxidation and
nitration of tyrosine residues on proteins [8]. Disappointingly,
there is no conclusive evidence of a causal link between oxidative
stress and the development of disease, and there is no successful
therapeutic application targeting oxidative stress. To date, clinical
attempts to scavenge ROS by applying antioxidants did not result
in clinical benefit [9] or even caused harm [10,11]. However, the
characterization of the relevant enzymatic sources of oxidative
stress may allow therapeutic targeting of oxidative stress by
preventing the formation of ROS in the first place, instead of
scavenging ROS after they have been formed. Nox42/2 but Neither Nox1y/2 nor Nox2y/2 Mice Are
Protected in Both Transient and Permanent Ischemic
Stroke We first subjected 6- to 8-wk-old male Nox42/2 mice to tMCAO
and, after 24 h, assessed infarct volumes by staining brain sections
with 2,3,5-triphenyltetrazolium chloride (TTC) (Figure 2A). Infarct
volumes were significantly smaller, by approximately 75%, in male
Nox42/2
mice
than
in
sex-matched
wild-type
controls
(25.5614.8 mm3 versus 78.7619.5 mm3, respectively). The smaller
infarct volume was functionally relevant: compared with wild-type
mice, Nox42/2 mice had significantly better overall neurological
function (Bederson score 1.260.7 in Nox42/2 mice versus 3.761.1
in wild-type mice) as well as better basal motor function and
coordination (grip test score 4.360.7 in Nox42/2 mice versus
1.761.3 in wild-type mice) 24 h after tMCAO (Figure 2B). Gender
can significantly influence stroke outcome in rodents [4,16,17]. Therefore, we also subjected female Nox42/2 mice to 60 min of
tMCAO. In line with the results in male mice, Nox4-deficient
female
mice
also developed significantly
smaller infarctions A potential source of ROS are NADPH oxidases, the only
known enzyme family that is only dedicated to ROS production
[12]. Four rodent genes of the catalytic subunit NOX, Nox1, Nox2,
Nox3, and Nox4, have been identified, of which Nox1, Nox2, and
Nox4 are expressed in the vasculature. NOX4 is the most
abundant vascular isoform; its expression is even higher in
cerebral than in peripheral blood vessels [13] and, further,
induced in stroke [14]. Therefore, we hypothesized that NOX4 is
the most relevant source of ROS in stroke. Author Summary Stroke is the second leading cause of death worldwide. Today, only one approved therapy exists—a drug that
breaks down blood clots—the effectiveness of which is
moderate, and it can only be used in about 10% of
patients because of contraindications. New therapeutic
strategies that are translatable to humans and more rigid
thresholds of relevance in pre-clinical stroke models are
needed. One candidate mechanism is oxidative stress,
which is the damage caused by reactive oxygen species
(ROS). Antioxidant approaches that specifically target ROS
have thus far failed in clinical trials. For a more effective
approach, we focus here on targeting ROS at its source by
investigating an enzyme involved in generating ROS,
known as NADPH oxidase type 4, or NOX4. We found that
NOX4 causes oxidative stress and death of nerve cells after
a stroke. Deletion of the NOX4-coding gene in mice, as
well as inhibiting the ROS-generating activity of NOX with
a pharmacological inhibitor, reduces brain damage and
improves neurological function, even when given hours
after a stroke. Importantly, neuroprotection was preserved
in old male and female Nox42/2 mice as well as in Nox42/2
mice
subjected
to
permanent
ischemia. NOX4
thus
represents a most promising new therapeutic target for
reducing oxidative stress in general, and in brain injury due
to stroke in particular. Introduction [3], only one therapy approved by the United States Food and
Drug Administration is available, i.e., thrombolysis using recom-
binant tissue plasminogen activator (rt-PA). However, the efficacy
of rt-PA on functional outcomes is moderate at best, and more
than 90% of all stroke patients must be excluded from rt-PA
treatment because of over 25 labeled contraindications. Therefore,
there is an unmet need for more effective therapies in acute stroke. Ischemic stroke has outstanding medical relevance as it is the
second leading cause of death in industrialized countries [1]. Due
to the aging of the population, the incidence of stroke is projected
to rise even further in the future [2]. Despite tremendous research
activity, with more than 100 clinical trials in human stroke patients PLoS Biology | www.plosbiology.org September 2010 | Volume 8 | Issue 9 | e1000479 September 2010 | Volume 8 | Issue 9 | e1000479 1 Role of NOX4 in Stroke has been implicated in the regulation of systemic and hypoxic
vascular responses. Therefore, we had to exclude systemic vascular
effects of NOX4 deletion on blood pressure, which may affect
stroke outcome independent of a specific neuronal or neurovas-
cular mechanism. Finally, to examine the therapeutic potential of
NOX4 as a drug target, we infused the specific NADPH oxidase
inhibitor VAS2870 [15] after ischemia, thus mirroring the clinical
scenario. PLoS Biology | www.plosbiology.org September 2010 | Volume 8 | Issue 9 | e1000479 NOX4 Is Induced during Ischemic Stroke in Mice and
Humans However, in contrast to Nox42/2 mice, we observed no protection
in these animals, neither in terms of infarct volumes nor on
functional outcomes on day 1 after tMCAO, even with large
subject sample sizes (n = 19 for Nox2y/2 mice, p.0.05; Figure 2A). Ischemic stroke is usually a disease of the elderly and,
consequently, one should verify any stroke-protective effects
observed in young adult laboratory animals also in an older
cohort [4,16,17]. Indeed, 18- to 20-wk-old Nox42/2 mice also
developed significantly smaller brain infarctions (27.8615.1 mm3
versus 81.8619.0 mm3, respectively) and less severe neurological
deficits than age-matched controls, thereby confirming our results
in young animals (Figure 2B). We also determined the functional
outcome and mortality of 6- to 8-wk-old male Nox42/2 mice and
matched wild-type controls over a longer time period after
ischemic stroke (Figure 2D). Five days after 60 min of tMCAO, 15
of 15 wild-type mice (100%) had died, which is in line with
previous reports [19]. In contrast, seven of ten Nox42/2 mice
(70%) survived until day 5, and five of these were still alive after
1 wk (p = 0.0039) (Figure 2D). In line with these findings, Nox4-
deficient mice showed significantly better Bederson scores than
controls over the whole observation period, and neurological
deficits remained low until day 7 (Figures 2D and S4). (30.166.7 mm3 versus 89.5622.2 mm3, respectively) and less
severe
neurological
deficits
(p,0.001)
than
female
controls
(Figure 2A and 2B). Histological analysis revealed that all infarcts
in Nox42/2 mice were restricted to the basal ganglia (arrow in
Figure 2A and 2C), whereas in wild-type mice, the neocortex was
also consistently affected. Serial magnetic resonance imaging of
living mice up to 6 d after stroke showed that in Nox42/2 mice the
infarct volume did not increase over time, thus indicating that
deletion of the Nox4 gene provides sustained protection against
stroke (Figure 2C). Moreover, infarcts always appeared hyperin-
tense on blood-sensitive constructive interference in steady state
(CISS) sequences. Hypointense areas, which typically indicate
intracerebral hemorrhage, were absent from Nox42/2 mice and
wild-type controls. This finding excludes the possibility of an
increased rate of bleeding complications caused by Nox4 deficiency. are restricted to the cortex and highly reproducible in size and
location. Moreover, photothrombosis has been shown to induce
early and profound ROS formation and blood-brain-barrier
leakage [20,21], two key readout parameters of the present
investigation. No Apparent Vascular Phenotype of Nox42/2 Mice Other
Than in Stroke In line with these findings, Nox4-
deficient mice showed significantly better Bederson scores than
controls over the whole observation period, and neurological
deficits remained low until day 7 (Figures 2D and S4). Protection from Ischemic Stroke in Nox42/2 Mice Is a
Result of Reduced Oxidative Stress, Neuronal Apoptosis,
and Blood-Brain-Barrier Leakage NOX4 Is Induced during Ischemic Stroke in Mice and
Humans Importantly, photothrombosis-induced infarct vol-
umes were as reduced in Nox42/2 mice relative to wild-type mice
(3.364.6 mm3 versus 25.0612.8 mm3, respectively, a difference
of 86.8%; Figure 2F) as they were in the tMCAO model. PLoS Biology | www.plosbiology.org Protection from Ischemic Stroke in Nox42/2 Mice Is a
Result of Reduced Oxidative Stress, Neuronal Apoptosis,
and Blood-Brain-Barrier Leakage Next we sought to elucidate the underlying mechanisms of this
NOX4-specific neurotoxicity in stroke. NOX4 can form superox-
ide or H2O2, which can interact with nitric oxide to form reactive
nitrogen species. Therefore, we stained brain sections with broad-
spectrum indicators of oxidative/nitrative stress, i.e., dihydroethi-
dium [28] and nitrotyrosine [8]. At 12 h and 24 h after tMCAO,
brains from wild-type mice exhibited a significantly larger amount
(by a factor of 2.5–3.5) of ROS in neurons than brains from sham-
operated animals, as quantified by dihydroethidium staining
(Figure 3A). Neurons from Nox42/2 mice, in contrast, showed
only very small ischemia-induced increases in ROS relative to
those in sham-operated controls (p.0.05). ROS formation from
neurons after 24 h was also significantly reduced in Nox42/2 mice
subjected to pMCAO (Figure S6). Because the dihydroethidium
stain may also indicate oxidative chemistry events, including
formation of ONOO2 and nitration of protein tyrosine residues
[8], we analyzed the extent of protein nitration in Nox42/2 and
wild-type mice subjected to tMCAO. In agreement with our
findings on the generation of ROS, tissue nitration occurred to a
lesser extent in ischemic brains from Nox42/2 mice than in those
from wild-type controls (Figure 3B). Oxidative chemistry events
such as the formation of ROS and peroxynitrite, as detected by According
to
the
current
experimental
stroke
guidelines
[4,16,17], any protective effect also requires evaluation in models
of both transient and permanent ischemia. We therefore subjected
Nox42/2 mice to filament-induced permanent middle cerebral
artery occlusion (pMCAO), a procedure in which no tissue
reperfusion occurs (Figure 2E). In the absence of Nox4, infarct
volumes (66.7628.6 mm3 versus 120.1615.6 mm3, p,0.05) and
neurological deficits (Bederson score 2.361.7 versus 3.460.8,
p,0.05) at day 1 after pMCAO were significantly reduced
compared with those in wild-type controls, although to a lesser
extent than they were in the tMCAO model (Figures 2E and S5). Brain infarctions following filament-induced pMCAO are large,
and the infarct borders are often not very well defined, which
limits the accuracy of any estimation on infarct volumes. We
therefore used another model of permanent stroke, cortical
photothrombosis, to further verify our findings. Here, the lesions According
to
the
current
experimental
stroke
guidelines
[4,16,17], any protective effect also requires evaluation in models
of both transient and permanent ischemia. We therefore subjected
Nox42/2 mice to filament-induced permanent middle cerebral
artery occlusion (pMCAO), a procedure in which no tissue
reperfusion occurs (Figure 2E). NOX4 Is Induced during Ischemic Stroke in Mice and
Humans To test this hypothesis, we generated constitutively NOX4-
deficient (Nox42/2) mice and directly compared them to NOX1-
deficient (Nox1y/2) and NOX2-deficient (Nox2y/2) mice. NOX4 September 2010 | Volume 8 | Issue 9 | e1000479 2 PLoS Biology | www.plosbiology.org Role of NOX4 in Stroke PLoS Biology | www.plosbiology.org September 2010 | Volume 8 | Issue 9 | e1000479 PLoS Biology | www.plosbiology.org September 2010 | Volume 8 | Issue 9 | e1000479 3 Role of NOX4 in Stroke Role of NOX4 in Stroke Figure 1. Induction of NOX4 expression after ischemic stroke in mice and humans. (A) Relative gene expression of Nox4 in the ischemic
basal ganglia (left) and cortex (right) of wild-type mice after sham operation and 4 h, 12 h, and 24 h after tMCAO (n = 5). *, p,0.05, one-way ANOVA,
Bonferroni post-hoc test, compared with sham-treated controls. (B) Immunohistochemical detection of NOX4 protein in ischemic brains of wild-type
mice (after sham operation or tMCAO, day 1) and humans (samples from stoke patients, after routine autopsy). We compared NOX4 immunolabeling
in the ischemic forebrain cortex and the unaffected contralateral side. In ischemic samples, NOX4 was predominantly expressed in neurons
(arrowheads) and endothelial cells (arrows). This distribution was confirmed by visualization of NOX4 and NeuN or NOX4 and von Willebrand Factor in
the same structures. All scale bars represent 100 mm. doi:10.1371/journal.pbio.1000479.g001 (30.166.7 mm3 versus 89.5622.2 mm3, respectively) and less
severe
neurological
deficits
(p,0.001)
than
female
controls
(Figure 2A and 2B). Histological analysis revealed that all infarcts
in Nox42/2 mice were restricted to the basal ganglia (arrow in
Figure 2A and 2C), whereas in wild-type mice, the neocortex was
also consistently affected. Serial magnetic resonance imaging of
living mice up to 6 d after stroke showed that in Nox42/2 mice the
infarct volume did not increase over time, thus indicating that
deletion of the Nox4 gene provides sustained protection against
stroke (Figure 2C). Moreover, infarcts always appeared hyperin-
tense on blood-sensitive constructive interference in steady state
(CISS) sequences. Hypointense areas, which typically indicate
intracerebral hemorrhage, were absent from Nox42/2 mice and
wild-type controls. This finding excludes the possibility of an
increased rate of bleeding complications caused by Nox4 deficiency. To establish any potential specificity of this function for NOX4
compared to NOX1 and NOX2 in stroke, we carried out identical
experiments in 6- to 8-wk-old Nox1y/2 and Nox2y/2 mice. No Apparent Vascular Phenotype of Nox42/2 Mice Other
Than in Stroke Based on the physiological distribution of NOX4 in kidney [22],
lung [23], and aorta [24], as well as cell biology data obtained
using small interfering RNA approaches [23], one would predict
basal
phenotypes
in
a
Nox42/2
mouse,
such
as
arterial
hypotension, reduced hypoxic pulmonary hypertension, and
altered renal function. Importantly, these effects could potentially
modulate or interfere with stroke outcome even in the absence of a
specific neuronal or neurovascular mechanism. Surprisingly,
systemic elimination of Nox4 did not result in any apparent
abnormal vascular phenotype (Text S1; Figures S1 and S2; Table
S1). In particular, blood pressure was normal, and hypoxic
pulmonary hypertension still occurred despite a 20-fold induction
of NOX4 in wild-type animals [23]. In contrast, Nox1- and
p47phox-deficient mice (a Nox2 subunit) have a lower basal blood
pressure, and their blood-pressure response to angiotensin II is
reduced [25–27]. Our data suggest that any phenotype caused by
deleting Nox4, unlike those caused by deleting Nox1 and Nox2,
would indeed be brain-specific. To establish any potential specificity of this function for NOX4
compared to NOX1 and NOX2 in stroke, we carried out identical
experiments in 6- to 8-wk-old Nox1y/2 and Nox2y/2 mice. However, in contrast to Nox42/2 mice, we observed no protection
in these animals, neither in terms of infarct volumes nor on
functional outcomes on day 1 after tMCAO, even with large
subject sample sizes (n = 19 for Nox2y/2 mice, p.0.05; Figure 2A). Ischemic stroke is usually a disease of the elderly and,
consequently, one should verify any stroke-protective effects
observed in young adult laboratory animals also in an older
cohort [4,16,17]. Indeed, 18- to 20-wk-old Nox42/2 mice also
developed significantly smaller brain infarctions (27.8615.1 mm3
versus 81.8619.0 mm3, respectively) and less severe neurological
deficits than age-matched controls, thereby confirming our results
in young animals (Figure 2B). We also determined the functional
outcome and mortality of 6- to 8-wk-old male Nox42/2 mice and
matched wild-type controls over a longer time period after
ischemic stroke (Figure 2D). Five days after 60 min of tMCAO, 15
of 15 wild-type mice (100%) had died, which is in line with
previous reports [19]. In contrast, seven of ten Nox42/2 mice
(70%) survived until day 5, and five of these were still alive after
1 wk (p = 0.0039) (Figure 2D). Protection from Ischemic Stroke in Nox42/2 Mice Is a
Result of Reduced Oxidative Stress, Neuronal Apoptosis,
and Blood-Brain-Barrier Leakage In the absence of Nox4, infarct
volumes (66.7628.6 mm3 versus 120.1615.6 mm3, p,0.05) and
neurological deficits (Bederson score 2.361.7 versus 3.460.8,
p,0.05) at day 1 after pMCAO were significantly reduced
compared with those in wild-type controls, although to a lesser
extent than they were in the tMCAO model (Figures 2E and S5). September 2010 | Volume 8 | Issue 9 | e1000479 4 Role of NOX4 in Stroke Role of NOX4 PLoS Biology | www.plosbiology.org
5
September 2010 | Volume 8 | Issue 9 | September 2010 | Volume 8 | Issue 9 | e1000479 PLoS Biology | www.plosbiology.org September 2010 | Volume 8 | Issue 9 | e1000479 PLoS Biology | www.plosbiology.org Role of NOX4 in Stroke Figure 2. Nox4 deficiency confers long-term neuroprotection and reduces mortality after acute ischemic stroke in young adult and
aged mice of either sex. (A) Upper panel shows representative TTC staining of three corresponding coronal brain sections of 6- to 8-wk-old male
and female wild-type (WT) mice, male Nox1y/2 mice, male Nox2y/2 mice, and male and female Nox42/2 mice, as well as 18- to 20-wk-old male wild-
type and Nox42/2 mice on day 1 after tMCAO. The ischemic infarcts (white) appear smallest in the Nox42/2 mice of either age or sex (arrows), and this
result was confirmed by infarct volumetry (lower panel). ***, p,0.0001, and **, p,0.001, one-way ANOVA, Bonferroni post-hoc test compared with
wild-type mice (n = 8–19 per group). (B) Neurological Bederson score (upper panel) and motor score (lower panel) on day 1 after tMCAO in the eight
mouse groups indicated above. (C) Serial magnetic resonance images of cerebral infarcts 1 d and 6 d after tMCAO in wild-type and Nox42/2 mice
(lower panel). The broken white lines show hyperintense ischemic lesions on day 1 after tMCAO in wild-type and Nox42/2 mice. Infarcts on day 1 are
smaller in Nox42/2 mice than in wild-type mice and remain restricted to the basal ganglia on day 6. Hematoxylin and eosin staining confirmed
neuronal damage in the cortex of wild-type mice 24 h after tMCAO (top panel, left), whereas cortical integrity was preserved in Nox42/2 mice (top
panel, right). (D) Mortality (upper panel) and long-term functional outcome (Bederson score, lower panel) in 6- to 8-wk-old male Nox42/2 mice and
wild-type controls. Survival curve (upper panel): **, p = 0.0039, log-rank test compared with wild-type mice (n = 10–15 per group). Protection from Ischemic Stroke in Nox42/2 Mice Is a
Result of Reduced Oxidative Stress, Neuronal Apoptosis,
and Blood-Brain-Barrier Leakage Again, post-stroke application of
VAS2870 to Nox42/2 mice had no additive neuroprotective or
superoxide-lowering effect compared to the outcomes in wild-type
animals treated with VAS2870 or untreated Nox42/2 mice
(Figure 4B–4D). This observation is consistent with our ex vivo
findings in ischemic brain slices and reaffirms that NOX4 rather than
NOX1 or NOX2 is critically involved in the pathophysiology of
ischemic stroke. Another, less specific inhibitor that also targets
molecules other than NADPH oxidases [36,37], apocynin, had no
effect on infarct size or functional outcome when given post-stroke
and did not reduce the formation of ROS in vivo (Figure 4B and 4C). To further examine whether the neuroprotective effect observed
in Nox42/2 mice is specifically related to reduced ROS formation
and not due to other nonspecific or developmental defects, we
performed a rescue experiment by restoring cerebral ROS levels in
Nox42/2 mice during the course of ischemic stroke by applying
exogenous H2O2 (Figure 4B–4D). Indeed, intrathecal administra-
tion of H2O2 rescued the phenotype in Nox42/2 mice, and infarct
volumes, functional deficits, and stroke-induced ROS formation
returned to the levels observed in wild-type mice (Figure 4B–4D). To determine whether VAS2870 is also active when applied in
vivo, we administered 2 mg of VAS2870 intrathecally to wild-type
mice 2 h and 12 h after tMCAO. This experimental therapeutic
approach significantly reduced brain infarct volumes (20.764.0 mm3
in VAS2870-treated mice versus 82.466.4 mm3 in vehicle-treated
controls) and significantly improved neurological function, to the
same extent as observed for the deletion of Nox4 in mice (Figure 4B
and 4C). Moreover, less oxidative stress was detected in ischemic
brains from VAS2870-treated animals than in those from vehicle-
treated controls (Figure 4D). Again, post-stroke application of
VAS2870 to Nox42/2 mice had no additive neuroprotective or
superoxide-lowering effect compared to the outcomes in wild-type
animals treated with VAS2870 or untreated Nox42/2 mice
(Figure 4B–4D). This observation is consistent with our ex vivo
findings in ischemic brain slices and reaffirms that NOX4 rather than
NOX1 or NOX2 is critically involved in the pathophysiology of
ischemic stroke. Another, less specific inhibitor that also targets
molecules other than NADPH oxidases [36,37], apocynin, had no
effect on infarct size or functional outcome when given post-stroke
and did not reduce the formation of ROS in vivo (Figure 4B and 4C). g
p
(p
) (
g
)
We also detected NOX4 in cerebral blood vessels (Figure 1B,
white arrow indicates endothelial cells). Treatment with the NOX Inhibitor VAS2870 Effectively
Protects Ischemic Brain Damage Even When Applied
After Stroke Treatment with the NOX Inhibitor VAS2870 Effectively
Protects Ischemic Brain Damage Even When Applied
After Stroke Finally, we wanted to examine whether these genetic insights
into the biology of oxidative stress in stroke and the role of NOX4
in general can be translated into a therapeutic intervention. Importantly, this intervention would have to be effective post-
stroke and ideally it would be pharmacological. Therefore, we
examined the efficacy of a validated, low-molecular-weight
NADPH oxidase inhibitor, VAS2870 [15,32–34], in vital brain
slices and in vivo. VAS2870 equally inhibits the ROS-generating
activity of all NOX subunits, i.e., NOX1, NOX2, and NOX4. Vital brain slices [35] taken from wild-type mice 12 h after
tMCAO produced significantly less ROS after pretreatment with
10 mM VAS2870, as did brain slices from untreated Nox42/2 mice
(Figure 4A). Importantly, incubating ischemic slices from Nox42/2
mice with VAS2870 had no additional inhibitory effect on
superoxide formation (Figure 4A). This finding further underlines
the extraordinary role of NOX4 in generating oxidative stress Protection from Ischemic Stroke in Nox42/2 Mice Is a
Result of Reduced Oxidative Stress, Neuronal Apoptosis,
and Blood-Brain-Barrier Leakage Therefore, we hypothe-
sized that Nox4 deficiency also influences the disruption of the
blood–brain barrier and edema formation mediated by ROS [31]. Integrity of the blood–brain barrier was preserved in Nox42/2
mice on day 1 after tMCAO. This finding correlated with
significantly less brain edema in Nox42/2 mice than in wild-type
controls, as assessed by the extent of extravasation of Evans blue
stain (8.065.9 mm3 in Nox42/2 mice versus 96.265.9 mm3 in
wild-type mice). Importantly, almost no brain edema was seen in
the brain regions where infarcts were regularly present in Nox42/2
mice (basal ganglia; Figure 3D, area delineated by the broken
white line). This result indicates that the lesser edema seen in the
Nox42/2 mice was a specific phenomenon and mechanistically
relevant but was not due to smaller infarct volumes. To further examine whether the neuroprotective effect observed
in Nox42/2 mice is specifically related to reduced ROS formation
and not due to other nonspecific or developmental defects, we
performed a rescue experiment by restoring cerebral ROS levels in
Nox42/2 mice during the course of ischemic stroke by applying
exogenous H2O2 (Figure 4B–4D). Indeed, intrathecal administra-
tion of H2O2 rescued the phenotype in Nox42/2 mice, and infarct
volumes, functional deficits, and stroke-induced ROS formation
returned to the levels observed in wild-type mice (Figure 4B–4D). PLoS Biology | www.plosbiology.org PLoS Biology | www.plosbiology.org Protection from Ischemic Stroke in Nox42/2 Mice Is a
Result of Reduced Oxidative Stress, Neuronal Apoptosis,
and Blood-Brain-Barrier Leakage Long-term
outcome (lower panel): ***, p,0.0001, and *, p,0.05, one-way ANOVA, Bonferroni post-hoc test compared with wild-type mice (n = 10–15 per group). (E) Upper panel shows representative TTC staining of three corresponding coronal brain sections of 6- to 8-wk-old male wild-type mice (left) and
matching Nox42/2 mice (right) on day 1 after pMCAO. Lower panel: Infarct volumes as measured by infarct volumetry (left) and Neurological
Bederson score (right). Nox4 deficiency also protects the brain from permanent ischemia. **, p,0.001, and *, p,0.05, two-tailed Student’s t-test
compared with wild-type mice (n = 7–11 per group). (F) Representative coronal brain sections of wild-type and Nox42/2 mice stained with TTC on day
1 after permanent cortical photothrombosis (PT) (upper panel). Cortical infarctions are smaller in the absence of NOX4 (arrow). The lower panel shows
infarct volumes in wild-type and Nox42/2 mice on day 1 after cortical photothrombosis. **, p,0.001, two-tailed Student’s t-test compared with wild-
type mice (n = 7 per group). All scale bars represent 100 mm. doi:10.1371/journal.pbio.1000479.g002 during the course of ischemic stroke, while other NOX isoforms
such as NOX1 or NOX2 are obviously less relevant. dihydroethidium staining and nitrotyrosine immunolabeling, can
induce neuronal apoptosis, which is a well-established mechanism
of tissue damage in ischemic stroke [29,30]. Indeed, superimposed
TUNEL and NeuN immunolabeling revealed widespread apop-
tosis of neurons in wild-type mice 24 h after stroke onset
(Figure 3C). In contrast, the number of apoptotic neurons in
Nox42/2 mice subjected to tMCAO was significantly lower, and
the basal apoptotic turnover rate in Nox42/2 mice fell within the
range found in sham-operated mice (p.0.05) (Figure 3C). suc
as NO
o NO 2 a e obv ous y ess e eva t. To determine whether VAS2870 is also active when applied in
vivo, we administered 2 mg of VAS2870 intrathecally to wild-type
mice 2 h and 12 h after tMCAO. This experimental therapeutic
approach significantly reduced brain infarct volumes (20.764.0 mm3
in VAS2870-treated mice versus 82.466.4 mm3 in vehicle-treated
controls) and significantly improved neurological function, to the
same extent as observed for the deletion of Nox4 in mice (Figure 4B
and 4C). Moreover, less oxidative stress was detected in ischemic
brains from VAS2870-treated animals than in those from vehicle-
treated controls (Figure 4D). PLoS Biology | www.plosbiology.org Discussion Finally, protein expression levels of NOX1 and NOX2
were almost unchanged in the brains of Nox42/2 mice (Figure
S3C), underlining that the profound neuroprotection we observed
is mediated by deficiency or blockade of NOX4 itself and not by
secondary effects. The hypothesis that free radicals are involved in acute ischemic
stroke and account for secondary infarct growth dates back to the
1970s [38] but has remained unproven [38,39]. The extent of
neuroprotection that we observed is exceptional compared with
that seen in many other pre-clinical stroke studies, in which the
reduction of infarct size usually does not exceed 30%–40% [40]. Such moderate reductions of infarct volume have not translated
into improvement of neurological status [3]. Most notably,
continuous assessment of functional deficits until 7 d after stroke
revealed that Nox4-null mice indeed showed a better amplitude
rather than simply altered kinetics of recovery. This protection in
Nox42/2 mice was further underlined by a significantly reduced
post-stroke long-term mortality. Secondary infarct growth mediated for example by edema
formation or hemorrhagic transformation is common during the
course of brain ischemia and can lead to worsening of neurological
symptoms [39]. Serial magnetic resonance imaging revealed that
infarcts in Nox42/2 mice remain small, even at later stages of
infarct development, and signs of intracerebral hemorrhage were
consistently absent, thus indicating that NOX4 inhibition is likely
to be safe and persistently effective. A plethora of compounds have provided neuroprotection in
animal models of brain ischemia, but they all failed in human
clinical trials [4]. This translational roadblock has been attributed
mainly to inadequate pre-clinical study design and severe
methodological shortcomings. Important confounding factors are
a lack of randomization or rater-blinded evaluation of study
results, and use of only one stroke model [16]. Strictly adhering to
current expert recommendations for basic stroke trials, we here
demonstrate that in the absence of NOX4, brain tissue can be
salvaged after ischemia or reperfusion injury (as occurs in the
tMCAO model). Most importantly, neuroprotection was preserved
in old male and female Nox42/2 mice as well as in Nox42/2 mice
subjected to permanent ischemia (i.e., cortical photothrombosis or
pMCAO). Compared to in the tMCAO model, however, the
reduction of infarct size in the pMCAO model was less
pronounced though still significant. Discussion The right panel shows the extent of extravasation (i.e., edema volume) as determined by planimetry in the wild-type and Nox42/2 mice 24 h after
tMCAO (n = 6 per group). For (A–C), ###, p,0.0001, and ##, p,0.001, compared with sham-treated mice; ***, p,0.0001, and **, p,0.001,
compared with wild-type mice by two-way ANOVA, Bonferroni post-hoc test. For (D), **, p,0.001, Two-tailed Student9s t-test, compared with wild-
type mice. All scale bars represent 100 mm. doi:10.1371/journal.pbio.1000479.g003 cerebral vessels of Nox42/2 mice subjected to tMCAO but not
pMCAO (unpublished data). Clearly, elimination of NOX4
remains beneficial in the absence of arterial recanalization, a
condition frequently observed in human stroke. finding for the wider concept of oxidative stress, which might also
be of relevance for other disease states, such as neurotrauma and
neuroinflammation, where oxidative stress, blood–brain barrier
damage, and neurotoxicity are involved. Rather than focusing on
antioxidants and the disappointing outcomes of their application,
the identification of the relevant source of oxidative stress and
preventing its formation may represent an approach with clinical
potential. q
y
In our experiments, deficiency of NOX1 or NOX2 had no
impact on infarct size or functional outcome after tMCAO. Although others have described protective effects of NOX2
deficiency after experimental stroke [42–44], we could not
reproduce those findings. The exact reasons for this discrepancy
are unclear at present. Differences in the experimental protocols
and middle cerebral artery occlusion times, which varied between
30 min and 120 min in previous investigations, might play a role
here [42–44]. In contrast to these previous studies, however, we
used especially high numbers (n = 19) of Nox2y/2 mice to verify our
findings. Moreover, type-II (beta) error of the differences between
infarct volumes in Nox2y/2 mice and wild-type controls was only
7% in our study (93% power, respectively) (Tables S3–S5), which
is a very powerful result compared to the positive reports on Nox2
deficiency in cerebral ischemia [42–44] as well as to many other
experimental stroke studies in general [4,45]. Moreover, the fact
that VAS2870, which specifically inhibits NADPH oxidases, could
not further decrease infarct size and ROS formation in Nox42/2
mice ex vivo and in vivo (Figure 4) clearly argues against a major
role of NOX1 or NOX2 in the pathophysiology of acute ischemic
stroke. Discussion Here we identify NOX4 as a relevant molecular source of
oxidative stress in cerebral ischemia, including some cases of
human stroke. Our data suggest that NOX4-mediated oxidative
stress leads to neuronal damage via leakage of the blood–brain
barrier and neuronal apoptosis—two pathophysiological hallmarks
of ischemic stroke. The extent of neuroprotection conferred by the
absence of NOX4 in male and female Nox42/2 mice was
exceptional and preserved in old animals. Importantly, the
outcomes of these genetic experiments were mimicked when we
pharmacologically inhibited NADPH oxidases within a clinically
relevant time after induction of stroke. We consider this a key PLoS Biology | www.plosbiology.org September 2010 | Volume 8 | Issue 9 | e1000479 6 Role of NOX4 in Stroke September 2010 | Volume 8 | Issue 9 | e1000479 September 2010 | Volume 8 | Issue 9 | e1000479 PLoS Biology | www.plosbiology.org 7 Role of NOX4 in Stroke Figure 3. Nox4 deficiency confers neuroprotection by reducing oxidative stress, neuronal apoptosis, and disruption of the blood–
brain barrier. (A and B) Left panels show representative brain sections from sham-operated wild-type (WT) mice and wild-type and Nox42/2 mice
24 h after tMCAO. Sections were stained for ROS and oxidative chemistry using dihydroethidium (DHE) (A), or stained for reactive nitrogen species by
using nitrotyrosine (B). Right panels show the number of cells per square millimeter that are positive for ROS or oxidative stress (A) or reactive
nitrogen species (B) in the ischemic hemispheres of sham-operated wild-type mice and wild-type and Nox42/2 mice 12 h and 24 h after tMCAO (n = 4
per group). (C) Left panels show representative brain sections from sham-operated wild-type mice and wild-type and Nox42/2 mice 24 h after
tMCAO, immunolabeled for the neuronal marker NeuN and subjected to TUNEL to show apoptosis. Right panel shows the number of TUNEL-positive
neurons per square millimeter in the ischemic hemispheres of sham-operated wild-type mice and wild-type and Nox42/2 mice 24 h after tMCAO
(n = 4 per group). (D) Left panels show corresponding coronal brain sections of wild-type and Nox42/2 mice on day 1 after tMCAO and injection of
Evans blue. Extravasation of Evans blue was reduced in areas where infarcts were regularly present in Nox42/2 mice (basal ganglia, broken white line). September 2010 | Volume 8 | Issue 9 | e1000479 PLoS Biology | www.plosbiology.org Discussion Right panel shows number of ROS-positive cells per square millimeter in brain slices from mice in the different treatment groups (n = 5 per
group). (B) Upper panel shows representative TTC staining of three corresponding coronal brain sections of wild-type mice treated with (left to right)
carrier solution (10% DMSO; control), 100 mg of apocynin intravenously 1 h after tMCAO, or 2 mg of VAS2870 intrathecally 2 h and 12 h after tMCAO,
untreated Nox42/2 mice, Nox42/2 mice treated with 2 mg of VAS2870 intrathecally 2 h and 12 h after tMCAO, and Nox42/2 mice treated with H2O2
intrathecally (15 mg/kg) immediately after the occlusion of the middle cerebral artery and then every hour until 6 h after stroke induction. Ischemic
infarcts (white) appear smaller (arrows) in VAS2870-treated wild-type mice and Nox42/2 mice than in control mice, but those in apocynin-treated
mice are similar to those in control mice. VAS2870 could not further decrease infarct volumes in Nox42/2 mice. Exogenous H2O2 reversed the stroke-
protective phenotype in Nox42/2 mice. These results were confirmed by infarct volumetry (lower panel) (n = 7–10 per group). (C) Neurological
Bederson score (upper panel) and motor score (lower panel) on day 1 after tMCAO in the different animal groups indicated in (B) (n = 7–10 per group). (D) Left panel shows representative brain sections from the different animal groups indicated in (B) stained for ROS by using dihydroethidium. Right
panel shows corresponding number of ROS-positive cells per square millimeter in the ischemic hemispheres (n = 3–5 per group). For (A), ###,
p,0.0001, compared with sham-operated mice, ***, p,0.0001 compared with control mice, ns, not significant, one-way ANOVA, Bonferroni post-hoc
test. For (B–D), ***, p,0.0001, **, p,0.001, *, p,0.05, ns, not significant, one-way ANOVA, Bonferroni post-hoc test, compared with controls. All scale
bars represent 100 mm. p
m
doi:10.1371/journal.pbio.1000479.g004 formation of ROS or of functional outcome after experimental
stroke in vivo. formation of ROS or of functional outcome after experimental
stroke in vivo. apocynin 1 h after the occlusion of the middle cerebral artery. In
order to restore ROS levels in Nox42/2 mice, animals received
repetitive intrathecal injections of H2O2 (15 mg/kg) immediately
after the occlusion of the middle cerebral artery and then every
hour until 6 h after stroke induction. Discussion Distinct pathomechanisms
that can be positively influenced only in the presence of tissue
reperfusion, i.e., after tMCAO but not pMCAO, such as
progressive thrombus formation in the cerebral microvasculature
[41], might account for this quantitative difference. Indeed,
preliminary results suggest that clotting is attenuated in the Nevertheless, we cannot completely rule out contributions of
other sources of ROS. Referring to this, Block et al. recently
reported that a functional NOX4 is present and regulated in
mitochondria, indicating the existence of a hitherto undescribed
source of mitochondrial ROS [46]. An unprecedented need exists for more effective therapies for
acute stroke, the second leading cause of death worldwide [1]. We
have demonstrated that pharmacological inhibition of NADPH
oxidases using the specific NADPH oxidase inhibitor VAS2870
[15,32–34] protects mice from brain ischemia within a clinically
meaningful 2-h time window. In contrast, the commonly used
organic compound apocynin may not be a NOX inhibitor in
vascular cells but rather acts as a nonspecific antioxidant [36]. It
also inhibits Rho kinase inhibitor [37], an activity that increases its
nonspecific actions. If apocynin inhibits NADPH oxidases at all, it
supposedly blocks the migration of the cellular NADPH oxidase
complex subunit p47phox to the membrane, thus interfering with
assembly of the functional NOX complex [47]. Therefore, it is
unlikely to inhibit the NOX4-containing NADPH oxidase, which
acts independently of any cytosolic subunits [12]. Indeed, in our
experiments, application of apocynin had no effect on the PLoS Biology | www.plosbiology.org September 2010 | Volume 8 | Issue 9 | e1000479 PLoS Biology | www.plosbiology.org 8 Role of NOX4 in Stroke PLoS Biology | www.plosbiology.org
9
September 2010 | Volume 8 | Issue 9 | e10004 September 2010 | Volume 8 | Issue 9 | e1000479 September 2010 | Volume 8 | Issue 9 | e1000479 PLoS Biology | www.plosbiology.org PLoS Biology | www.plosbiology.org Role of NOX4 in Stroke Figure 4. The NADPH oxidase inhibitor VAS2870 protects the brain from damage during acute ischemic stroke. (A) Left panel shows
representative images of vital brain slices from sham-operated wild-type (WT) mice and wild-type mice and Nox42/2 mice 12 h after tMCAO. Slices
were incubated ex vivo with VAS2870 (10 mM) or carrier solution (1% DMSO; control) for 30 min and stained with dihydroethidium (DHE) to detect
ROS. Discussion In summary, we have demonstrated that NOX4-derived oxida-
tive stress is a crucial player in the pathophysiology of acute ischemic
stroke, while Nox4 deletion does not affect basal vascular or renal
function. Nox4 gene reconstitution experiments in Nox42/2 mice
and studies of the effects of different, structurally unrelated NOX
inhibitors—should they become available—would be desirable to
further substantiate the causality between NOX4 deficiency and
protection from cerebral ischemia. Pharmacological inhibition of
NADPH oxidases using specific compounds may also pave new
avenues for the treatment of ischemic brain injury in humans. Because NADPH oxidase–mediated production of ROS may
represent a general mechanism of neurotoxicity, our findings may
extend to other ischemic disorders and neurodegenerative or
inflammatory diseases. Further studies in relevant disease models
are warranted. Cortical photothrombosis was induced in 6- to 8-wk-old wild-
type or Nox42/2 mice as described previously [51,52]. Stroke Analysis Stroke analysis was performed as described previously [53,54]. To
determine infarct size, mice were killed 24 h after tMCAO, pMCAO,
or cortical photothrombosis. Brains were cut in 2-mm-thick coronal
sections using a mouse brain slice matrix (Harvard Apparatus). The
slices were stained with 2% TTC (Sigma-Aldrich) to visualize the
infarcts. Planimetric measurements (ImageJ software, United States
National Institutes of Health), calculating lesion volumes, were
corrected for brain edema as described previously [55]. Determination of brain edema using Evans blue dye was
performed as described previously [19]. Stroke Models If not otherwise mentioned, we performed 60 min of tMCAO in
6- to 8-wk-old male mice weighing 20–25 g, as described
previously [48,49]. To exclude age- and gender-specific effects,
18- to 20-wk-old male and 6- to 8-wk-old female mice were used in
some subgroups. For pMCAO the occluding filament was left in
situ until sacrificing the animals [41]. Stroke Study Design Detailed study characteristics are provided in Table S2. We
strictly followed the recent international expert recommendations
for conducting research in mechanism-driven basic stroke studies
[4,6,7,16,17,40]. Human Specimens Specimens from patients who had experienced a stroke were
collected during routine autopsy at the Department of Neuropa-
thology, University of Wu¨rzburg, Germany. Materials and Methods Magnetic resonance imaging was performed repeatedly at 24 h
and 6 d after stroke on a 1.5-T magnetic resonance unit (Vision
Siemens) as described previously [56]. We used a custom-made
dual
channel
surface
coil
designed
for
examining
mice
(A063HACG; Rapid Biomedical). The imaging protocol com-
prised a coronal T2-weighted sequence (slice thickness 2 mm) and
a blood-sensitive coronal three-dimensional T2-weighted gradient
echo CISS (slice thickness 1 mm) sequence. Magnetic resonance
images were assessed with respect to infarct morphology and the
occurrence of intracerebral bleeding. Refer to the Text S1 for more detailed methodology. The
generation of the Nox4-null mice is described in Figure S3. Vital Brain Slices Vital brain slices from infarcted mouse brains (between –2 mm
and –4 mm from bregma) were prepared as described previously
[57]. Quantitative PCR Analysis After RNA isolation, we quantified NOX4 mRNA expression
using real-time PCR and the TaqMan system (TaqMan Gene
Expression Arrays for murine NOX4, assay ID Mm00479246_m1,
Applied Biosystems), using 18s rRNA (TaqMan Predeveloped Assay
Reagents, part number 4319413E, Applied Biosystems) to normal-
ize the amount of sample RNA. At 2 h and12 h after the induction of tMCAO, subgroups of
wild-type mice or Nox42/2 mice were randomly selected to receive
either 2 mg of the NOX-specific inhibitor VAS2870 (Vasopharm
GmbH [32,33]) or carrier solution (10% dimethyl sulfoxide,
Sigma) intrathecally, as described previously [50]. In another
group, wild-type mice were injected intravenously with 100 mg of PLoS Biology | www.plosbiology.org Accession Numbers The GenBank (http://www.ncbi.nlm.nih.gov/Genbank) acces-
sion numbers for the genes discussed in this paper are NOX1,
NM_172203; NOX2, NM_007807; and NOX4, NM_015760. Quantification of Protein Expression Quantification of Protein Expression We quantified amounts of NOX1, NOX2, and NOX4 protein
in the cortex and basal ganglia by Western blot analysis. (
p
)
Found at: doi:10.1371/journal.pbio.1000479.s002 (1.64 MB TIF) Histology and Immunohistochemistry Histology was performed by using formalin-fixed mouse brains
on day 1 after tMCAO. Samples were embedded in paraffin and PLoS Biology | www.plosbiology.org September 2010 | Volume 8 | Issue 9 | e1000479 10 Role of NOX4 in Stroke cut into 4-mm-thick sections (0.5 mm anterior from bregma). After
deparaffinization and rehydration, tissues were stained with
hematoxylin and eosin or Nissl staining solution (Sigma-Aldrich). Immunohistochemical detection of NOX4 was performed on
formalin-fixed human brain slices or cryopreserved mouse brain
slices. A NOX4-specific primary antibody [58] was applied at a
dilution of 1:200 overnight at 4uC. To identify the cellular origin
we performed double staining of NOX4 with the neuronal marker
NeuN (1:1,000) and the endothelial marker von Willebrand Factor
(1:25). arterial pressure (PAP) in isolated perfused lungs during normoxic
(21%
O2)
ventilation. (E)
Strength
of
hypoxic
pulmonary
vasoconstriction (HPV) as indicated by the maximum increase in
PAP (DPAP) upon acute hypoxic ventilation (10 min, 1% O2) in
isolated perfused lungs. No significant differences were observed
between wild-type and Nox42/2 mice. Data are derived from six
mice in each case. (F) Renal hypertrophy as assessed by kidney
weight per body surface area (BSA) (g/m2). There was no
significant difference in terms of renal mass between wild-type and
Nox42/2 mice at 17 wk of age. (G) Albuminuria at 17 wk of age
(mg/24 h). There was no significant difference in 24-h urinary
albumin excretion between wild-type and Nox42/2 mice at 17 wk
of age. Statistical Analysis Figure
S3
Generation of Nox4 knockout mice and
counter-regulation of NOX1 and NOX2. (A) Construct
development for Nox4 knockout mice. Exons 14 and 15 are flanked
by loxP sites and followed by a floxed neomycin resistance gene
(neo) and a negative-selection cassette coding for diphtheria toxin A
(dta) as described in the Text S1. Embryonic stem cell clones were
generated by homologous recombination with the targeting vector. Transient expression of Cre recombinase results in three different
recombination events. Type 1 results in deletion of the neo cassette
and thus floxed exons 14 and 15. These cells can be used to
generate conditional Nox4 knockout. Type 2 results in deletion of
the floxed exons, and type 3 results in the deletion of exons 14 and
15 and the neo cassette. These cells were used to generate the Nox4
knockout mice. (B) Western blot demonstrating the absence of the
64-kDa NOX4 band in the aorta, lung, and kidney of Nox42/2
mice. (C) Expression of NOX1 and NOX2 is not upregulated in
Nox42/2 mice. The uppermost left panel shows results of
densitometric analysis of the NOX1 134-kDa band in brain
samples of the cortex and basal ganglia from Nox42/2 (pale bar)
and wild-type mice (black bar). Data are presented as the relative
amount of the NOX1 band normalized to GAPDH and represent
the mean 6 standard error of three samples. The right panel
shows a Western blot comparison of brain and aorta samples from
wild-type mice demonstrating the presence of the 134-kDa band in
both samples. The center and lowest panels show results of
densitometric analysis of the 91- and 53-kDa NOX2 bands seen in
brain samples from the cortex and basal ganglia of Nox42/2 (pale
bar) and wild-type mice (black bar). Data are presented as the Data are expressed as mean 6 standard deviation and were
analyzed statistically using the PrismGraph 4.0 software package
(GraphPad Software). In the case of multiple group comparisons,
data were tested for Gaussian distribution with the D’Agostino and
Pearson omnibus normality test and then analyzed by Bonferroni-
corrected one-way ANOVA or two-way ANOVA. Otherwise, the
two-tailed Student’s t-test was applied. For comparison of survival
curves the log-rank test was used. P-values less than 0.05 were
considered significant. Detailed power and type-II (beta) error
calculations on infarct volumes are provided in Tables S3–S5. Oxidative Chemistry Biomarkers The presence of ROS and other oxidants such as ONOO2 was
visualized on frozen mouse brain sections 12 h and 24 h after
tMCAO or 24 h after pMCAO using dihydroethidium (Sigma;
2 mM stock) staining, as described previously [59], in coronal brain
sections taken from identical regions (–0.5 mm from bregma) of
sham-operated controls, wild-type and Nox42/2 mice that had
undergone stroke, and wild-type mice and Nox42/2 mice treated
with VAS2870 or H2O2. Found at: doi:10.1371/journal.pbio.1000479.s001 (1.23 MB TIF) Found at: doi:10.1371/journal.pbio.1000479.s001 (1.23 MB TIF) Figure S2
Cerebral blood flow, cerebral vasculature,
and brain structure are normal in Nox42/2 mice. (A)
Regional cerebral blood flow (rCBF) in the right territory of the
middle cerebral artery as measured by laser Doppler flowmetry in
wild-type (WT) mice and in Nox1y/2, Nox2y/2, and Nox42/2 mice
(n = 4 per group) at baseline levels, after insertion of the thread
(ischemia) and again 10 min after removal of the thread
(reperfusion). No significant differences were observed between
the groups at any time point. p.0.05, two-way ANOVA,
Bonferroni post-hoc test, compared with baseline rCBF. (B)
Assessment of the cerebral vasculature in wild-type and Nox42/2
mice. A complete circle of Willis (white arrows) was identified in all
animals studied, and the distribution of the trunk and branch of
the middle cerebral artery appeared to be anatomically identical
among the genotypes. (C) Normal brain structure in Nox42/2
mice. Representative Nissl-stained 5-mm coronal paraffin-wax-
embedded brain sections of 3-mo-old wild-type and Nox42/2 mice
(n = 3 each), showing a macroscopic view (uppermost panel),
formation of the hippocampus formation (center panel), and
somatomotor areas of the neocortex (lowermost panel). Found at: doi:10.1371/journal.pbio.1000479.s002 (1.64 MB TIF) Immunohistochemical staining for nitrotyrosine to visualize
additional reactive nitrogen species was conducted on cryopre-
served brain sections taken from identical regions of the mouse
brain (–0.5 mm from bregma) 12 h and 24 h after tMCAO, using
a polyclonal nitrotyrosine antibody. Apoptotic neurons in the ischemic hemisphere 24 h after
tMCAO were visualized by TUNEL on paraffin-wax-embedded
slices, using the TUNEL in situ cell death detection kit, TMR red
(Roche). NeuN/TUNEL double staining was performed on
cryopreserved brain slices. Author Contributions Found
at:
doi:10.1371/journal.pbio.1000479.s006
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TIF) Found
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doi:10.1371/journal.pbio.1000479.s006
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doi:10.1371/journal.pbio.1000479.s006
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TIF) The author(s) have made the following declarations about their
contributions: Conceived and designed the experiments: CK AS MHdA
KJD NW HHS. Performed the experiments: CK HG MEA EJ MM DB
TS CG PK KB MKS AMH SGM GS SM AS LB VGD HF TK. Analyzed
the data: CK MEA EJ MM DB LB VGD HF TK MHdA KJD NW
HHHWS. Contributed reagents/materials/analysis tools: CK HG KW
AMS HHHWS. Wrote the paper: CK KW HHHWS. References 1. World Health Organization (2008) The top ten causes of death. Fact sheet
number 310. Geneva: World Health Organization. Available: http://www.who. int/mediacentre/factsheets/fs310_2008.pdf. 5 p. 1. World Health Organization (2008) The top ten causes of death. Fact sheet
number 310. Geneva: World Health Organization. Available: http://www.who. int/mediacentre/factsheets/fs310_2008.pdf. 5 p. 1. World Health Organization (2008) The top ten causes of death. Fact sheet
number 310. Geneva: World Health Organization. Available: http://www.who. int/mediacentre/factsheets/fs310_2008.pdf. 5 p. 11. Omenn GS (2007) Chemoprevention of lung cancers: lessons from CARET, the
beta-carotene and retinol efficacy trial, and prospects for the future. Eur J Cancer
Prev 16: 184–191. 2. Elkins JS, Johnston SC (2003) Thirty-year projections for deaths from ischemic
stroke in the United States. Stroke 34: 2109–2112. 12. Opitz N, Drummond GR, Selemidis S, Meurer S, Schmidt HH (2007) The ‘A’s
and ‘O’s of NADPH oxidase regulation: a commentary on ‘‘Subcellular
localization and function of alternatively spliced Noxo1 isoforms’’. Free Radic
Biol Med 42: 175–179. 2. Elkins JS, Johnston SC (2003) Thirty-year projections for deaths from ischemic
stroke in the United States. Stroke 34: 2109–2112. 3. O’Collins VE, Macleod MR, Donnan GA, Horky LL, van der Worp BH, et al. (2006) 1,026 experimental treatments in acute stroke. Ann Neurol 59: 467–477. 3. O’Collins VE, Macleod MR, Donnan GA, Horky LL, van der Worp BH, et al. (2006) 1,026 experimental treatments in acute stroke. Ann Neurol 59: 467–477. 13. Miller AA, Drummond GR, Schmidt HH, Sobey CG (2005) NADPH oxidase
activity and function are profoundly greater in cerebral versus systemic arteries. Circ Res 97: 1055–1062. 4. Dirnagl U (2006) Bench to bedside: the quest for quality in experimental stroke
research. J Cereb Blood Flow Metab 26: 1465–1478. 5. Whalley K (2006) Slicing into stroke therapeutics. Nat Rev Drug Discov 5:
632–632. 14. McCann SK, Dusting GJ, Roulston CL (2008) Early increase of Nox4 NADPH
oxidase and superoxide generation following endothelin-1-induced stroke in
conscious rats. J Neurosci Res 86: 2524–2534. 6. Sena ES, van der Worp HB, Bath PM, Howells DW, Macleod MR (2010)
Publication bias in reports of animal stroke studies leads to major overstatement
of efficacy. PLoS Biol 8: e1000344. doi:10.1371/journal.pbio.1000344. 15. Niethammer P, Grabher C, Look AT, Mitchison TJ (2009) A tissue-scale
gradient of hydrogen peroxide mediates rapid wound detection in zebrafish. Nature 459: 996–999. 7. van der Worp HB, Howells DW, Sena ES, Porritt MJ, Rewell S, et al. Text S1
Supplementary results, supplementary meth-
ods, and supplementary references. Text S1
Supplementary results, supplementary meth-
ods, and supplementary references. Found
at:
doi:10.1371/journal.pbio.1000479.s012
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DOC) Found
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doi:10.1371/journal.pbio.1000479.s012
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DOC) Figure S5
Motor function after pMCAO. Motor function
was assessed by the grip test in 6- to 8-wk-old male Nox42/2 mice
(n = 7) and wild-type (WT) controls (n = 11) 24 h after pMCAO. Two-tailed Student’s t-test compared with wild-type mice. ns, not
significant. Found at: doi:10.1371/journal.pbio.1000479.s005 (0.18 MB TIF) Found at: doi:10.1371/journal.pbio.1000479.s007 (0.04 MB PDF) Found at: doi:10.1371/journal.pbio.1000479.s007 (0.04 MB PDF) Acknowledgments We thank Ba¨rbel Fu¨hler and Courtney Jackson for their help in breeding
the Nox42/2 colonies in Gießen and Melbourne, respectively; Prof. Bettie
Sue Masters, University of Texas Health Science Center San Antonio,
United States, and Prof. Lutz Hein, University of Freiburg, Germany, for
their advice in designing the knockout strategy; Prof. Karl-Heinz Krause,
University of Geneva, Switzerland, for providing Nox1y/2 mice; Prof. Wolfgang Roggendorf, Department of Neuropathology and Brain Bank
Center, University of Wu¨rzburg, Germany, and Andreas Reif, Department
of Psychiatry, University of Wu¨rzburg, Germany, for providing human
stroke specimens; Marissa Bowden and Melanie Glaser for technical
assistance; Sandra Cox for carefully editing the manuscript, and
Vasopharm GmbH, Wu¨rzburg, Germany, for providing VAS2870. We
also thank the members of the German Mouse Clinic for comprehensive
phenotyping of the mice and fruitful discussions. H.H.H.W.S. would like to
dedicate this work to his mother, Renate Schmidt, who while this paper
was in preparation on December 31, 2009, died of a severe stroke. Figure S6
Oxidative stress is reduced in brains from
Nox42/2 mice after pMACO. Left panels show representative
brain sections from wild-type (WT) and Nox42/2 mice 24 h after
sham operation of pMCAO. Sections were stained for ROS and
oxidative chemistry using dihydroethidium. Right panel shows the
number of cells per square millimeter that are positive for ROS or
oxidative stress in the ischemic hemisphere of wild-type and Nox42/2
mice 24 h after sham operation or pMCAO (n= 3–5 per group). ##, p,0.001 compared with sham-treated mice; **, p,0.001
compared with wild-type mice by one-way ANOVA, Bonferroni
post-hoc test. Supporting Information Figure S1
Systemic and pulmonary blood pressure as
well as kidney function in Nox42/2 mice are unchanged. (A and B) Radiotelemetry recordings of basal mean arterial
pressure (MAP) and heart rate (HR) of wild-type (WT) (open
circles, n = 10) and Nox42/2 (filled squares, n = 14) mice. Data are
represented as 1-h (A) and 24-h (B) averages of mean arterial
pressure (left panels) and heart rate (right panels). Dark and light
periods are denoted by black and white bars, respectively. (C)
Right ventricular systolic pressure (RVSP) as assessed in vivo in
anaesthetized Nox42/2 and wild-type mice. (D) Mean pulmonary PLoS Biology | www.plosbiology.org September 2010 | Volume 8 | Issue 9 | e1000479 11 Role of NOX4 in Stroke Found at: doi:10.1371/journal.pbio.1000479.s008 (0.09 MB PDF) Found at: doi:10.1371/journal.pbio.1000479.s008 (0.09 MB PDF) relative amount of either the 91-kDa band or 53 k-Da band
normalized to GAPDH and represent the mean 6 standard error
of three samples. The bottom right panel shows a Western blot
comparison of NOX2 expression in the brain and aorta of wild-
type mice, demonstrating the presence of the 91-kDa and 53-kDa
bands in both tissues. Found at: doi:10.1371/journal.pbio.1000479.s003 (24.75 MB
TIF) Table S3
Power and type-II (beta) error calculations on
infarct volumes depicted in Figure 2A. Found at: doi:10.1371/journal.pbio.1000479.s009 (0.06 MB PDF) Table S4
Power and type-II (beta) error calculations on
infarct volumes depicted in Figure 2E. Found at: doi:10.1371/journal.pbio.1000479.s010 (0.05 MB PDF) Table S4
Power and type-II (beta) error calculations on
infarct volumes depicted in Figure 2E. Found at: doi:10.1371/journal.pbio.1000479.s003 (24.75 MB
TIF) Found at: doi:10.1371/journal.pbio.1000479.s010 (0.05 MB PDF) Table S5
Power and type-II (beta) error calculations on
infarct volumes depicted in Figure 4B. Figure S4
Long-term outcomes are improved in Nox42/2
mice after tMCAO. Long-term outcome of motor function (grip
test) in 6- to 8-wk-old male Nox42/2 mice (n =10) and wild-type (WT)
controls (n= 15) after tMCAO. Nox42/2 mice performed better over
the whole observation period. **, p,0.001 and *, p,0.05, one-way
ANOVA, Bonferroni post-hoc test compared with wild-type mice. Found
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doi:10.1371/journal.pbio.1000479.s004
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TIF) Found at: doi:10.1371/journal.pbio.1000479.s011 (0.06 MB PDF) Table S1
Results of blood gas analysis and posterior
communicating artery (PComA) score in wild-type and
Nox42/2 mice. Found at: doi:10.1371/journal.pbio.1000479.s007 (0.04 MB PDF) References Bobba A, Atlante A, Moro L, Calissano P, Marra E (2007) Nitric oxide has dual
opposite roles during early and late phases of apoptosis in cerebellar granule
neurons. Apoptosis 12: 1597–1610. yp
g
50. Wu WP, Xu XJ, Hao JX (2004) Chronic lumbar catheterization of the spinal
subarachnoid space in mice. J Neurosci Methods 133: 65–69. 51. Kleinschnitz C, Braeuninger S, Pham M, Austinat M, Nolte I, et al. (2008)
Blocking of platelets or intrinsic coagulation pathway-driven thrombosis does not
prevent cerebral infarctions induced by photothrombosis. Stroke 39: 1262–1268. 30. Loh KP, Huang SH, De Silva R, Tan BK, Zhu YZ (2006) Oxidative stress:
apoptosis in neuronal injury. Curr Alzheimer Res 3: 327–337. 31. Sandoval KE, Witt KA (2008) Blood-brain barrier tight junction permeability
and ischemic stroke. Neurobiol Dis 32: 200–219. p
y p
52. Schroeter M, Jander S, Stoll G (2002) Non-invasive induction of focal cerebral
ischemia in mice by photothrombosis of cortical microvessels: characterization of
inflammatory responses. J Neurosci Methods 117: 43–49. 32. Stielow C, Catar RA, Muller G, Wingler K, Scheurer P, et al. (2006) Novel Nox
inhibitor of oxLDL-induced reactive oxygen species formation in human
endothelial cells. Biochem Biophys Res Commun 344: 200–205. 53. Kleinschnitz C, Hofstetter HH, Meuth SG, Braeuninger S, Sommer C, et al. (2006) T cell infiltration after chronic constriction injury of mouse sciatic nerve is
associated with interleukin-17 expression. Exp Neurol 200: 480–485. 33. ten Freyhaus H, Huntgeburth M, Wingler K, Schnitker J, Baumer AT, et al. (2006) Novel Nox inhibitor VAS2870 attenuates PDGF-dependent smooth
muscle cell chemotaxis, but not proliferation. Cardiovasc Res 71: 331–341. 54. Kleinschnitz C, Pozgajova M, Pham M, Bendszus M, Nieswandt B, et al. (2007)
Targeting platelets in acute experimental stroke: impact of glycoprotein Ib, VI,
and IIb/IIIa blockade on infarct size, functional outcome, and intracranial
bleeding. Circulation 115: 2323–2330. 34. Lange S, Heger J, Euler G, Wartenberg M, Piper HM, et al. (2009) Platelet-
derived growth factor BB stimulates vasculogenesis of embryonic stem cell-
derived endothelial cells by calcium-mediated generation of reactive oxygen
species. Cardiovasc Res 81: 159–168. 55. Ginsberg MD, Becker DA, Busto R, Belayev A, Zhang Y, et al. (2003)
Stilbazulenyl nitrone, a novel antioxidant, is highly neuroprotective in focal
ischemia. Ann Neurol 54: 330–342. 35. Meuth SG, Budde T, Kanyshkova T, Broicher T, Munsch T, et al. References (2010)
Can animal models of disease reliably inform human studies? PLoS Med 7:
e1000245. doi:10.1371/journal.pmed.1000245. 16. Fisher M, Feuerstein G, Howells DW, Hurn PD, Kent TA, et al. (2009) Update
of the stroke therapy academic industry roundtable preclinical recommenda-
tions. Stroke 40: 224–2250. 8. Eliasson MJ, Huang Z, Ferrante RJ, Sasamata M, Molliver ME, et al. (1999)
Neuronal nitric oxide synthase activation and peroxynitrite formation in
ischemic stroke linked to neural damage. J Neurosci 19: 5910–5918. 17. Macleod MR, Fisher M, O’Collins V, Sena ES, Dirnagl U, et al. (2009) Good
laboratory practice: preventing introduction of bias at the bench. Stroke 40:
e50–e52. 9. Steinhubl SR (2008) Why have antioxidants failed in clinical trials? Am J Cardiol
101: 14D–19D. 18. Vallet P, Charnay Y, Steger K, Ogier-Denis E, Kovari E, et al. (2005) Neuronal
expression of the NADPH oxidase NOX4, and its regulation in mouse
experimental brain ischemia. Neuroscience 132: 233–238. 10. Dotan Y, Pinchuk I, Lichtenberg D, Leshno M (2009) Decision analysis supports
the paradigm that indiscriminate supplementation of vitamin E does more harm
than good. Arterioscler Thromb Vasc Biol 29: 1304–1309. PLoS Biology | www.plosbiology.org September 2010 | Volume 8 | Issue 9 | e1000479 12 Role of NOX4 in Stroke Role of NOX4 in Stroke 19. Austinat M, Braeuninger S, Pesquero JB, Brede M, Bader M, et al. (2009)
Blockade of bradykinin receptor B1 but not bradykinin receptor B2 provides
protection from cerebral infarction and brain edema. Stroke 40: 285–293. 40. Crossley NA, Sena E, Goehler J, Horn J, van der Worp B, et al. (2008) Empirical
evidence of bias in the design of experimental stroke studies: a metaepidemio-
logic approach. Stroke 39: 929–934. 20. Watson BD, Dietrich WD, Busto R, Wachtel MS, Ginsberg MD (1985)
Induction of reproducible brain infarction by photochemically initiated
thrombosis. Ann Neurol 17: 497–504. 41. Pham M, Kleinschnitz C, Helluy X, Bartsch AJ, Austinat M, et al. (2010)
Enhanced cortical reperfusion protects coagulation factor XII-deficient mice
from ischemic stroke as revealed by high-field MRI. Neuroimage 49:
2907–2914. 21. Kleinschnitz C, Bendszus M, Frank M, Solymosi L, Toyka KV, et al. (2003) In
vivo monitoring of macrophage infiltration in experimental ischemic brain
lesions by magnetic resonance imaging. J Cereb Blood Flow Metab 23:
1356–1361. 42. Chen H, Song YS, Chan PH (2009) Inhibition of NADPH oxidase is
neuroprotective after ischemia-reperfusion. J Cereb Blood Flow Metab 29:
1262–1272. 43. References Jackman KA, Miller AA, De Silva TM, Crack PJ, Drummond GR, et al. (2009)
Reduction of cerebral infarct volume by apocynin requires pretreatment and is
absent in Nox2-deficient mice. Br J Pharmacol 156: 680–688. 22. Geiszt M, Kopp JB, Varnai P, Leto TL (2000) Identification of renox, an
NAD(P)H oxidase in kidney. Proc Natl Acad Sci U S A 97: 8010–8014. 23. Mittal M, Roth M, Konig P, Hofmann S, Dony E, et al. (2007) Hypoxia-
dependent regulation of nonphagocytic NADPH oxidase subunit NOX4 in the
pulmonary vasculature. Circ Res 101: 258–267. 44. Walder CE, Green SP, Darbonne WC, Mathias J, Rae J, et al. (1997) Ischemic
stroke injury is reduced in mice lacking a functional NADPH oxidase. Stroke 28:
2252–2258. p
y
24. Griendling KK (2004) Novel NAD(P)H oxidases in the cardiovascular system. Heart 90: 491–493. 45. van der Worp HB, de Haan P, Morrema E, Kalkman CJ (2005) Methodological
quality of animal studies on neuroprotection in focal cerebral ischaemia. J Neurol
252: 1108–1114. 25. Gavazzi G, Banfi B, Deffert C, Fiette L, Schappi M, et al. (2006) Decreased
blood pressure in NOX1-deficient mice. FEBS Lett 580: 497–504. 46. Block K, Gorin Y, Abboud HE (2009) Subcellular localization of Nox4 and
regulation in diabetes. Proc Natl Acad Sci U S A 106: 14385–14390. 26. Landmesser U, Cai H, Dikalov S, McCann L, Hwang J, et al. (2002) Role of
p47(phox) in vascular oxidative stress and hypertension caused by angiotensin II. Hypertension 40: 511–515. 47. Touyz RM (2008) Apocynin, NADPH oxidase, and vascular cells: a complex
matter. Hypertension 51: 172–174. 27. Matsuno K, Yamada H, Iwata K, Jin D, Katsuyama M, et al. (2005) Nox1 is
involved in angiotensin II-mediated hypertension: a study in Nox1-deficient
mice. Circulation 112: 2677–2685. 48. Elvers M, Stegner D, Hagedorn I, Kleinschnitz C, Braun A, et al. (2010)
Impaired alpha(IIb)beta(3) integrin activation and shear-dependent thrombus
formation in mice lacking phospholipase D1. Sci Signal 3: ra1. 28. Veresh Z, Racz A, Lotz G, Koller A (2008) ADMA impairs nitric oxide-
mediated arteriolar function due to increased superoxide production by
angiotensin II-NAD(P)H oxidase pathway. Hypertension 52: 960–966. 49. Berna-Erro A, Braun A, Kraft R, Kleinschnitz C, Schuhmann MK, et al. (2009)
STIM2 regulates capacitive Ca2+ entry in neurons and plays a key role in
hypoxic neuronal cell death. Sci Signal 2: ra67. 29. References (2003)
Contribution of TWIK-related acid-sensitive K+ channel 1 (TASK1) and
TASK3 channels to the control of activity modes in thalamocortical neurons. J Neurosci 23: 6460–6469. 56. Kleinschnitz C, De Meyer SF, Schwarz T, Austinat M, Vanhoorelbeke K, et al. (2009) Deficiency of von Willebrand factor protects mice from ischemic stroke. Blood 113: 3600–3603. 36. Heumuller S, Wind S, Barbosa-Sicard E, Schmidt HH, Busse R, et al. (2008)
Apocynin is not an inhibitor of vascular NADPH oxidases but an antioxidant. Hypertension 51: 211–217. 57. Meuth SG, Kleinschnitz C, Broicher T, Austinat M, Braeuninger S, et al. (2009)
The neuroprotective impact of the leak potassium channel TASK1 on stroke
development in mice. Neurobiol Dis 33: 1–11. 37. Schluter T, Steinbach AC, Steffen A, Rettig R, Grisk O (2008) Apocynin-
induced vasodilation involves Rho kinase inhibition but not NADPH oxidase
inhibition. Cardiovasc Res 80: 271–279. p
58. Anilkumar N, Weber R, Zhang M, Brewer A, Shah AM (2008) Nox4 and nox2
NADPH oxidases mediate distinct cellular redox signaling responses to agonist
stimulation. Arterioscler Thromb Vasc Biol 28: 1347–1354. 38. Flamm ES, Demopoulos HB, Seligman ML, Poser RG, Ransohoff J (1978) Free
radicals in cerebral ischemia. Stroke 9: 445–447. 59. Murakami K, Kondo T, Kawase M, Li Y, Sato S, et al. (1998) Mitochondrial
susceptibility to oxidative stress exacerbates cerebral infarction that follows
permanent focal cerebral ischemia in mutant mice with manganese superoxide
dismutase deficiency. J Neurosci 18: 205–213. 39. Dirnagl U, Iadecola C, Moskowitz MA (1999) Pathobiology of ischaemic stroke:
an integrated view. Trends Neurosci 22: 391–397. PLoS Biology | www.plosbiology.org September 2010 | Volume 8 | Issue 9 | e1000479 PLoS Biology | www.plosbiology.org 13
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Parameterization of downward longwave radiation based on long-
term baseline surface radiation measurements in China odeling and Prediction Centre (CEMC), China Meteorological Administration, Beijing, 10008 10 Correspondence to: Weijun Quan (quanquan78430@163.com) Abstract. Downward longwave radiation (DLR) affects energy exchange between the land surface and the atmosphere, and
plays an important role in weather forecasting, agricultural activities, and the development of climate models. Because DLR
is seldom observed at conventional radiation stations, numerous empirical parameterizations have been presented to estimate Abstract. Downward longwave radiation (DLR) affects energy exchange between the land surface and the atmosphere, and
plays an important role in weather forecasting, agricultural activities, and the development of climate models. Because DLR
is seldom observed at conventional radiation stations, numerous empirical parameterizations have been presented to estimate DLR from screen-level meteorological variables. The reliability and representativeness of parameterization depend on the
15
coefficients regressed from the simultaneous observations of DLR and meteorological variables. Only a few previous studies
have attempted to build parameterizations over regions in China such as the Tibetan Plateau and East China. In this study, a
long-term (2011–2022) hourly dataset of DLR and meteorological elements, obtained from seven stations of the China
Baseline Surface Radiation Network, was used to recalculate the coefficients of the Brunt and Weng models, and to develop a new model. Results showed that the mean bias error (MBE) and relative MBE (rMBE) between the measured clear-sky
20
DLR and that estimated using the Brunt, Weng, and new models were −4.3, −5.1, and 3.7 W m−2 and −1.5 %, −1.8 %, and
1.3 %, respectively. The root mean squared errors (RMSEs) where in the range of 13.8–14.3 W m−2 and the relative RMSEs
(rRMSEs) were approximately 5.0 %. The MBEs (rMBEs) of the Brunt, Weng, and new models under all-sky conditions
were −2.8 W m−2 (−1.0 %), −6.1 W m−2 (−2.1 %), and −1.5 W m−2 (−0.5 %), respectively. The RMSE (rRMSE) of the
parameterization models in retrieving all-sky DLR was ~17.5 W m−2 (~6.1 %). Therefore, the models are considered suitable
25
for retrieval of DLR over China parameterization models in retrieving all-sky DLR was ~17.5 W m−2 (~6.1 %). Therefore, the models are considered suitable
25
for retrieval of DLR over China. https://doi.org/10.5194/acp-2022-794
Preprint. Discussion started: 6 January 2023
c⃝Author(s) 2023. CC BY 4.0 License. Parameterization of downward longwave radiation based on long-
term baseline surface radiation measurements in China
Junli Yang1,2, Jianglin Hu1,2, Qiying Chen1,2, and Weijun Quan3,4
1CMA Earth System Modeling and Prediction Centre (CEMC), China Meteorological Administration, Beijing, 100081,
China
5
2State Key Laboratory of Severe Weather (LaSW), China Meteorological Administration, Beijing, 100081, China
3Beijing Weather Forecast Centre, Beijing Meteorological Service, Beijing, 100089, China
4Environmental Meteorology Forecast Centre of Beijing-Tianjin-Hebei, Beijing, 100089, China Parameterization of downward longwave radiation based on long-
term baseline surface radiation measurements in China
Junli Yang1,2, Jianglin Hu1,2, Qiying Chen1,2, and Weijun Quan3,4
1CMA Earth System Modeling and Prediction Centre (CEMC), China Meteorological Administration, Beijing, 100081,
China
5
2State Key Laboratory of Severe Weather (LaSW), China Meteorological Administration, Beijing, 100081, China
3Beijing Weather Forecast Centre, Beijing Meteorological Service, Beijing, 100089, China
4Environmental Meteorology Forecast Centre of Beijing-Tianjin-Hebei, Beijing, 100089, China 1 Introduction Many studies have demonstrated that all-sky emissivity can be well
predicted from clear-sky emissivity with correction for cloud effects (Crawford and Duchon, 1999; Bilbao and De Miguel,
2007; Wang and Liang, 2009; Alados, et al., 2012; Li et al., 2017; Liu et al., 2020). Because the regression coefficients of empirical parameterization models exhibit spatial dependence (Goss and Brooks, 2
Because the regression coefficients of empirical parameterization models exhibit spatial dependence (Goss and Brooks,
1956; Brutsaert, 1975; Marthews et al., 2012; Liu et al., 2020), they should be recalculated on the basis of observations over
50
wider regions to ensure their accuracy and representativeness in estimating DLR. For instance, Wang and Liang (2009)
assessed the performance of clear-sky DLR parameterization models presented by Brunt (1932) and Brutsaert (1975) at 36
global sites. However, owing to the shortage of high-quality DLR measurements in China, most previous works focused on
retrieval of DLR over only a few regions, e.g., the Tibetan Plateau (Weng et al., 1993; Zhu et al., 2017; Liu et al., 2020) and
East China (Wang and Liang, 2009). Therefore, these models might not represent optimal parameterizations suited to
55
retrieval of DLR over other areas of China. Fortunately, the China Meteorological Administration has established the China
Baseline Surface Radiation Network (CBSRN) in 2007 (Li et al., 2013), which currently comprises seven stations (Mohe,
Xilinhot, Yanqi, Shangdianzi, Xuchang, Wenjiang, and Dali). Nine radiometric components including DLR are measured at
1-min intervals at CBSRN stations. The purpose of this study was to recalculate the coefficients of the Brunt (1932) model
and the Weng (1993) model, and to develop a new parametric formula in terms of a long-term (2011–2022) hourly dataset
60
obtained from the CBSRN stations. 1 Introduction This study represents an advance in comparison with previous work in terms of the
following aspects: 1) it not only recalculated the regression coefficients of the Brunt and Weng models, but also developed a
new parametric formula suited to estimation of DLR over China; 2) the hourly cloud fraction (CF) measured by a HY-WP1A
Intelligent Weather Observation System was incorporated to considerably improve the handling of cloud effects in DLR Because the regression coefficients of empirical parameterization models exhibit spatial dependence (Goss and Brooks,
1956; Brutsaert, 1975; Marthews et al., 2012; Liu et al., 2020), they should be recalculated on the basis of observations over
50
wider regions to ensure their accuracy and representativeness in estimating DLR. For instance, Wang and Liang (2009)
assessed the performance of clear-sky DLR parameterization models presented by Brunt (1932) and Brutsaert (1975) at 36
global sites. However, owing to the shortage of high-quality DLR measurements in China, most previous works focused on
retrieval of DLR over only a few regions, e.g., the Tibetan Plateau (Weng et al., 1993; Zhu et al., 2017; Liu et al., 2020) and 1956; Brutsaert, 1975; Marthews et al., 2012; Liu et al., 2020), they should be recalculated on the basis of observations over
50
wider regions to ensure their accuracy and representativeness in estimating DLR. For instance, Wang and Liang (2009)
assessed the performance of clear-sky DLR parameterization models presented by Brunt (1932) and Brutsaert (1975) at 36
global sites. However, owing to the shortage of high-quality DLR measurements in China, most previous works focused on
retrieval of DLR over only a few regions, e.g., the Tibetan Plateau (Weng et al., 1993; Zhu et al., 2017; Liu et al., 2020) and East China (Wang and Liang, 2009). Therefore, these models might not represent optimal parameterizations suited to
55
retrieval of DLR over other areas of China. Fortunately, the China Meteorological Administration has established the China
Baseline Surface Radiation Network (CBSRN) in 2007 (Li et al., 2013), which currently comprises seven stations (Mohe,
Xilinhot, Yanqi, Shangdianzi, Xuchang, Wenjiang, and Dali). Nine radiometric components including DLR are measured at
1-min intervals at CBSRN stations. The purpose of this study was to recalculate the coefficients of the Brunt (1932) model East China (Wang and Liang, 2009). Therefore, these models might not represent optimal parameterizations suited to
55
retrieval of DLR over other areas of China. 1 Introduction Many studies have demonstrated that all-sky emissivity can be well
predicted from clear-sky emissivity with correction for cloud effects (Crawford and Duchon, 1999; Bilbao and De Miguel,
2007; Wang and Liang, 2009; Alados, et al., 2012; Li et al., 2017; Liu et al., 2020). In comparison with other radiation components, DLR is seldom observed at conventional radiation stations (Iziomon et
al., 2003; Stephens et al., 2012). Therefore, considerable effort has been made to develop simple parameterization methods
to calculate DLR from easily measured meteorological variables (Duarte et al., 2006). As identified by Ångström (1915),
35
clear-sky DLR can be determined from the emissivity and effective temperature of the atmosphere. Under clear-sky
conditions, as much as 60 % (90 %) of atmospheric emission is derived from the atmosphere within the first 100 m (1 km). When the sky is overcast, more than 90 % originates from within first 1-km layer between the ground and the bottom of the
cloud (Ohmura, 2001). Following the pioneering work of Ångström, numerous investigators have presented empirical 35 relationships between effective atmospheric emissivity (hereinafter refer to emissivity) under clear-sky conditions and
40
meteorological elements such as vapor pressure (e) (Brunt, 1932; Weng et al., 1993; Niemelä et al., 2001), screen-level
temperature (Ta) (Swinbank, 1963; Idso and Jackson, 1969), dewpoint temperature (Td) (Berdahl and Fromberg, 1982), e and
Ta (Brutsaert, 1975; Satterlund, 1979; Idso, 1981; Iziomon et al., 2003), and relative humidity (φ) and Ta (Carmona et al.,
2014). Under all-sky conditions, the presence of cloud can increase emissivity and atmospheric radiation. Clouds generally consist of water vapor, water droplets, or ice crystals. They absorb thermal radiation very strongly and radiate similar to a
45
black body in the infrared range (Heitor et al., 1991). Many studies have demonstrated that all-sky emissivity can be well
predicted from clear-sky emissivity with correction for cloud effects (Crawford and Duchon, 1999; Bilbao and De Miguel,
2007; Wang and Liang, 2009; Alados, et al., 2012; Li et al., 2017; Liu et al., 2020). Because the regression coefficients of empirical parameterization models exhibit spatial dependence (Goss and Brooks, consist of water vapor, water droplets, or ice crystals. They absorb thermal radiation very strongly and radiate similar to a
45
black body in the infrared range (Heitor et al., 1991). 1 Introduction Downward longwave radiation (DLR) on the ground is one of the fluxes involved in the exchange of energy between Earth’s
surface and the atmosphere (Konzelmann et al., 1994; Gabathuler et al., 2001; Sridhar and Elliot, 2002). Consequently, DLR
plays a vital role in weather forecasting, agricultural production (e.g., prediction of frost and crop temperature), climate
30 30 1 1 simulations, and water cycle modeling (Crawford and Duchon, 1999; Bilbao and De Miguel, 2007; Li et al., 2017; Liu et al.,
2020). https://doi.org/10.5194/acp-2022-794
Preprint. Discussion started: 6 January 2023
c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/acp-2022-794
Preprint. Discussion started: 6 January 2023
c⃝Author(s) 2023. CC BY 4.0 License. simulations, and water cycle modeling (Crawford and Duchon, 1999; Bilbao and De Miguel, 2007; Li et al., 2017; Liu et al., simulations, and water cycle modeling (Crawford and Duchon, 1999; Bilbao and De Miguel, 2007; Li et al., 2017; Liu et al.,
2020). In comparison with other radiation components, DLR is seldom observed at conventional radiation stations (Iziomon et
al., 2003; Stephens et al., 2012). Therefore, considerable effort has been made to develop simple parameterization methods
to calculate DLR from easily measured meteorological variables (Duarte et al., 2006). As identified by Ångström (1915),
35
clear-sky DLR can be determined from the emissivity and effective temperature of the atmosphere. Under clear-sky
conditions, as much as 60 % (90 %) of atmospheric emission is derived from the atmosphere within the first 100 m (1 km). When the sky is overcast, more than 90 % originates from within first 1-km layer between the ground and the bottom of the
cloud (Ohmura, 2001). Following the pioneering work of Ångström, numerous investigators have presented empirical
relationships between effective atmospheric emissivity (hereinafter refer to emissivity) under clear-sky conditions and
40
meteorological elements such as vapor pressure (e) (Brunt, 1932; Weng et al., 1993; Niemelä et al., 2001), screen-level
temperature (Ta) (Swinbank, 1963; Idso and Jackson, 1969), dewpoint temperature (Td) (Berdahl and Fromberg, 1982), e and
Ta (Brutsaert, 1975; Satterlund, 1979; Idso, 1981; Iziomon et al., 2003), and relative humidity (φ) and Ta (Carmona et al.,
2014). Under all-sky conditions, the presence of cloud can increase emissivity and atmospheric radiation. Clouds generally
consist of water vapor, water droplets, or ice crystals. They absorb thermal radiation very strongly and radiate similar to a
45
black body in the infrared range (Heitor et al., 1991). 1 Introduction Fortunately, the China Meteorological Administration has established the China
Baseline Surface Radiation Network (CBSRN) in 2007 (Li et al., 2013), which currently comprises seven stations (Mohe,
Xilinhot, Yanqi, Shangdianzi, Xuchang, Wenjiang, and Dali). Nine radiometric components including DLR are measured at
1-min intervals at CBSRN stations. The purpose of this study was to recalculate the coefficients of the Brunt (1932) model and the Weng (1993) model, and to develop a new parametric formula in terms of a long-term (2011–2022) hourly dataset
60
obtained from the CBSRN stations. This study represents an advance in comparison with previous work in terms of the
following aspects: 1) it not only recalculated the regression coefficients of the Brunt and Weng models, but also developed a
new parametric formula suited to estimation of DLR over China; 2) the hourly cloud fraction (CF) measured by a HY-WP1A
Intelligent Weather Observation System was incorporated to considerably improve the handling of cloud effects in DLR and the Weng (1993) model, and to develop a new parametric formula in terms of a long-term (2011–2022) hourly dataset
60
obtained from the CBSRN stations. This study represents an advance in comparison with previous work in terms of the
following aspects: 1) it not only recalculated the regression coefficients of the Brunt and Weng models, but also developed a
new parametric formula suited to estimation of DLR over China; 2) the hourly cloud fraction (CF) measured by a HY-WP1A
Intelligent Weather Observation System was incorporated to considerably improve the handling of cloud effects in DLR 2 retrieval under all-sky conditions; and 3) the spatial representativeness of the parameterization models over China was
65
improved through use of measurements from the seven CBSRN stations in China. https://doi.org/10.5194/acp-2022-794
Preprint. Discussion started: 6 January 2023
c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/acp-2022-794
Preprint. Discussion started: 6 January 2023
c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/acp-2022-794
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c⃝Author(s) 2023. CC BY 4.0 License. retrieval under all-sky conditions; and 3) the spatial representativeness of the parameterization models over China was
65
improved through use of measurements from the seven CBSRN stations in China. 65 2.1 Site description Figure 1 shows the geographical locations of the seven CBSRN stations: Mohe (MH; 52 Figure 1 shows the geographical locations of the seven CBSRN stations: Mohe (MH; 52.97° N, 122.52° E; 438.5 m a.s.l.), Figure 1 shows the geographical locations of the seven CBSRN stations: Mohe (MH; 52.97° N, 122.52° E; 438.5 m a.s.l.),
Xilinhot (XL; 44.13° N, 116.33° E; 1003.0 m a.s.l.), Yanqi (YQ; 42.05° N, 86.61° E; 1056.5 m a.s.l.), Shangdianzi (SDZ;
70
40.65° N, 117.12° E; 293.3 m a.s.l.), Xuchang (XC; 34.07° N, 113.93° E; 67.2 m a.s.l.), Wenjiang (WJ; 30.75° N, 103.86° E;
547.7 m a.s.l.), and Dali (DL; 25.71° N, 100.18° E; 1990.5 m a.s.l.). It can be seen from Table 1 that these stations are
distributed in seven representative climatic zones, i.e., the cold temperate zone (MH), middle temperate semiarid zone (XL),
middle temperate arid zone (YQ), warm temperature semihumid zone (SDZ), northern subtropical humid zone (XC), middle
subtropical humid zone (WJ), and subtropical humid zone (DL). Additionally, the elevation of three stations (i.e., XC, SDZ,
75
and MH) is <500 m a.s.l., one station (WJ) has medium elevation (547.7 m a.s.l.), and the other three stations (i.e., XL, YQ,
and DL) have elevation >1000 m a.s.l. (Table 1). Figure 1 shows the geographical locations of the seven CBSRN stations: Mohe (MH; 52.97° N, 122.52° E; 438.5 m a.s.l.),
Xilinhot (XL; 44.13° N, 116.33° E; 1003.0 m a.s.l.), Yanqi (YQ; 42.05° N, 86.61° E; 1056.5 m a.s.l.), Shangdianzi (SDZ;
70
40.65° N, 117.12° E; 293.3 m a.s.l.), Xuchang (XC; 34.07° N, 113.93° E; 67.2 m a.s.l.), Wenjiang (WJ; 30.75° N, 103.86° E;
547.7 m a.s.l.), and Dali (DL; 25.71° N, 100.18° E; 1990.5 m a.s.l.). 2.1 Site description It can be seen from Table 1 that these stations are
distributed in seven representative climatic zones, i.e., the cold temperate zone (MH), middle temperate semiarid zone (XL),
middle temperate arid zone (YQ), warm temperature semihumid zone (SDZ), northern subtropical humid zone (XC), middle
subtropical humid zone (WJ) and subtropical humid zone (DL) Additionally the elevation of three stations (i e XC SDZ
75 Xilinhot (XL; 44.13° N, 116.33° E; 1003.0 m a.s.l.), Yanqi (YQ; 42.05° N, 86.61° E; 1056.5 m a.s.l.), Shangdianzi (SDZ;
70
40.65° N, 117.12° E; 293.3 m a.s.l.), Xuchang (XC; 34.07° N, 113.93° E; 67.2 m a.s.l.), Wenjiang (WJ; 30.75° N, 103.86° E;
547.7 m a.s.l.), and Dali (DL; 25.71° N, 100.18° E; 1990.5 m a.s.l.). It can be seen from Table 1 that these stations are
distributed in seven representative climatic zones, i.e., the cold temperate zone (MH), middle temperate semiarid zone (XL),
middle temperate arid zone (YQ), warm temperature semihumid zone (SDZ), northern subtropical humid zone (XC), middle subtropical humid zone (WJ), and subtropical humid zone (DL). Additionally, the elevation of three stations (i.e., XC, SDZ,
75
and MH) is <500 m a.s.l., one station (WJ) has medium elevation (547.7 m a.s.l.), and the other three stations (i.e., XL, YQ,
and DL) have elevation >1000 m a.s.l. (Table 1). subtropical humid zone (WJ), and subtropical humid zone (DL). Additionally, the elevation of three stations (i.e., XC, SDZ,
75
and MH) is <500 m a.s.l., one station (WJ) has medium elevation (547.7 m a.s.l.), and the other three stations (i.e., XL, YQ,
and DL) have elevation >1000 m a.s.l. (Table 1). Figure 1. Geographical locations of seven CBSRN stations in China. Terrain data represent GTOP30 digital
elevation model (ftp://edcftp.cr.usgs.gov/data/gtopo30/global/). Figure 1. Geographical locations of seven CBSRN stations in China. Terrain data represent GTOP30 digital
elevation model (ftp://edcftp.cr.usgs.gov/data/gtopo30/global/). 80 3 3 https://doi.org/10.5194/acp-2022-794
Preprint. Discussion started: 6 January 2023
c⃝Author(s) 2023. CC BY 4.0 License. 85 85 Table 1. Basic descriptions of CBSRN stations in China. 2.1 Site description Station name Station ID Latitude(° N) Longitude(° E) Altitude(m)
Climatic zones
Mohe
50136
52.97
122.52
438.5
Cold temperate zone
Xilinhot
54102
44.13
116.33
1107.0
Middle temperate semiarid zone
Yanqi
51567
42.05
86.61
1056.5
Middle temperate arid zone
Shangdianzi
54421
40.65
117.12
293.3
Warm temperate semihumid zone
Xuchang
57089
34.07
113.93
67.2
Northern subtropical humid zone
Wenjiang
56187
30.75
103.86
547.7
Middle subtropical humid zone
Dali
56751
25.71
100.18
1990.5
Subtropical humid zone Station name Station ID Latitude(° N) Longitude(° E) Altitude(m)
Clima 2.2 Instruments and data The CBSRN stations use IR02 pyrgeometers (Huksflux, the Netherlands) to measure DLR. The spectral range of the IR02
instrument is 4.5–42 μm, which covers most of spectral range of atmospheric longwave radiation, making it a suitable
instrument for measuring DLR in most cases. Moreover, to avoid influencing from solar radiation, the IR02 is shaded by a
90
ball mounted on a FS-ST22 automatic solar tracker (Jiangsu Radio Science Research Institute Co. Ltd., China) during
observation. Its temperature dependence is within ±3 % (−10 to 40 °C), and a ventilation/heating system is installed to
reduce the influence of environmental temperature and to prevent dew/dust fall on its window. Note that the field of view
(FOV) of the IR02 instrument is 150° rather than the desired 180°, which means its price is attractive, while the accuracy instrument is 4.5–42 μm, which covers most of spectral range of atmospheric longwave radiation, making it a suitable
instrument for measuring DLR in most cases. Moreover, to avoid influencing from solar radiation, the IR02 is shaded by a
90
ball mounted on a FS-ST22 automatic solar tracker (Jiangsu Radio Science Research Institute Co. Ltd., China) during
observation. Its temperature dependence is within ±3 % (−10 to 40 °C), and a ventilation/heating system is installed to
reduce the influence of environmental temperature and to prevent dew/dust fall on its window. Note that the field of view
(FOV) of the IR02 instrument is 150° rather than the desired 180°, which means its price is attractive, while the accuracy loss is relatively minor (Hukseflux, 2022). The IR02 sampling frequency is 1 Hz and the 1-min averaged data are stored
95
using a WUSH-BR data logger (Jiangsu Radio Science Research Institute Co. Ltd., China). The IR02 pyrgeometers are
carefully maintained and regularly calibrated by the manufactures to guarantee that the observations meet the standards
specified by the China National Centre for Meteorological Metrology (Huo et al., 2017). A fisheye camera, mounted on top of the HY-WP1A Intelligent Weather Observation System (Huayun Sounding loss is relatively minor (Hukseflux, 2022). The IR02 sampling frequency is 1 Hz and the 1-min averaged data are stored
95
using a WUSH-BR data logger (Jiangsu Radio Science Research Institute Co. Ltd., China). The IR02 pyrgeometers are
carefully maintained and regularly calibrated by the manufactures to guarantee that the observations meet the standards
specified by the China National Centre for Meteorological Metrology (Huo et al., 2017). 2.2 Instruments and data A fisheye camera, mounted on top of the HY-WP1A Intelligent Weather Observation System (Huayun Sounding A fisheye camera, mounted on top of the HY-WP1A Intelligent Weather Observation System (Huayun Sounding
Meteorological Technology Inc., China), is used to automatically record CF data. Full-sky photographs with a FOV of 180°
100
are acquired at 1-min intervals. The photographs are then processed using artificial intelligence image detecting technology
to yield hourly CF data with uncertain <10 % (Hua et al., 2021). y
,
p
g
y
(
y
g
Meteorological Technology Inc., China), is used to automatically record CF data. Full-sky photographs with a FOV of 180°
100
are acquired at 1-min intervals. The photographs are then processed using artificial intelligence image detecting technology
to yield hourly CF data with uncertain <10 % (Hua et al., 2021). Meteorological elements (i.e., Ta, e, and φ) are observed by an automatic weather station (AWS) at 1-min intervals and
the data are stored using a HY3000 data logger (Huayun Sounding Meteorological Technology Inc China) Meteorological Technology Inc., China), is used to automatically record CF data. Full-sky photographs with a FOV of 180°
100
are acquired at 1-min intervals. The photographs are then processed using artificial intelligence image detecting technology
to yield hourly CF data with uncertain <10 % (Hua et al., 2021). Meteorological elements (i.e., Ta, e, and φ) are observed by an automatic weather station (AWS) at 1-min intervals and
the data are stored using a HY3000 data logger (Huayun Sounding Meteorological Technology Inc., China). Meteorological elements (i.e., Ta, e, and φ) are observed by an automatic weather station (AWS) at 1-min intervals and
the data are stored using a HY3000 data logger (Huayun Sounding Meteorological Technology Inc., China). The data used in this study, which were downloaded from the China Meteorological Administration Data Service
105
(http://idata.cma/cmadaas/), undergo strictly quality controlled by meteorological experts and trained engineers of the
National Meteorological Information Centre of China. Note that the DLR data measured by the IR02 instruments at high-
elevation stations (i.e., MH and YQ) under extremely dry and cold synoptic conditions, in which irrational DLR The data used in this study, which were downloaded from the China Meteorological Administration Data Service
105
(http://idata.cma/cmadaas/), undergo strictly quality controlled by meteorological experts and trained engineers of the
National Meteorological Information Centre of China. 2.2 Instruments and data Note that the DLR data measured by the IR02 instruments at high-
elevation stations (i.e., MH and YQ) under extremely dry and cold synoptic conditions, in which irrational DLR The data used in this study, which were downloaded from the China Meteorological Administration Data Service
105
(http://idata.cma/cmadaas/), undergo strictly quality controlled by meteorological experts and trained engineers of the
National Meteorological Information Centre of China. Note that the DLR data measured by the IR02 instruments at high-
elevation stations (i.e., MH and YQ) under extremely dry and cold synoptic conditions, in which irrational DLR 4 measurements might be produced due to the high temperature dependency of the IR02 pyrgeometer (Hukseflux, 2022), were
not involved in this study. 110
https://doi.org/10.5194/acp-2022-794
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c⃝Author(s) 2023. CC BY 4.0 License. measurements might be produced due to the high temperature dependency of the IR02 pyrgeometer (Hukseflux, 2022), were
not involved in this study. 110 measurements might be produced due to the high temperature dependency of the IR02 pyrgeometer (Hukseflux, 2022), were
not involved in this study
110 110 3.1 Emissivity calculation Effective atmospheric emissivity () is defined as the ratio of incoming long-wave radiation to blackbody radiation at screen-
level air temperature (Monteith, 1961; Rodgers, 1967; Prata, 1996): ε =
DLR
σ𝑇𝑎4 , (1) ε =
DLR
σ𝑇𝑎4 , 115 115 (1) where DLR is the downward hemispheric longwave irradiance (W m−2) at the ground, which can be observed directly by a
pyrgeometer; Ta is the screen air temperature (K) measured by the AWS; and σ is the Stefan–Boltzmann constant
(5.6697×10−8 W m−2 K−4). where DLR is the downward hemispheric longwave irradiance (W m−2) at the ground, which can be observed directly by a
pyrgeometer; Ta is the screen air temperature (K) measured by the AWS; and σ is the Stefan–Boltzmann constant
(5.6697×10−8 W m−2 K−4). 3.2 Statistical methods This study used the nonlinear curve fitting method, orthogonal distance regression (ODR) iteration algorithm and
120
Levenberg–Marquardt iteration algorithm to regress the coefficients of the parameterization models. Parameterizations were
assessed by means of statistical parameters such as mean bias error (MBE), relative MBE (rMBE), root mean squared error
(RMSE), relative RMSE (rRMSE), and the correlation coefficient (r). The MBE is an indicator adopted to denote whether
predictions from the parameterization are overestimates (positive values) or underestimates (negative values) in comparison with the measurements. The RMSE accounts for the average magnitude of the errors but it does not provide an indication of
125
the direction of the errors. The correlation coefficient r reflects the linear agreement between the observed parameter and the
estimated variable (Gubler et al., 2012; Zhou et al., 2021). 4.1 Clear-sky emissivity parameterization where clr,B, clr,W, and clr,Y represent the clear-sky emissivity retrieved from the Brunt model, Weng model, and new model
145
developed in this study, respectively, e (hPa) is vapor pressure, and Ta (K) is screen-level air temperature. The coefficients of
determination (R2) of Eqs. (2)–(4) were 0.999, 0.999, and 0.930, respectively. The Brunt model (denoted by the black thick curve in Fig. 2a) can well fit all data pairs under most cases (0 < e ≤ 45
hPa), especially those data pairs observed at low-elevation (<1000 m) stations such as XC (67.2 m a.s.l.) and SDZ (293.3 m The Brunt model (denoted by the black thick curve in Fig. 2a) can well fit all data pairs under most cases (0 < e ≤ 45
hPa), especially those data pairs observed at low-elevation (<1000 m) stations such as XC (67.2 m a.s.l.) and SDZ (293.3 m a.s.l.), whereas the Weng model (denoted by the red thick curve in Fig. 2a) appears to fit the data pairs better than the Brunt
150
model under dry conditions (e ≤17.5 hPa). Note that the Weng model was proposed in terms of radiation data observed over
the Tibetan Plateau, where the atmospheric vapor pressure is lower than that in other regions in China. Therefore, it can be
inferred that the Weng model is suitable for estimating clear-sky emissivity over arid regions. The parametric formula
developed in this study (denoted by the red thick curve in Fig. 2b) fitted the data pairs reasonably and was considered to have
basis in physics because it uses both e and Ta as input. 155 a.s.l.), whereas the Weng model (denoted by the red thick curve in Fig. 2a) appears to fit the data pairs better than the Brunt
150
model under dry conditions (e ≤17.5 hPa). Note that the Weng model was proposed in terms of radiation data observed over
the Tibetan Plateau, where the atmospheric vapor pressure is lower than that in other regions in China. Therefore, it can be
inferred that the Weng model is suitable for estimating clear-sky emissivity over arid regions. The parametric formula
developed in this study (denoted by the red thick curve in Fig. 2b) fitted the data pairs reasonably and was considered to have
basis in physics because it uses both e and Ta as input. 155 160 160 160 Figure 2. 4.1 Clear-sky emissivity parameterization In this study, the coefficients of the Brunt model and the Weng model were calibrated using the nonlinear curve fitting
130
method with 12,368 clear-sky hourly data pairs (DLR and e) observed at seven CBSRN stations between January 2011 and
December 2017. Note that both the Brunt model and the Weng model are single-parameter parameterization models because
only one parameter (e) is adopted as input in these models. The Brunt model is a power function of e with an exponent of 1/2,
whereas the Weng model is a natural logarithm function of e. A two-parameter model such as the Brutsaert (1975) model, in which e and Ta are both used as input parameters, is recognized to be more reasonable than a single-parameter model in
135
terms of the physical mechanism, especially under warm and wet conditions (Culf and Gash, 1993; Prata, 1996). In this which e and Ta are both used as input parameters, is recognized to be more reasonable than a single-parameter model in
135
terms of the physical mechanism, especially under warm and wet conditions (Culf and Gash, 1993; Prata, 1996). In this 5 https://doi.org/10.5194/acp-2022-794
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c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/acp-2022-794
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c⃝Author(s) 2023. CC BY 4.0 License. study, we developed a two-parameter parametric formula (hereinafter refer to the parametric formula) that is similar to the
Brutsaert model except the exponent of the function is set to 1/3 rather than 1/7. The coefficients of the parametric formulae
were computed on the basis of the clear-sky hourly dataset (DLR, e, and Ta) using the nonlinear curve fitting method
together with the ODR iteration algorithm. 140 the parameterization models for retrieving clear-sky emissivity can be expressed as follows: The formulae of the parameterization models for retrieving clear-sky emissivity can be expressed a ε𝑐𝑙𝑟,𝐵= 0.599+0.053√𝑒 , ε𝑐𝑙𝑟,𝐵= 0.599+0.053√𝑒 , (2)
ε𝑐𝑙𝑟,𝑊= 0.590+0.075ln(1+𝑒) , (3)
ε𝑐𝑙𝑟,𝑌= 0.532+0.808√𝑒/𝑇𝑎
3
, (4) (4) where clr,B, clr,W, and clr,Y represent the clear-sky emissivity retrieved from the Brunt model, Weng model, and new model
145
developed in this study, respectively, e (hPa) is vapor pressure, and Ta (K) is screen-level air temperature. The coefficients of
determination (R2) of Eqs. (2)–(4) were 0.999, 0.999, and 0.930, respectively. 4.1 Clear-sky emissivity parameterization 180
Reference
Network/Site
Number
of Sites Elevation(m)
a
b
Resolution Country
Brunt [1932]
Benson
1
6
0.520 0.065 Monthly
UK
Anderson [1954]
Laker Hefner
1
369
0.680 0.036 Monthly
USA
Goss and Brooks
[1956]
Davis
1
14
0.660 0.039 Monthly
USA
DeCoster and
Schuepp [1957]
Kinshasa
1
321
0.645 0.048 Daily
Zaire
Monteith [1961]
Kew
1
-
0.530 0.065 Hourly
England
Swinbank [1963]
Aspendale, Kerang
2
-
0.640 0.037 Hourly
Australia
Berger et al. [1984] Carpentras
1
-
0.660 0.040 Hourly
France
Berdahl and Martin
[1984]
Tucson, Gaithersburg,
San Antornio, Boulder
St. Louis, West palm
beach
6
-
0.564 0.059 Hourly
USA
Heitor et al. [1991]
Sacavem
1
-
0.590 0.044 Hourly
Portugal
Iziomon et al.[2003] Bremgarten, Feldberg
2
212, 1489
0.600 0.064 Hourly
Germany
Wang and Liang
[2009]
SURFRAD
AsiaFlux
FLUXNET
AmeriFlux
GAME AAN
36
98–4700
0.605 0.048 Hourly
USA
Indonesia
Japan
China
Thailand
Australia
Botswana
Canada
Germany
Li et al. [2017]
SURFRAD
7
98–1689
0.598 0.057 Hourly
USA
Liu et al. [2020]
Naqu, Nyingchi, Ali
3
2290–4507
0.560 0.070 Minute
China The values of coefficients a and b of the well-known Brunt model reported by previous authors as well as those derived
in this study are listed in Table 2. The values of coefficients a (0.599) and b (0.053) derived in this study are in consistent
170
with those presented both by Li et al. (2017) and by Wang and Liang (2009), but differ from those provided by other
investigators. Discrepancies in the coefficients might result from different atmospheric conditions (e.g., water vapor content,
CF, and temperature profiles) and the temporal resolution (hourly, daily, or monthly) of the data used in establishing the
parameterization models. The greater the values of coefficient b, the larger the dependences of the parameterization formula
on water vapor (Iziomon et al., 2003). Note that the value of coefficients b was > 0.05 in the Brunt model, Monteith (1961)
175
model, Iziomon model, Berdahl and Martin (1984) model, Li model, and the model developed in this study, which means
that these models have greater sensitivity to water vapor in comparison with other models. 7
Table 2. Coefficients of the Brunt model reported by previous investigators as well as those derived in this study. The temporal resolution of the data used to derive the coefficients and the details of network/sites at which the
observations were performed are also listed. 4.1 Clear-sky emissivity parameterization Scatter plots of measured clear-sky emissivity versus coincident measurements of (a) vapor pressure
165
and (b) the ratio of vapor pressure to screen-level air temperature. Circles represent data pairs observed at
seven CBSRN stations. Black and red thick curves in (a) denote the Brunt model (Eq. 2) and the Weng model
(Eq. 3), respectively. Red thick curve in (b) denotes the parametric formula developed in this study (Eq. 4). Figure 2. Scatter plots of measured clear-sky emissivity versus coincident measurements of (a) vapor pressure
165
and (b) the ratio of vapor pressure to screen-level air temperature. Circles represent data pairs observed at
seven CBSRN stations. Black and red thick curves in (a) denote the Brunt model (Eq. 2) and the Weng model
(Eq. 3), respectively. Red thick curve in (b) denotes the parametric formula developed in this study (Eq. 4). 6 https://doi.org/10.5194/acp-2022-794
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c⃝Author(s) 2023. CC BY 4.0 License. 7
The values of coefficients a and b of the well-known Brunt model reported by previous authors as well as those deriv
in this study are listed in Table 2. The values of coefficients a (0.599) and b (0.053) derived in this study are in consiste
170
with those presented both by Li et al. (2017) and by Wang and Liang (2009), but differ from those provided by oth
investigators. Discrepancies in the coefficients might result from different atmospheric conditions (e.g., water vapor conte
CF, and temperature profiles) and the temporal resolution (hourly, daily, or monthly) of the data used in establishing t
parameterization models. The greater the values of coefficient b, the larger the dependences of the parameterization formu
on water vapor (Iziomon et al., 2003). Note that the value of coefficients b was > 0.05 in the Brunt model, Monteith (196
175
model, Iziomon model, Berdahl and Martin (1984) model, Li model, and the model developed in this study, which mea
that these models have greater sensitivity to water vapor in comparison with other models. Table 2. Coefficients of the Brunt model reported by previous investigators as well as those derived in this study. The temporal resolution of the data used to derive the coefficients and the details of network/sites at which the
observations were performed are also listed. 4.1 Clear-sky emissivity parameterization Reference
Network/Site
Number
of Sites Elevation(m)
a
b
Resolution Country
Brunt [1932]
Benson
1
6
0.520 0.065 Monthly
UK
Anderson [1954]
Laker Hefner
1
369
0.680 0.036 Monthly
USA
Goss and Brooks
[1956]
Davis
1
14
0.660 0.039 Monthly
USA
DeCoster and
Schuepp [1957]
Kinshasa
1
321
0.645 0.048 Daily
Zaire
Monteith [1961]
Kew
1
-
0.530 0.065 Hourly
England
Swinbank [1963]
Aspendale, Kerang
2
-
0.640 0.037 Hourly
Australia
Berger et al. [1984] Carpentras
1
-
0.660 0.040 Hourly
France
Berdahl and Martin
[1984]
Tucson, Gaithersburg,
San Antornio, Boulder
St. Louis, West palm
beach
6
-
0.564 0.059 Hourly
USA
Heitor et al. [1991]
Sacavem
1
-
0.590 0.044 Hourly
Portugal
Iziomon et al.[2003] Bremgarten, Feldberg
2
212, 1489
0.600 0.064 Hourly
Germany
Wang and Liang
[2009]
SURFRAD
AsiaFlux
FLUXNET
AmeriFlux
GAME AAN
36
98–4700
0.605 0.048 Hourly
USA
Indonesia
Japan
China
Thailand
Australia
Botswana
Canada
Germany
Li et al. [2017]
SURFRAD
7
98–1689
0.598 0.057 Hourly
USA
Liu et al. [2020]
Naqu, Nyingchi, Ali
3
2290–4507
0.560 0.070 Minute
China 7 This work
CBSRN
7
67–1991
0.599 0.053 Hourly
China
https://doi.org/10.5194/acp-2022-794
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c⃝Author(s) 2023. CC BY 4.0 License. 4.2 All-sky emissivity parameterization All-sky emissivity can be derived from clear-sky emissivity with correction for clouds and other meteorological elements. In
this study, the parameterizations of all-sky emissivity were derived using the dataset of observations recorded at seven
CBSRN stations between January 2011 and December 2020. The dataset comprises 71,204 hourly measurements of DLR, e, Ta, CF, and φ under all-sky conditions. The formulae derived for all-sky emissivity are as follo
5 F, and φ under all-sky conditions. The formulae derived for all-sky emissivity are as follows: Ta, CF, and φ under all-sky conditions. The formulae derived for all-sky emissivity are as follows:
185
ε𝑎𝑙𝑙,𝐵= ε𝑐𝑙𝑟,𝐵(1 −0.178CF0.339) + 0.075CF0.395∅0.253, (5)
ε𝑎𝑙𝑙,𝑊= ε𝑐𝑙𝑟,𝑊(1 + 0.186CF0.499) −0.298CF0.424∅−0.360, (6)
ε𝑎𝑙𝑙,𝑌= ε𝑐𝑙𝑟,𝑌(1 −0.201CF0.796) + 0.088CF1.038∅0.221. (7) ε𝑎𝑙𝑙,𝐵= ε𝑐𝑙𝑟,𝐵(1 −0.178CF0.339) + 0.075CF0.395∅0.253, (5)
ε𝑎𝑙𝑙,𝑊= ε𝑐𝑙𝑟,𝑊(1 + 0.186CF0.499) −0.298CF0.424∅−0.360, (6)
ε𝑎𝑙𝑙,𝑌= ε𝑐𝑙𝑟,𝑌(1 −0.201CF0.796) + 0.088CF1.038∅0.221. (7) (7) where all,B, all,W, and all,Y represent all-sky emissivity; clr,B, clr,W, and clr,Y are clear-sky e where all,B, all,W, and all,Y represent all-sky emissivity; clr,B, clr,W, and clr,Y are clear-sky emissivity calculated using Eqs. (2)–(4), respectively; CF is the cloud fraction (0–1); φ is relative humidity (%); and the coefficients of determination for Eqs. 190
(5)-(7) are 0.745, 0.748, and 0.750, respectively. where all,B, all,W, and all,Y represent all-sky emissivity; clr,B, clr,W, and clr,Y are clear-sky emissivity calculated using Eqs. (2)–(4), respectively; CF is the cloud fraction (0–1); φ is relative humidity (%); and the coefficients of determination for Eqs. 190
(5)-(7) are 0.745, 0.748, and 0.750, respectively. 190 (2)–(4), respectively; CF is the cloud fraction (0–1); φ is relative humidity (%); and the coefficients of determination for Eqs. 190
(5)-(7) are 0.745, 0.748, and 0.750, respectively. 4.3 Emissivity validation Measurements versus calculations of effective atmospheric emissivity by the Brunt
model, the Weng model, and the model developed in this study for (a)–(c) clear-sky and (d)–(f)
all-sky conditions. Black lines denote the 1:1 line. 5 Figure 3. Measurements versus calculations of effective atmospheric emissivity by the Brunt
model, the Weng model, and the model developed in this study for (a)–(c) clear-sky and (d)–(f)
all-sky conditions. Black lines denote the 1:1 line. Figure 3. Measurements versus calculations of effective atmospheric emissivity by the Brunt
model, the Weng model, and the model developed in this study for (a)–(c) clear-sky and (d)–(f)
all-sky conditions. Black lines denote the 1:1 line. Figure 3. Measurements versus calculations of effective atmospheric emissivity by the Brunt
model, the Weng model, and the model developed in this study for (a)–(c) clear-sky and (d)–(f)
all-sky conditions. Black lines denote the 1:1 line. To illustrate the performance of each of the parameterization models in estimating both clear- and all-sky emissivity in
different seasons, several statistics are summarized in Table 3 and plotted in Fig. 4. The model developed in this study can
overestimate clear-sky emissivity in all seasons except winter (with rRMBE of −1.1 %), whereas, both the Brunt model and
the Weng model can underestimate clear-sky emissivity in all seasons (Fig. 4a). The influence of involving Ta in the model would be more noteworthy during summer and winter because Ta reaches its maximum and minimum value in these seasons,
220
respectively. Furthermore, all parametrization models exhibited apparent negative rMBEs in winter, unlike in other seasons. In winter, the CO2 content over China usually reaches its annual maximum (Fang et al., 2014). Therefore, underestimation of
clear-sky emissivity using parameterization models would be greater in winter owing to the effect of neglecting CO2 in the
models. Under all-sky conditions, the rMBEs of the parameterization models were negative in all seasons, except for the
B
t
d l ( MBE f 0 6 %)
d th
d l d
l
d i thi
t d ( MBE f 1 5 %) i
t
(Fi
4b)
225 would be more noteworthy during summer and winter because Ta reaches its maximum and minimum value in these seasons,
220
respectively. Furthermore, all parametrization models exhibited apparent negative rMBEs in winter, unlike in other seasons. In winter, the CO2 content over China usually reaches its annual maximum (Fang et al., 2014). 4.3 Emissivity validation To verify the clear-sky emissivity parameterization models (Eqs. (2)–(4)) defined in section 4.1, this study used an
independent clear-sky dataset comprising 1,706 hourly clear-sky measurements of DLR, e, and Ta at four CBSRN stations 195 (YQ, XL, SDZ, and XC) acquired between January 2018 and July 2021. The MBEs (rMBEs) between the measured clear-
195
sky emissivity and that estimated by the Brunt model, the Weng model, and model developed in this study were −0.013
(−1.8 %), −0.015 (−2.1 %), and 0.007 (1.0 %), respectively (Fig. 3a–c). The small positive MBE of the model developed in
this study might be attributable to the fact that two parameters (e and Ta) are involved in the equation. Meanwhile, all models
yielded analogous RMSEs (~0.039) and rRMSEs (~5.3 %), which might be a reflection of the dataset and selected independent variables used to establish the formulae. For example, the effects of CO2, O3, and aerosols on emissivity were
200
not considered in these formulae (Staley and Jurica, 1972; Kjaersgaard et al., 2007; Gubler et al., 2012). The parameterization models used to estimate all-sky emissivity (Eqs. (5)–(7)) were validated on the basis of an
independent dataset comprising 20,970 hourly all-sky measurements (DLR, e, Ta, CF, and φ) acquired at three CBSRN
stations (XL, SDZ, XC) between January 2021 and April 2022. The MBEs (rMBEs) between the measured all-sky
emissivity and that calculated by the Brunt model, the Weng model, and model developed in this study were −0.006
205
(−0.8 %), −0.017 (−2.2 %), and −0.004 (−0.5 %), respectively (Fig. 3d–f). Note that the MBEs (rMBEs) of the all-sky
emissivity were close to or even less than those of the clear-sky emissivity. One possible reason is that more samples (20,970)
were adopted in verifying the all-sky emissivity than were adopted in validating the clear-sky emissivity (i.e., 1,706). Another reason is that more input parameters (e.g., CF and RH) other than e and Ta were included in the all-sky emissivity 8 https://doi.org/10.5194/acp-2022-794
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c⃝Author(s) 2023. CC BY 4.0 License. formulae, which alleviated the abrupt variations of e or Ta. However, the RMSE of the all-sky emissivity parameterization
210
model was ~0.049, which is higher than that (~0.039) of the clear-sky emissivity model. Figure 3. 4.3 Emissivity validation Therefore, underestimation of
clear-sky emissivity using parameterization models would be greater in winter owing to the effect of neglecting CO2 in the
models. Under all-sky conditions, the rMBEs of the parameterization models were negative in all seasons, except for the Brunt model (rMBE of 0.6 %) and the model developed in this study (rMBE of 1.5 %) in autumn (Figure 4b). 225
For clear-sky emissivity (Fig. 4c), the rRMSEs between the measurements and the estimations of three models were ~5 %
in spring (March–May), summer (June–August), and autumn (September–November), but >5.7 % in winter (December– (
)
p
y (
)
(
g
)
For clear-sky emissivity (Fig. 4c), the rRMSEs between the measurements and the estimations of three models were ~5 %
in spring (March–May), summer (June–August), and autumn (September–November), but >5.7 % in winter (December– 9 9 https://doi.org/10.5194/acp-2022-794
Preprint. Discussion started: 6 January 2023
c⃝Author(s) 2023. CC BY 4.0 License. February). For all-sky emissivity, the rRMSEs between the measurements and the estimations of three models were closer
with values of ~6.5 %, ~5.0 %, ~6.3 %, and ~7.4 % in spring, summer, autumn, and winter, respectively (Fig. 4d, Table 3). February). For all-sky emissivity, the rRMSEs between the measurements and the estimations of three models were closer
with values of ~6.5 %, ~5.0 %, ~6.3 %, and ~7.4 % in spring, summer, autumn, and winter, respectively (Fig. 4d, Table 3). February). For all-sky emissivity, the rRMSEs between the measurements and the estimations of three models were closer
with values of ~6.5 %, ~5.0 %, ~6.3 %, and ~7.4 % in spring, summer, autumn, and winter, respectively (Fig. 4d, Table 3). February). For all-sky emissivity, the rRMSEs between the measurements and th
with values of ~6.5 %, ~5.0 %, ~6.3 %, and ~7.4 % in spring, summer, autumn, an 30
Figure 4. Seasonal statistics of rMBEs for three parameterization models (a) under clear-sky
conditions and (b) under all-sky conditions; the corresponding rRMSEs for three models (c) under
clear-sky conditions and (d) under all-sky conditions. Dashed line denotes the rRMSE value of 5 %. Figure 4. Seasonal statistics of rMBEs for three parameterization models (a) under clear-sky
conditions and (b) under all-sky conditions; the corresponding rRMSEs for three models (c) under
clear-sky conditions and (d) under all-sky conditions. Dashed line denotes the rRMSE value of 5 %. Table 3. 4.3 Emissivity validation Comparison between the measured emissivity and those estimated using three models in four
seasons under clear- and all-sky conditions. ble 3. Comparison between the measured emissivity and those estimated using three models in fou
asons under clear- and all-sky conditions. Table 3. Comparison between the measured emissivity and those estimated using three models in four
seasons under clear- and all-sky conditions. Table 3. Comparison between the measured emissivity and those estimated using three models in four
seasons under clear- and all-sky conditions. 10
Season Sky condition
Model
MBE
rMBE
(%)
RMSE
rRMSE
(%)
r
Sample
number
Spring
Clear sky
Brunt
−0.012
−1.7
0.038
5.1
0.762
445
Weng
−0.012
−1.5
0.037
4.9
0.772
This work 0.011
1.4
0.038
5.0
0.772
All sky
Brunt
−0.022
−2.8
0.051
6.5
0.815
4389
Weng
−0.025
−3.2
0.052
6.6
0.819
This work −0.018
−2.3
0.049
6.3
0.816
Summer Clear sky
Brunt
−0.001
−0.1
0.029
3.6
0.769
254
Weng
−0.011
−1.3
0.030
3.7
0.750
This work 0.023
2.9
0.037
4.6
0.769
All sky
Brunt
−0.007
−0.9
0.041
4.8
0.810
5036
Weng
−0.016
−1.9
0.047
5.5
0.763
This work 0.000
−0.1
0.040
4.6
0.822
Autumn Clear sky
Brunt
−0.008
−1.1
0.032
4.2
0.841
369
Weng
−0.008
−1.0
0.033
4.3
0.841
This work 0.017
2.2
0.035
4.5
0.848 10 https://doi.org/10.5194/acp-2022-794
Preprint. Discussion started: 6 January 2023
c⃝Author(s) 2023. CC BY 4.0 License. All sky
Brunt
0.005
0.6
0.049
6.3
0.873
5106
Weng
−0.001
−0.2
0.048
6.1
0.886
This work 0.012
1.5
0.050
6.5
0.878
Winter Clear sky
Brunt
−0.022
−3.1
0.047
6.6
0.670
638
Weng
−0.024
−3.5
0.047
6.6
0.688
This work −0.007
−1.1
0.040
5.7
0.695
All sky
Brunt
−0.003
−0.5
0.054
7.6
0.592
6439
Weng
−0.024
−3.4
0.054
7.5
0.709
This work −0.010
−1.4
0.052
7.2
0.657 4.4 DLR validation Based on the Eq. (1), DLR can be calculated in terms of the measurements of screen-level temperature and the corresponding
emissivity estimated using the parameterization models. Statistical results (Table 3) indicated that the MBEs (rMBEs)
between the measured clear-sky DLR and that estimated using the Brunt model, the Weng model, and model developed in
this study were −4.3 W m−2 (−1.5 %), −5.1 W m−2 (−1.8 %), and 3.7 W m−2 (1.3 %), respectively (Fig. 5a−c). The RMSE
240
(rRMSE) of both the Brunt model and the Weng model was 13.8 W m−2 (4.9 %), i.e., slightly lower than that of the model
developed in this study (RMSE: 14.3 W m−2, rRMSE: 5.1 %). Figure 5. Same as Fig. 3 except for the DLR. 260 It can be seen from Table 4 that both the Brunt model and the Weng model could underestimate clear-sky DLR in all
seasons (MBEs in the range of −7.1 to 0.0 W m−2), while the model developed in this study could overestimate clear-sky
DLR in all seasons except winter (MBE of −1.8 W m−2). Additionally, all models could underestimate all-sky DLR (MBEs
in the range of −9.4 to −0.1 W m−2) in all cases, except the Brunt model and the model developed in this study could
255
overestimate all-sky DLR in autumn (MBE of 1.2 and 4.0 W m−2, respectively). The RMSEs (rRMSEs) of clear-sky DLR
estimated by the parameterization models were approximately 14.8 W m−2 (5.0 %), 14.4 W m−2 (4.0 %), 13.0 W m−2 (4.2 %),
and 13.6 W m−2 (6.2 %) in spring, summer, autumn, and winter, respectively; the counterparts of all-sky DLR were
approximately 18.6 W m−2 (6.3 %), 18.4 W m−2 (4.9 %), 17.7 W m−2 (6.1 %), and 15.6 W m−2 (7.3 %) in spring, summer, and
winter, respectively. 260 in the range of −9.4 to −0.1 W m−2) in all cases, except the Brunt model and the model developed in this study could
255
overestimate all-sky DLR in autumn (MBE of 1.2 and 4.0 W m−2, respectively). The RMSEs (rRMSEs) of clear-sky DLR
estimated by the parameterization models were approximately 14.8 W m−2 (5.0 %), 14.4 W m−2 (4.0 %), 13.0 W m−2 (4.2 %),
and 13.6 W m−2 (6.2 %) in spring, summer, autumn, and winter, respectively; the counterparts of all-sky DLR were
approximately 18.6 W m−2 (6.3 %), 18.4 W m−2 (4.9 %), 17.7 W m−2 (6.1 %), and 15.6 W m−2 (7.3 %) in spring, summer, and
winter, respectively. 260 Table 4. Comparison between the measured DLR and those estimated using three models in four
seasons under clear- and all-sky conditions. Figure 5. Same as Fig. 3 except for the DLR. Figure 5. Same as Fig. 3 except for the DLR. Under all-sky conditions, the MBEs (rMBEs) of the Brunt model, the Weng model, and parametric formula developed
245
in this study were −2.8 W m−2 (−1.0 %), −6.1 W m−2 (−2.1 %), and −1.5 W m−2 (−0.5 %), respectively (Fig. 5d−f). The Under all-sky conditions, the MBEs (rMBEs) of the Brunt model, the Weng model, and parametric formula developed
in this study were −2.8 W m−2 (−1.0 %), −6.1 W m−2 (−2.1 %), and −1.5 W m−2 (−0.5 %), respectively (Fig. 5d−f). The 245 11 https://doi.org/10.5194/acp-2022-794
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c⃝Author(s) 2023. CC BY 4.0 License. RMSEs (rRMSEs) between the measured all-sky DLR and that retrieved using the Brunt model, the Weng model, and model
developed in this study were 17.3 W m−2 (6.0 %), 18.1 W m−2 (6.3 %), and 17.0 W m−2 (5.9 %), respectively. It can be seen
that the RMSE (rRMSE) of the all-sky DLR retrieved from the parameterization models was ~3.5 W m−2 (1.0 %) greater than
that of the clear-sky DLR retrieval. Occurrence of clouds and wider ranges of temperature and humidity under all-sky
conditions would disperse the relationship between the observations and the predictions of DLR. 250 It can be seen from Table 4 that both the Brunt model and the Weng model could underestimate clear-sky DLR in all
seasons (MBEs in the range of −7.1 to 0.0 W m−2), while the model developed in this study could overestimate clear-sky
DLR in all seasons except winter (MBE of −1.8 W m−2). Additionally, all models could underestimate all-sky DLR (MBEs
in the range of −9.4 to −0.1 W m−2) in all cases, except the Brunt model and the model developed in this study could
255
overestimate all-sky DLR in autumn (MBE of 1.2 and 4.0 W m−2, respectively). The RMSEs (rRMSEs) of clear-sky DLR
estimated by the parameterization models were approximately 14.8 W m−2 (5.0 %), 14.4 W m−2 (4.0 %), 13.0 W m−2 (4.2 %),
and 13.6 W m−2 (6.2 %) in spring, summer, autumn, and winter, respectively; the counterparts of all-sky DLR were
approximately 18.6 W m−2 (6.3 %), 18.4 W m−2 (4.9 %), 17.7 W m−2 (6.1 %), and 15.6 W m−2 (7.3 %) in spring, summer, and
winter, respectively. Figure 5. Same as Fig. 3 except for the DLR. Season Sky condition
Model
MBE
(Wm−2)
rMBE
(%)
RMSE
(Wm−2)
rRMSE
(%)
r
Sample
number
Spring
Clear sky
Brunt
−4.6
−1.5
14.8
4.9
0.954
445
Weng
−4.3
−1.4
14.3
4.8
0.956
This work
4.7
1.6
15.2
5.1
0.955
All sky
Brunt
−8.3
−2.8
18.7
6.4
0.948
4389
Weng
−9.4
−3.2
19.1
6.5
0.948
This work −6.6
−2.3
18.0
6.1
0.947
Summer Clear sky
Brunt
−0.0
−0.0
12.9
3.5
0.921
254
Weng
−4.6
−1.3
13.5
3.6
0.913
This work 10.9
2.9
16.9
4.6
0.919
All sky
Brunt
−3.1
−0.8
17.8
4.7
0.922
5036
Weng
−7.0
−1.9
20.5
5.4
0.902
This work −0.1
−0.0
17.0
4.5
0.924
Autumn Clear sky
Brunt
−3.1
−1.0
12.4
4.0
0.966
369
Weng
−3.0
−1.0
12.9
4.2
0.963
This work
7.1
2.3
13.9
4.5
0.967
All sky
Brunt
1.2
0.4
17.4
6.0
0.965
5106
Weng
−0.8
−0.3
17.5
6.0
0.965
This work
4.0
1.4
18.2
6.3
0.963
Winter Clear sky
Brunt
−6.6
−3.0
14.2
6.5
0.940
638
Weng
−7.1
−3.3
14.2
6.5
0.946
This work −1.8
−0.8
12.5
5.7
0.947
All sky
Brunt
−1.9
−0.9
15.7
7.3
0.941
6439 Table 4. Comparison between the measured DLR and those estimated using three models in four
seasons under clear- and all-sky conditions. 12 Weng
−7.4
−3.4
15.9
7.4
0.947
This work −3.4
−1.6
15.2
7.1
0.942
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c⃝Author(s) 2023. CC BY 4.0 License. 5 Discussion and conclusions On the basis of the long-term (2011–2022) hourly data measured at the seven CBSRN stations adopted in this study, it
is reasonable to suggest that the parameterization models considered in this study have reasonable spatial representation and
robustness. 275 robustness. 275
This study used continuous hourly CF observations from the HY-WP1A, which remarkably improved the consideration
of cloud effects on estimations of emissivity and DLR. For example, CF data with high temporal resolution can improve
accuracy in estimating emissivity and DLR, and provide the opportunity to study diurnal variations in DLR. In spite of seven CBSRN stations located in various representative regions in China were adopted in this study, however, This study used continuous hourly CF observations from the HY-WP1A, which remarkably improved the consideration
of cloud effects on estimations of emissivity and DLR. For example, CF data with high temporal resolution can improve
accuracy in estimating emissivity and DLR, and provide the opportunity to study diurnal variations in DLR. In spite of seven CBSRN stations located in various representative regions in China were adopted in this study, however,
more CBSRN stations in China are still expected to establish to improve the spatial homogeneity of the parameterization
280
models. On the other hand, the IR02 pyrgeometers currently used at the CBSRN stations should be replaced with more
precise instruments such as the CGR4 pyrgeometer. The IR02 was found usually to produce irrational positive records of
DLR under extreme cold and dry synoptic conditions, which might be caused by its large temperature dependency (within
±3 % under −10 to 40 °C). more CBSRN stations in China are still expected to establish to improve the spatial homogeneity of the parameterization
280
models. On the other hand, the IR02 pyrgeometers currently used at the CBSRN stations should be replaced with more
precise instruments such as the CGR4 pyrgeometer. The IR02 was found usually to produce irrational positive records of
DLR under extreme cold and dry synoptic conditions, which might be caused by its large temperature dependency (within
±3 % under −10 to 40 °C). Though the dominant emitter of longwave radiation in the atmosphere is water vapor, other gases (e.g., CO2 and O3) and
285
aerosols also emit longwave radiation. The effects of gases and aerosols on DLR, whereas, are not considered sufficiently in
the parameterization models. 5 Discussion and conclusions To date, several empirical parameterization models to derive DLR from near-surface mete To date, several empirical parameterization models to derive DLR from near-surface meteorological elements have been
developed on the basis of field observations obtained at a few sites in China. In this study, we utilized a long-term dataset of
265
hourly observations from seven CBSRN stations to recalculate the coefficients of the Brunt model, the Weng model, and a
new parameterization model to estimate atmospheric effective emissivity and DLR under clear-sky and all-sky conditions. The main conclusions of this study are as follows. developed on the basis of field observations obtained at a few sites in China. In this study, we utilized a long-term dataset of
265
hourly observations from seven CBSRN stations to recalculate the coefficients of the Brunt model, the Weng model, and a
new parameterization model to estimate atmospheric effective emissivity and DLR under clear-sky and all-sky conditions. The main conclusions of this study are as follows. Generally, three parameterization models can reliably estimate emissivity and DLR under clear- and all-sky conditions, Generally, three parameterization models can reliably estimate emissivity and DLR under clear- and all-sky conditions,
i.e., the MBEs between the measured clear-sky DLR and that estimated using the Brunt model, the Weng model, and new
70
model developed in this study were −4.3, −5.1, and 3.7 W m−2, respectively; for all-sky DLR, the corresponding MBEs were
−2.8, −6.1, and −1.5 W m−2, respectively. i.e., the MBEs between the measured clear-sky DLR and that estimated using the Brunt model, the Weng model, and new
270
model developed in this study were −4.3, −5.1, and 3.7 W m−2, respectively; for all-sky DLR, the corresponding MBEs were
−2.8, −6.1, and −1.5 W m−2, respectively. On the basis of the long-term (2011–2022) hourly data measured at the seven CBSRN stations adopted in this study, it
is reasonable to suggest that the parameterization models considered in this study have reasonable spatial representation and i.e., the MBEs between the measured clear-sky DLR and that estimated using the Brunt model, the Weng model, and new
270
model developed in this study were −4.3, −5.1, and 3.7 W m−2, respectively; for all-sky DLR, the corresponding MBEs were
−2.8, −6.1, and −1.5 W m−2, respectively. 5 Discussion and conclusions It is expected that the influences of atmospheric components on the relationships between
clear-sky emissivity and screen-level meteorological variables will be further explored by means of the comprehensive
observations at Global Atmosphere Watch station such as SDZ. Data availability. The data used in this study can be provided by the corresponding author upon reasonable request. 290 Data availability. The data used in this study can be provided by the corresponding author upon reasonable request. 290 Data availability. The data used in this study can be provided by the corresponding author upon reasonable request. 290 13 https://doi.org/10.5194/acp-2022-794
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c⃝Author(s) 2023. CC BY 4.0 License. Author contributions. JH and QC contributed to shaping the ideas and presenting research goal and constructive comments
on the research. JY and WQ presented the construction of the paper. JY contributed to processing and analysis of the data as
well as preparing the manuscript. WQ contributed the ideas, organized the research, performed the review, edited the
manuscript, and provided the funding acquisitions. 95 295 Review statement. This paper was edited by ××× and reviewed by ××× anonymous referees. Review statement. This paper was edited by ××× and reviewed by ××× anonymous referees. Competing interests. The authors declare that they have no conflict of interest. Acknowledgements. We greatly appreciate Qifeng Lu of the CEMC for providing valuable and stimulating comments. We
also thank Na Liu of the National Meteorological Information Centre for her helpful suggestions on how to correctly use the
300
radiation data and meteorological data. We wish to thank reviewers and the editor for their valuable insights that helped
greatly strengthen the manuscript. Acknowledgements. We greatly appreciate Qifeng Lu of the CEMC for providing valuable and stimulating comments. We
also thank Na Liu of the National Meteorological Information Centre for her helpful suggestions on how to correctly use the
300
radiation data and meteorological data. We wish to thank reviewers and the editor for their valuable insights that helped
greatly strengthen the manuscript. Acknowledgements. We greatly appreciate Qifeng Lu of the CEMC for providing valuable and stimulating comments. We
also thank Na Liu of the National Meteorological Information Centre for her helpful suggestions on how to correctly use the
300
radiation data and meteorological data. We wish to thank reviewers and the editor for their valuable insights that helped
greatly strengthen the manuscript. Financial support. This study was funded by the China Scholarship Council (No. 202205330024), National Key Research Financial support. This study was funded by the China Scholarship Council (No. 202205330024), National Key Research
and Development Program of China (Grant No. 2017YFB0504002), National Science and Technology Infrastructure
305
Platform Project (2017), and the Special Fund for Basic Scientific Research of Institute of Urban Meteorology (Grant No. IUMKY201735). and Development Program of China (Grant No. 2017YFB0504002), National Science and Technology Infrastructure
305
Platform Project (2017), and the Special Fund for Basic Scientific Research of Institute of Urban Meteorology (Grant No. IUMKY201735). References
310 References
310
Alados, I., Foyo-Moreno, I., and Alados-Arboledas, L.: Estimation of downwelling longwave irradiance under all-sky
conditions, Int. J. Climatol., 32, 781-793, https://doi.org/10.1002/joc.2307, 2012. Anderson, E. R.: Energy budget studies, water loss investigations: LakeHefner studies, Technical Report, U. S. Geol. Surv. Prof. Pap., 269, 71-119, 1954. Alados, I., Foyo-Moreno, I., and Alados-Arboledas, L.: Estimation of downwelling longwave irradiance under all-sky
conditions, Int. J. Climatol., 32, 781-793, https://doi.org/10.1002/joc.2307, 2012. Anderson, E. R.: Energy budget studies, water loss investigations: LakeHefner studies, Technical Report, U. S. Geol. Surv. Prof. Pap., 269, 71-119, 1954. Ångström, A.: A study of the radiation of the atmosphere, Smithsonian Miscellaneous Collection, 65, 1-159, 1915. 315 Ångström, A.: A study of the radiation of the atmosphere, Smithsonian Miscellaneous Collection, 65, 1-159, 1915. 315
Berdahl, P. and Fromberg, R.: The thermal radiance of clear skies, Sol. Energy, 29, 299-314, https://doi.org/10.1016/0038-
092X(82)90245-6, 1982. Berdahl, P. and Martin, M.: Emissivity of clear skies, Sol. Energy, 32, 663-664, https://doi.org/10.1016/0038-
092X(84)90144-0, 1984. Berdahl, P. and Fromberg, R.: The thermal radiance of clear skies, Sol. Energy, 29, 299-314, https://doi.org/10.1016/0038-
092X(82)90245-6, 1982. 092X(82)90245-6, 1982. Berdahl, P. and Martin, M.: Emissivity of clear skies, Sol. Energy, 32, 663-664, https://doi.org/10.1016/0038-
092X(84)90144-0, 1984. Berdahl, P. and Martin, M.: Emissivity of clear skies, Sol. Energy, 32, 663-664, https://doi.org/10.1016/0038-
092X(84)90144-0, 1984. Berger, X., Buriot, D., and Garnier, F.: About the equivalent radiative temperature for clear skies, Sol. Energy, 32, 725-733,
320
https://doi.org/10.1016/0038-092X(84)90247-0, 1984. Berger, X., Buriot, D., and Garnier, F.: About the equivalent radiative temperature for clear skies, Sol. Energy, 32, 725-733,
320
https://doi.org/10.1016/0038-092X(84)90247-0, 1984. Bilbao, J. and De Miguel, A. H.: Estimation of daylight downward longwave atmospheric irradiance under clear-sky and all- Berger, X., Buriot, D., and Garnier, F.: About the equivalent radiative temperature for clear skies, Sol. Energy, 32, 725-733,
320
https://doi.org/10.1016/0038-092X(84)90247-0, 1984. Bilbao, J. and De Miguel, A. H.: Estimation of daylight downward longwave atmospheric irradiance under clear-sky and all-
sky conditions, J. Appl. Meteorol. Clim., 46, 878-889, https://doi.org/10.1175/JAM2503.1, 2007. Bilbao, J. and De Miguel, A. H.: Estimation of daylight downward longwave atmospheric irradiance under clear-sky and all-
sky conditions, J. Appl. Meteorol. Clim., 46, 878-889, https://doi.org/10.1175/JAM2503.1, 2007. 14 https://doi.org/10.5194/acp-2022-794
Preprint. Discussion started: 6 January 2023
c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/acp-2022-794
Preprint. Discussion started: 6 January 2023
c⃝Author(s) 2023. CC BY 4.0 License. Brunt,
D.:
Notes
on
radiation
in
the
atmosphere,
Quart. J. Roy. Meteorol. Soc.,
58,
389-418,
https://doi.org/10.1002/qj.49705824704, 1932. References
310 325 Brutsaert, W.: On a derivable formula for long-wave radiation from clear skies, Water Resour. Res., 11, 742-744,
https://doi.org/10.1029/WR011i005p00742, 1975. Carmona, F., Rivas, R., and Caselles, V.: Estimation of daytime downward longwave radiation under clear and cloudy skies
conditions over a sub-humid region, Theor. Appl. Climatol., 115, 281-295, https://doi.org/10.1007/s00704-013-0891-3,
2014. 2014. 330
Crawford, T. M., and Duchon, C. E.: An improved parameterization for estimating effective atmospheric emissivity for use
in calculating daytime downwelling longwave radiation, J. Appl. Meteorol., 38, 474-480, 1999. Culf, A. D. and Gash, J. H.: Longwave radiation from clear skies in Niger: a comparison of observations with simple
formulas, J. Appl. Meteorol., 32, 539-547, https://doi.org/ 10.1175/1520-0450(1993)0322.0.CO;2, 1993. 2014. 30
Crawford, T. M., and Duchon, C. E.: An improved parameterization for estimating effective atmospheric emissivity for use
in calculating daytime downwelling longwave radiation, J. Appl. Meteorol., 38, 474-480, 1999. Culf, A. D. and Gash, J. H.: Longwave radiation from clear skies in Niger: a comparison of observations with simple Crawford, T. M., and Duchon, C. E.: An improved parameterization for estimating effective atmospheric emissivity for use
in calculating daytime downwelling longwave radiation, J. Appl. Meteorol., 38, 474-480, 1999. Culf, A. D. and Gash, J. H.: Longwave radiation from clear skies in Niger: a comparison of observations with simple
formulas, J. Appl. Meteorol., 32, 539-547, https://doi.org/ 10.1175/1520-0450(1993)0322.0.CO;2, 1993. DeCoster, M. and Schuepp, W.: Measures de rayonnement effectif à Leopoldville, Acad. Roy. Sci. Colon. Brussels Bull. 335
Seances, 3, 642-651, 1957. Duarte, H. F., Dias, N. L., and Maggiotto, S. R.: Assessing daytime downward longwave radiation estimates for clear and
cloudy skies in Southern Brazil, Agr. Forest. Meteorol., 139, 171-181, https://doi.org/10.1016/j.agrformet.2006.06.008,
2006. DeCoster, M. and Schuepp, W.: Measures de rayonnement effectif à Leopoldville, Acad. Roy. Sci. Colon. Brussels Bull. 35
Seances, 3, 642-651, 1957. Duarte, H. F., Dias, N. L., and Maggiotto, S. R.: Assessing daytime downward longwave radiation estimates for clear and
cloudy skies in Southern Brazil, Agr. Forest. Meteorol., 139, 171-181, https://doi.org/10.1016/j.agrformet.2006.06.008,
2006. Fang, S. X., Zhou, L. X., Tans, P. P., Ciais, P., Steinbacher, M., Xu, L., and Luan, T.: In situ measurement of atmospheric
340
CO2 at the four WMO/GAW stations in China, Atmos. Chem. Phys., 14, 2541-2554, https://doi.org/10.5194/acp-14-
2541-2014, 2014. Gabathuler, M., Marty, C. A., and Hanselmann, K. W.: Parameterization of incoming longwave radiation in high-mountain
environments, Phys. Geogr., 22, 99-114, https://doi.org/10.1080/02723646.2001.10642732, 2001. Fang, S. X., Zhou, L. X., Tans, P. References
310 P., Ciais, P., Steinbacher, M., Xu, L., and Luan, T.: In situ measurement of atmospheric
340
CO2 at the four WMO/GAW stations in China, Atmos. Chem. Phys., 14, 2541-2554, https://doi.org/10.5194/acp-14-
2541-2014, 2014. Gabathuler, M., Marty, C. A., and Hanselmann, K. W.: Parameterization of incoming longwave radiation in high-mountain
environments, Phys. Geogr., 22, 99-114, https://doi.org/10.1080/02723646.2001.10642732, 2001. Goss, J. R. and Brooks, F. A.: Constants for empirical expressions for downcoming atmospheric radiation under cloudless
345
sky, J. Meteorol., 13, 482-488, 1956. Gubler, S., Gruber, S., and Purves, R. S.: Uncertainties of parameterized surface downward clear-sky shortwave and all-sky
longwave radiation, Atmos. Chem. Phys., 12, 5077-5098, https://doi.org/10.5194/acp-12-5077-2012, 2012. Heitor, A., Biga, A. J., and Rosa, R.: Thermal radiation components of the energy balance at the ground. Agr. Forest. Goss, J. R. and Brooks, F. A.: Constants for empirical expressions for downcoming atmospheric radiation under cloudless
345
sky, J. Meteorol., 13, 482-488, 1956. Gubler, S., Gruber, S., and Purves, R. S.: Uncertainties of parameterized surface downward clear-sky shortwave and all-sky
longwave radiation, Atmos. Chem. Phys., 12, 5077-5098, https://doi.org/10.5194/acp-12-5077-2012, 2012. Heitor, A., Biga, A. J., and Rosa, R.: Thermal radiation components of the energy balance at the ground. Agr. Forest. Meteorol., 54, 29-48, https://doi.org/10.1016/0168-1923(91)90039-S, 1991. 350 Heitor, A., Biga, A. J., and Rosa, R.: Thermal radiation components of the energy balance at the ground. Agr. Forest. Meteorol., 54, 29-48, https://doi.org/10.1016/0168-1923(91)90039-S, 1991. 350
Hua, H. X., Zhang, R. Q., Li, Z. H., and Tian, X.: Installation, application and maintenance of HY-WP1 intelligent weather
observation instrument. Henan Sci. Tech., 31, 6-8, doi:10.3969/j.issn.1003-5168.2021.31.011, 2021 (in Chinese). Meteorol., 54, 29-48, https://doi.org/10.1016/0168-1923(91)90039-S, 1991. 350
Hua, H. X., Zhang, R. Q., Li, Z. H., and Tian, X.: Installation, application and maintenance of HY-WP1 intelligent weather
observation instrument. Henan Sci. Tech., 31, 6-8, doi:10.3969/j.issn.1003-5168.2021.31.011, 2021 (in Chinese). Hukseflux: User manual IR02 pyrgeometer with heater, User mannual, v2207, 1-43, Hukseflux Thermal Sensors B. V.,
Delftechpark 31, 2628 XJ Delft, The Netherlands, 2022. Hua, H. X., Zhang, R. Q., Li, Z. H., and Tian, X.: Installation, application and maintenance of HY-WP1 intelligent weather
observation instrument. Henan Sci. Tech., 31, 6-8, doi:10.3969/j.issn.1003-5168.2021.31.011, 2021 (in Chinese). Hukseflux: User manual IR02 pyrgeometer with heater, User mannual, v2207, 1-43, Hukseflux Thermal Sensors B. V.,
Delftechpark 31, 2628 XJ Delft, The Netherlands, 2022. Huo, Q., Ren, Z. References
310 L., and Hansen, S.: Comparison of models for calculating daytime long-wave irradiance
365
using long term data set. Agr. Forest. Meteorol., 143, 49-63, https://doi.org/10.1016/j.agrformet.2006.11.007, 2007. using long term data set. Agr. Forest. Meteorol., 143, 49 63, https://doi.org/10.1016/j.agrformet.2006.11.007, 2007. Konzelmann, T., van de Wal, R. S. W., Greuell, W., Bintanja, R. Henneken, E. A. C. and Abe-Ouchi, A.: Parameterization of
global and longwave incoming radiation for the Greenland Ice Sheet, Global. Planet. Change, 9, 143-164,
https://doi.org/10.1016/0921-8181(94)90013-2, 1994. Li, F., Ji, C. L., Mo, Y. Q., and Guo, Z. J.: Examination and evaluation of integrality and rationality of China basic radiation
370
station data, Plateau Meteorol., 32, 1203-1213, https://doi.org/10.7522/j.issn.1000-0534.2012.00113, 2013 (in
Chinese). Li, F., Ji, C. L., Mo, Y. Q., and Guo, Z. J.: Examination and evaluation of integrality and rationality of China basic radiation
370
station data, Plateau Meteorol., 32, 1203-1213, https://doi.org/10.7522/j.issn.1000-0534.2012.00113, 2013 (in
Chinese). Liu, M. Q., Zheng, X. D., Zhang, J. Q. and Xia, X. A.: A revisiting of the parameterization of downward longwave radiation
in summer over the Tibetan Plateau based on high-temporal-resolution measurements, Atmos. Chem. Phys., 20, 4415- Liu, M. Q., Zheng, X. D., Zhang, J. Q. and Xia, X. A.: A revisiting of the parameterization of downward longwave radiation
in summer over the Tibetan Plateau based on high-temporal-resolution measurements, Atmos. Chem. Phys., 20, 4415-
4426, http://dx.doi.org/10.5194/acp-20-4415-2020, 2020. 375 4426, http://dx.doi.org/10.5194/acp-20-4415-2020, 2020. 375
Li, M. Y., Jiang, Y. J., and Coimbra, C. F. M.: On the determination of atmospheric longwave irradiance under all-sky
conditions, Sol. Energy, 144, 40-48, https://dx.doi.org/10.1016/j.solener.2017.01.006, 2017. Marthews, T. R., Malhi, Y., and Iwata, H.: Calculating downward longwave radiation under clear and cloudy conditions over
a tropical lowland forest site: an evaluation of model schemes for hourly data, Theor. Appl. Climatol., 107, 461-477, Li, M. Y., Jiang, Y. J., and Coimbra, C. F. M.: On the determination of atmospheric longwave irradiance under all-sky
conditions, Sol. Energy, 144, 40-48, https://dx.doi.org/10.1016/j.solener.2017.01.006, 2017. Marthews, T. R., Malhi, Y., and Iwata, H.: Calculating downward longwave radiation under clear and cloudy conditions over
a tropical lowland forest site: an evaluation of model schemes for hourly data, Theor. Appl. Climatol., 107, 461-477,
https://doi.org/10.1007/s00704-011-0486-9, 2012. 380 https://doi.org/10.1007/s00704-011-0486-9, 2012. 380
Niemelä, S., Räisänen, P. and Savijärvi, H.: Comparison of surface radiative flux parameterizations Part I: Longwave
radiation, Atmos. Res., 58, 1-18, https://doi.org/10.1016/S0169-8095(01)00084-9, 2001. Monteith, J. L.: An empirical method for estimating long-wave radiation exchange in the British Isles, Quart. J. R. Meteorol. References
310 H., Liu, N., Sun, C., and Li, X.: Comparative evaluation of observations from the meteorological radiation
355
station and the baseline surface radiation station. Hubei Agri. Sci., 56, 4056-4061, doi:10.14088/j.cnki.issn0439-
8114.2017.21.015, 2017 (in Chinese). Idso, S. B.: A set of equations for full spectrum and 8- to 14-μm and 10.5- to 12.5-μm thermal radiation from cloudless skies,
Water Resour. Res., 17, 295-304, https://doi.org/10.1029/WR017i002p00295, 1981. Huo, Q., Ren, Z. H., Liu, N., Sun, C., and Li, X.: Comparative evaluation of observations from the meteorological radiation
355
station and the baseline surface radiation station. Hubei Agri. Sci., 56, 4056-4061, doi:10.14088/j.cnki.issn0439-
8114.2017.21.015, 2017 (in Chinese). Idso, S. B.: A set of equations for full spectrum and 8- to 14-μm and 10.5- to 12.5-μm thermal radiation from cloudless skies,
Water Resour. Res., 17, 295-304, https://doi.org/10.1029/WR017i002p00295, 1981. 15 https://doi.org/10.5194/acp-2022-794
Preprint. Discussion started: 6 January 2023
c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/acp-2022-794
Preprint. Discussion started: 6 January 2023
c⃝Author(s) 2023. CC BY 4.0 License. Idso, S. B. and Jackson, R. D.: Thermal radiation from the atmosphere, J. Geophy. Res., 74, 5397-5403
360
https://doi.org/10.1029/JC074i023p05397, 1969. Idso, S. B. and Jackson, R. D.: Thermal radiation from the atmosphere, J. Geophy. Res., 74, 5397-5403,
360
https://doi.org/10.1029/JC074i023p05397, 1969. Iziomon, M. G., Mayer, H., and Matzarakis, A.: Downward atmospheric longwave irradiance under clear and cloudy skies:
M
t
d
t i
ti
J
At
S l
T
Ph
65
1107 1116 Idso, S. B. and Jackson, R. D.: Thermal radiation from the atmosphere, J. Geophy. Res., 74, 5397-5403,
360
https://doi.org/10.1029/JC074i023p05397, 1969. Iziomon, M. G., Mayer, H., and Matzarakis, A.: Downward atmospheric longwave irradiance under clear and cloudy skies:
Measurement
and
parameterization,
J. Atmos. Sol. Terr. Phys.,
65,
1107-1116,
https://doi.org/10.1016/j.jastp.2003.07.007, 2003. Iziomon, M. G., Mayer, H., and Matzarakis, A.: Downward atmospheric longwave irradiance under clear and cloudy skies:
Measurement
and
parameterization,
J. Atmos. Sol. Terr. Phys.,
65,
1107-1116,
https://doi.org/10.1016/j.jastp.2003.07.007, 2003. p
g
j j
p
Kjaersgaard, J. H., Plauborg, F. L., and Hansen, S.: Comparison of models for calculating daytime long-wave irradiance
365
using long term data set. Agr. Forest. Meteorol., 143, 49-63, https://doi.org/10.1016/j.agrformet.2006.11.007, 2007. Konzelmann, T., van de Wal, R. S. W., Greuell, W., Bintanja, R. Henneken, E. A. C. and Abe-Ouchi, A.: Parameterization of
global and longwave incoming radiation for the Greenland Ice Sheet, Global. Planet. Change, 9, 143-164,
https://doi.org/10.1016/0921-8181(94)90013-2, 1994. Kjaersgaard, J. H., Plauborg, F. References
310 Soc., 87,171-179, https://doi.org/10.1002/qj.49708837615, 1961. Niemelä, S., Räisänen, P. and Savijärvi, H.: Comparison of surface radiative flux parameterizations Part I: Longwave
radiation, Atmos. Res., 58, 1-18, https://doi.org/10.1016/S0169-8095(01)00084-9, 2001. Monteith, J. L.: An empirical method for estimating long-wave radiation exchange in the British Isles, Quart. J. R. Meteorol. Soc., 87,171-179, https://doi.org/10.1002/qj.49708837615, 1961. Ohmura, A.: Physical basis for the temperature-based melt-index method, J. Appl. Meteorol., 40, 753-761,
385
https://doi.org/10.1175/1520-0450(2001)040%3C0753:PBFTTB%3E2.0.CO;2, 2001. Prata, A. J.: A new long-wave formula for estimating downward clear-sky radiation at the surface, Q. J. R. Meteorol. Soc.,
122, 1127-1151, https://doi.org/10.1002/qj.49712253306, 1996. Rodgers, C. D.: The use of emissivity in atmospheric radiation calculations, Q. J. R. Meteorol. Soc., 93, 43-54, Ohmura, A.: Physical basis for the temperature-based melt-index method, J. Appl. Meteorol., 40, 753-761,
385
https://doi.org/10.1175/1520-0450(2001)040%3C0753:PBFTTB%3E2.0.CO;2, 2001. Prata, A. J.: A new long-wave formula for estimating downward clear-sky radiation at the surface, Q. J. R. Meteorol. Soc.,
122, 1127-1151, https://doi.org/10.1002/qj.49712253306, 1996. Rodgers, C. D.: The use of emissivity in atmospheric radiation calculations, Q. J. R. Meteorol. Soc., 93, 43-54,
https://doi.org/10.1002/qj.49709339504, 1967. 390
Satterlund, D. R.: An improved equation for estimating longwave radiation from the atmosphere, Water Resour. Res., 15,
1649-1650, https://doi.org/10.1029/wr015i006p01649, 1979. Sridhar, V., and Elliott, R. L.: On the development of a simple downwelling longwave radiation scheme. Agr. Forest. Meteorol., 112, 237-243, https://doi.org/10.1016/S0168-1923(02)00129-6, 2002. Ohmura, A.: Physical basis for the temperature-based melt-index method, J. Appl. Meteorol., 40, 753-761,
385
https://doi.org/10.1175/1520-0450(2001)040%3C0753:PBFTTB%3E2.0.CO;2, 2001. https://doi.org/10.1175/1520-0450(2001)040%3C0753:PBFTTB%3E2.0.CO;2, 2001. Prata, A. J.: A new long-wave formula for estimating downward clear-sky radiation at the surface, Q. J. R. Meteorol. Soc.,
122, 1127-1151, https://doi.org/10.1002/qj.49712253306, 1996. Prata, A. J.: A new long-wave formula for estimating downward clear-sky radiation at the surface, Q. J. R. Meteorol. Soc.,
122, 1127-1151, https://doi.org/10.1002/qj.49712253306, 1996. Rodgers, C. D.: The use of emissivity in atmospheric radiation calculations, Q. J. R. Meteorol. Soc., 93, 43-54,
https://doi.org/10.1002/qj.49709339504, 1967. 0 Satterlund, D. R.: An improved equation for estimating longwave radiation from the atmosphere, Water Resour. Res., 15,
1649-1650, https://doi.org/10.1029/wr015i006p01649, 1979. Sridhar, V., and Elliott, R. L.: On the development of a simple downwelling longwave radiation scheme. Agr. Forest. Meteorol., 112, 237-243, https://doi.org/10.1016/S0168-1923(02)00129-6, 2002. 16 https://doi.org/10.5194/acp-2022-794
Preprint. Discussion started: 6 January 2023
c⃝Author(s) 2023. CC BY 4.0 License. https://doi.org/10.5194/acp-2022-794
Preprint. Discussion started: 6 January 2023
c⃝Author(s) 2023. CC BY 4.0 License. Staley, D. O. and Jurica, G. M.: Effective atmospheric emissivity under clear skies, J. Appl. Meteorol., 11, 349-356,
95
https://doi.org/10.1175/1520-0450(1972)011%3C0349:EAEUCS%3E2.0.CO;2, 1972. Staley, D. O. and Jurica, G. References
310 M.: Effective atmospheric emissivity under clear skies, J. Appl. Meteorol., 11, 349-356,
395
https://doi.org/10.1175/1520-0450(1972)011%3C0349:EAEUCS%3E2.0.CO;2, 1972. Stephens, G. L., Wild, M., Stackhouse Jr, P. W., L’Ecuyer, T., Kato, S., and Henderson, D. S.: The global character of the
flux of downward longwave radiation, J. Climate, 25, 2329-2340, https://doi.org/10.1175/jcli-d-11-00262.1, 2012. Swinbank, W. C.: Long-wave radiation from clear skies, Quart. J. Roy. Meteorol. Soc., 89, 339-348,
https://doi.org/10.1002/qj.49708938105, 1963. 400
Wang, K. C., and Liang, S. L.: Global atmospheric downward longwave radiation over land surface under all-sky conditions
from 1973 to 2008, J. Geophys. Res., 114, D19101, https://doi.org/10.1029/2009JD011800, 2009. Weng, D. M., Sun, Z. A. and Wen, Z. Z.: Climatological study on downward atmospheric radiation in China, J. Nanjing Inst. Meteorol., 16, 1-5, https://doi.org/10.13878/j.cnki.dqkxxb.1993.01.001, 1993 (in Chinese). Staley, D. O. and Jurica, G. M.: Effective atmospheric emissivity under clear skies, J. Appl. Meteorol., 11, 349-356,
395
https://doi.org/10.1175/1520-0450(1972)011%3C0349:EAEUCS%3E2.0.CO;2, 1972. https://doi.org/10.1175/1520-0450(1972)011%3C0349:EAEUCS%3E2.0.CO;2, 1972. Stephens, G. L., Wild, M., Stackhouse Jr, P. W., L’Ecuyer, T., Kato, S., and Henderson, D. S.: The global character of the
flux of downward longwave radiation, J. Climate, 25, 2329-2340, https://doi.org/10.1175/jcli-d-11-00262.1, 2012. S i b
k
W
C
L
di ti
f
l
ki
Q
t
J
R
M t
l
S
89
339 348 https://doi.org/10.1175/1520 0450(1972)011%3C0349:EAEUCS%3E2.0.CO;2, 1972. Stephens, G. L., Wild, M., Stackhouse Jr, P. W., L’Ecuyer, T., Kato, S., and Henderson, D. S.: The global character of the
flux of downward longwave radiation, J. Climate, 25, 2329-2340, https://doi.org/10.1175/jcli-d-11-00262.1, 2012. Swinbank, W. C.: Long-wave radiation from clear skies, Quart. J. Roy. Meteorol. Soc., 89, 339-348,
https://doi.org/10.1002/qj.49708938105, 1963. 400
Wang, K. C., and Liang, S. L.: Global atmospheric downward longwave radiation over land surface under all-sky conditions
f
1973
2008 J G
h
R
114 D19101 h
//d i
/10 1029/2009JD011800 2009 Stephens, G. L., Wild, M., Stackhouse Jr, P. W., L’Ecuyer, T., Kato, S., and Henderson, D. S.: The global character of the
flux of downward longwave radiation, J. Climate, 25, 2329-2340, https://doi.org/10.1175/jcli-d-11-00262.1, 2012. Stephens, G. L., Wild, M., Stackhouse Jr, P. W., L’Ecuyer, T., Kato, S., and Henderson, D. S.: The global character of the
flux of downward longwave radiation, J. Climate, 25, 2329-2340, https://doi.org/10.1175/jcli-d-11-00262.1, 2012. Swinbank, W. C.: Long-wave radiation from clear skies, Quart. J. Roy. Meteorol. Soc., 89, 339-348,
https://doi.org/10.1002/qj.49708938105, 1963. 400 Swinbank, W. C.: Long-wave radiation from clear skies, Quart. J. Roy. Meteorol. Soc., 89, 339-348,
https://doi.org/10.1002/qj.49708938105, 1963. 00 Wang, K. C., and Liang, S. References
310 L.: Global atmospheric downward longwave radiation over land surface under all-sky conditions
from 1973 to 2008, J. Geophys. Res., 114, D19101, https://doi.org/10.1029/2009JD011800, 2009. Wang, K. C., and Liang, S. L.: Global atmospheric downward longwave radiation over land surface under all-sky conditions
from 1973 to 2008, J. Geophys. Res., 114, D19101, https://doi.org/10.1029/2009JD011800, 2009. Weng, D. M., Sun, Z. A. and Wen, Z. Z.: Climatological study on downward atmospheric radiation in China, J. Nanjing Inst. Meteorol., 16, 1-5, https://doi.org/10.13878/j.cnki.dqkxxb.1993.01.001, 1993 (in Chinese). Weng, D. M., Sun, Z. A. and Wen, Z. Z.: Climatological study on downward atmospheric radiation in China, J. Nanjing Inst. Meteorol., 16, 1-5, https://doi.org/10.13878/j.cnki.dqkxxb.1993.01.001, 1993 (in Chinese). Zhou, H. G., Quan, W. J., Wang, Z. F., Li, X. L., Li, Y. R., and Zhao, H. J.: Comparison of sunshine duration measurements
405
between a Jordan sunshine recorder and three automatic sensors at Shangdianzi GAW station, J. Meteorol. Res., 35,
716-728, https://doi.org/10.1007/s13351-021-0158-3, 2021. Zhou, H. G., Quan, W. J., Wang, Z. F., Li, X. L., Li, Y. R., and Zhao, H. J.: Comparison of sunshine duration measurements
405
between a Jordan sunshine recorder and three automatic sensors at Shangdianzi GAW station, J. Meteorol. Res., 35,
716-728, https://doi.org/10.1007/s13351-021-0158-3, 2021. Zhu, M. L., Yao, T. D., Yang, W., Xu, B. Q., and Wang, X. J.: Evaluation of parameterizations of incoming longwave
radiation in the high-mountain region of the Tibetan Plateau, J. Appl. Meteorol. Climatol., 56, 833-848,
https://doi.org/10.1175/JAMC-D-16-0189.1, 2017. 410 Zhou, H. G., Quan, W. J., Wang, Z. F., Li, X. L., Li, Y. R., and Zhao, H. J.: Comparison of sunshine duration measurements
405
between a Jordan sunshine recorder and three automatic sensors at Shangdianzi GAW station, J. Meteorol. Res., 35,
716-728, https://doi.org/10.1007/s13351-021-0158-3, 2021. 716-728, https://doi.org/10.1007/s13351-021-0158-3, 2021. Zhu, M. L., Yao, T. D., Yang, W., Xu, B. Q., and Wang, X. J.: Evaluation of parameterizations of incoming longwave
radiation in the high-mountain region of the Tibetan Plateau, J. Appl. Meteorol. Climatol., 56, 833-848,
https://doi.org/10.1175/JAMC-D-16-0189.1, 2017. 410 410 17 17
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Weed Suppression in Only-Legume Cover Crop Mixtures
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Agronomy
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Received: 16 September 2019; Accepted: 15 October 2019; Published: 17 October 2019 Abstract: Weed suppression is a potential benefit of cover crop mixtures, as species diversity may
allow for combining early and late-season competition with weeds. Here, we studied if this is possible
for only-legume mixtures containing species with different growth rates, by testing two legumes,
alsike clover (AC; Trifolium hybridum L.) and black medic (BM; Medicago lupulina L.) in two field
trials sown in 2016 and 2017. Five AC:BM ratios (100:0, 67:33, 50:50, 33:67, and 0:100) were grown
at three densities (50%, 100%, and 150% of recommended seed density). Cover crop and weed
aboveground biomass (CCB and WB, respectively) were harvested three times, after establishment
in spring (H1), in summer (H2), and in autumn after mulching (H3). Compared to fallow plots, all
monocultures and mixtures showed early-season weed suppression in terms of biomass production
and more efficiency over time with an average reduction of 42%, 52%, and 96% in 2016, and 39%,
55%, and 89% in 2017 at H1, H2, and H3, respectively. Out of 54 mixture treatments, only eight
mixtures showed stronger weed suppression than monocultures. Mixtures reduced WB by 28%, as
an average value, in 2017 compared to the respective monocultures, but not significantly in 2016,
indicating that the crop diversity effect on weeds was dependent on the growing environment. Weed
suppression was significantly higher at 100% and 150% seed density than 50%, but no significant
differences were determined between 100% and 150% seed density. After mulching, no density effect
was observed on CCB and WB. In conclusion, AC and BM can be used as a keystone species on weed
suppression for sustainable agriculture as they possess plasticity to suppress weeds when higher
biomass productivity is limited by environmental conditions. However, their diversity effects are
time and condition dependent. Appropriate seed density and mulching can successfully be employed
in weed management, but seed density may not have an effect after mulching. Keywords: asynchrony; crop diversification; forage legume; functional traits; interspecific interaction;
mixed cropping; weed control Weed Suppression in Only-Legume Cover
Crop Mixtures Heba Elsalahy 1,2,*
, Thomas Döring 3
, Sonoko Bellingrath-Kimura 1,4
and
Danny Arends 5 Heba Elsalahy 1,2,*
, Thomas Döring 3
, Sonoko Bellingrath-Kimura 1,4
and
D
A
d
5 1
Department of Agronomy and Crop Science, Humboldt-University of Berlin, Albrecht-Thaer-Weg 5,
14195 Berlin, Germany; belks@zalf.de 1
Department of Agronomy and Crop Science, Humboldt-University of Berlin, Albrecht-Thaer-Weg 5,
14195 Berlin, Germany; belks@zalf.de 1
Department of Agronomy and Crop Science, Humboldt-University of Berlin, Albrecht-Thaer-Weg 5,
14195 Berlin, Germany; belks@zalf.de y
2
Faculty of Science, Botany and Microbiology Department, Assiut University, Assiut 71516, Egypt 2
Faculty of Science, Botany and Microbiology Department, Assiut University, Assiut 71516, Egypt
3
Agroecology and Organic Farming Group, University of Bonn, Auf dem Hügel 6, 53121 Bonn, Germany;
tdoering@uni-bonn.de y
y
gy
p
y
gyp
3
Agroecology and Organic Farming Group, University of Bonn, Auf dem Hügel 6, 53121 Bonn, Germany;
tdoering@uni-bonn.de 4
Research Area “Land Use and Governance”, Leibniz Centre for Agricultural Landscape Research (ZALF),
Eberswalder Str. 84, 15374 Müncheberg, Germany 4
Research Area “Land Use and Governance”, Leibniz Centre for Agricultural Landscape Research (ZALF),
Eberswalder Str. 84, 15374 Müncheberg, Germany g
y
5
Animal Breeding Biology and Molecular Genetics, Albrecht Daniel Thaer-Institute for Agricultural and
Horticultural Sciences, Humboldt University of Berlin, 14195 Berlin, Germany; danny.arends@gmail.com 5
Animal Breeding Biology and Molecular Genetics, Albrecht Daniel Thaer-Institute for Agricultural and
Horticultural Sciences, Humboldt University of Berlin, 14195 Berlin, Germany; danny.arends@gmail.com
*
Correspondence: Heba.elsalahy@agrar.hu-berlin.de; Tel.: +49-030-2093-46396 Horticultural Sciences, Humboldt University of Berlin, 14195 Berlin, Germany; danny.arends@gmail.com
*
Correspondence: Heba.elsalahy@agrar.hu-berlin.de; Tel.: +49-030-2093-46396 *
Correspondence: Heba.elsalahy@agrar.hu-berlin.de; Tel.: +49-030-2093-46396 agronomy agronomy 1. Introduction Weeds are a serious biotic threat in agroecosystems affecting crop productivity and crop quality [1,2]. The estimated crop yield loss due to weeds is about 34% worldwide [3]. In conventional arable farming Agronomy 2019, 9, 648; doi:10.3390/agronomy9100648 www.mdpi.com/journal/agronomy www.mdpi.com/journal/agronomy 2 of 19 Agronomy 2019, 9, 648 systems, the strategy of weed control relies heavily on synthetic herbicides. However, the reliance on
herbicides in agriculture is currently posing problems such as risks to human health, environmental
pollution, and the evolution of weeds that are resistant to herbicides [4]. In contrast, low-input
agriculture and organic farming exploit approaches that aim for high crop competitiveness such as
intercropping or cover crop mixtures to suppress weeds. These approaches provide an alternative
pathway of self-regulation and resilience of the agroecosystems to meet environmental perturbations
and secure sustainable agriculture [5–7]. g
Cover crops are introduced into the cropping system in two major ways: By growing them when
the soil is not cultivated, or, alternatively, intercropping them with the main crop for a part of the
growing season as living mulch [8,9]. In both systems, cover crops interfere with the development of
weed populations through two mechanisms. First, the rapid and successful establishment of cover
crops will hamper weed growth and development through niche pre-emption and competition [10,11]. However, the initial weed seedbank [12], weed response to the ambient conditions [13,14], and soil
fertility [15,16] may interfere with the successful establishment of cover crops. Second, incorporated
(green manures) or surface application of crop residues as mulch can effectively inhibit or retard
germination and establishment of weeds [17,18]. This effect is due to allelopathic or physical effects,
the stimulation of soil-borne pathogens, or a combination of these mechanisms [19,20]. Therefore,
designing or optimizing a mixture of cover crops where the two mechanisms could be operating
overtime would allow for an effective weed control. Diversification of cropping systems by increasing the number of crop species grown together has
been proposed as a means of ecological weed management in agroecosystems [6]. Reduced weed
biomass in intercropping systems has been reported by several workers for cereal-grain legume [21],
for pea-barley [22], for maize–legume [23], and for maize-faba bean [24]. 1. Introduction These studies confirmed that
species diversity maintains a highly asymmetric competition over weeds [25], but these relations still
need to be investigated in further detail and compared with the effects of other agronomic options of
weed control, e.g., increased seed density or mulching. Other studies have reported that species identity
(species performance in a particular ecosystem function) outweighs the effect of species diversity
on weed suppression [26,27]. Species identity is affected by species traits (species characteristics),
e.g., species’ growth rate and species´ response to environmental conditions, which may reflect the
overall performance of the species [28,29]. In this context, mixing species with different traits, e.g.,
different growth rates may create a temporal asynchrony in species growth dynamics, where the
fast-growing species competes with the early-season weeds and the slow-growing species competes
with the late-season weeds. This approach may provide an effective weed management over the whole
growing season while decreasing the direct competition for resources between the mixed-species. Perennial forage legumes in mixtures have demonstrated their ability to provide important
ecosystem services [30], mainly via reducing the dependency on mineral fertilizer by fixing atmospheric
nitrogen [23,31]. In addition, legume cover crop mixtures have also been observed to contribute to
weed control [32–34]. In practice, perennial legumes are often combined with grasses, especially
when they are grown for forage production. In this situation, the competition among legumes and
non-legumes on weed suppression is likely to be largely influenced by nitrogen dynamics, thereby
masking potential effects of any other traits that are related to competitive ability [21]. For this reason,
we chose to focus on legume-only crops to understand the potential of forage legumes to suppress
weeds, and, in this context, to study the potential of increasing crop diversity for better weed control. Weed suppression by cover crops is the result of internal and external factors such as crop species
and genotype, crop density, plant species diversity, and composition, then management practice such
as cutting and mulching. The relative proportion of species can modify the crop species’ relative
competitive strength [35,36]. According to the complementarity hypothesis, mixtures with more equal
proportions (i.e., seed mixtures with greater species evenness) may be more productive [37] and less
prone to weeds infestation [38–40]. Contrary to this hypothesis, many studies did not observe a pattern
of greater biomass in equiproportional mixtures [25,35,41]. 1. Introduction Moreover, increasing crop density usually 3 of 19 Agronomy 2019, 9, 648 resulted in decreased weed biomass [42,43]. In this context, a properly managed mixture is expected to
have higher biomass productivity that intimately related to weed suppression [44,45]. In this study, we investigated two legume species alsike clover (AC; Trifolium hybridum L.) and
black medic (BM; Medicago lupulina L.) in 15 treatments consists of five mixing ratios of AC:BM
100:0, 67:33, 50:50, 33:67, and 0:100 sown at three seed densities representing 50%, 100%, and
150% of the recommended seed density. The species were chosen based on several contrasting
characteristics regarding growth rates, response to temperature, and water and growth habit. BM is a
fast-growing perennial legume [29,46] that is well adapted to warm and dry conditions [47,48], has a
short-medium growth habit that forms a superior ground cover, and regrows fast after cutting [49–51]. AC is a slow-growing perennial legume that is drought-sensitive and well adapted to cool and wet
conditions [52,53] and has an upright growth habit with a single crown from which multiple florets are
produced [54]. Therefore, the two legume species are supposed to represent asynchrony in growth
rates and asynchrony in response to environmental conditions; thus, strongly suppressing weeds. Specifically, we aimed to test whether a mixture of AC and BM has a potential ability to reduce the
risk of yield losses and suppress weeds in varied environmental conditions (e.g., areas with uncertain
rainfall). Here, we refer to weed suppression by reducing weed biomass, but we acknowledge that an
important component of weed suppression is not covered by any measurements in our study, refers
to the weed seedbank. Thus, our hypotheses are: (1) The two legume species differ in their ability to
suppress weeds. (2) Mixtures of the two legume species suppress weeds better than expected from the
average of the monocultures. (3) Equiproportional mixtures suppress weeds more strongly than other
mixtures. (4) Higher seed densities lead to stronger weed suppression. (5) Mulching intensifies density
effects as the mulch residues that result from planting high seed density allows for a thick cover above
the ground, consequently higher weed suppression. (6) Weed biomass is negatively related to crop
biomass productivity. 2.1. Study Sites Two field trials were conducted on 2016 (field FU9) and on 2017 (field S5), respectively, at the
experimental field station of the Humboldt University of Berlin in Dahlem (52◦28′ N, 13◦18′ E, 51 m
above sea level). The two fields varied in soil fertility and soil type in the top 20-cm soil layer (Table 1). The crop sequence in both field trials was cereals—potato—winter wheat—legumes. All meteorological
data at each harvest (H1, H2, and H3) during the growing season of each year are presented in Figure 1. Table 1. Summary of soil physical and chemical properties at FU9 and S5 fields for the top 20 cm of the
soil profile. Site (Year)
Soil Type
pH
Organic Matter
N
P
K
Mg
Ca
Fe
Soil Texture
Sand Clay
Silt
%
mg kg−1
%
FU9 (2016)
Sandy clay
loam
6.3
1.24
0.13
251
90
52
1471
7354
69
22
9
S5 (2017)
Sandy loam
5.6
0.72
0.09
121
83
37
1242
5044
76
14
10
2 2 E
i
l D i 2.2. Experimental Design 2.2. Experimental Design The experimental design in both years was a randomized complete block design with three
replicates in 2016 and four replicates in 2017. Plot size was 3 m × 9 m including plot margins. Each
plot consisted of 20 rows with 13.5 cm spacing between rows. The selected species were alsike clover
(cv. Dawn) and black medic (cv. Ekola). Five mixing ratios (MR) of AC:BM including 100:0, 67:33,
50:50, 33:67, and 0:100 were grown at three different sowing densities (Den) representing 50%, 100%,
and 150% of recommended seed density. 4 of 19
4 of 20 Agronomy 2019, 9, 648
Agronomy 2019, 9, 648 Agronomy 2019, 9, 648
Agronomy 2019, 9, 648 The trial employed a response surface design (Figure S1) and included 16 variants per block,
representing the monocultures of the two species at three densities (six variants), the three proportions
at each seed density (nine variants), and a fallow plot as one further variant; this bare ground was
used as a control plot for weed growth (Figure S2). Each of the 16 variants was assigned to one plot
within a block. When the fallow is not included in the analysis, our design can also be considered as a
two-factorial design with mixing ratios as one factor (with five levels), determined by the presence
and proportion of the two legume species and seed density as the other factor (with three levels)
(see Figure S1). The seeds were bought from the companies Deutsche Saatveredelung AG (DSV) and
Camena-Samen for AC and BM, respectively. representing the monocultures of the two species at three densities (six variants), the three
proportions at each seed density (nine variants), and a fallow plot as one further variant; this bare
ground was used as a control plot for weed growth (Figure S2). Each of the 16 variants was assigned
to one plot within a block. When the fallow is not included in the analysis, our design can also be
considered as a two-factorial design with mixing ratios as one factor (with five levels), determined
by the presence and proportion of the two legume species and seed density as the other factor (with
three levels) (see Figure S1). The seeds were bought from the companies Deutsche Saatveredelung
AG (DSV) and Camena-Samen for AC and BM, respectively. 2.2. Experimental Design Seed rates of AC and BM in monoculture in practice are reported as 10 and 15 kg ha−1, p
y
Seed rates of AC and BM in monoculture in practice are reported as 10 and 15 kg ha−1,
respectively [29]. These rates were modified to 7.6 and 17.6 kg ha−1 of AC and BM, respectively,
according to their thousand grain weight (0.80 g for AC and 1.85 g for BM) in order to obtain an equal
number of plants in the two monocultures. As a result, the absolute number of seeds in the monocultures
at 100% relative seed density was 950 seeds m−2 for both species. This number of seeds was corrected
for the percent of germination in each year. Based on this, the seeds were weighed for each plot, were
thoroughly mixed in case of the mixtures, and were kept at room temperature (approximately 20 ◦C)
for a few days before sowing in late April. Sowing depth was approximately 0.5 cm and was adjusted
precisely in the field by using a sowing machine (Wintersteiger AG, Ried im Innkreis, Austria). Mulching
in summer was done by cutting the crop and the weeds in all treatments including the zero-seed density
control plots at approximately 4 cm height with a Uni-Mäher UMK 18 (from Gerhard Dücker KG
Maschinenfabrik, Stadtlohn, Germany) and leaving the cut material on the plots. respectively [29]. These rates were modified to 7.6 and 17.6 kg ha−1 of AC and BM, respectively,
according to their thousand grain weight (0.80 g for AC and 1.85 g for BM) in order to obtain an equal
number of plants in the two monocultures. As a result, the absolute number of seeds in the
monocultures at 100% relative seed density was 950 seeds m-2 for both species. This number of seeds
was corrected for the percent of germination in each year. Based on this, the seeds were weighed for
each plot, were thoroughly mixed in case of the mixtures, and were kept at room temperature
(approximately 20 °C) for a few days before sowing in late April. Sowing depth was approximately
0.5 cm and was adjusted precisely in the field by using a sowing machine (Wintersteiger AG, Ried
im Innkreis, Austria). 2.2. Experimental Design Mulching in summer was done by cutting the crop and the weeds in all
treatments including the zero-seed density control plots at approximately 4 cm height with a Uni-
Mäher UMK 18 (from Gerhard Dücker KG Maschinenfabrik, Stadtlohn, Germany) and leaving the
cut material on the plots. Th
i
l l
i
d b ff
l
di
ll
i
l
f
h
id
d The experimental layout contained buffer plots surrounding all main plots from the outside
edge of the field with 1.5 m width. These buffer plots were planted with the highest density of the
two-species equiproportional mixture. Dead seeds were used to be cultivated in the control plots
to ensure consistent application of the sowing operation across all plots; here, seeds were killed by
applying heat to the seeds in an oven at 100 ◦C for three consecutive days. The experimental layout contained buffer plots surrounding all main plots from the outside edge
of the field with 1.5 m width. These buffer plots were planted with the highest density of the two-
species equiproportional mixture. Dead seeds were used to be cultivated in the control plots to ensure
consistent application of the sowing operation across all plots; here, seeds were killed by applying
heat to the seeds in an oven at 100 °C for three consecutive days. Figure 1. Schedule of the two field experiments and the meteorological data including the
accumulated precipitation (mm), average mean temperature (°C), and average radiation (MJ m−2)
during three harvest times (H1, H2, and H3) in the years 2016 and 2017 (Source: Agricultural
Climatology of the HU-Berlin, Dahlem; [55]). DAS: Days After Sowing, DAM: Days After Mulching. * p < 0.05. Figure 1. Schedule of the two field experiments and the meteorological data including the accumulated
precipitation (mm), average mean temperature (◦C), and average radiation (MJ m−2) during three
harvest times (H1, H2, and H3) in the years 2016 and 2017 (Source: Agricultural Climatology of the
HU-Berlin, Dahlem; [55]). DAS: Days After Sowing, DAM: Days After Mulching. “*” was mentioned
to clarify the reason for the delay in mulching in 2017. Figure 1. Schedule of the two field experiments and the meteorological data including the
accumulated precipitation (mm), average mean temperature (°C), and average radiation (MJ m−2)
during three harvest times (H1, H2, and H3) in the years 2016 and 2017 (Source: Agricultural
Climatology of the HU-Berlin, Dahlem; [55]). 2.3. Weed Biomass and Cover Crop Biomass We quantified cover crop and weed aboveground biomass (CCB and WB, respectively) in three
harvest times, during spring, summer, and early autumn of each year. The time of harvest was
based on calculating the growing degree days (GDD; ◦Cd) of BM by using a base temperature of
0.6 ◦C [56] to compare plants in similar growth stages across experimental years. Depending on these
calculations the plants were specifically harvested at 670 ± 100 ◦Cd (40 ± 10 Days after Sowing; DAS)
and 1100 ± 100 ◦Cd (64 ± 10 DAS) at H1 and H2, respectively. y
After mulching, the degree days were calculated again for the regrowth of the plants; they
represented 1480 ◦Cd in 2016 and 1164 ◦Cd in 2017. The difference in growing degree days (316 ◦Cd)
was due to a delay in mulching by around 24 d after H2 in 2017 (Figure 1). The reason for this delay
was related to rainy weather in 2017. In addition, the lower temperature during this period of plant
regrowth extended the time required to reach the same degree days as in 2016. Despite all this, H3 was
conducted around the same number of days after mulching in the two years (74 ± 6). At each harvest, sampling was performed in a number of selected 0.5 m long rows only taken
from the central seven m of the total nine m plot length. The total number of samples per each of the
16 variants was n = 12 (4 rows × 3 blocks) in 2016 and n = 16 (4 rows × 4 blocks) in 2017. The fresh
samples were manually separated into three fractions of AC, BM, and all other species (which were
considered as weeds). These sample fractions were then dried separately at 85 ◦C for 48 h to obtain the
dry content. In addition, only in 2016, the weeds were identified as the species level in the selected
0.5 m of fallow plots and the monocultures of AC and BM at a density of 150% (Table S2) to characterize
weed species composition on the site. Mixture effect is one of the basic metrics to judge the performance of a mixture and its efficiency
in biomass production and weed suppression. 2.3. Weed Biomass and Cover Crop Biomass Here, we were interested in calculating the mixture
effect only on weed suppression by subtracting the weed biomass in the mixture from the average
weed biomass in the monoculture; Equation (1). When the weed biomass in the mixture is smaller than
the weed biomass in the monoculture, it creates a negative size effect that indicates an efficiency of the
mixture in weed suppression. (1) Mixture Effectweed suppression = WBMixture −WBAvg. Monoculture
(1) Mixture Effectweed suppression = WBMixture −WBAvg. Monoculture
(1) Mixture Effectweed suppression = WBMixture −WBAvg. Monoculture 2.2. Experimental Design DAS: Days After Sowing, DAM: Days After Mulching. * p < 0.05. Figure 1. Schedule of the two field experiments and the meteorological data including the accumulated
precipitation (mm), average mean temperature (◦C), and average radiation (MJ m−2) during three
harvest times (H1, H2, and H3) in the years 2016 and 2017 (Source: Agricultural Climatology of the
HU-Berlin, Dahlem; [55]). DAS: Days After Sowing, DAM: Days After Mulching. “*” was mentioned
to clarify the reason for the delay in mulching in 2017. Figure 1. Schedule of the two field experiments and the meteorological data including the
accumulated precipitation (mm), average mean temperature (°C), and average radiation (MJ m−2)
during three harvest times (H1, H2, and H3) in the years 2016 and 2017 (Source: Agricultural
Climatology of the HU-Berlin, Dahlem; [55]). DAS: Days After Sowing, DAM: Days After Mulching. * p < 0.05. Figure 1. Schedule of the two field experiments and the meteorological data including the accumulated
precipitation (mm), average mean temperature (◦C), and average radiation (MJ m−2) during three
harvest times (H1, H2, and H3) in the years 2016 and 2017 (Source: Agricultural Climatology of the
HU-Berlin, Dahlem; [55]). DAS: Days After Sowing, DAM: Days After Mulching. “*” was mentioned
to clarify the reason for the delay in mulching in 2017. 5 of 19 Agronomy 2019, 9, 648 3.1. Cover Crop Biomass A strong and significant Yr effect was observed on mean CCB at each harvest time, with higher
biomass values in 2016 than in 2017 by 12.9%, 45.0%, and 17.4% at H1, H2, and H3, respectively
(Figure 2, Figure S5). In both years, the Den effect was significant only at H1 and H2. However, the MR
effect was inconsistently significant at the different harvest times and not significant at all at H2 in 2016. At H1, in both years, BM-monoculture was dominant at each given seed density, producing higher
biomass up to 40.4% and 33.8 % in 2016 and 2017, respectively, than the other treatments. In contrast,
AC-monoculture produced significantly the lowest CCB at each given seed density in comparison with
the other treatments representing a reduction by 46.1% and 29.2% in 2016 and 2017, respectively. In the
mixtures, a trend of producing less CCB was observed as the BM proportion decreased in the mixture. However, there was no significant difference between the three binary mixtures at any of the different
densities (Figure 2A,D). The significant Den effect showed higher CCB at 100% and 150% seed density
by 27.6% and 31.8%, respectively than 50%. At H2, in both years, after crop establishment (ca. 30% of plants flowering), the dynamics
of biomass productivity was highly depending on the growing conditions (weather and/or soil). Specifically, in 2016, no MR effect was observed while the Den effect was still significant and showed
an increase in the CCB in an average of 20.5% and 24.4% at 100% and 150% seed density, respectively,
in comparison to 50% seed density (Figure 2B). However, in 2017, significant effects were observed for
MR and Den. In particular, AC-monoculture was significantly more productive than BM-monoculture
by 20.2% and 34.5% at 50% and 100% seed density, respectively but not at density 150%. In the average
of all mixing ratios, the Den effect was significant and showed an increase in CCB by 24.9% and 20.6%
at 100% and 150% seed density, respectively, than the 50% seed density (Figure 2E). At H3, in both years, the MR effect was again significant but stronger in 2016 than in 2017. The dynamics of plant regrowth after mulching showed similar dynamics as in H1, i.e., BM-monoculture
produced the highest biomass than almost all the other treatments. 3.1. Cover Crop Biomass An exception was in 2016 at
density 150% when the equiproportional mixture produced higher CCB than the other mixtures, but
significantly higher than the average monocultures by 24.4%. No density effect was observed after
mulching and all the mixing ratios produced almost the same biomass at each given density. However,
the CCB was significantly higher in 2016 than 2017 by 22.1%, 17.7%, and 12.2% at 50%, 100%, and 150%,
respectively (Figure 2C,F). 3. Results Depending on the climate data during the growing season, the experimental year of 2016 was
characterized by a dry season with precipitation almost 30% less than the long-term average, while in
2017, it was a relatively wet season with precipitation almost 36% higher than the long-term average
(Figure 1). 2.4. Statistical Analysis Aiming to provide an easier interpretation of the results, data from each harvest were analyzed
separately in each year (Yr) to determine the mixing ratio (MR) and the density (Den) effects and the
interaction between them. CCB was normally distributed and homogenous with respect to variances,
according to Shapiro test and Bartlett’s test, respectively. Weed biomass was not normally distributed
and failed the homogeneity of variance test and was square-root-transformed to satisfy normality
criteria and to homogenize variances. To determine the Yr effect on the dependent variable (CCB and WB) at each harvest, a mixed
linear model was used with Yr, MR, Den, Yr × MR, Yr × Den, MR × Den as fixed effects and block
nested with row as random effects. Here, MR consisted of five levels, comprising the two monocultures
and three mixtures with different species proportions. Comparing different models using the plot nested in the block did not improve the model
and showed higher AIC (Akaike Information Criterion) values [57]. Further statistical analysis was
conducted for each year separately in each harvest by using mixed linear models with MR, Den and
MR x Den as fixed effects and block nested with row as a random effect. The weedy fallow treatment
was only included in analyzing WB at each harvest time to evaluate WB in the fallow treatments. After applying ANOVA, Tukey’s HSD tests at the 0.05 probability level were used to determine
differences among the mean values of the mixing ratios at a given density and among the groups of
seed densities by using the Agricola R-package [58]. Agronomy 2019, 9, 648 6 of 19 Agronomy 2019, 9, 648 To determine the mixture effect on weed suppression, we calculated contrasts of mixtures versus
the average of their respective proportion-weighted monocultures. These absolute mixture effects were
calculated for both CCB and WB. Absolute mixture effects were tested for significance by subjecting the
contrasts to two-sided Welch’s t-tests against zero. Further, the effect of absolute mixture effect in the
crops on the absolute mixture effects in weeds was tested with linear regression models. All statistical
tests were performed using R (version 3.5.3) with R studio (version 1.1.383) [59]. 3.2. Weed Biomass The effect of the Yr on WB was gradually less significant over time, i.e., at H1, H2, and H3,
respectively. The MR effect depended on the growing conditions and its strength increased over time
(Figure 3A–F). However, in both years, the Den effect on weed suppression was always significant at
each harvest with higher densities being associated with lower WB. The MR × Den interactions were
not significant on WB, with the exception of those at H1 and H3 in 2017 (Figure 3). 7 of 19 Agronomy 2019, 9, 648 At H1 in 2016, the MR effect was not significant, but the Den effect was significant and all the
mixing ratios suppressed weeds effectively as the seed density increased. On average of the different
mixing ratios, WB was reduced up to 44.1% at 150% seed density in comparison with the fallow plots
(Figure 3A). On the contrary, in 2017, the MR effect was significant where AC-monoculture was the
less suppressive at all seed densities and showed higher WB up to 49.9% at density 150% than the
average of the other mixing ratios. In 2017, at the low seed density, WB did not differ significantly
from the WB in the fallow plot. However, significant weed suppression has been observed at density
100% and 150% by 49.3% and 48.8%, respectively in comparison with the fallow plots (Figure 3D). At H2, in 2016, only the Den effect was significant and WB decreased as the seed density increased,
up to 61.6% at 150% seed density in comparison with the fallow plots (Figure 3B). In 2017, both
MR and Den effects were significant and showed the same trend as in H1 of the same year; where
again AC-monoculture was less suppressive and showed up to 45% more WB at 150% than the other
treatments. The Den effect was significant at low density and reduced WB by 42.5%, in comparison
with the fallow plot, but highly significant at density 100% and 150% and reduced WB by 62.9% and
60.3%, respectively (Figure 3E). After mulching, at H3, WB decreased compared to H2 by 59.0% and 64.1% as mean values in 2016
and 2017, respectively (Figure 3B versus C, E versus F). The MR effect was significant in 2016; where the
equiproportional mixture (50:50) was the most suppressive and showed up to 83.4% reduction in WB
than the other mixing ratios. 3.2. Weed Biomass While, in 2017, the MR effect was highly significant but AC-monoculture
was the most suppressive and reduced WB up to 73.8% than the other mixing ratios. Despite the high
weed development in fallow plots (Figure 3C,F); all the cover crop treatments strongly reduced WB by
96% and 89% in 2016 and 2017, respectively. However, in an average of the different mixing ratios, the
Den effect was not significant between the different treatments as seed density increased (Figure 3C,F). In Figure 4, the linear correlation between CCB and WB revealed that the relationship between
these two variables was dependent on the environment (a combination of trial year and trial site) and
on the harvest time. In 2016, at H1 and H3, no significant relationship was found between CCB and
WB, while at H2, the strength of the negative relationship between CCB and WB explained 96% of the
variation (Figure 4B). Conversely in 2017, at H1 the linear relationship between WB and CCB explained
49% of the variation. No relation was observed at H2 and H3 (Figure 4D–F). Out of 54 contrasts between mixtures and monocultures (2 trial years × 3 mixing proportions × 3
harvests × 3 seed densities), only eight contrasts showed significantly stronger weed suppression in
mixtures than in their respective monocultures (Table 2). Within these contrasts, there was no obvious
pattern with regard to the experimental factors, i.e., none of the variables (proportion, density and
harvest time) showed a consistent influence on the mixture effect on weeds. However, pooled across all
other factors (i.e., proportion, density and harvest time), mixing the legumes significantly reduced WB
by 28%, as an average value, in 2017 compared to the respective monocultures, but not significantly in
2016 (Figure 5). 8 of 19
8 of 20 Agronomy 2019, 9, 648
Agronomy 2019 9 648 Figure 2. Cover crop aboveground biomass (CCB) of alsike clover (AC) and black medic (BM) containe
15 treatments of five mixing ratios (MR) of AC:BM (100:0, 67:33, 50:50, 33:67, and 0:100) sown at three see
densities (Den) represent 50%, 100%, and 150% of the recommended seed density and harvested three time
(H1; panel A and D, H2; Panel B and E, and H3; Panel C and F) per growing seasons of 2016 (Panel A, B
and C) and 2017 (Panel D, E, and F). Vertical bars represent Tukey’s HSD.test (p < 0.05) at a given see
density. 3.2. Weed Biomass Different letters above the vertical bars indicate significant differences among the densities, base
on ANOVA followed by Tukey’s HSD test (p < 0.05). Asterisks indicate significant effects of the MR, Den
and the interaction between them according to ANOVA; *** = p < 0.001, ** = p < 0.01, * = p < 0.05, ns = no
significant. Figure 2. Cover crop aboveground biomass (CCB) of alsike clover (AC) and black medic (BM) contained
15 treatments of five mixing ratios (MR) of AC:BM (100:0, 67:33, 50:50, 33:67, and 0:100) sown at
three seed densities (Den) represent 50%, 100%, and 150% of the recommended seed density and
harvested three times (H1; panel A and D, H2; Panel B and E, and H3; Panel C and F) per growing
seasons of 2016 (Panel A, B, and C) and 2017 (Panel D, E, and F). Vertical bars represent Tukey’s
HSD.test (p < 0.05) at a given seed density. Different letters above the vertical bars indicate significant
differences among the densities, based on ANOVA followed by Tukey’s HSD test (p < 0.05). Asterisks
indicate significant effects of the MR, Den, and the interaction between them according to ANOVA;
*** = p < 0.001, ** = p < 0.01, * = p < 0.05, ns = not significant. Figure 2. Cover crop aboveground biomass (CCB) of alsike clover (AC) and black medic (BM) conta
15 treatments of five mixing ratios (MR) of AC:BM (100:0 67:33 50:50 33:67 and 0:100) sown at three
Figure 2. Cover crop aboveground biomass (CCB) of alsike clover (AC) and black medic (BM) contained Figure 2. Cover crop aboveground biomass (CCB) of alsike clover (AC) and black medic (BM) contai
15 treatments of five mixing ratios (MR) of AC:BM (100:0, 67:33, 50:50, 33:67, and 0:100) sown at three s
densities (Den) represent 50%, 100%, and 150% of the recommended seed density and harvested three ti
(H1; panel A and D, H2; Panel B and E, and H3; Panel C and F) per growing seasons of 2016 (Panel A
and C) and 2017 (Panel D, E, and F). Vertical bars represent Tukey’s HSD.test (p < 0.05) at a given s
density. Different letters above the vertical bars indicate significant differences among the densities, ba
on ANOVA followed by Tukey’s HSD test (p < 0.05). 3.2. Weed Biomass Asterisks indicate significant effects of the MR, D
and the interaction between them according to ANOVA; *** = p < 0.001, ** = p < 0.01, * = p < 0.05, ns =
significant. Figure 2. Cover crop aboveground biomass (CCB) of alsike clover (AC) and black medic (BM) contained
15 treatments of five mixing ratios (MR) of AC:BM (100:0, 67:33, 50:50, 33:67, and 0:100) sown at
three seed densities (Den) represent 50%, 100%, and 150% of the recommended seed density and
harvested three times (H1; panel A and D, H2; Panel B and E, and H3; Panel C and F) per growing
seasons of 2016 (Panel A, B, and C) and 2017 (Panel D, E, and F). Vertical bars represent Tukey’s
HSD.test (p < 0.05) at a given seed density. Different letters above the vertical bars indicate significant
differences among the densities, based on ANOVA followed by Tukey’s HSD test (p < 0.05). Asterisks
indicate significant effects of the MR, Den, and the interaction between them according to ANOVA;
*** = p < 0.001, ** = p < 0.01, * = p < 0.05, ns = not significant. Figure 2. Cover crop aboveground biomass (CCB) of alsike clover (AC) and black medic (BM) contai
15 treatments of five mixing ratios (MR) of AC:BM (100:0, 67:33, 50:50, 33:67, and 0:100) sown at three s
densities (Den) represent 50%, 100%, and 150% of the recommended seed density and harvested three ti
(H1; panel A and D, H2; Panel B and E, and H3; Panel C and F) per growing seasons of 2016 (Panel A
and C) and 2017 (Panel D, E, and F). Vertical bars represent Tukey’s HSD.test (p < 0.05) at a given s
density. Different letters above the vertical bars indicate significant differences among the densities, ba
on ANOVA followed by Tukey’s HSD test (p < 0.05). Asterisks indicate significant effects of the MR, D
and the interaction between them according to ANOVA; *** = p < 0.001, ** = p < 0.01, * = p < 0.05, ns =
significant. Figure 2. 3.2. Weed Biomass Cover crop aboveground biomass (CCB) of alsike clover (AC) and black medic (BM) contained
15 treatments of five mixing ratios (MR) of AC:BM (100:0, 67:33, 50:50, 33:67, and 0:100) sown at
three seed densities (Den) represent 50%, 100%, and 150% of the recommended seed density and
harvested three times (H1; panel A and D, H2; Panel B and E, and H3; Panel C and F) per growing
seasons of 2016 (Panel A, B, and C) and 2017 (Panel D, E, and F). Vertical bars represent Tukey’s
HSD.test (p < 0.05) at a given seed density. Different letters above the vertical bars indicate significant
differences among the densities, based on ANOVA followed by Tukey’s HSD test (p < 0.05). Asterisks
indicate significant effects of the MR, Den, and the interaction between them according to ANOVA;
*** = p < 0.001, ** = p < 0.01, * = p < 0.05, ns = not significant. 9 of 19
9 of 20 Agronomy 2019, 9, 648
Agronomy 2019, 9, 648 Figure 3. Weed biomass (WB) emerged in the fallow plots and with cover crop of alsike clover (AC)
and black medic (BM) contained 15 treatments of five mixing ratios (MR) of AC:BM (100:0, 67:33,
50:50, 33:67, and 0:100) sown at three seed densities (Den) represent 50%, 100%, and 150% of the
recommended seed density and harvested three times (H1; panel A and D, H2; panel B and E, and
H3; panel C and F) per growing season of 2016 (Panel A, B, and C) and 2017 (Panel D, E, and F). Vertical bars represent Tukey’s HSD test (p < 0.05) at a given seed density. Different letters above the
vertical bars indicate significant differences among the densities, based on ANOVA followed by
Tukey’s HSD test (p < 0.05). Asterisks indicate significant effects of MR, Den, and the interaction
between them according to ANOVA; *** = p < 0.001, ** = p < 0.01, * = p < 0.05, and ns = not significant. Figure 3. 3.2. Weed Biomass Weed biomass (WB) emerged in the fallow plots and with cover crop of alsike clover (AC)
and black medic (BM) contained 15 treatments of five mixing ratios (MR) of AC:BM (100:0, 67:33,
50:50, 33:67, and 0:100) sown at three seed densities (Den) represent 50%, 100%, and 150% of the
recommended seed density and harvested three times (H1; panel A and D, H2; panel B and E, and H3;
panel C and F) per growing season of 2016 (Panel A, B, and C) and 2017 (Panel D, E, and F). Vertical
bars represent Tukey’s HSD test (p < 0.05) at a given seed density. Different letters above the vertical
bars indicate significant differences among the densities, based on ANOVA followed by Tukey’s HSD
test (p < 0.05). Asterisks indicate significant effects of MR, Den, and the interaction between them
according to ANOVA; *** = p < 0.001, ** = p < 0.01, * = p < 0.05, and ns = not significant. Figure 3. Weed biomass (WB) emerged in the fallow plots and with cover crop of alsike clover (AC)
and black medic (BM) contained 15 treatments of five mixing ratios (MR) of AC:BM (100:0, 67:33,
50:50, 33:67, and 0:100) sown at three seed densities (Den) represent 50%, 100%, and 150% of the
recommended seed density and harvested three times (H1; panel A and D, H2; panel B and E, and
H3; panel C and F) per growing season of 2016 (Panel A, B, and C) and 2017 (Panel D, E, and F). Vertical bars represent Tukey’s HSD test (p < 0.05) at a given seed density. Different letters above the
vertical bars indicate significant differences among the densities, based on ANOVA followed by
Tukey’s HSD test (p < 0.05). Asterisks indicate significant effects of MR, Den, and the interaction
between them according to ANOVA; *** = p < 0.001, ** = p < 0.01, * = p < 0.05, and ns = not significant. Figure 3. 3.2. Weed Biomass Weed biomass (WB) emerged in the fallow plots and with cover crop of alsike clover (AC)
and black medic (BM) contained 15 treatments of five mixing ratios (MR) of AC:BM (100:0, 67:33,
50:50, 33:67, and 0:100) sown at three seed densities (Den) represent 50%, 100%, and 150% of the
recommended seed density and harvested three times (H1; panel A and D, H2; panel B and E, and H3;
panel C and F) per growing season of 2016 (Panel A, B, and C) and 2017 (Panel D, E, and F). Vertical
bars represent Tukey’s HSD test (p < 0.05) at a given seed density. Different letters above the vertical
bars indicate significant differences among the densities, based on ANOVA followed by Tukey’s HSD
test (p < 0.05). Asterisks indicate significant effects of MR, Den, and the interaction between them
according to ANOVA; *** = p < 0.001, ** = p < 0.01, * = p < 0.05, and ns = not significant. Figure 3. Weed biomass (WB) emerged in the fallow plots and with cover crop of alsike clover (AC)
and black medic (BM) contained 15 treatments of five mixing ratios (MR) of AC:BM (100:0, 67:33,
50:50, 33:67, and 0:100) sown at three seed densities (Den) represent 50%, 100%, and 150% of the
recommended seed density and harvested three times (H1; panel A and D, H2; panel B and E, and
H3; panel C and F) per growing season of 2016 (Panel A, B, and C) and 2017 (Panel D, E, and F). Vertical bars represent Tukey’s HSD test (p < 0.05) at a given seed density. Different letters above the
vertical bars indicate significant differences among the densities, based on ANOVA followed by
Tukey’s HSD test (p < 0.05). Asterisks indicate significant effects of MR, Den, and the interaction
between them according to ANOVA; *** = p < 0.001, ** = p < 0.01, * = p < 0.05, and ns = not significant. Figure 3. 3.2. Weed Biomass Linear correlation between the cover crop aboveground biomass (CCB) and weed biomass
(WB); the cover crop species are alsike clover (AC) and black medic (BM) contained 15 treatments of
five mixing ratios (MR) of AC:BM (100:0, 67:33, 50:50, 33:67, and 0:100; colored in symbols blue, violet,
green, orange, and red, respectively), sown at three seed densities (Den) represent 50%, 100%, and
150% of the recommended seed density, and harvested three times (H1; panel A and D, H2; panel B
and E and H3; panel C and F) per growing season of 2016 (Panel A, B, and C) and 2017 (Panel D, E,
and F)
Figure 4. Linear correlation between the cover crop aboveground biomass (CCB) and weed biomass
(WB); the cover crop species are alsike clover (AC) and black medic (BM) contained 15 treatments of
five mixing ratios (MR) of AC:BM (100:0, 67:33, 50:50, 33:67, and 0:100; colored in symbols blue, violet,
green, orange, and red, respectively), sown at three seed densities (Den) represent 50%, 100%, and 150%
of the recommended seed density, and harvested three times (H1; panel A and D, H2; panel B and E
and H3; panel C and F) per growing season of 2016 (Panel A, B, and C) and 2017 (Panel D, E, and F). Figure 4. Linear correlation between the cover crop aboveground biomass (CCB) and weed biomass
(WB); the cover crop species are alsike clover (AC) and black medic (BM) contained 15 treatments of
five mixing ratios (MR) of AC:BM (100:0, 67:33, 50:50, 33:67, and 0:100; colored in symbols blue, violet,
green, orange, and red, respectively), sown at three seed densities (Den) represent 50%, 100%, and
150% of the recommended seed density, and harvested three times (H1; panel A and D, H2; panel B
and E and H3; panel C and F) per growing season of 2016 (Panel A, B, and C) and 2017 (Panel D, E,
and F)
Figure 4. 3.2. Weed Biomass Weed biomass (WB) emerged in the fallow plots and with cover crop of alsike clover (AC)
and black medic (BM) contained 15 treatments of five mixing ratios (MR) of AC:BM (100:0, 67:33,
50:50, 33:67, and 0:100) sown at three seed densities (Den) represent 50%, 100%, and 150% of the
recommended seed density and harvested three times (H1; panel A and D, H2; panel B and E, and H3;
panel C and F) per growing season of 2016 (Panel A, B, and C) and 2017 (Panel D, E, and F). Vertical
bars represent Tukey’s HSD test (p < 0.05) at a given seed density. Different letters above the vertical
bars indicate significant differences among the densities, based on ANOVA followed by Tukey’s HSD
test (p < 0.05). Asterisks indicate significant effects of MR, Den, and the interaction between them
according to ANOVA; *** = p < 0.001, ** = p < 0.01, * = p < 0.05, and ns = not significant. 10 of 19
10 of 20 Agronomy 2019, 9, 648
Agronomy 2019, 9, 648 Figure 4. Linear correlation between the cover crop aboveground biomass (CCB) and weed biomass
(WB); the cover crop species are alsike clover (AC) and black medic (BM) contained 15 treatments of
five mixing ratios (MR) of AC:BM (100:0, 67:33, 50:50, 33:67, and 0:100; colored in symbols blue, violet,
green, orange, and red, respectively), sown at three seed densities (Den) represent 50%, 100%, and
150% of the recommended seed density, and harvested three times (H1; panel A and D, H2; panel B
and E and H3; panel C and F) per growing season of 2016 (Panel A, B, and C) and 2017 (Panel D, E,
and F)
Figure 4. Linear correlation between the cover crop aboveground biomass (CCB) and weed biomass
(WB); the cover crop species are alsike clover (AC) and black medic (BM) contained 15 treatments of
five mixing ratios (MR) of AC:BM (100:0, 67:33, 50:50, 33:67, and 0:100; colored in symbols blue, violet,
green, orange, and red, respectively), sown at three seed densities (Den) represent 50%, 100%, and 150%
of the recommended seed density, and harvested three times (H1; panel A and D, H2; panel B and E
and H3; panel C and F) per growing season of 2016 (Panel A, B, and C) and 2017 (Panel D, E, and F). Figure 4. 3.2. Weed Biomass Linear correlation between the cover crop aboveground biomass (CCB) and weed biomass
(WB); the cover crop species are alsike clover (AC) and black medic (BM) contained 15 treatments of
five mixing ratios (MR) of AC:BM (100:0, 67:33, 50:50, 33:67, and 0:100; colored in symbols blue, violet,
green, orange, and red, respectively), sown at three seed densities (Den) represent 50%, 100%, and 150%
of the recommended seed density, and harvested three times (H1; panel A and D, H2; panel B and E
and H3; panel C and F) per growing season of 2016 (Panel A, B, and C) and 2017 (Panel D, E, and F). 11 of 19 11 of 19 Agronomy 2019, 9, 648 Table 2. Mixture effect on weed suppression for three binary mixtures of alsike clover (AC) and black medic (BM) in comparison with average monocultures in two
years 2016 and 2017 at three harvest times (H1: 40 ± 10 days after sowing (DAS), H2: 64 ± 10 DAS, and H3: 146 ± 26 DAS). The mixtures included three mixing ratios
of AC:BM 67:33, 50:50, and 33:67 sown at three seed densities (50%, 100%, and 150% of the recommended seed density). Abbreviations are MixB: A mixture dominated
by BM, MixC: A mixture of equal proportions of the two species, MixA: AMixture dominated by AC, Avg_mono: Average monocultures of both species. Asterisks
indicate a significant difference according to Welch’s t-test at p < 0.05. Mixture effect = WBMixture – WBAvg. Monoculture. All negative values of size effect indicate
suppressive mixtures. Density (%)
Contrast: Mixture
vs. 3.2. Weed Biomass Average of
Respective
Monocultures
2016
2017
H1
H2
H3
H1
H2
H3
Effect
Size
p
Effect
Size
p
Effect
Size
p
Effect
Size
p
Effect
Size
p
Effect
Size
P
50
MixB-Avg_mono
−7.0
0.49
−65.1
0.49
27.4
0.13
−7.9
0.48
−46.5
0.05 *
−2.3
0.81
MixC-Avg_mono
4.7
0.81
−94.0
0.30
4.3
0.27
−2.6
0.87
−49.3
0.05 *
−6.7
0.50
MixA-Avg_mono
43.6
0.19
−91.1
0.34
−2.4
0.48
15.9
0.31
−37.9
0.34
26.6
0.14
100
MixB-Avg_mono
−2.4
0.89
27.8
0.79
12.8
0.58
15.7
0.02 *
10.5
0.73
−10.5
0.14
MixC-Avg_mono
−32.2
0.01 **
43.0
0.59
−25.9
0.07
−19.6
0.03 *
−55.9
0.05 *
−19.9
0.07
MixA-Avg_mono
11.8
0.53
81.6
0.44
−25.6
0.07
−18.3
0.03 *
−23.5
0.37
−12.5
0.24
150
MixB-Avg_mono
32.1
0.09
−15.8
0.79
−6.3
0.23
−17.2
0.17
−50.3
0.05 *
−7.9
0.15
MixC-Avg_mono
28.2
0.02 *
45.8
0.36
−12.5
<0.01 **
−13.4
0.09
−48.1
0.06
4.7
0.48
MixA-Avg_mono
19.2
0.17
10.2
0.90
−3.0
0.63
−4.4
0.57
−46.4
0.09
9.3
0.46
*** = p < 0.001, ** = p < 0.01, * = p < 0.05. 12 of 19
12 of 20 Agronomy 2019, 9, 648
g
y Figure 5. Weed biomass (WB) in the fallow plots, monocultures, and mixtures of two legume cover
crops of alsike clover (AC) and black medic (BM) in two years 2016 and 2017. The treatments of the
cover crop contained five mixing ratios of AC:BM (100:0, 67:33, 50:50, 33:67, and 0:100) sown at three
seed densities (50%, 100%, and 150% of the recommended seed densities). The WB was harvested
three times during the season at H1: In spring (40 ± 10 days after sowing; DAS), H2: In summer (64 ±
10 DAS), and H3: In autumn (146 ± 26 DAS). The vertical error bars represent standard error (SD/10). Different letters above the vertical bars indicate significant differences among the means of the fallow
plots, monocultures, and mixtures across the three harvest times, based on ANOVA followed by
Tukey’s HSD test (p < 0.05). Figure 5. Weed biomass (WB) in the fallow plots, monocultures, and mixtures of two legume cover
crops of alsike clover (AC) and black medic (BM) in two years 2016 and 2017. The treatments of the
cover crop contained five mixing ratios of AC:BM (100:0, 67:33, 50:50, 33:67, and 0:100) sown at three
seed densities (50%, 100%, and 150% of the recommended seed densities). 4 1 Cover Crop Species Effect
4.1. Cover Crop Species Effect In 2016, the relatively dry conditions supported a
better performance of both legume species in monocultures and mixtures than in 2017. Therefore,
all treatments produced more or less equal biomass and suppressed weeds with the same efficiency. This finding was contrary to expectations for AC, as this species was supposed to be negatively affected
by drought conditions [29,52,53] and consequently its strength to suppress weeds would be lower. The unexpectedly outstanding performance of AC in the dry season, in 2016, suggests that the effect p
p
ff
Plant growth dynamics were similar in both years at the early stage of the establishment (H1),
where BM-monoculture was dominant. This may be partly due to the fast growth rate of BM than
AC, therefore it was expected for AC to be less competitive at the early growth stage [29,46]. The
efficiency of BM in suppressing weed in both years likely confirms that rapid growth rate is a key
trait for weed suppression [10,11]. However, the efficiency of AC in weed suppression at H1 in 2016
was comparable to the other treatments. This indicates that legume species with a slow growth rate
may use alternative mechanisms such as allelopathy to suppress weeds at the early stage. It was
widely reported that many species belonging to the genera Trifolium and Medicago release allelopathic
Plant growth dynamics were similar in both years at the early stage of the establishment (H1),
where BM-monoculture was dominant. This may be partly due to the fast growth rate of BM than AC,
therefore it was expected for AC to be less competitive at the early growth stage [29,46]. The efficiency
of BM in suppressing weed in both years likely confirms that rapid growth rate is a key trait for weed
suppression [10,11]. However, the efficiency of AC in weed suppression at H1 in 2016 was comparable
to the other treatments. This indicates that legume species with a slow growth rate may use alternative
mechanisms such as allelopathy to suppress weeds at the early stage. It was widely reported that
many species belonging to the genera Trifolium and Medicago release allelopathic compounds [60,61]. widely reported that many species belonging to the genera Trifolium and Medicago release allelopathic
compounds [60,61]. The dynamics of CCB at H2 after successful crop establishment were remarkably different, in
both years, depending on the growing conditions. 4. Discussion
4. Discussion The overarching goal of this study was to evaluate the ability of binary mixtures of only-legume
forage species to suppress weeds, measured in the response of WB. We were also interested in
learning whether the benefits of these mixtures would be consistent across different environmental
conditions represented by three harvest times across the growing seasons (spring, summer, and early
autumn seasons, the last after cover crop mulching, of two years 2016 and 2017). Here, we summarize
and discuss the effects of the field trials with respect to cover crop species effect, diversity effects,
proportion effects density effects mulching effects site and year effects
The overarching goal of this study was to evaluate the ability of binary mixtures of only-legume
forage species to suppress weeds, measured in the response of WB. We were also interested in learning
whether the benefits of these mixtures would be consistent across different environmental conditions
represented by three harvest times across the growing seasons (spring, summer, and early autumn
seasons, the last after cover crop mulching, of two years 2016 and 2017). Here, we summarize and
discuss the effects of the field trials with respect to cover crop species effect, diversity effects, proportion
effects, density effects, mulching effects, site, and year effects. 3.2. Weed Biomass Weed biomass (WB) in the fallow plots, monocultures, and mixtures of two legume cover
crops of alsike clover (AC) and black medic (BM) in two years 2016 and 2017. The treatments of the
cover crop contained five mixing ratios of AC:BM (100:0, 67:33, 50:50, 33:67, and 0:100) sown at three
seed densities (50%, 100%, and 150% of the recommended seed densities). The WB was harvested
three times during the season at H1: In spring (40 ± 10 days after sowing; DAS), H2: In summer (64 ±
10 DAS), and H3: In autumn (146 ± 26 DAS). The vertical error bars represent standard error (SD/10). Different letters above the vertical bars indicate significant differences among the means of the fallow
plots, monocultures, and mixtures across the three harvest times, based on ANOVA followed by
Tukey’s HSD test (p < 0.05). Figure 5. Weed biomass (WB) in the fallow plots, monocultures, and mixtures of two legume cover
crops of alsike clover (AC) and black medic (BM) in two years 2016 and 2017. The treatments of the
cover crop contained five mixing ratios of AC:BM (100:0, 67:33, 50:50, 33:67, and 0:100) sown at three
seed densities (50%, 100%, and 150% of the recommended seed densities). The WB was harvested
three times during the season at H1: In spring (40 ± 10 days after sowing; DAS), H2: In summer
(64 ± 10 DAS), and H3: In autumn (146 ± 26 DAS). The vertical error bars represent standard error
(SD/10). Different letters above the vertical bars indicate significant differences among the means of the
fallow plots, monocultures, and mixtures across the three harvest times, based on ANOVA followed by
Tukey’s HSD test (p < 0.05). 3.2. Weed Biomass The WB was harvested
three times during the season at H1: In spring (40 ± 10 days after sowing; DAS), H2: In summer
(64 ± 10 DAS), and H3: In autumn (146 ± 26 DAS). The vertical error bars represent standard error
(SD/10). Different letters above the vertical bars indicate significant differences among the means of the
fallow plots, monocultures, and mixtures across the three harvest times, based on ANOVA followed by
Tukey’s HSD test (p < 0.05). Figure 5. Weed biomass (WB) in the fallow plots, monocultures, and mixtures of two legume cover
Figure 5. Weed biomass (WB) in the fallow plots, monocultures, and mixtures of two legume cover Figure 5. Weed biomass (WB) in the fallow plots, monocultures, and mixtures of two legume cover
crops of alsike clover (AC) and black medic (BM) in two years 2016 and 2017. The treatments of the
cover crop contained five mixing ratios of AC:BM (100:0, 67:33, 50:50, 33:67, and 0:100) sown at three
seed densities (50%, 100%, and 150% of the recommended seed densities). The WB was harvested
three times during the season at H1: In spring (40 ± 10 days after sowing; DAS), H2: In summer (64 ±
10 DAS), and H3: In autumn (146 ± 26 DAS). The vertical error bars represent standard error (SD/10). Different letters above the vertical bars indicate significant differences among the means of the fallow
plots, monocultures, and mixtures across the three harvest times, based on ANOVA followed by
Tukey’s HSD test (p < 0.05). Figure 5. Weed biomass (WB) in the fallow plots, monocultures, and mixtures of two legume cover
crops of alsike clover (AC) and black medic (BM) in two years 2016 and 2017. The treatments of the
cover crop contained five mixing ratios of AC:BM (100:0, 67:33, 50:50, 33:67, and 0:100) sown at three
seed densities (50%, 100%, and 150% of the recommended seed densities). The WB was harvested
three times during the season at H1: In spring (40 ± 10 days after sowing; DAS), H2: In summer
(64 ± 10 DAS), and H3: In autumn (146 ± 26 DAS). The vertical error bars represent standard error
(SD/10). Different letters above the vertical bars indicate significant differences among the means of the
fallow plots, monocultures, and mixtures across the three harvest times, based on ANOVA followed by
Tukey’s HSD test (p < 0.05). Figure 5. 4 1 Cover Crop Species Effect
4.1. Cover Crop Species Effect 4.1. Cover Crop Species Effect
Plant growth dynamics were similar in both years at the early stage of the establishment (H1),
where BM-monoculture was dominant. This may be partly due to the fast growth rate of BM than
AC, therefore it was expected for AC to be less competitive at the early growth stage [29,46]. The
efficiency of BM in suppressing weed in both years likely confirms that rapid growth rate is a key
trait for weed suppression [10,11]. However, the efficiency of AC in weed suppression at H1 in 2016
was comparable to the other treatments. This indicates that legume species with a slow growth rate
may use alternative mechanisms such as allelopathy to suppress weeds at the early stage. It was
widely reported that many species belonging to the genera Trifolium and Medicago release allelopathic
compounds [60,61]. The dynamics of CCB at H2 after successful crop establishment were remarkably different, in
both years, depending on the growing conditions. In 2016, the relatively dry conditions supported a
better performance of both legume species in monocultures and mixtures than in 2017. Therefore, all
treatments produced more or less equal biomass and suppressed weeds with the same efficiency. This finding was contrary to expectations for AC, as this species was supposed to be negatively
affected by drought conditions [29,52,53] and consequently its strength to suppress weeds would be
Plant growth dynamics were similar in both years at the early stage of the establishment (H1),
where BM-monoculture was dominant. This may be partly due to the fast growth rate of BM than AC,
therefore it was expected for AC to be less competitive at the early growth stage [29,46]. The efficiency
of BM in suppressing weed in both years likely confirms that rapid growth rate is a key trait for weed
suppression [10,11]. However, the efficiency of AC in weed suppression at H1 in 2016 was comparable
to the other treatments. This indicates that legume species with a slow growth rate may use alternative
mechanisms such as allelopathy to suppress weeds at the early stage. It was widely reported that
many species belonging to the genera Trifolium and Medicago release allelopathic compounds [60,61]. The dynamics of CCB at H2 after successful crop establishment were remarkably different, in both
years, depending on the growing conditions. 4.2. Diversity Effects Diversity effects (mixing versus non-mixing) are expected to be strong in the case of mixing species
with contrasting traits regarding growth rates, response to water and temperature [28,29]. This approach
of diversification potentially entails higher efficiency of weed suppression via complementary and
consistent biomass production across the whole season under variable environmental conditions [44,45]. In our study, despite the contrasting traits of the two legume species regarding growth rates, the
observed diversity effects on weed suppression were mostly weak and inconsistent during the growing
season (Table 2). However, as an average over the whole season, mixtures significantly reduced WB compared to
the monocultures only in 2017 (Figure 5). Therefore, our results show that diversity effects of legumes
on weed suppression are partly in agreement with the expectations and observations made in previous
studies [21–24]. These contradictory results make it more difficult to give any clear recommendations
for farmers about whether or not mixing crops helps to suppress weeds. In our case, the results shown
in Figure 5 and Table 2 suggest that although mixing species has the potential for weed suppression,
the mixing effect is not always reliable and other practices need to be employed for effective weed
control. Despite this, finding out a significant cumulative diversity effect of only-legume mixtures
on weed suppression at least in one year is likely important to the intrinsic and functional value of
legumes in agroecosystems. It indicates that the legacy effect of legume mixtures on weed suppression
can be left during one growing season, but it depends on the growing conditions. A potential explanation for the inconsistence of the diversity effect in the tested forage legume
mixture on weed suppression is that species identity effects of the two legumes may have been
comparatively strong, superseding any mixture effects [26,27]. In contrast to [25], our results present
empirical evidence that species identity in legume mixtures is more important than species diversity. This may also include differential effects of the two legume species on some weed species. In both
field trials, fat hen (Chenopodium album L.) and barnyard grass (Echinochloa crus galli L.) were dominant. Height measurements of the two weed species conducted after H2 in 2017 showed significant
correlations with total WB (Figure S3) and confirmed that crop species identity effects on the weeds
are strong. 4 1 Cover Crop Species Effect
4.1. Cover Crop Species Effect In 2016, the relatively dry conditions supported a
better performance of both legume species in monocultures and mixtures than in 2017. Therefore, all
treatments produced more or less equal biomass and suppressed weeds with the same efficiency. This finding was contrary to expectations for AC, as this species was supposed to be negatively
affected by drought conditions [29 52 53] and consequently its strength to suppress weeds would be
The dynamics of CCB at H2 after successful crop establishment were remarkably different, in both
years, depending on the growing conditions. In 2016, the relatively dry conditions supported a
better performance of both legume species in monocultures and mixtures than in 2017. Therefore,
all treatments produced more or less equal biomass and suppressed weeds with the same efficiency. This finding was contrary to expectations for AC, as this species was supposed to be negatively affected
by drought conditions [29,52,53] and consequently its strength to suppress weeds would be lower. The unexpectedly outstanding performance of AC in the dry season, in 2016, suggests that the effect 13 of 19 Agronomy 2019, 9, 648 of water level in limiting the growth of AC might be alleviated by other factors (e.g., soil physical,
chemical, and biological properties). In 2017, the significant increase in the biomass of AC-monoculture compared to the other
treatments was a result of a heavy rain event experienced just 10 days before H2. This caused temporary
waterlogging, which strongly affected the growth of BM in a negative way (e.g., plant yellowing). The
negative response of BM in the wet conditions is in accordance with the literature [47,48]. However,
the relative advantage of AC in these conditions did not result in a higher weed suppression capacity. g
p
he relative advantage of AC in these conditions did not result in a higher weed suppression capa After mulching (H3), the dynamics of crop regrowth after mulching in both years were similar
to H1, with the dominance of BM-monoculture over the other treatments. This suggests that the
inherent characteristics of the plant species drive its growth and regrowth after mulching. Actually,
BM is able to regrow after cutting through stoloniferous growth forming horizontal branched stem-like
structure [49–51], whereas AC regrows after cutting by forming tiller-like branches at the crown that
frequently develop into floral stem under favorable climatic and soil conditions [54]. 4 1 Cover Crop Species Effect
4.1. Cover Crop Species Effect In 2017, the strong
ability of BM to recover, after mulching, after the stress event of heavy rains clearly demonstrated
its resilient capacity to such events. However, BM is widely known for its better performance in dry
conditions [29,46]. One of the main findings in this study suggests that AC and BM can be used as
keystone species on weed suppression for sustainable agriculture in comparison with previous studies
reported weed-suppressive effects of other legumes [32–34]. 4.3. Proportion Effects Our study further provided an opportunity to test the potential effects of the variations of legume
proportions within the mixtures on weed suppression. It was hypothesized that the increasing crop
species evenness supports stronger weed suppression [38,39]. Possible mechanisms include the fact
that the species that are evenly distributed in space may use resources more equitably and produce a
competitive environment that is difficult for weeds to invade [36,38–40]. The results reported here only
partially agree with this hypothesis. At H1, species proportions played no role in weed suppression at the early growth stage. This
may be related to the slow interaction between the two species at the early stage AC has a slow growth
rate. This means that the approach of increasing evenness for efficient weed suppression might be not
reliable at the early stage. At H2, in 2017, the trend of the equiproportional mixture to be more suppressive than the other
mixtures is in agreement with previous studies reporting that mixtures with greater species evenness
are less prone to weeds infestation [7,39]. At H3, after mulching, the equiproportional mixture was almost, but not consistently more
suppressive than the other mixtures. Here, the proportion effect is in line with some studies reported
that evenness in mixtures is less likely to be a factor affecting WB [25,35,41]. From an agronomic
viewpoint, our results across the whole season show that the changing species proportion in legume
mixtures is not a hugely influential force on weed suppression. 4.2. Diversity Effects Moreover, the plant height of both weed species decreased with increasing crop density
suggesting a strong competitive ability of AC and BM with fat hen and barnyard grass (Figure S4). Further, pot trials conducted with these weed species indicate that the two tested legumes showed
differential allelopathic effects on these weeds [62]. Agronomy 2019, 9, 648 14 of 19 Agronomy 2019, 9, 648 A likely reason why we did not observe stronger diversity effects on weeds was the interference
of temporal complementarity of the two legumes’ growth characteristics with mulching [28]. The
potential of the two legumes to suppress weeds more strongly than is expected from their respective
monocultures stems from their complementary growth characteristics over time: While BM grows fast
and suppresses early weeds; later weed growth is inhibited by the slow-growing AC. However, by the
time AC’s potential can be exploited, mulching takes place, thereby essentially resetting the conditions,
and favouring BM, which in comparison to AC is faster to regrow after being cut. 4.4. Density Effects At H1 and H2, we found that doubling crop density from 50% to 100% significantly increased
weed suppression, confirming that high density increases the competitive interactions and resource use
between cover crop species to ensure effective weed suppression [36,42,43]. However, no significant
difference has been found between the seed densities 100% and 150% indicates that the recommended
seed density of the selected legume species is sufficient for weed suppression. The efficiency of weed
suppression at H2 (Figure 3B,E) is mainly a result of the deprivation of weeds from sunlight and space
by increasing crop competition after successful establishment specifically at higher seed density [36,41]. Our results confirm that legume cover crops are able to suppress weeds efficiently as living mulches at
high density. At H3, after mulching, observing no density effect on CCB and WB is additional evidence for
species-specific characteristics of AC and BM to compensate for low density. This finding is of practical
importance in sustainable agriculture, as it confirms that after mulching adjusting seed density is of
less importance on productivity and weed suppression. Based on this finding less concern can be given
to seed density when mulching of cover crop is planned within the season or in long-term agricultural
management practices (e.g., managed grassland), but on the condition that the species can compensate
after cutting or after mulching. 4.6. Mulching Effects In both years, at H3, weeds in the legume-plots were inhibited by the mulching and did not grow
up; therefore, WB was significantly lower than H2. Comparing the fallow with any of the treatments
with the cover crop confirms that mulching a legume cover crop strongly reduces WB. Three general
mechanisms can be distinguished that may underlie this observation: (1) Cutting may lead to direct
mortality, or reduced growth, of weeds in particular if the top-growth is removed at lower levels
below 7 cm [63]; the reduction in biomass is evident from the comparison between H2 and H3 in
the fallow plots. (2) Differential recovery from cutting between weeds, especially annual ones, and
the forage legumes may lead to a shift in the competitive balance towards the crop; and (3) residues
of legumes (and weeds) left as a dead mulch may contribute to weed suppression; either (a) as a
physical barrier, (b) through reduced light transmittance to the soil surface [18], and (c) by release
of allelochemicals [17,19,20] that reduce weed seedling growth. Due to this last mechanism, caution
should be taken when the mulch of these two legume species come to practice in crop rotation because
some unwanted consequences on the subsequent cash crop might be implemented. In fact, it has been
reported that species that are suppressive to weeds might be suppressive to other crops [21]. An explanation as for why we did observe weeds after mulching in the fallow plots, (Figure 3C,F)
despite the physical barrier of the dead plant residues above the soil surface may be related to three main
potential reasons: (1) Some late-season weeds may have started to germinate immediately after cutting;
(2) small weeds may have been left untouched by the mower and continued to grow; (3) recalcitrant
weeds may have regrown even after cutting; indeed, we did observe that some species were able to
regrow after mulching, e.g., barnyard grass (Echinochloa crus galli L.), mostly from individual un-cut
tillers of cut plants. 4.5. Relationship between Crop Biomass and Weed Biomass Although many studies hypothesized that the increase in biomass production was intimately
related to greater weed suppression [44,45], we have found that this relationship between CCB and WB
can be weak and inconsistent in forage legume crops (Figure 4). A key mechanism behind a rigorous 15 of 19 15 of 19 Agronomy 2019, 9, 648 relationship between CCB and WB is complementary, which occurs when there is asynchrony in species
growth rates and/or in species responses to environmental. In our study, the contrasting traits of legume
species in the mixture allowed for complementary that were time and condition dependent. However,
the efficient weed suppression in the two years reveals that there is a parallel mechanism used by AC
and BM independent of time and condition which is mostly allelopathy. Our finding indicates that the
allelopathic ability of these two species is constitutive and it gives the two species plasticity to suppress
weeds when the higher biomass productivity is limited by the environmental conditions. 5. Conclusions Both studied species, AC and BM, provide evidence for their ability to suppress weeds in
monocultures and mixtures. However, the effectiveness of weed control by these legumes is
time-dependent, species-dependent, and density-dependent (only before mulching). Further, species
identity (or functional identity) in legume mixtures had a stronger effect than species diversity, with
weed suppression in mixtures being stronger than in the respective monocultures only in one of the
two study years. Thus, the crop diversity effect on weeds was dependent on the growing environment. The equiproportional mixture of legumes showed a weak trend to be more suppressive to weeds and
was inconsistently more productive than a mixture dominated by either of the component species. In addition, the effect of seed density on weed suppression depends on harvest time; higher
density reduces weeds but no density effect is observed after mulching, indicating that compensatory
growth against low density increases over time. Weed suppression in only-legume binary mixtures
is partially related to high CCB productivity and depends on time and the environment. Moreover,
mixture effects in the crop and weed suppression are time-dependent (within one season), but not all
time-dependent effects are consistent. On the basis of this finding, the strength of weed suppression in
the average of all treatments over the two years (40% shortly after establishment, 50% at the flowering
stage, and 90% after mulching) indicates that legumes develop different mechanisms to suppress
weeds, potentially including chemical (allelopathy) and physical effects. It also suggests that legumes
have wide flexibility to be used in different locations for the purpose of weed suppression in marginal
areas or ecological revegetated areas as biofertilizers. Further research is needed to investigate the potential use of allelopathy at the early stage of
legume growth to support our new finding and to determine if it is dependent on species, cultivar, and
environmental conditions. In addition, it would be of great importance to understand which factors in
the soil would alleviate the drought effect on drought-sensitive species such as AC to consider these
factors for higher crop productivity and efficient weed suppression in dry-prone areas. Moreover,
future studies with more focus on weed suppression are recommended to investigate an appropriate
approach to use these two legumes in agriculture systems (e.g., by comparing leaving and removing the
cut plant materials). 4.7. Site and Year Effects The variation in CCB and WB between the two fields in the study site was primarily the result of
the different growing conditions for the cover crops and the weeds in the two years. In all treatments,
at H1, CCB was generally lower in 2017 than 2016, whereas WB was higher even in the fallow plot
(Figure 2A,D). The reduced CCB may be due to the low soil fertility in the field S5 (2017) than in FU9
(2016), specifically phosphorous that was 48.2% lower in 2017 than in 2016 (Table 1). This may have
directly or indirectly affected legume growth as it is known that legumes need adequate amounts of
phosphorous (P) for N2-fixing nodules (e.g., [16]). However, the higher WB in the same year, even in
the fallow, indicates that the limited nutrient levels in 2017 did not reduce WB (Figure 3D). This finding
is in line with studies reported that low soil fertility could be less resistant to weed invasion if it
supports small competitive biomass leading to an increase in weed growth and establishment [15,39]. In addition, greater weed suppression in low-productive soil was also reported in a previous study [23]. An alternative explanation to the variation in CCB and WB between the two fields could be related
to differences in the abundance of weeds in the seedbank as the initial seedbank can influence the
success of cover crops on weed management [12]. Additionally, in both years the plants experienced
different growth conditions that may have affected weed population dynamics as a function of
ecological interactions within and between plant populations, nutrient, and water limitation, rainfall,
and temperature [13]. Specifically, in 2017 the low temperature (Figure 1) at the early growth stage may
have increased the prevalence of competitive weed species and this effect is in agreement with [14]. Agronomy 2019, 9, 648 16 of 19 16 of 19 Conflicts of Interest: The authors declare no competing financial interests. Acknowledgments: The Jameel Scholarship at Humboldt University, Berlin, Germany, is greatly appreciated for
its financial support. We thank Timo Kautz and Christina-Luise Roß (Humboldt-University of Berlin, Department
of Agronomy and Crop Science) for valuable suggestions and comments on the manuscript and the staffat the
Humboldt-University research station in Dahlem for technical support during the two-field experiment. Funding: This research was funded by the Jameel Scholarship at Humboldt-University, Berlin, Germany
[Ph.D. project]. References 1. Chauhan, B.S.; Gill, G.S. Ecologically based weed management strategies. In Recent Advances in Weed
Management; Chauhan, B.S., Mahajan, G., Eds.; Springer: New York, NY, USA, 2014; pp. 1–11. 2. Abouziena, H.F.; Haggag, W.M. Weed Control in Clean Agriculture: A Review. Planta Daninha 2016, 34,
377–392. [CrossRef] 3. Oerke, E.C. Crop losses to pests. J. Agric. Sci. 2006, 144, 31–43. [CrossRef] 4. Westwood, J.H.; Charudattan, R.; Duke, S.O.; Fennimore, S.A.; Marrone, P.; Slaughter, D.C.; Swanton, C.;
Zollinger, R. Weed Management in 2050: Perspectives on the Future of Weed Science. Weed Sci. 2018, 66,
275–285. [CrossRef] . Lithourgidis, A.S.; Dordas, C.A.; Damalas, C.A.; Vlachostergios, D.N. Annual intercrops: An alterna
pathway for sustainable agriculture. Aust. J. Crop Sci. 2011, 5, 396–410. 6. Lemessa, F.; Wakjira, M. Cover crops as a means of ecological weed management in agroecosystems. J. Crop
Sci. Biotechnol. 2015, 18, 133–145. [CrossRef] 7. Finney, D.M.; Kaye, J.P. Functional diversity in cover crop polycultures increases multifunctionality of an
agricultural system. J. Appl. Ecol. 2017, 54, 509–517. [CrossRef] 8. Lorin, M.; Jeuffroy, M.H.; Butier, A.; Valantin-Morison, M. Undersowing winter oilseed rape with
Frost-Sensitive legume living mulches to improve weed control. Eur. J. Agron. 2015, 71, 96–105. [CrossRef] 9. Hiltbrunner, J.; Jeanneret, P.; Liedgens, M.; Stamp, P.; Streit, B. Response of weed communities to legume
living mulches in winter wheat. J. Agron. Crop Sci. 2007, 193, 93–102. [CrossRef] 10. Fradgley, N.S.; Creissen, H.E.; Pearce, H.; Howlett, S.A.; Pearce, B.D.; Döring, T.F.; Girling, R.D. Weed
Suppression and Tolerance in Winter Oats. Weed Technol. 2017, 31, 740–751. [CrossRef] 11. Weidlich, E.W.A.; von Gillhaussen, P.; Delory, B.M.; Blossfeld, S.; Poorter, H.; Temperton, V.M. The Importance
of Being First: Exploring Priority and Diversity Effects in a Grassland Field Experiment. Front. Plant Sci. 2017, 7, 1–12. [CrossRef] 12. Teasdale, J.R.; Mangum, R.W.; Radhakrishnan, J.; Cavigelli, M.A. Weed seedbank dynamics in three organic
farming crop rotations. Agron. J. 2004, 96, 1429–1435. [CrossRef] 13. Ramesh, K.; Matloob, A.; Aslam, F.; Florentine, S.K.; Chauhan, B.S. Weeds in a Changing Climate:
Vulnerabilities, Consequences, and Implications for Future Weed Management. Front. Plant Sci. 2017, 8, 95. [CrossRef] [PubMed] 14. Ferrero, R.; Lima, M.; Davis, A.S.; Gonzalez-Andujar, J.L. Weed Diversity Affects Soybean and Maize Yield in
a Long Term Experiment in Michigan, USA. Front. Plant Sci. 2017, 8, 1–10. [CrossRef] [PubMed] 15. Ruisi, P.; Frangipane, B.; Amato, G.; Frenda, A.S.; Plaia, A.; Giambalvo, D.; Saia, S. 5. Conclusions We also suggest a research question regarding using AC and BM in environmental
engineering and landscape management to suppress weeds or to use them as natural herbicides while
increasing soil fertility. Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4395/9/10/648/s1,
Figure S1: The Mixture design response surface methodology for different combinations of AC and BM, Figure
S2: The design of the two field experiments, Figure S3: Relationship between weeds’ heights of Fat-hen
(Chenopodium album L.), and Barnyard grass (Echinochloa crus-galli L.; Ech) and total WB, Figure S4: Weed height of
Fat-hen (Chenopodium album L.) and Barnyard grass (Echinochloa crus-galli L.) in response to cover crops of AC and
BM in monocultures and three binary mixtures, Figure S5: WB and CCB averaged across all treatments at three
harvest times in 2016 and 2017; Table S1: Model output of the linear mixed model describing the mixing effects of
AC and BM on CCB and WB, Table S2: List of the most common weeds in 2016 recorded in the fallow plots and at
high density (150% of the recommended seed density) of AC and BM in monocultures. Author Contributions: H.E. and T.D. conceived and designed the experiments, conducted the experiments, and
collected the data. T.D. provided the facilities and advised on the preparation of materials. H.E. and T.D. wrote
the manuscript. H.E., T.D., S.B.-K., and D.A. performed the statistical tests. S.B.-K. read and edited the manuscript. All authors approved the final manuscript. Funding: This research was funded by the Jameel Scholarship at Humboldt-University, Berlin, Germany
[Ph.D. project]. ding: This research was funded by the Jameel Scholarship at Humboldt-University, Berlin, Germany
D. project]. Acknowledgments: The Jameel Scholarship at Humboldt University, Berlin, Germany, is greatly appreciated for
its financial support. We thank Timo Kautz and Christina-Luise Roß (Humboldt-University of Berlin, Department
of Agronomy and Crop Science) for valuable suggestions and comments on the manuscript and the staffat the
Humboldt-University research station in Dahlem for technical support during the two-field experiment. Conflicts of Interest: The authors declare no competing financial interests. 17 of 19 17 of 19 Agronomy 2019, 9, 648 References Effects of intercropping on yield, weed incidence, forage quality and soil residual N in
organically grown forage maize (Zea mays L.) and faba bean (Vicia faba L.). Field Crop. Res. 2014, 169, 21–29. p
y
pp
effects of Maize-Legume intercropping in organic farming. Int. J. Pest Manag. 2010, 56, 173–181. [CrossRef]
24. Stoltz, E.; Nadeau, E. Effects of intercropping on yield, weed incidence, forage quality and soil residual N in
organically grown forage maize (Zea mays L.) and faba bean (Vicia faba L.). Field Crop. Res. 2014, 169, 21–29. [CrossRef] 24. Stoltz, E.; Nadeau, E. Effects of intercropping on yield, weed incidence, forage quality and soil residual N in
organically grown forage maize (Zea mays L.) and faba bean (Vicia faba L.). Field Crop. Res. 2014, 169, 21–29. [CrossRef] 25. Connolly, J.; Sebastià, M.T.; Kirwan, L.; Finn, J.A.; Llurba, R.; Suter, M.; Collins, R.P.; Porqueddu, C.;
Helgadóttir, Á.; Baadshaug, O.H.; et al. Weed suppression greatly increased by plant diversity in intensively
managed grasslands: A Continental-Scale Experiment. J. Appl. Ecol. 2018, 55, 852–862. [CrossRef] [PubMed]
26. Smith, R.G.; Atwood, L.W.; Warren, N.D. Increased productivity of a cover crop mixture is not associated
with enhanced agroecosystem services PLoS ONE 2014 9 e97351 [CrossRef] 25. Connolly, J.; Sebastià, M.T.; Kirwan, L.; Finn, J.A.; Llurba, R.; Suter, M.; Collins, R.P.; Porqueddu, C.;
Helgadóttir, Á.; Baadshaug, O.H.; et al. Weed suppression greatly increased by plant diversity in intensively
managed grasslands: A Continental-Scale Experiment. J. Appl. Ecol. 2018, 55, 852–862. [CrossRef] [PubMed] Helgadóttir, Á.; Baadshaug, O.H.; et al. Weed suppression greatly increased by plant diversity in intensively
managed grasslands: A Continental-Scale Experiment. J. Appl. Ecol. 2018, 55, 852–862. [CrossRef] [PubMed]
26. Smith, R.G.; Atwood, L.W.; Warren, N.D. Increased productivity of a cover crop mixture is not associated
with enhanced agroecosystem services. PLoS ONE 2014, 9, e97351. [CrossRef] 26. Smith, R.G.; Atwood, L.W.; Warren, N.D. Increased productivity of a cover crop mixture is not associated
with enhanced agroecosystem services. PLoS ONE 2014, 9, e97351. [CrossRef] 27. Cong, W.F.; Suter, M.; Lüscher, A.; Eriksen, J. Species interactions between forbs and Grass-Clover contribute
to yield gains and weed suppression in forage grassland mixtures. Agric. Ecosyst. Environ. 2018, 268, 154–161. [CrossRef] 28. Yu, Y.; Makowski, D.; Stomph, T.J.; van der Wopke, W. Robust increases of land equivalent ratio with
temporal niche differentiation: A Meta-Quantile regression. Agron. J. 2016, 108, 2269–2279. [CrossRef] 29. References Döring, T.F.; Baddeley, J.A.; Brown, R.; Collins, R.; Crowley, O.; Cuttle, S.; Howlett, S.A.; Jones, H.E.;
Mccalman, H.; Measures, M.; et al. Using Legume-Based mixtures to enhance the nitrogen use efficiency and
economic viability of cropping systems: Project Report (RD-2007-3447). HGCA/Agric. Hort. Dev. Board 2013,
190. 30. Finn, J.A.; Kirwan, L.; Connolly, J.; Sebastià, M.T.; Helgadottir, A.; Baadshaug, O.H.; Bélanger, G.; Black, A.;
Brophy, C.; Collins, R.P.; et al. Ecosystem function enhanced by combining four functional types of plant
species in intensively managed grassland mixtures: A 3-Year Continental-Scale field experiment. J. Appl. Ecol. 2013, 50, 365–375. [CrossRef] 31. Stagnari, F.; Maggio, A.; Galieni, A.; Pisante, M. Multiple benefits of legumes for agriculture sustainability:
An overview. Chem. Biol. Technol. Agric. 2017, 4, 1–13. [CrossRef] 32. Storkey, J.; Döring, T.; Baddeley, J.; Marshall, A.; Roderick, S.; Jones, H. Modelling the ability of legumes to
suppress weeds. Asp. Appl. Biol. 2011, 109, 53–58. 33. Döring, T.F.; Storkey, J.; Baddeley, J.A.; Collins, R.P.; Crowley, O.; Howlett, S.A.; Jones, H.E.; McCalman, H.;
Measures, M.; Pearce, H.; et al. Weeds in Organic Fertility-Building Leys: Aspects of Species Richness and
Weed Management. Org. Farming 2017, 3. [CrossRef] 34. Dhamala, N.R.; Eriksen, J.; Carlsson, G.; Søegaard, K.; Rasmussen, J. Highly productive forage legume stands
show no positive biodiversity effect on yield and N2-Fixation. Plant Soil 2017, 417, 169–182. [CrossRef] 35. Emery, S.M.; Gross, K.L. Dominant species identity, not community evenness, regulates invasion in
experimental grassland plant communities. Ecology 2007, 88, 954–964. [CrossRef] [PubMed] 36. Hauggaard-Nielsen, H.; Andersen, M.K.; Jørnsgaard, B.; Jensen, E.S. Density and relative frequency effects
on competitive interactions and resource use in pea-barley intercrops. Field Crop. Res. 2006, 95, 256–267. [CrossRef] 37. Kirwan, L.; Lüscher, A.; Sebastià, M.T.; Finn, J.A.; Collins, R.P.; Porqueddu, C.; Helgadottir, A.;
Baadshaug, O.H.; Brophy, C.; Coran, C.; et al. Evenness drives consistent diversity effects in intensive
grassland systems across 28 European sites. J. Ecol. 2007, 95, 530–539. [CrossRef] 8. Tracy, B.F.; Renne, I.J.; Gerrish, J.; Sanderson, M.A. Effects of plant diversity on invasion of weed specie
experimental pasture communities. Basic Appl. Ecol. 2004, 5, 543–550. [CrossRef] 9. Tracy, B.F.; Sanderson, M.A. Forage productivity, species evenness and weed invasion in pasture commun
Agric. Ecosyst. Environ. 2004, 102, 175–183. [CrossRef] 40. Polley, H.W.; Wilsey, B.J. Reductions in grassland species evenness increase dicot seedling invasion and
spittle bug infestation. Ecol. Lett. 2002, 5, 676–684. 41. Sanderson, M.A.; Brink, G.; Stout, R.; Ruth, L. References Nitrogen uptake and
nitrogen fertilizer recovery in old and modern wheat genotypes grown in the presence or absence of
interspecific competition. Front. Plant Sci. 2015, 6, 1–10. [CrossRef] [PubMed] p
p
16. Sulieman, S.; Tran, L.S.P. Phosphorus homeostasis in legume nodules as an adaptive strategy to phosphorus
deficiency. Plant Sci. 2015, 239, 36–43. [CrossRef] 17. Mahmood, A.; Ihsan, M.Z.; Khaliq, A.; Hussain, S.; Cheema, Z.A.; Naeem, M.; Daur, I.; Hussain, H.A.;
Alghabari, F. Crop Residues Mulch as Organic Weed Management Strategy in Maize. Clean-Soil Air Water
2016, 44, 317–324. [CrossRef] 18. Ranaivoson, L.; Naudin, K.; Ripoche, A.; Affholder, F.; Rabeharisoa, L.; Corbeels, M. Agro-Ecological functions
of crop residues under conservation agriculture. A review. Agron. Sustain. Dev. 2017, 37, 1–17. [CrossRef] 19. Caamal-Maldonado, J.A.; Jiménez-Osornio, J.J.; Torres-Barragán, A.; Anaya, A.L. The use of allelopathic
legume cover and mulch species for weed control in cropping systems. Agron. J. 2001, 93, 27–36. [CrossRef] legume cover and mulch species for weed control in cropping systems. Agron. J. 2001, 93, 27–36. [CrossRef]
20. Reddy, P.P. Crop Residue Management and Organic Amendments. In Agro-Ecological Approaches to Pest
Management for Sustainable Agriculture; Springer: Singapore, 2017; pp. 29–41. 20. Reddy, P.P. Crop Residue Management and Organic Amendments. In Agro-Ecological Approaches to Pest
Management for Sustainable Agriculture; Springer: Singapore, 2017; pp. 29–41. 21. Bedoussac, L.; Journet, E.P.; Hauggaard-Nielsen, H.; Naudin, C.; Corre-Hellou, G.; Jensen, E.S.; Prieur, L.;
Justes, E. Ecological principles underlying the increase of productivity achieved by Cereal-Grain legume
intercrops in organic farming. A review. Agron. Sustain. Dev. 2015, 35, 911–935. [CrossRef] 22. Corre-Hellou, G.; Dibet, A.; Hauggaard-Nielsen, H.; Crozat, Y.; Gooding, M.; Ambus, P.; Dahlmann, C.;
von Fragstein, P.; Pristeri, A.; Monti, M.; et al. The competitive ability of Pea-Barley intercrops against
weeds and the interactions with crop productivity and soil N availability. Field Crop. Res. 2011, 122, 264–272. [CrossRef] 18 of 19 18 of 19 Agronomy 2019, 9, 648 23. Bilalis, D.; Papastylianou, P.; Konstantas, A.; Patsiali, S.; Karkanis, A.; Efthimiadou, A. Weed-Suppressive
effects of Maize-Legume intercropping in organic farming. Int. J. Pest Manag. 2010, 56, 173–181. [CrossRef] 23. Bilalis, D.; Papastylianou, P.; Konstantas, A.; Patsiali, S.; Karkanis, A.; Efthimiadou, A. Weed-Suppressive
effects of Maize-Legume intercropping in organic farming. Int. J. Pest Manag. 2010, 56, 173–181. [CrossRef]
24. Stoltz, E.; Nadeau, E. References Grass-Legume proportions in forage seed mixtures and effects
on herbage yield and weed abundance. Agron. J. 2013, 105, 1289–1297. [CrossRef] 42. Bulson, H.A.J.; Snaydon, R.W.; Stopes, C.E. Effects of plant density on intercropped wheat and field beans in
an organic farming system. J. Agric. Sci. 1997, 128, 59–71. [CrossRef] 43. Weiner, J.; Andersen, S.B.; Wille, W.K.M.; Griepentrog, H.W.; Olsen, J.M. Evolutionary Agroecology: The
potential for cooperative, high density, Weed-Suppressing cereals. Evol. Appl. 2010, 3, 473–479. [CrossRef]
[PubMed] 19 of 19 19 of 19 Agronomy 2019, 9, 648 44. Finney, D.M.; White, C.M.; Kaye, J.P. Biomass production and carbon/nitrogen ratio influence ecosystem
services from cover crop mixtures. Agron. J. 2016, 108, 39–52. [CrossRef] 45. Bybee-Finley, K.; Mirsky, S.B.; Ryan, M.R. Crop Biomass Not Species Richness Drives Weed Suppression in
Warm-Season Annual Grass–Legume Intercrops in the Northeast. Weed Sci. 2017, 65, 669–680. [CrossRef] 45. Bybee-Finley, K.; Mirsky, S.B.; Ryan, M.R. Crop Biomass Not Species Richness Drives Weed Suppression in
W
S
A
l G
L
I t
i
th N
th
t W d S i 2017 65 669 680 [C
R f] Warm-Season Annual Grass–Legume Intercrops in the Northeast. Weed Sci. 2017, 65, 669–680. [CrossRef]
46. Clark, A. Managing Cover Crops Profitably. In Sustainable Agriculture Research and Education, 3rd ed.;
Sustainable Agriculture Network: Beltsville, MD, USA, 2007; pp. 1–244. 46. Clark, A. Managing Cover Crops Profitably. In Sustainable Agriculture Research and Education, 3rd ed.;
Sustainable Agriculture Network: Beltsville, MD, USA, 2007; pp. 1–244. 47. FAO. Ecocrop, Data Sheet, Black medic (Medicago Lupulina). Available online: http://ecocrop.fao.org/ecocrop/
srv/en/dataSheet?id=7654 (accessed on 15 September 2019). 48. Floulds, W. Response To Soil Moisture Supply in Three Leguminous Species: I.; Growth, Reproduction and
Mortality. New Phytol. 1978, 80, 535–545. [CrossRef] 49. Dzyubenko, N.I.; Dzyubenko, E.A. Crops. Medicago lupulina L.-Black medic. In Interactive Agricultural
Ecological Atlas of Russia and Neighboring Countries. Economic Plants and their Diseases, Pests and Weeds; 2009;
A
il bl
li
htt
//
tl
/
/
t
t/
lt
l/M di
l
li
K/i d
ht
l (
d 49. Dzyubenko, N.I.; Dzyubenko, E.A. Crops. Medicago lupulina L.-Black medic. In Interactive Agricultural
Ecological Atlas of Russia and Neighboring Countries. Economic Plants and their Diseases, Pests and Weeds; 2009;
Available online: http://www.agroatlas.ru/en/content/cultural/Medicago_lupulina_K/index.html (accessed
on 15 September 2019). 0. Watschke, T.L.; Dernoeden, P.H.; Shetlar, D.J. Managing Turfgrass Pests, 2nd ed.; CRC Press: London,
2013; p. 519. 51. Heuzé, V.; Thiollet, H.; Tran, G.; Hassoun, P.; Lebas, F. References Feedipedia, Black medic (Medicago lupulina), a program
by INRA, CIRAD, AFZ, and FAO. Available online: https://www.feedipedia.org/node/277 (accessed on
15 September 2019). 52. Chapman, H.M.; Dodds, K.G.; Keoghan, J.M. Evaluation of the agronomic potential of pasture legume
introductions on droughty outwash soils. N. Z. J. Agric. Res. 1990, 33, 21–27. [CrossRef] 53. Sheaffer, C.C.; Ehlke, N.J.; Albrecht, K.A.; Peterson, P.R. Forage Legumes: Clovers, Birdsfoot Trefoil, Cicer
Milkvetch, Crownvetch and Alfalfa, 2nd ed.; Agric. Exp. Stn.: Saint Paul, Minnesota, 2003; pp. 608–2003. 55. Agricultural Climatology of the Humboldt-University of Berlin. Available online: https://www.agrar.hu-
berlin.de/de/institut/departments/dntw/agrarmet/service/wo (accessed on 15 September 2018). 56. Tribouillois, H.; Dürr, C.; Demilly, D.; Wagner, M.H.; Justes, E. Determination of germination response
to temperature and water potential for a wide range of cover crop species and related functional groups. PLoS ONE 2016, 11, 1–16. [CrossRef] 57. Burnham, K.P.; Anderson, D.R.; Huyvaert, K.P. AIC model selection and multimodel inference in behavioral
ecology: Some background, observations, and comparisons. Behav. Ecol. Sociobiol. 2011, 65, 23–35. [CrossRef] 57. Burnham, K.P.; Anderson, D.R.; Huyvaert, K.P. AIC model selection and multimodel inference in behavioral
ecology: Some background, observations, and comparisons. Behav. Ecol. Sociobiol. 2011, 65, 23–35. [CrossRef]
58. De Mendiburu, F. Agricolae: Statistical Procedures for Agricultural Research. R package version 1.3-0. 2019. Available online: https://CRAN.R-project.org/package=agricolae (accessed on 15 September 2019). 58. De Mendiburu, F. Agricolae: Statistical Procedures for Agricultural Research. R package version 1.3-0. 2019. Available online: https://CRAN.R-project.org/package=agricolae (accessed on 15 September 2019). 59. R Core Team language and environment for statistical computing. R Foundation for Statistical Computing;
R Core Team language and environment for statistical computing: Vienna, Austria, 2019; Available online:
https://www.R-project.org/. (accessed on 1 August 2019). 60. Chon, S.U.; Jennings, J.A.; Nelson, C.J. Alfalfa (Medicago sativa L.) autotoxicity: Current Status. Allelopath. J. 2006, 18, 57–80. 61. Liu, Q.; Xu, R.; Yan, Z.; Jin, H.; Cui, H.; Lu, L.; Zhang, D.; Qin, B. Phytotoxic allelochemicals from roots and
root exudates of Trifolium pratense. J. Agric. Food Chem. 2013, 61, 6321–6327. [CrossRef] 62. Elsalahy, H.; Bellingrath-Kimura, S.; Döring, T. Allelopathic effects in species mixtures of legumes. © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/). References In Proceedings of the Improving Sown Grasslands through Breeding and Management, Proceedings of the Joint 20th
Symposium of the European Grassland Federation and the 33rd Meeting of the EUCARPIA, Zurich, Switzerland,
24–27 June 2019; Huguenin-Elie, O., Studer, B., Kölliker, R., Reheul, D., Probo, M., Barre, P., Feuerstein, U.,
Roldán-Ruiz, I., Mariotte, P., Hopkins, A., Eds.; Organising Committee: Zurich, Switzerland, 2019; p. 128. 63. Abu-Dieyeh, M.; Watson, A. Impact of mowing and weed control on broadleaf weed population dynamics
in turf. J. Plant Interact. 2005, 1, 239–252. [CrossRef] © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/). © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Design and Implementation of a Dual-Input Single-Output Photovoltaic Converter
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Energies
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Received: 9 June 2020; Accepted: 13 July 2020; Published: 16 July 2020 Abstract: In many solar inverters, a dc/dc converter is mainly located between the solar arrays and
the inverter. This study presents an enhanced maximum power point tracking (MPPT) algorithm for
photovoltaic (PV) systems that drives solar array voltages to track a reference value and decreases
fluctuations and oscillations in PV voltage. Different from the previously presented methods, a novel
MPPT method is proposed that ensures tracking accuracy by considering output voltage in addition
to input voltage and currents. The proposed method detects dI/dV variations, compares the output
voltage with the desired reference to shift operation mode and refreshes step size. The digital filtering,
enhanced PI, and perturb-and-observe (P&O) tracking features of the proposed MPPT method make
it robust to mitigate source fluctuations and sensitivity to partial shading based oscillations. In order
to validate the success of the proposed method, a test rig has been installed with dual boost converters. The performance improvements have been verified by both simulation and experimental results that
are compared to InCon and P&O MPPT methods. It is also confirmed by experimental results that the
proposed MPPT method provides robust control capability in terms of tracking the reference voltage
and rejecting the effects of various shading situations on solar arrays. Keywords: dc/dc power conversion; solar power generation; inverters; power control; microprocessor
applications energies
Article
Design and Implementation of a Dual-Input
Single-Output Photovoltaic Converter energies Design and Implementation of a Dual-Input
Single-Output Photovoltaic Converter Ersan Kabalci 1,*
, Yasin Kabalci 2
and Josep M. Guerrero 3,* 1
Department of Electrical and Electronics Engineering, Faculty of Engineering and Architecture,
Nevsehir Haci Bektas Veli University, Nevsehir 50300, Turkey
2
Department of Electrical and Electronics Engineering, Faculty of Engineering,
Nigde Ömer Halisdemir University, Nigde 51240, Turkey; yasinkabalci@ohu.edu.tr
3
Center for Research on Microgrids (CROM), Department of Energy Technology, Aalborg University,
9220 Aalborg, Denmark 1
Department of Electrical and Electronics Engineering, Faculty of Engineering and Architecture,
Nevsehir Haci Bektas Veli University, Nevsehir 50300, Turkey
2
Department of Electrical and Electronics Engineering, Faculty of Engineering,
Nigde Ömer Halisdemir University, Nigde 51240, Turkey; yasinkabalci@ohu.edu.tr
3
Center for Research on Microgrids (CROM), Department of Energy Technology, Aalborg University
9220 Aalborg, Denmark *
Correspondence: kabalci@nevsehir.edu.tr (E.K.); joz@et.aau.dk (J.M.G.) 1. Introduction The increased energy demand and the integration of renewable energy sources (RESs) have
brought several challenges to the existing power grid in recent decades. The grid-tie inverters are
being widely used for the integration of RES in microgrid and distributed generation applications [1–3]. The improvements of microgrids have improved the efficiency and power electronics requirements due
to a wide variety of applications in low-voltage (LV) [4]. The novel converter and inverter topologies
including non-isolated or capacitively isolated dc/dc converters and multilevel inverters have been
extensively researched [5–8]. The solar photovoltaic (PV) systems have gained increased interest
among other renewable sources up to 2013 [9], and they have reached to 177 GW total capacity all over
the world [10]. The power converters are involved to integrate PV sources to the utility grid. The dc/dc
converters, inverters and control methods used in solar inverters are reviewed in [11,12]. The most
widely used inverter type that has been used for many years in PV plants was a traditional centralized
one due to its higher power rates in a single inverter block. However, the efficiency and technological
improvements have promoted the use of string and multi-string inverters as state-of-the-art in PV www.mdpi.com/journal/energies www.mdpi.com/journal/energies Energies 2020, 13, 3679; doi:10.3390/en13143679 Energies 2020, 13, 3679 2 of 17 plants. On the other hand, the residential solar plants that have lower rated power are equipped
with micro-inverters due to their low cost and increased efficiency in maximum power point tracking
(MPPT) [11]. The most important issues in the improvement of solar inverters are related to efficiency of MPPT
algorithms, decreasing the overall cost, improving the power density regarding size and volumes, and
ensuring the isolation. The recent solar inverters are configured without galvanic isolation by removing
the bulky line frequency of high frequency transformers, but assembling with capacitive isolation at the
PV array connection. Another recent improvement in solar inverters is provided by MPPT algorithms
that are capable of coping with partial shading situations [13,14]. Figure 1 represents a set of PV inverter
topologies of centralized, string and multi-string configurations. The first topology is convenient for
connecting high number of solar arrays to a single inverter which operates as a centralized power
converter and interfaces the highest power rate among others. 1. Introduction Although the centralized inverter
is capable of converting the obtained power of solar arrays, it lacks in MPPT efficiency since the
limited number of tracking inputs are available in the inverter configuration. The second topology
seen in Figure 1 is improved as a solution to decreased MPPT efficiency of the centralized inverter
where many string inverters are used to interface solar arrays, and the number of MPPT algorithms is
increased. Thus, the limited number of controllers in a centralized inverter is tackled, and increased
MPPT controllers in a string inverter provides more successful tracking capability. The string inverters
are comprised by single-stage or two-stage where the latter is built with a boost converter that ensures
to obtain higher values of dc-bus voltage before the inverter section. The last configuration provides
an increased number of boost converters in a solar plant due to the architecture of a multi-string
inverter. The voltage level of dc-bus is adjusted by many boost converters to ensure obtaining a stable
dc voltage level. Moreover, the advantages of centralized and string inverter topologies are combined
in multi-string inverters due to increased number of MPPT controllers and robust dc-bus voltage
which enables the smooth conversion at the inverter section [12]. PV Array
dc-ac
Three Phase
dc-ac
PV String
dc-ac
PV String
Single Phase
dc-dc
dc-dc
dc-ac
Single Phase/Three Phase
Utility Grid
Centralized PV Inverter
String PV Inverter
Multi String PV Inverter
Figure 1. Photovoltaic (PV) inverter configurations. The latest researches in single-phase solar inverter topologies are focused on removing the
transformer requirement, and providing alternative isolation methods for maintaining the security
requirements [7,11,12,15]. Although the galvanic isolation was an obligation in terms of safety
regulations for many years, the recent arrangements have led to the use of capacitive isolation which
allows to design lighter inverters. The transformerless solar inverters have proved their efficiency
and reliability with several applications in many residential and industrial areas. The advantages of PV Array
dc-ac
Three Phase
dc-ac
PV String
dc-ac
PV String
Single Phase
dc-dc
dc-dc
dc-ac
Single Phase/Three Phase
Utility Grid
Centralized PV Inverter
String PV Inverter
Multi String PV Inverter
Figure 1. Photovoltaic (PV) inverter configurations. Figure 1. Photovoltaic (PV) inverter configurations. The latest researches in single-phase solar inverter topologies are focused on removing the
transformer requirement, and providing alternative isolation methods for maintaining the security
requirements [7,11,12,15]. 1. Introduction Although the galvanic isolation was an obligation in terms of safety
regulations for many years, the recent arrangements have led to the use of capacitive isolation which
allows to design lighter inverters. The transformerless solar inverters have proved their efficiency
and reliability with several applications in many residential and industrial areas. The advantages of 3 of 17 Energies 2020, 13, 3679 transformerless inverter topologies have been analyzed in [15] where many industrial inverters are
compared in terms of overall efficiency, volume, power density and size. In this paper, a robust control algorithm has been proposed for dc/dc converters in a multi-string
PV inverter that includes a dual boost converter integrating solar strings to a dc-ac converter on the
dc interface. Dual boost converters that are operated and controlled by the proposed MPPT system
regulate the dc-link voltage required for the unfolding inverter. The existing MPPT controllers in the
literature are based on feedforward control of dc converters regarding measured current and voltage
values. On the other hand, some MPPT methods operating with only feedback signals have been
proposed. The main contribution of this paper is to propose a novel MPPT method that combines
advantages of feedforward and feedback based control in a single algorithm. The proposed MPPT
algorithm is applied and is tested on the dc/dc converters of a single-phase two-stage transformerless
multi-string inverter. The simulation and experimental results confirm that the proposed MPPT control
method not only ensures the tracking of reference voltage closely but also guarantees the rejecting of
shading effects. It is also important to note that the unfolding inverter and its control stages are out of
the scope of this work since this paper is mainly focused on the implementation of MPPT control and
the dc interface of a multi-string inverter. This paper is organized as follows. In Section 2, a brief description of typical and recent
single-phase transformerless PV inverters is presented, while Section 3 provides a brief analysis
of the most widely used and recent MPPT algorithms. In addition, the design and contributions of
proposed topology compared with the existing PV inverters are explained in Section 3. The design
methodology and control strategy of the dc/dc converter is presented in Section 4. In Section 5,
the modeling and simulation analyses are shown, and experimental studies and results are given in
Section 6. Finally, conclusions are presented in Section 7. 2. Single-Phase Transformerless PV Inverters The most important outcomes of recent discussions on transformerless inverters are to increase
the overall efficiency due to removal of the transformer, to improve efficient MPPT control, and to
decrease the leakage current of this new isolation type. The increased integration of solar plants at
the LV medium have promoted the research and improvements of single-phase transformerless PV
inverters as a commercial technology. The extensive researches on single-phase solar inverters have
focused on novel inverter topologies such as highly efficient and reliable concept (HERIC), T-type,
H5 and H6 in addition to traditional H-bridge and neutral point clamp (NPC) configurations [16]. The literature survey has shown that solar inverters are configured either in single-stage or in two-stage
structure owing to their dc-bus limits and grounding requirements. The single-stage topologies are
commonly comprised of an inverting section which interfaces solar arrays and generates ac output
while the two-stage topologies are improved with a dc/dc converter at the first stage and inverter
section in the second stage. Figure 2 represents the most widely known topologies of novel solar
inverters as single or two-stage configurations [9,11,15,17–21]. Figure 2a shows a transformerless solar
inverter in single-stage configuration with a dc link capacitor (Clink) acting as the input source of the
MPPT controller [19]. The inverting stage of this topology is comprised by an asymmetrical half-bridge
network. On the other hand, an NPC topology configured in H-bridge structure is seen in Figure 2b in
which the device is titled as H-NPC topology due to configuration, and it is operated in single-stage
architecture. Another common mode of transformerless inverter topology has been given in Figure 2c
where the leakage currents are eliminated [17,21]. The MPPT controller of this topology requires
high input voltage which is supplied by a boost converter comprising the first stage of topology. Although the switching device numbers are lower than other inverters, this common-mode inverter
requires increased number of passive devices compared to other configurations. A recent application of
NPC in a two-stage PV inverter is shown in Figure 2d where a boost converter has comprised a dc/dc
converter and the grounding has been provided by the middle point of the half-bridge NPC branch [11]. Although resonant converter topologies have been proposed in this configuration, the efficiency of 4 of 17 Energies 2020, 13, 3679 the inverter has been limited and thus it is not accepted in industrial and commercial applications. 2. Single-Phase Transformerless PV Inverters The most efficient and commercially accepted two-stage HERIC and H5 topologies are presented in
Figure 2e,f, respectively. Both of these topologies are widely researched due to increased efficiency
and leading structure in managing the coupling of solar arrays with the utility grid. The H5 and
H6 topologies are commonly derived from HERIC topology by integrating fifth and sixth switching
devices on positive and negative input terminals of conventional H-bridge topology [18]. S1
S2
VLink
+
-
CLink
Lb
Linv
Rload
Cload
+
-
Vg
S3
S4
Cin
vin
+
-
ib
iinv
ig
iC
(a)
Sa1
Vdc/2
C1
Da1
1
-Vdc/2
C2
Da1
PV
Db1
1
Db1
Sa2
Sa1
1
Sa2
1
Sb1
Sb2
Sb1
1
Sb2
1
P
RS/2
RS/2
LS/2
LS/2
VS
N
0
a
b
(b)
SB
S2
CPV
L2
Lf
PV
LB
Cin
DB
L1
S1
CDC
Cf
Grid
(c)
C1
Qb
PV
Grid
Lf
Lb
Db
Cp
Cp
Q1
Q2
Q3
Q4
Filter
(d)
CPV
S1
S2
S3
S4
PV
Lf1
Grid
Lf2
S5
S6
D5
D6
Cf
(e)
Cdc
S1
S2
S3
S4
Lf1
Grid
S5
Lf2
PV
Cf
(f)
Figure 2. Single phase solar inverter configurations, (a) single-stage structure [19], (b) single-stage
H-NPC [20], (c) common-mode transformerless [17], (d) boost converter+NPC two-stage [11],
(e) two-stage highly efficient and reliable concept (HERIC) inverter [18], (f) H5 inverter [18]. Th
d
h
i i
d
d i S
i
4 i d
i
d i
i
l
h
f
l Sa1
Vdc/2
C1
Da1
1
-Vdc/2
C2
Da1
PV
Db1
1
Db1
Sa2
Sa1
1
Sa2
1
Sb1
Sb2
Sb1
1
Sb2
1
P
RS/2
RS/2
LS/2
LS/2
VS
N
0
a
b
(b) S1
S2
VLink
+
-
CLink
Lb
Linv
Rload
Cload
+
-
Vg
S3
S4
Cin
vin
+
-
ib
iinv
ig
iC
(a) (b) (a) d
C1
Qb
PV
Grid
Lf
Lb
Db
Cp
Cp
Q1
Q2
Q3
Q4
Filter
(d) SB
S2
CPV
L2
Lf
PV
LB
Cin
DB
L1
S1
CDC
Cf
Gr
(c) (c) (d) CPV
S1
S2
S3
S4
PV
Lf1
Grid
Lf2
S5
S6
D5
D6
Cf
(e)
Cdc
S1
S2
S3
S4
Lf1
Grid
S5
Lf2
PV
Cf
(f)
Figure 2. 3. Maximum Power Point Tracking (MPPT) Algorithms The deviation of input voltage and current of a solar array is detected by using an MPPT algorithm
in solar inverters. The MPPT algorithm is responsible for forcing the converter to use maximum
available power. However, the stable and unstable conditions of solar array should be closely tracked
and reacted timely. The efficiency of a solar power conversion system depends to the load variations
as well as source deviations. The unstable and changing operation circumstances should be dealt by
an MPPT algorithm for ensuring the reliable and efficient operation in a power converter. Many MPPT
methods and approaches have been proposed in the current literature [12,22,23]. The hill-climbing,
perturb-and-observe (P&O) and incremental conductance (InCon) MPPT methods have been used
for many years due to their simple and low cost structure in implementation. In addition to research
studies, lots of the commercial solar inverters also take advantage of hill-climbing and P&O MPPTs. The novel MPPT methods have been improved with a number of soft-computing methods such as
artificial neural networks (ANN), particle swarm optimization (PSO), and genetic algorithms (GA). The soft-computing methods ensure more precise calculations, but they lack in simplicity and low-cost
structure due to high-performance processor requirements [12,22]. The increased mathematical
calculation requirement of soft-computing methods decreases the response time while the cost of the
processor is increased due to performance conditions. The capacitor control, dP/dV and dP/dI MPPT
algorithms are improved with feedback signal based control [16,22,24–26]. The MPPT algorithms are expected to deal with decreased efficiency of the solar plant by detecting
the maximum power point. However, algorithms are not mostly able to detect the difference in local
MPP (LMPP) and global MPP (GMPP) under partial shading circumstances. Therefore, traditional
MPPT methods are not capable to efficiently operate in partial shading situations [25,26]. The proposed control algorithm in this study is based on the adoption of the conventional
P&O algorithm and output voltage feedback. In other words, the configurations of feedforward
and feedback type controllers are combined in terms of hardware, and a featured control algorithm
has been implemented with low-cost requirements. The proposed MPPT method is improved by
considering the output voltage of dc/dc converter that is drastically decreased under partial shading
conditions. Furthermore, the proposed MPPT method integrates an improved P&O algorithm with a
PI controller for voltage feedback. 2. Single-Phase Transformerless PV Inverters Single phase solar inverter configurations, (a) single-stage structure [19], (b) single-stage
H-NPC [20], (c) common-mode transformerless [17], (d) boost converter+NPC two-stage [11],
(e) two-stage highly efficient and reliable concept (HERIC) inverter [18], (f) H5 inverter [18]. Cdc
S1
S2
S3
S4
Lf1
Grid
S5
Lf2
PV
Cf
(f) CPV
S1
S2
S3
S4
PV
Lf1
Grid
Lf2
S5
S6
D5
D6
Cf
(e) (f) (e) Figure 2. Single phase solar inverter configurations, (a) single-stage structure [19], (b) single-stage
H-NPC [20], (c) common-mode transformerless [17], (d) boost converter+NPC two-stage [11],
(e) two-stage highly efficient and reliable concept (HERIC) inverter [18], (f) H5 inverter [18]. The proposed converter that is introduced in Section 4 is designed in a single-phase transformerless
two-stage configuration. The dc/dc conversion stage has been comprised of boost converters to achieve a
multi-string topology. On the other hand, the dc-ac conversion has been performed by single-phase
H-bridge topology to decrease the passive components and switching losses while it increases the 5 of 17 Energies 2020, 13, 3679 inverter efficiency. A brief description of MPPT methods that are state-of-the-art is presented in the
following section. Thus, it will facilitate expressing our contribution to a controlling strategy by
comparing existing literature. 4. Design and Control Strategy of The Converter The presented converter is composed of a two-stage structure where boost dc/dc converters
comprise the first stage while the second stage is a fundamental single-phase H-bridge inverter. The converters are shunt connected to enclose the point of common coupling (PCC) at dc-bus, as shown
in Figure 3. The solar modules cause parasitic capacitance at their positive and negative terminals
due to its grounded frame. In addition to this, the potential difference occurred during switching
transitions of converter injects leakage current on dc and ac side along the solar inverter. The leakage
current increases the electromagnetic interferences (EMI), harmonic rates, grid distortions and distorts
the safety. One of the most common solutions is to connect a parasitic capacitance between positive
and negative terminals of solar modules and ground in transformerless topologies. The value of
capacitance is suggested to be around 50–220 nF/kW in a single-capacitor model [20,27,28]. Therefore,
each positive and negative terminal of solar arrays are equipped with 1 µF film capacitors, as shown in
dashed lines in Figure 3. 6 of 17 Energies 2020, 13, 3679 Figure 3. Block diagram of proposed two-stage PV inverter and control system. Figure 3. Block diagram of proposed two-stage PV inverter and control system. An STM32F407 microprocessor that is one from the 32-bit ARM family manages the control
operations. Each boost converter includes a hall-effect current sensor to track PV currents while resistor
networks perform the voltage sensing of capacitors at the input and output of the converter. The sensed
values are supplied to analog-digital converter (ADC) ports of the microprocessor by the signal
conditioning circuits that are composed of isolation amplifiers. In other words, each boost converter
provides three measurement signals to the microprocessor that are PV current, input capacitor voltage,
and output capacitor voltage. Although the signal conditioning circuits include passive low-pass
filters, the acquired signals are filtered by a software-based finite impulse response (FIR) filter in the
microprocessor. Once the inherited digital signals are filtered, input measurements are transferred to
the P&O section, and output capacitor measurement is applied to the PI controller as illustrated in the
block diagram. The control algorithm is dependent on three input signals obtained from a hall-effect
sensor for current and resistor networks for voltages. Therefore, the additional circuit required for
the control algorithm is achieved with a low-cost design. 4. Design and Control Strategy of The Converter The design studies are presented in two
sections, one is on data acquisition and signal conditioning networks, and the other one is on the
control algorithm. 4.1. Design Criteria of Proposed Control Method The control algorithm requires a data acquisition interface transmitting the measured signals to
the microprocessor. The measurement points at the boost converter, as shown in Figure 4a, inherit
the input parameters that are involved in the control algorithm, and the signal conditioning circuit is
designed as presented in Figure 4b. The input capacitor CI1 is responsible for mitigating the switching
ripples that cause fluctuations in inductor current IL1 and ensures achieving an average current flow
along with the inductor. The input capacitor and inductor share the current provided by solar array
that is shown as iPV1 with respect to the Kirchoff’s current law as follows; (1) iPV1 = iCI1 + iL1
(1) The driving signal α1 of semiconductors is the function of the error signal. Therefore, the reference
current iDC_re f provided by the current controller in the MPPT block is as, The driving signal α1 of semiconductors is the function of the error signal. Therefore, the reference
current iDC_re f provided by the current controller in the MPPT block is as, iDC_re f = iPV1 + i∗
d
(2) (2) where iPV1 is the instant current of solar array and i∗
d is the deviation value of current from the reference
of the controller. In this case, Equation (2) is revised by taking into consideration the current of parasitic 7 of 17 Energies 2020, 13, 3679 Energies 2020, 13, 3679 capacitance C1; the relation between input current, parasitic capacitance current iC1, and reference
current of controller i∗
d is used to determine the feedforward error rate by using Equations (1) and (2), (3) iDC_re f = iC1 + iPV1 + i∗
d
(3)
eIdc = iC1 + iPV1 + i∗
d −iPV1
(4)
eIdc = iC1 + i∗
d
(5) (4) (5) which yields an error of zero when iDC_re f equals to iPV1 and thus iC1 = −i∗
d. The capacitor current
iPV1 will be zero in steady-state operation. The parasitic capacitance current iC1 will be zero in
steady-state operation. which yields an error of zero when iDC_re f equals to iPV1 and thus iC1 = −i∗
d. The capacitor current
iPV1 will be zero in steady-state operation. The parasitic capacitance current iC1 will be zero in
steady-state operation. 4.1. Design Criteria of Proposed Control Method PV
Q1
L1
D1
CI1
CO1
α1
VPV1
iPV1
VO1
id
*
Carrier
>
x
Zero Order
Hold
Delay
Zero Order
Hold
PI P&O MPPT
Controller
Voltage
Limiter
e*
v
i
p
k
k + s
PI
+
-
Switch
Vref
*
Vdc
+
-
ev
el
-el
Delay
+
-
Zero
Order
Hold
D1
GND
Vc
Vout Vref
Outputs
VCC
+
-
VCC
R1
R2
3V3
VCI1+
C1
iPV1
R11
R12
(a)
PV
PV
Q1
L1
D1
CI1
CO1
α1
VPV1
iPV1
VO1
id
*
Carrier
>
x
Zero Order
Hold
Delay
Zero Order
Hold
PI P&O MPPT
Controller
Voltage
Limiter
e*
v
i
p
k
k + s
PI
+
-
Switch
Vref
*
Vdc
+
-
ev
el
-el
Delay
+
-
Zero
Order
Hold
D1
CASR 25
GND
Vc
Vout Vref
10
9
8
Outputs
1
2
3
Inputs
VPV1+
VCC
+
-
VCC
R1
R2
10 kΩ
RV1
3V3
R3
R4
D2
+
-
R5
R8
VCI1+
C1
R10
R9
iPV1
VPV1-
VCI1-
R6
R7
3V3
VCC
VO1+
R11
R12
D2
+
-
R13
R14
R18
R17
VO1-
R15
R16
3V3
VCC
VPV1
VO1
(b)
Figure 4. Block diagram of proposed converter and control system, (a) boost converter with control
system, (b) signal conditioning interface along converter and microprocessor. 4.1. Design Criteria of Proposed Control Method PV
Q1
L1
D1
CI1
CO1
α1
VPV1
iPV1
VO1
id
*
Carrier
>
x
Zero Order
Hold
Delay
Zero Order
Hold
PI P&O MPPT
Controller
Voltage
Limiter
e*
v
i
p
k
k + s
PI
+
-
Switch
Vref
*
Vdc
+
-
ev
el
-el
Delay
+
-
Zero
Order
Hold
(a)
PV
PV
Q1
L1
D1
CI1
CO1
α1
VPV1
iPV1
VO1
id
*
Carrier
>
x
Zero Order
Hold
Delay
Zero Order
Hold
PI P&O MPPT
Controller
Voltage
Limiter
e*
v
i
p
k
k + s
PI
+
-
Switch
Vref
*
Vdc
+
-
ev
el
-el
Delay
+
-
Zero
Order
Hold Zero Order
Hold Zero Order
Hold
Delay (a) D1
VCC
+
-
VCC
R1
VCI1+
C1
iPV1
R11
R12
D1
CASR 25
GND
Vc
Vout Vref
10
9
8
Outputs
1
2
3
Inputs
VPV1+
VCC
+
-
VCC
R1
R2
10 kΩ
RV1
3V3
R3
R4
D2
+
-
R5
R8
VCI1+
C1
R10
R9
iPV1
VPV1-
VCI1-
R6
R7
3V3
VCC
VO1+
R11
R12
D2
+
-
R13
R14
R18
R17
VO1-
R15
R16
3V3
VCC
VPV1
VO1
(b)
Figure 4. Block diagram of proposed converter and control system, (a) boost converter with control
system, (b) signal conditioning interface along converter and microprocessor. VCC
R1
VCI1+
C1
D2
+
-
R5
R8
R10
R9
VCI1
R6
R7
3V3
VCC
VPV1
(b) (b) D1
GND
V
V
t V
f
O
+
-
3V3
iPV1
R1
Figure 4. Block diagram of proposed converter and control system, (a) boost converter with control
system, (b) signal conditioning interface along converter and microprocessor. Energies 2020, 13, 3679 8 of 17 On the other hand, the second control stage of the MPPT controller is based on a PI compensator
that is improved regarding to output voltage. The instant value of output voltage is achieved from
the nodes of the output capacitor that is CO1 for the first boost converter. The transfer function of the
output PI compensator is presented in Equation (6), the voltage error that is used as the reference
for the compensation process is given in Equation (7), and the transfer function of the outer loop in
Equation (8) as follows in s-domain. 4.1. Design Criteria of Proposed Control Method GPI = kP + kI
s
(6)
eV(s) = Vre f (s) −VO1(s)
(7)
T(s) = VPV1(s)
Vre f (s) =
kPs + kI
CO1s2 + kPs + kI
(8) (6) (7) (8) where the transfer function given in Equation (8) is derived by considering the settling time tS
and minimum switching time Ts_min as given in Equations (9) and (10) for kP and kI parameters,
respectively [24,29]. kP = 2CO1ζωn
(9)
kI = CO1ω2
n
(10) (9) (10) where the angular frequency is dependent on the time constant as ζωn = 1/τ. By selecting the
damping ratio ζ as 0.707, switching frequency at 25 kHz, and output capacitor as 1000 µF, the natural
frequency ωn is calculated as 5657.7 rad/s. The kP and kI values are obtained as 7.99 and 32, 009.56 by
employing Equations (9) and (10), respectively. The outer compensator loop is arranged according to
calculated values and additional tuning is performed to decrease the error band during experimental
studies. The voltage reference of solar array is tracked in the PI P&O MPPT controller, and the switch
is triggered to select the P&O algorithm or PI compensator regarding the MPP operation. 4.2. Design Criteria of MPPT Controller Figure 5 shows the flowchart of the proposed PI P&O MPPT algorithm where the main program
consists of two subroutines as PI and P&O functions. The initial step sets counters and registers instant
power, instant voltage, instant current, and MPPT power to zero in Step 1. In the next step, the counter
is increased by one and then MPPT calculations get started in Step 3. The voltage and current of PV,
and the output voltage with the V(n) parameter are acquired, and PV power is calculated in Step 4. After storing the values, the algorithm compares the actual voltage and current with previously stored
values for detecting power fluctuations. The measured PV power is compared with previous global MPP,
and change in GMPP is detected in Step 5. The measured output voltage is compared with a reference
boundary in Step 6 where the program jumps to the P&O function if the achieved voltage is not higher or
lower than 90% or 110% of the desired output voltage. This operation is accomplished by the multiport
switch seen in the orange block of Figure 4a. The switch is driven by the middle port connection which
triggers the main program for calling PI or P&O subroutines. Once the achieved output voltage gets out
of the boundary band, then the program calls the PI control subroutine starting from Step 7. Once the PI compensation subroutine is called, the error voltage that implies the difference
between reference and instant voltages is calculated in Step 7.1. In the case in which the error is
positive according to the calculation in Step 7.2, it is detected that the output voltage is lower than the
reference. Therefore, the duty cycle is increased in Step 7.4 to increase the output voltage. When the
output voltage of the converter is higher than the reference, the comparison in Step 7.2 results negative
and the duty cycle is decreased as denoted in Step 7.3. The reference voltage tracking operation is
handled when the desired output voltage gets out of the boundary. Otherwise, the algorithm sustains
the operation in the lower section of Figure 5 where the P&O MPPT is operated. The conventional 9 of 17 Energies 2020, 13, 3679 P&O maintains operation at GMPP point until partial shading occurs at any time. The GMPP and MPP
points were detected until Step 8.1, and the algorithm uses stored data to operate Step 8.2. 4.2. Design Criteria of MPPT Controller When a
partial shading occurs, the ∆V value in Step 4 changes and the main program operates the P&O
function again to track the most recent MPP. The changes on ∆P are checked in Step 8.2 and the
comparison results define the ∆V operation to select at Step 8.3 or Step 8.4. The required change on
duty cycle is performed according to comparisons and the algorithm detects the recent GMPP to track
starting from Step 2. There are several MPPT algorithms that have been proposed to perform similar
operations with much more complex calculations and increased comparisons [29–33]. However, it is
important to note that the proposed PI P&O flowchart provides refined algorithms and performs rapid
comparisons when compared to others. 1. Initialize
n=PMPPT=PPV(t)=VPV(t)=IPV(t)=0
2. Step counter n=n+1
3. Acquire and Calculate
VPV(t), IPV(t), PPV(t)=VPV(t)*IPV(t),
4. Calculate power&voltage change
ΔP=P(n+1)-P(n), ΔV=V(n+1)-V(n)
5. Calculate GMPP
ΔPGMPP=P(n)-PGMPP
6. Vref
Comparison
0.9*V(n)˂Vref˂1.1*V(n)
7.1. Calculate error for PI
eV_ref=Vref -VO
7.2
eV_ref ˃ 0
Y
N
N
8.2. P&O
ΔP ≥ 0
8.1. Call the P&O Function
Y
8.3. ΔV ˃ 0
Y
N
8.4. ΔV ≥ 0
Y
N
Y
N
8.5. Increase duty
D(n+1)=D(n)+c
8.7. Decrease duty
D(n+1)=D(n)−c
8.6. Decrease duty
D(n+1)=D(n)-c
8.8. Increase duty
D(n+1)=D(n)+c
7.3. Decrease duty
D(n+1)=D(n)−c
7.4. Increase duty
D(n+1)=D(n)+c
Figure 5. Flowchart of the proposed PI perturb-and-observe (P&O) maximum power point tracking
(MPPT) algorithm. 8.4. ΔV ≥ 0 Figure 5. Flowchart of the proposed PI perturb-and-observe (P&O) maximum power point tracking
(MPPT) algorithm. 5. Modeling Studies Several Matlab/Simulink modeling studies have been performed in order to evaluate the
performance of the proposed PI P&O MPPT algorithm and to compare it with widely used algorithms. Therefore, a PV energy conversion system has been designed considering the block diagram given
in Figure 3. Each dc/dc converter is supplied by parallel-connected solar arrays that are composed
of four series-connected PV modules at each array. Thus, eight PV modules comprise a PV string
connected to a dc/dc converter in Figure 3. The PV modules are modeled referring to a commercial one
with 54.7 V maximum power voltage and 5.58 A maximum power current [34]. Two dc/dc converters
operating in boost mode have been designed to interface the modeled PV plant. The design criteria of
converters have been carried out regarding the schematic diagram presented in Figure 4a that the input 10 of 17 Energies 2020, 13, 3679 and output capacitors are set as CI1 = 100 µF and CO1 = 1000 µF while the inductor is L1 = 1.2 mH. The switching frequency, steady-state current ripple, and voltage ripple values have been calculated by
using the following equations where the instant duty cycle D(t) and switching frequency are obtained
with Equations (11) and (12), respectively [29]; D(t) = 1 −
VPV
VCO1 + ∆VCO1(t)
(11)
fS(t) = VPV · D(t)
∆iL · L1
(12) (11) (12) The VPV denotes PV voltage as a supply of boost converter, while VCO is output capacitor voltage,
and ∆VCO (t) is capacitor voltage ripple. The PV voltage is assumed as VPV = 218.8 V due to solar array,
VCO = 500 V and ∆VCO (t) = 50 V that yields D (t) = 0.6. The switching frequency fS(t) oscillates
around nominal duty cycle DN(t) and nominal switching frequency fSN(t) is calculated considering
Equation (12) where the inductor current ripple is limited to ∆iL = 4 A. Under these assumptions,
fS(t) = 27.35 kHz is achieved. On the other hand, the nominal values of the duty cycle and switching
frequency at steady-state operation have been calculated by using Equations (13) and (14), respectively. DN = 1 −VPV
VCO1
(13)
fSN = VPV · DN
∆iL · L1
(14) (13) (14) that yield DN = 0.56 and fSN = 25.526 kHz. In order to put into evidence, the MPP tracking
performance of the proposed method has been compared with widely used P&O and InCon MPPT
methods. 5. Modeling Studies The modeled power converter system has been designed with the aforementioned values for
passive devices and each of the MPPT algorithms has been tested by using identical Matlab/Simulink
models. The irradiations G1 and G2 applied to each solar array have been arbitrarily varied to
analyze partial shading conditions. The irradiations and variations are presented in Table 1. Figure 6
illustrates the simulation results of modeled MPPT controllers where the converter parameters and
irradiation values have been applied as aforementioned. The array powers, converter and dc bus
voltages, and duty cycles corresponding to presented G1 and G2 values have been measured for InCon
(Figure 6a), P&O (Figure 6b) and proposed PI P&O MPPT (Figure 6c) algorithms. The first axis of each
figure shows the supplied power of first and second array, P1 and P2, respectively. The output voltages
of converters and dc bus have been indicated with VO1, VO2, and Vdc_bus titles in the second axis. The duty cycle ratios and variations have been illustrated in the third axis as D1 and D2. The output
power of solar arrays has been rapidly forced by irradiation when the converters are driven by InCon
and P&O MPPT methods. It is exhibited that the power losses of the InCon controlled converter are
higher than the P&O controlled converter. Therefore, the output voltage levels of VO1 and VO2 that are
expected to be equal deviate due to variation of duty cycle ratios generated by the InCon and P&O
MPPT controller. On the other hand, the output voltage levels of the P&O algorithm are more robust
against irradiation changes while its oscillations are much more visible. Table 1. Irradiation changes and intervals. Interval (s) [t1 −t2]
Irradiation (W/m2) (G1)
Interval (s) [t1 −t2]
Irradiation (W/m2) (G2)
[0 −1.35]
1000
[0 −1.3]
945
[1.35 −3.2]
[649 −600]
[1.3 −2.3]
670
[3.2 −4.5]
[695 −810]
[2.3 −3.55]
[945 −810]
[4.5 −5.85]
950
[3.55 −4.85]
735
[5.85 −7.3]
890
[4.85 −6.45]
915
[7.3 −8.55]
790
[6.45 −8.7]
980
[8.55 −10]
[935 −975]
[8.7 −10]
905 Table 1. Irradiation changes and intervals. Table 1. Irradiation changes and intervals. 11 of 17 Energies 2020, 13, 3679 The presented figures show that the highest voltage of any converter supplies the output voltage
where voltage oscillation ∆V reaches up to 80 V in the second interval covering [1.3 −3.2] seconds
of simulation. 5. Modeling Studies The proposed MPPT controller satisfactorily tracks the irradiation changes and shifts
the operation mode between the P&O and PI controller in the case of ∆V exceeding the predefined
hysteresis band. This operation mode control is shown in the presented simulation results of Figure 6c
that the output voltage of the dc bus is limited around the 5 V hysteresis band. The detailed view of
output voltage fluctuations and power rates has been presented in Figure 6d. The output power of
solar arrays is measured around 2.21 and 2.35 kW at various operation intervals where the rated total
power of solar arrays is 4.8 kW. Once the operation conditions and prototype optimizations have been
carried out in simulation studies, the proposed MPPT method has been experimentally investigated. The test rig has been installed referring to simulation parameters and converters have been configured
with calculated values. (a)
(b)
(c)
(d)
Figure 6. Simulation results of modeled power converter and MPPT methods, (a) Incremental
Conductance MPPT, (b) P&O MPPT, (c) Proposed PI P&O MPPT, (d) detailed ∆P and ∆VO measurements
for proposed method. (a)
(b) (a) (b) (a) (b) (c) (d) (d) (c) Figure 6. Simulation results of modeled power converter and MPPT methods, (a) Incremental
Conductance MPPT, (b) P&O MPPT, (c) Proposed PI P&O MPPT, (d) detailed ∆P and ∆VO measurements
for proposed method. 6. Experimental Results The MPPT algorithm proposed in this study has been experimentally tested and the success of
the proposed algorithm has been compared with regular PI, InCon MPPT, and P&O MPPT algorithms. To verify the validity of the proposed algorithm, several partial shading conditions and varying
irradiations have been applied by using a solar array simulator (Chroma 62050H-600S SAS) and
programmable dc power supply as the second array input. The dual dc/dc converter has been 12 of 17 Energies 2020, 13, 3679 implemented with the FBA75BA50 dual MOSFET module at 5 kW rated power. The operation
conditions and device parameters used in the dc/dc converter test rig have been shown in Table 2. The current measurements have been realized by using hall-effect current transducers (CASR25-NP)
that provide strict dv/dt immunity while the voltage measurements have been performed by resistor
networks with low cost. The fast recovery diodes (FREDs) used in the dc/dc converters are DSEI
60–10A diodes with 35 ns reverse recovery time trr. The accuracy and reliability of implemented
measurement sections including hall-effect transducers and resistor networks have been tested
in a wide operation range, and they have been integrated to dc converters after verifications. The implemented current and voltage measurement sections provide 98.33% accuracy for current
and 99.85% for voltage measurement [1]. The achieved high accurate signals have been applied
to ADC ports of the microprocessor where an FIR filter to ensure pure measurement signals have
been detected to track if MPP initially filters the measurement signals. The sampling time ts of a
microprocessor that has 168 MHz core speed has been set to 1 µs and sampling times of PI and
P&O algorithms tS_MPP has been set to 10 ms. The dual converter structure has been loaded with
R-L loads in order to carry out several power rates. The implemented test rig has been shown in
Figure 7 where the SAS interface of solar array configuration and shading analysis screen is depicted
in Figure 7a, Chroma 62050H-600S SAS device in Figure 7b, dual boost converter setup in Figure 7c
and measurement setup in Figure 7d. The partial shading analyses and solar array test have been
performed by configuring SAS as predefined conditions as presented in simulation studies. Table 2. Components and values of dual boost converter. Table 2. Components and values of dual boost converter. 6. Experimental Results Parameter
Value
Input voltage range
150–500 V
Rated power (P)
5 kW
Switching frequency ( fSN)
30 kHz
Input and Output DC capacitors (CI1, CI2, CO1, CO2)
2200 µF/250 V
Inductor
1.2 mH/20 A
Power switches (FBA75BA50)
MOSFET (500 V/75 A)
Fast recovery diodes (FREDs)
DSEI 60-10A (60 A–1000 V)
Current sensors (Hall effect)
CASR-25
Microprocessor
STM32F407-VGT ARM
Solar array simulator
Chroma 62050H-600S The experimental study results have been shown in Figures 8 and 9 where each scope screen
depicts 375 s of measurements (time/div = 25 s in Figure 8b, time/div = 50 s in Figure 8a,c). The second
channel of scope (CH2, 50 V/div) presents the first solar array input, third channel of scope (CH3,
50 V/div) presents the third solar array input, and fourth channel (CH4, 100 V/div) denotes the dc
bus voltage of the converter. Figure 8 shows experimental results of the dc converter that is controlled
by the PI controller, InCon MPPT, and P&O MPPT, respectively, in Figure 8a–c. The dc input voltages have been applied as a dual solar array as seen in Figure 8a. Although
the PI controller provides stable operation during a single supply, it was not capable of tracking the
desired reference voltage (Vre f = 200 V) when both of the solar arrays were operated simultaneously
or at different times. The overshoots or dips of output voltage have been extended up to 40% of the
reference, as depicted in Figure 8a. On the other hand, the settling time takes around 40s to stabilize
the desired output when one of the sources is stopped to provide supply (Vdc2 = 0 V). The step time
(tS_MPP = 10 ms) causes fluctuations on dc supply while InCon algorithm searches and tracks the
MPP as seen in Figure 8b. The reference error is measured around 10% to 20% according to single or
dual supply conditions. On the other hand, the output voltage oscillations reach up to 120 V during
InCon MPPT control. Another comparison of the proposed algorithm has been carried out with regular
P&O MPPT where Figure 8c shows the experimental results. In the case in which both inputs are
supplied, the oscillations on the second input have been decreased as seen in Figure 8c, where the 13 of 17 13 of 17 Energies 2020, 13, 3679 output voltage fluctuations sustain and the obtained output cannot track the desired reference voltage
satisfactorily. 6. Experimental Results Although the response time of P&O MPPT is lower than the InCon algorithm, it takes
around 5 s to track the input voltage. Moreover, the reference error of the P&O algorithm has been
measured as 5% referring to experimental results seen in Figure 8c. (a)
(b)
(c)
(d)
Figure 7. Hardware and setup used in experimental studies, (a) solar array simulator (SAS)
interface for partial shading configuration, (b) Chroma 62050H-600S SAS, (c) dual boost converter,
(d) measurement system. (b) (a) (b) (a) (c) (c)
(d)
Figure 7. Hardware and setup used in experimental studies, (a) solar array simulator (SAS)
interface for partial shading configuration, (b) Chroma 62050H-600S SAS, (c) dual boost converter,
(d) measurement system. (d) (c) (d) Figure 7. Hardware and setup used in experimental studies, (a) solar array simulator (SAS)
interface for partial shading configuration, (b) Chroma 62050H-600S SAS, (c) dual boost converter,
(d) measurement system. The performance of the proposed MPPT algorithm has been tested and the experimental results
have been given in Figure 9a,b under various operation conditions. In the first step shown in Figure 9a,
a single supply has been applied for a short time and then the second supply has been integrated
into the system. While a significant spike on output voltage has been observed in the previous three
control methods, the proposed MPPT algorithm compensates for the connection and disconnections
of any solar array at any time. The disconnection of the second solar array has been investigated
at the 270th second where the second supply is set to Vdc2 = 0 V. The first solar array has been
configured to operate under partial shading conditions while the second source has been connected
and disconnected to the system at stable irradiation. The proposed MPPT algorithm has provided
successful tracking performance regarding reference value (Vre f = 200 V) under highly and rapidly
changing supply voltages. The reference error of the proposed method has been detected lower than
1%. In the next analysis shown in Figure 9b, the sources have been configured in order to operate
under variously shading conditions. The first solar array has provided rapidly changing irradiations
while the second one has provided relatively lower changes. The voltage level of the first source has 14 of 17 Energies 2020, 13, 3679 been changed between 50 V and 190 V, and sudden increment and decrements have been applied to
the dc converter. 6. Experimental Results (a)
(b)
(c)
Figure 8. Experimental results of MISO converter with different control methods, (a) PI, (b) InCon
MPPT, (c) P&O MPPT. (a)
(b) (a) (b) (a) (b) (c) (c) Figure 8. Experimental results of MISO converter with different control methods, (a) PI, (b) InCon
MPPT, (c) P&O MPPT. (b) (a) (a)
(b)
Figure 9. Experimental results for PI P&O MPPT controlled converter (Vre f = 200 V) under rapidly
changing irradiations and partial shading. (a) Figure 9. Experimental results for PI P&O MPPT controlled converter (Vre f = 200 V) under rapidly
changing irradiations and partial shading. The experimental test results clearly verified that the proposed MPPT control algorithm
successfully tracks the desired output reference. Even under the worst operation conditions, the error
rate on tracking the reference is below 5%, and the algorithm compensates this error lower than 2 s. Eventually, it is also confirmed by experimental studies in addition to simulation studies that the
proposed MPPT algorithm is more robust against rapidly or slightly changing irradiations and more
successful on tracking the reference values. Energies 2020, 13, 3679 15 of 17 7. Conclusions A two-stage MPPT algorithm is proposed for solar inverters in this study. The main contribution
of the proposed method is to track voltage and current perturbation at the input and govern the output
voltage at the dc bus. The enhanced MPPT algorithm, namely PI P&O MPPT, detects the oscillations
that occurred on solar array power during partial shading and reacts to maintain providing stable dc
voltage at the point of common coupling of dual dc/dc converters. Initially, the mathematical and
simulation modeling of the proposed MPPT algorithm has been realized. After the modifications
and implementations of the proposed MPPT method have been verified by simulation studies, the
experimental results are achieved by using an implemented test rig of a dual dc/dc boost converter
in which each of converters has been controlled with dedicated MPPT controllers. The STM32F407
microprocessor has been utilized to manage control operations and MPPT algorithms of converters. The proposed MPPT algorithm has been compared with conventional P&O and InCon methods. As the simulation and experimental results verify, the PI P&O algorithm decreases oscillations and
fluctuations on solar array voltages, rejects the sudden or slight partial shading effects on input
voltages, provides more resilient dc bus voltage with very low reference error, and rapidly reacts
to source variations with the faster response time. It is also confirmed by experimental results that
the deviation between the reference voltage and actual output voltage is lower than 5% at the worst
operation conditions, and it provides the fastest tracking speed among other methods compared. hor Contributions: All authors are involved equally in developing the full research manuscript for its fina
entation. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by TUBITAK under grant number 7141079. This work was supported
by VILLUM FONDEN under the VILLUM Investigator Grant (no. 25920): Center for Research on Microgrids
(CROM); www.crom.et.aau.dk. References 1. Kabalci, E.; Kabalci, Y. A wireless metering and monitoring system for solar string inverters. Int. J. Electr. Power Energy Syst. 2018, 96, 282–295, doi:10.1016/j.ijepes.2017.10.013. [CrossRef] 2. Kabalci, Y.; Kabalci, E. Modeling and analysis of a smart grid monitoring system for renewable energy
sources. Solar Energy 2017, 153, 262–275, doi:10.1016/j.solener.2017.05.063. [CrossRef] 3. Wu, W.; Ji, J.; Blaabjerg, F. Aalborg Inverter—A New Type of “Buck in Buck, Boost in Boost” Grid-Tied
Inverter. IEEE Trans. Power Electron. 2015, 30, 4784–4793, doi:10.1109/TPEL.2014.2363566. [CrossRef] 4. Schweizer, M.; Kolar, J.W. Design and Implementation of a Highly Efficient Three-Level T-Type Converter
for Low-Voltage Applications. IEEE Trans. Power Electron. 2013, 28, 899–907, doi:10.1109/TPEL.2012.2203151. [CrossRef] 5. Wang, C.; Yang, L.; Wang, Y.; Meng, Z.; Li, W.; Han, F. Circuit Configuration and Control of a Grid-Tie
Small-Scale Wind Generation System for Expanded Wind Speed Range. IEEE Trans. Power Electron. 2017,
32, 5227–5247, doi:10.1109/TPEL.2016.2608909. [CrossRef] 6. Gnanasambandam, K.; Edpuganti, A.; Rathore, A.K.; Srinivasan, D. Modified Synchronous Pulsewidth
Modulation of Current-Fed Five-Level Inverter for Solar Integration. IEEE Trans. Power Electron. 2017,
32, 3370–3381, doi:10.1109/TPEL.2016.2585584. [CrossRef] 7. Dutta, S.; Debnath, D.; Chatterjee, K. A Grid-Connected Single-Phase Transformerless Inverter Controlling
Two Solar PV Arrays Operating Under Different Atmospheric Conditions. IEEE Trans. Ind. Electron. 2018,
65, 374–385, doi:10.1109/TIE.2017.2711577. [CrossRef] 8. Fuentes, C.D.; Rojas, C.A.; Renaudineau, H.; Kouro, S.; Perez, M.A.; Meynard, T. Experimental Validation of
a Single DC Bus Cascaded H-Bridge Multilevel Inverter for Multistring Photovoltaic Systems. IEEE Trans. Ind. Electron. 2017, 64, 930–934, doi:10.1109/TIE.2016.2619661. [CrossRef] 9. Sasidharan, N.; Singh, J.G. A Novel Single-Stage Single-Phase Reconfigurable Inverter Topology for a Solar
Powered Hybrid AC/DC Home. IEEE Trans. Ind. Electron. 2017, 64, 2820–2828, doi:10.1109/TIE.2016.2643602. [CrossRef] 10. Anthon, A.; Zhang, Z.; Andersen, M.A.E.; Holmes, D.G.; McGrath, B.; Teixeira, C.A. The Benefits of SiC
mosfets in a T-Type Inverter for Grid-Tie Applications. IEEE Trans. Power Electron. 2017, 32, 2808–2821,
doi:10.1109/TPEL.2016.2582344. [CrossRef] 11. Meneses, D.; Blaabjerg, F.; García, Ó.; Cobos, J.A. Review and Comparison of Step-Up Transformerless
Topologies for Photovoltaic AC-Module Application. IEEE Trans. Power Electron. 2013, 28, 2649–2663,
doi:10.1109/TPEL.2012.2227820. [CrossRef] 12. Romero-Cadaval, E.; Spagnuolo, G.; Franquelo, L.G.; Ramos-Paja, C.A.; Suntio, T.; Xiao, W.M. Grid-Connected Photovoltaic Generation Plants: Components and Operation. IEEE Ind. Electron. Mag. 2013,
7, 6–20, doi:10.1109/MIE.2013.2264540. [CrossRef] 13. El-Dein, M.Z.S.; Kazerani, M.; Salama, M.M.A. An Optimal Total Cross Tied Interconnection for
Reducing Mismatch Losses in Photovoltaic Arrays. IEEE Trans. Sustain. Energy 2013, 4, 99–107,
doi:10.1109/TSTE.2012.2202325. [CrossRef] 14. El-Dein, M.Z.S.; Kazerani, M.; Salama, M.M.A. Abbreviations The following abbreviations are used in this manuscript: Clink
dc link capacitor
CI1
input capacitor
fSN
nominal switching frequency
iC1
parasitic capacitance current
i∗
d
deviation value of current
iDC_re f
reference current
iL1
inductor current
iPV1
instant current of solar array
tS
settling time
Ts_min
minimum switching time
ζ
damping ratio
ωn
natural frequency
GMPP
Global MPP
HERIC
Highly Efficient and Reliable Concept
InCon
Incremental Conductance
LMPP
Local MPP
MPPT
Maximum power point tracking
NPC
Neutral Point Clamp
P&O
Perturb-and-observe 16 of 17 Energies 2020, 13, 3679 References Optimal Photovoltaic Array Reconfiguration to Reduce Partial
Shading Losses. IEEE Trans. Sustain. Energy 2013, 4, 145–153, doi:10.1109/TSTE.2012.2208128. [CrossRef] 15. Kerekes, T.; Teodorescu, R.; Rodríguez, P.; Vázquez, G.; Aldabas, E. A New High-Efficiency
Single-Phase Transformerless PV Inverter Topology. IEEE Trans. Ind. Electron. 2011, 58, 184–191,
doi:10.1109/TIE.2009.2024092. [CrossRef] 16. Kabalcı, E. Review on novel single-phase grid-connected solar inverters: Circuits and control methods. Solar Energy 2020, 198, 247–274. [CrossRef] 17. Azary, M.T.; Sabahi, M.; Babaei, E.; Meinagh, F.A.A. Modified Single-Phase Single-Stage Grid-Tied Flying
Inductor Inverter With MPPT and Suppressed Leakage Current. IEEE Trans. Ind. Electron. 2018, 65, 221–231,
doi:10.1109/TIE.2017.2719610. [CrossRef] 18. Chen, B.; Gu, B.; Zhang, L.; Zahid, Z.U.; Lai, J.; Liao, Z.; Hao, R. A High-Efficiency MOSFET Transformerless
Inverter for Nonisolated Microinverter Applications. IEEE Trans. Power Electron. 2015, 30, 3610–3622,
doi:10.1109/TPEL.2014.2339320. [CrossRef] 17 of 17 17 of 17 Energies 2020, 13, 3679 19. Xia, Y.; Roy, J.; Ayyanar, R. A Capacitance-Minimized, Doubly Grounded Transformer less Photovoltaic
Inverter With Inherent Active-Power Decoupling. IEEE Trans. Power Electron. 2017, 32, 5188–5201,
doi:10.1109/TPEL.2016.2606344. [CrossRef] 20. Rojas, C.A.; Aguirre, M.; Kouro, S.; Geyer, T.; Gutierrez, E. Leakage Current Mitigation in Photovoltaic
String Inverter Using Predictive Control With Fixed Average Switching Frequency. IEEE Trans. Ind. Electron. 2017, 64, 9344–9354, doi:10.1109/TIE.2017.2708003. [CrossRef] 21. Vázquez, N.; Rosas, M.; Hernández, C.; Vázquez, E.; Perez-Pinal, F.J. A New Common-Mode Transformerless
Photovoltaic Inverter. IEEE Trans. Ind. Electron. 2015, 62, 6381–6391, doi:10.1109/TIE.2015.2426146. [CrossRef] 21. Vázquez, N.; Rosas, M.; Hernández, C.; Vázquez, E.; Perez-Pinal, F.J. A New Common-Mode Transformerless
Photovoltaic Inverter. IEEE Trans. Ind. Electron. 2015, 62, 6381–6391, doi:10.1109/TIE.2015.2426146. [CrossRef]
22. Kabalci, E. Maximum power point tracking (MPPT) algorithms for photovoltaic systems. In Energy Harvesting
and Energy Efficiency; Springer: Berlin Germany 2017; pp 205 234 22. Kabalci, E. Maximum power point tracking (MPPT) algorithms for photovoltaic systems. In Energy Harvesting
and Energy Efficiency; Springer: Berlin, Germany, 2017; pp. 205–234. 23. Moon, S.; Yoon, S.; Park, J. A New Low-Cost Centralized MPPT Controller System for Multiply
Distributed Photovoltaic Power Conditioning Modules. IEEE Trans. Smart Grid 2015, 6, 2649–2658,
doi:10.1109/TSG.2015.2439037. [CrossRef] 24. Montoya, D.G.; Ramos-Paja, C.A.; Giral, R. Improved Design of Sliding-Mode Controllers Based on the Requirements
of MPPT Techniques. IEEE Trans. Power Electron. 2016, 31, 235–247, doi:10.1109/TPEL.2015.2397831. [CrossRef] 25. Li, X.; Wen, H.; Hu, Y.; Jiang, L.; Xiao, W. Modified Beta Algorithm for GMPPT and Partial Shading Detection
in Photovoltaic Systems. IEEE Trans. Power Electron. 2018, 33, 2172–2186, doi:10.1109/TPEL.2017.2697459. [CrossRef] 26. c⃝2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/). References Furtado, A.M.S.; Bradaschia, F.; Cavalcanti, M.C.; Limongi, L.R. A Reduced Voltage Range Global Maximum
Power Point Tracking Algorithm for Photovoltaic Systems Under Partial Shading Conditions. IEEE Trans. Ind. Electron. 2018, 65, 3252–3262, doi:10.1109/TIE.2017.2750623. [CrossRef] 27. Chen, W.; Yang, X.; Zhang, W.; Song, X. Leakage current calculation for PV inverter system based on a
parasitic capacitor model. IEEE Trans. Power Electron. 2016, 31, 8205–8217. [CrossRef] 28. Kouro, S.; Leon, J.I.; Vinnikov, D.; Franquelo, L.G. Grid-connected photovoltaic systems: An overview of
recent research and emerging PV converter technology. IEEE Ind. Electron. Mag. 2015, 9, 47–61. [CrossRef] 29. Bianconi, E.; Calvente, J.; Giral, R.; Mamarelis, E.; Petrone, G.; Ramos-Paja, C.A.; Spagnuolo, G.; Vitelli, M. A Fast Current-Based MPPT Technique Employing Sliding Mode Control. IEEE Trans. Ind. Electron. 2013,
60, 1168–1178, doi:10.1109/TIE.2012.2190253. [CrossRef] 30. Brito, M.A.G.d.; Galotto, L.; Sampaio, L.P.; Melo, G.d.A.E.; Canesin, C.A. Evaluation of the Main
MPPT Techniques for Photovoltaic Applications. IEEE Trans. Ind. Electron. 2013, 60, 1156–1167,
doi:10.1109/TIE.2012.2198036. [CrossRef] 31. Manickam, C.; Raman, G.P.; Raman, G.R.; Ganesan, S.I.; Chilakapati, N. Efficient global maximum power
point tracking technique for a partially shaded photovoltaic string. IET Power Electron. 2016, 9, 2637–2644,
doi:10.1049/iet-pel.2015.1040. [CrossRef] 32. Chen, K.; Tian, S.; Cheng, Y.; Bai, L. An Improved MPPT Controller for Photovoltaic System Under Partial
Shading Condition. IEEE Trans. Sustain. Energy 2014, 5, 978–985, doi:10.1109/TSTE.2014.2315653. [CrossRef] 33. Ahmed, J.; Salam, Z. An Accurate Method for MPPT to Detect the Partial Shading Occurrence in a PV
System. IEEE Trans. Ind. Inform. 2017, 13, 2151–2161, doi:10.1109/TII.2017.2703079. [CrossRef] 34. Technical Datasheet. SunPower 305 Solar Panel. Technical Report. 2007. Available online:
https://www.pocosolar.com/wp-content/themes/twentyfifteen/pdfs/Sunpower%20Solar%20Panels/
sunpower_305wht_spec_sheet.pdf (accessed on 13 July 2020). c⃝2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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English
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Differentiation of two types of mobilized peripheral blood stem cells by microRNA and cDNA expression analysis
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Journal of translational medicine
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cc-by
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Published: 22 July 2008 Received: 23 June 2008
Accepted: 22 July 2008 Journal of Translational Medicine 2008, 6:39
doi:10.1186/1479-5876-6-39 This article is available from: http://www.translational-medicine.com/content/6/1/39 is article is available from: http://www.translational-medicine.c © 2008 Jin et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Differentiation of two types of mobilized peripheral blood stem
cells by microRNA and cDNA expression analysis
Ping Jin1, Ena Wang1, Jiaqiang Ren1, Richard Childs2, Jeong Won Shin1,3,
Hanh Khuu1, Francesco M Marincola1 and David F Stroncek*1 Address: 1Department of Transfusion Medicine, Clinical Center, National Institutes of Health, Bethesda, Maryland, USA, 2Hematology Branch,
National Heart, Lung, and Blood Institute, Bethesda, Maryland, USA and 3Department of Laboratory Medicine, Soonchunhyang University
Hospital, Seoul, Korea Email: Ping Jin - pjin@cc.nih.gov; Ena Wang - EWang@cc.nih.gov; Jiaqiang Ren - renj@cc.nih.gov; Richard Childs - childsr@nhlbi.nih.gov;
Jeong Won Shin - jwshin@hosp.sch.ac.kr; Hanh Khuu - hkhuu@cc.nih.gov; Francesco M Marincola - Fmarincola@cc.nih.gov;
David F Stroncek* - Dstroncek@cc.nih.gov * Corresponding author Abstract Background: Mobilized-peripheral blood hematopoietic stem cells (HSCs) have been used for
transplantation, immunotherapy, and cardiovascular regenerative medicine. Agents used for HSC
mobilization include G-CSF and the CXCR4 inhibitor AMD3100 (plerixafor). The HSCs cells
mobilized by each agent may contain different subtypes and have different functions. To
characterize mobilized HSCs used for clinical applications, microRNA (miRNA) profiling and gene
expression profiling were used to compare AMD3100-mobilized CD133+ cells from 4 subjects,
AMD3100 plus G-CSF-mobilized CD133+ cells from 4 subjects and G-CSF-mobilized CD34+ cells
from 5 subjects. The HSCs were compared to peripheral blood leukocytes (PBLs) from 7 subjects. Results: Hierarchical clustering of miRNAs separated HSCs from PBLs. miRNAs up-regulated in
all HSCs included hematopoiesis-associated miRNA; miR-126, miR-10a, miR-221 and miR-17-92
cluster. miRNAs up-regulated in PBLs included miR-142-3p, -218, -21, and -379. Hierarchical
clustering analysis of miRNA expression separated the AMD3100-mobilized CD133+ cells from G-
CSF-mobilized CD34+ cells. Gene expression analysis of the HSCs naturally segregated samples
according to mobilization and isolation protocol and cell differentiation status. Conclusion: HSCs and PBLs have unique miRNA and gene expression profiles. miRNA and gene
expression microarrays maybe useful for assessing differences in HSCs. B
Journal of Translational Medicine Open Access Background enhance leukocyte recovery after immunosuppresive ther-
apy, and to mount cancer rejection by adoptive transfer of
tumor infiltrating lymphocytes (TIL) [2]. HSCs have also
been used to treat patients with ischemic cardiac disease
to improve revascularization and cardiac function follow-
ing acute myocardial ischemia [3,4]. However, due to the
diversity of stem cell sources, mobilization methods g
Hematopoietic stem cells (HSCs) have been used for
more than 35 years for transplantation therapy to treat
acute and chronic leukemia, lymphoma, marrow failure
and congenital immune deficiency. Advances in immuno-
therapy have lead to the use of HSCs to produce dendritic
cells (DCs) to enhance antigen presentation [1], to Page 1 of 12
(page number not for citation purposes) Page 1 of 12
(page number not for citation purposes) http://www.translational-medicine.com/content/6/1/39 Journal of Translational Medicine 2008, 6:39 http://www.translational-medicine.com/content/6/1/39 http://www.translational-medicine.com/content/6/1/39 employed, purity of cells, and the content of cell subsets,
there are many different types of HSCs and those that are
most beneficial for one application may not be best for
another. sity in HSC clinical products. Antibodies specific for
CD34 have been the standard agent for the isolation of
HSCs. In addition, anti-CD133 has also been used [24-
27]. Approximately 75% of G-CSF mobilized peripheral
blood stem cells (PBSCs) express CD34 as well as CD133,
but small populations express one or the other [28]. HSCs can be obtained from several different sources
including bone marrow, mobilized peripheral blood, and
umbilical cord blood. For transplantation, traditionally,
HSCs were obtained from the bone marrow. However,
umbilical cord blood has been found to be especially rich
in HSCs [5] and HSCs have been found in the peripheral
blood and their level in the circulation increases several-
fold after G-CSF administion [6,7]. For HSC transplanta-
tion all three types of HSCs are used, but for most other
applications mobilized peripheral blood HSCs are most
commonly used. MicroRNAs (miRNA) are short, 20–22 nucleotide long,
RNA molecules which negatively regulate protein transla-
tion in a variety of biological processes, including devel-
opmental
timing,
signal
transduction,
tissue
differentiation and stem cell renewal and differentiation. Some miRNAs are specifically expressed in stem cells and
control stem cell self-renewal and differentiation by nega-
tively regulating the expression of certain key genes in
stem cells. The diversity of HSCs used for clinical therapies has also
increased due to the development of new HSC mobilizing
agents. Background For many years granulocyte colony-stimulating
factor (G-CSF) has been the standard agent to increase the
level of circulating HSCs. The administration of G-CSF
daily for 4 to 6 days results in a 10- to 30-fold increase in
the number of circulating HSCs [8,9] and G-CSF-mobi-
lized HSCs collected by apheresis have been used for
transplantation, immune therapy and the treatment of
cardiac ischemia. Another HSC mobilizing agent,
AMD3100, has been used with G-CSF to mobilize stem
cells for autologous transplants [10] and is currently being
evaluated as a single agent to mobilize HSCs for alloge-
neic donor transplants [11,12]. To determine if miRNA and gene expression profiling
would be beneficial in distinguishing different types of
HSCs, we compared CD133+ cells isolated from
AMD3100- and AMD3100 plus G-CSF-mobilized PBSC
concentrates with CD34+ cells isolated from G-CSF-mobi-
lized PBSC concentrates. We applied miRNA profiling and
gene expression profiling analysis to assess these three dif-
ferent types of progenitor cell populations using periph-
eral blood T cells, B cells, monocytes and NK cells as a
reference. We hypothesized that miRNA and gene expres-
sion analysis would be useful for characterizing HSCs. Global gene and miRNA expression profiling was used to
compare HSCs and peripheral blood leukocytes. T cells, B
cells, monocytes and NK cells isolated from peripheral
blood mononuclear cells (PBMCs) were compared to the
HSCs. The mechanisms by which AMD3100 and G-CSF alter
HSC trafficking and mobilization are different suggesting
that HSCs with different intrinsic properties maybe be
mobilized by these agents. AMD3100, as a CXCR4 antag-
onist, mobilizes HSCs within 6 hours by disrupting the
engagement of stem cell surface CXCR4 with its ligand
SDF-1 (CXCL12) which is expressed on marrow osteob-
lasts [10,13-20]. In contrast G-CSF mobilizes stem cells
indirectly by down regulating the expression of SDF-1 on
marrow osteoblasts and by releasing neutrophil and
monocyte proteolytic enzymes including neutrophil
elastase, cathepsin G, and maxtrix metalloproteinase-9
which in turn degrade important HSC trafficking and
adhesion molecules c-kit, VCAM-1, CXCR4, and SDF-1
[21]. In animal studies AMD3100 mobilizes a CD34+ cell
population with a greater long-term marrow repopulating
capacity than G-CSF [12,22,23], possibly due to differ-
ences in mechanisms of mobilization. MicroRNA array isolated cells were cryopreserved. The proportion of iso-
lated cells that expressed CD133 ranged from 88% to
98%. isolated cells were cryopreserved. The proportion of iso-
lated cells that expressed CD133 ranged from 88% to
98%. y
A miRNA probe set was designed using mature antisense
miRNA sequences (Sanger data base, version 8.1) consist-
ing of 736 human, mouse, rat and virus plus two control
probes. The probes were 5' amine modified and printed in
duplicate in the Immunogenetics Section of the DTM on
CodeLink activated slides (General Electric, GE Health,
NJ, USA) via covalent bonding.Small RNA was enriched
from 10 ug total RNA by flashPAGE (Pre-cast Gel, Type A)
(Ambion, Austin, TX, USA) and purified using flashPAGE
reaction clean-up kit (Ambion, Austin, TX, USA) accord-
ing to manufacture's instruction. The same procedures
were applied to obtain small RNA from the Epstein-Barr
virus (EBV)-transformed lymphoblastoid cell lines that
was used as the reference for the miRNA expression array
assay. Fragmented small RNA were 3'-end tailed with
amine-modified nucleotides and chemically coupled to
CyDye fluors (Amersham Biosciences, piscatway, NJ,
USA), the test sample with Cy5 and the reference with
Cy3, using the mirVana miRNA Labeling Kit (Ambion)
following the manufacturer recommended protocol. After
labeling, the samples and the reference were co-hybrid-
ized to the miRNA array at room temperature over night. Both the processed cDNA and the miRNA array slides
were scanned by GenePix scanner Pro 4.0 (Axon, Sunny-
vale, CA, USA). G-CSF stem cell mobilization, collection, and isolation
Healthy subjects were given G-CSF (10 μg/kg) (Filgrastim,
Amgen Thousand Oaks, CA) subcutaneously daily for 5
days. A mobilized PBSC concentrate was collected by leu-
kapheresis (CS3000, Baxter Healthcare Corp) on the fifth
day and cryopreserved in 5% DMSO and 6% pentastarch
using a controlled rate freezer and stored in liquid nitro-
gen. The PBSC concentrate was thawed and CD34+ cells
were isolated by immunoaffinity chromatography with
CD34 monoclonal antibody and magnetic beads (Isolex,
Baxter Healthcare). The proportion of isolated cells that
expressed CD34 ranged from 87% to 98%. Non-mobilized PBMC collection and sample preparation
A non-mobilized PBMC concentrate was collected from 7
healthy subjects by apheresis (CS3000) at the Department
of Transfusion Medicine (DTM), Clinical Center (CC),
National Institutes of Health (NIH). All subjects signed an
informed consent approved by the NIH. Hematopoietic progenitor cell isolation
AMD3100 and AMD3100 plus G-CSF stem cell mobilization, For the mobilization of cells with AMD3100 alone, one
dose of AMD3100 (Plerixafor, Genzyme Corporation,
Cambridge, MA) (240 μg/kg) was given subcutaneously
and PBSC concentrate was collected by leukapheresis
(CS3000, Baxter Healthcare Corp., Fenwal Division, Deer-
field, IL) six hours later. For AMD3100 plus G-CSF-mobi-
lization G-CSF (10 μg/kg) (Filgrastim, Amgen, Thousand
Oaks, CA) was given subcutaneously to healthy subjects
daily for four days and on the fifth day in addition to G-
CSF, AMD3100 (240 μg/kg) was given subcutaneously. A
mobilized PBSC concentrate was collected by leukapher-
esis (CS3000, Baxter Healthcare Corp.) on day 5 twelve
hours after the administration of AMD3100 and 2 hours
after the last dose of G-CSF. CD133+ cells were isolated
from the PBSC concentrates by positive selection with
anti-CD133 and magnetic microparticles (CliniMacs,
Miltenyi Biotec, Bergisch Gladbach, Germany) and the Although commonly accepted HSC specific surface mark-
ers have been used for HSC characterization and purifica-
tion, differences in the specificities of monoclonal
antibodies used to isolate HSCs have contributed to diver- Page 2 of 12
(page number not for citation purposes) Page 2 of 12
(page number not for citation purposes) http://www.translational-medicine.com/content/6/1/39 http://www.translational-medicine.com/content/6/1/39 Journal of Translational Medicine 2008, 6:39 RNA preparation, amplification and labeling Total RNA was isolated from each sample by using TRI-
ZOL reagent (Invitrogen, Carlsbad, CA). MicroRNA array Mononuclear
leukocytes were separated from contaminating granulo-
cytes and red blood cells by Ficoll gradient separation, cry-
opreserved and stored in liquid nitrogen. T cells, B cells,
NK cells and monocytes were isolated by positive selec-
tion by using anti-CD3, anti-CD19, anti-CD56 and anti-
CD14 magnetic beads, respectively (AutoMACS, Miltenyi
Biotec) (purity ≥ 90%). cDNA expression array
l
A (
) Total RNA (3 μg) was amplified from 0.5 × 106 to 107 cells
into anti-sense RNA (aRNA), also, total RNA from PBMCs
pooled from six normal donors was extracted and ampli-
fied into aRNA to serve as the constant reference [29] Test
and reference RNAs were labeled with Cy5 (red) and Cy3
(green) dyes, respectively, and co-hybridized to the cus-
tom-made 17.5 K cDNA (UniGene cluster) microarrays
which were printed in the Immunogenetics Section of
DTM with a configuration of 32 × 24 × 23 [30]. Clones
used for printing included a combination of the Research
Genetics RG_HsKG_031901 8 k clone set and 9,000
clones selected from the RG_Hs_seq_ver_070700 40 k
clone set. The 17,500 spots included 12,072 uniquely-
named genes, 875 duplicated genes and about 4,000
expression sequence tags. The complete list of genes
included in the Hs-CCDTV-17.5k-1px printing is available
at the web site http://nciarray.nci.nih.gov/gal_files/Hs-
CCDTM17.5k-2px.gal. Data and statistical analyses The raw microarray data set was filtered according to a
standard procedure to exclude spots with minimum
intensity that was arbitrarily set to an intensity parameter
of ≥ 300 for cDNA expression data and ≥ 100 for the
miRNA microarray data in both fluorescence channels. If
the fluorescence intensity of one channel was below the
cut-off while the other was above, the lower channel
intensity was overridden. Spots with diameters < 25 μm
for cDNA expression array and < 10 μm for miRNA micro-
arry and flagged spots were also excluded from the analy-
ses. Then, the filtered data were normalized using Lowess
Smoother and retrieved by the BRB ArrayTool http://
linus.nci.nih.gov/BRB-ArrayTools.html developed at the
National Cancer Institute (NCI), Biometric Research
Branch, Division of Cancer Treatment and Diagnosis. Hierarchical cluster analysis and TreeView software [31]
were used for visualization [32]. All of the predictions of
miRNA gene targets were made using BRB ArrayTool
microRNA
targets
http://linus.nci.nih.gov/BRB-Array
Tools.html developed at the NCI, Biometric Research
Branch, Division of Cancer Treatment and Diagnosis. RNA preparation, amplification and labeling
Total RNA was isolated from each sample by using TRI-
ZOL reagent (Invitrogen, Carlsbad, CA). Peripheral blood leukocytes and hematopoietic progenitor
cells PBMC concentrates were collected by apheresis from 7
healthy subjects. B cells, T cells, NK cells, and monocytes Page 3 of 12
(page number not for citation purposes) http://www.translational-medicine.com/content/6/1/39 Journal of Translational Medicine 2008, 6:39 http://www.translational-medicine.com/content/6/1/39 were isolated from each PBMC concentrate. 92% to 98%
of the isolated B cells expressed CD21, 95% to 98% of T
cells expressed CD3, 90% to 97% of NK cells expressed
CD56 and 92% to 98% of monocytes expressed CD14. Mobilized peripheral blood HSCs were collected from 13
healthy subjects: 5 were given G-CSF and their HSCs iso-
lated with anti-CD34 while 4 were given AMD3100 alone
and 4 were given AMD3100 plus G-CSF and their HSCs
isolated with anti-CD133. In the four donors given
AMD3100 plus G-CSF the concentration of circulating
CD34+ cells increased from 62 ± 45 × 106/L prior to the
administration of AMD3100 to 215 ± 117 × 106/L twelve
hours after AMD3100 was given, suggesting that
AMD3100 was responsible for mobilizing a substantial were isolated from each PBMC concentrate. 92% to 98%
of the isolated B cells expressed CD21, 95% to 98% of T
cells expressed CD3, 90% to 97% of NK cells expressed
CD56 and 92% to 98% of monocytes expressed CD14. Mobilized peripheral blood HSCs were collected from 13
healthy subjects: 5 were given G-CSF and their HSCs iso-
lated with anti-CD34 while 4 were given AMD3100 alone
and 4 were given AMD3100 plus G-CSF and their HSCs
isolated with anti-CD133. In the four donors given
AMD3100 plus G-CSF the concentration of circulating
CD34+ cells increased from 62 ± 45 × 106/L prior to the
administration of AMD3100 to 215 ± 117 × 106/L twelve
hours after AMD3100 was given, suggesting that
AMD3100 was responsible for mobilizing a substantial portion of the circulating CD34+ cells. In the four donors
given AMD3100 the CD34+ cell counts increased from a
baseline level of 3 ± 4 × 106/L to 23 ± 4 × 106/L 6 hours
after the administration of AMD3100. Comparison of miRNA expression among hematopoietic
progenitor cells and peripheral blood leukocytes tained a mixture of B cells (n = 7), monocytes (n = 7), T
cells (n = 2) and NK cells (n = 4) (Figure 1A). Within the
HSC group the 4 AMD3100-mobilized and the 4
AMD3100 plus G-CSF-mobilized CD133+ cells clustered
together, but separate from the G-CSF-mobilized CD34+
cells. in HSCs. Both miR-126 and miR-10a and 7 others, miR-
19a, -19b, -17-5p, 20b, -93, -130a and -221, have been
previously reported to be expressed by HSCs [33-35]. Among the 13 miRNAs whose expression was increased in
HSCs were 3 miRNAs belonging to the cancer-associated
miR-17-92 cluster; miR-19a, 19b, and -17-5p. When the
expression of all the miRNAs in the miR-17-92 cluster (n
= 7) were compared between HSCs and PBLs, 5 of the 7
were increased in HSCs (Table 2). Regulated by the onco-
gene cMyc, the miR-17-92 cluster targets large numbers of
genes. Consistent with this notion, the expression of cMyc
gene was increased 3.3-fold in HSCs based on our gene
expression analysis. The expression of 3 miRNAs in a sec-
ond cancer-associated miR-106-303 cluster were also
increased in HSCs (Table 2). In contrast, expression of two
cancer suppressing miRNAs, miR-15a and miR-16, were
decreased in HSCs. in HSCs. Both miR-126 and miR-10a and 7 others, miR-
19a, -19b, -17-5p, 20b, -93, -130a and -221, have been
previously reported to be expressed by HSCs [33-35]. Among the 13 miRNAs whose expression was increased in
HSCs were 3 miRNAs belonging to the cancer-associated
miR-17-92 cluster; miR-19a, 19b, and -17-5p. When the
expression of all the miRNAs in the miR-17-92 cluster (n
= 7) were compared between HSCs and PBLs, 5 of the 7
were increased in HSCs (Table 2). Regulated by the onco-
gene cMyc, the miR-17-92 cluster targets large numbers of
genes. Consistent with this notion, the expression of cMyc
gene was increased 3.3-fold in HSCs based on our gene
expression analysis. The expression of 3 miRNAs in a sec-
ond cancer-associated miR-106-303 cluster were also
increased in HSCs (Table 2). In contrast, expression of two
cancer suppressing miRNAs, miR-15a and miR-16, were
decreased in HSCs. The make-up of the miRNAs that separated HSCs and
PBLs differed markedly. Eight signature miRNAs demon-
strated increased expression and 3 miRNAs decreased
expression in HSCs compared to PBLs (Figure 1B). (HSC) and 28 peripheral blood leukocyte (PBL) samples* * The expression of miRs between the two groups were compared with t-tests (p < 0.005) Comparison of miRNA expression among hematopoietic
progenitor cells and peripheral blood leukocytes Among the 13 HSC samples and the 28 peripheral blood
leukocyte (PBL) samples analyzed ≥ 80% of the samples
expressed 148 miRNAs of the 457 human miRNA in our
chip. Unsupervised hierarchical clustering analysis based
on the 148 miRNAs revealed 3 distinct groups: the exclu-
sively HSC cluster; the T (n = 5) and NK cell (n = 3) cluster;
and the B cell and monocyte dominant cluster which con- A MicroRNA (miRNA) expression profiles of hematopoietic stem cells (HSCs) and peripheral blood leukocytes (PBLs)
Figure 1
MicroRNA (miRNA) expression profiles of hematopoietic stem cells (HSCs) and peripheral blood leukocytes
(PBLs). RNA was isolated from T cells, B cells, monocytes and NK cells from 7 subjects, G-CSF-mobilized CD34+ cells from
5 subjects, AMD3100 (A)-mobilized CD133+ cells from 4 subjects, and AMD3100 plus G-CSF (A+G)-mobilized CD133+ cells
from 4 subjects and miRNA expression was analyzed using an expression array with 457 human miRNAs. A) Unsupervised
hierarchical clustering of Eisen was used to analyze the 148 miRNAs that remained after filtering (miRNA expressed in ≥ 80%
of samples). B) Signature miRNAs whose expression was markedly up-regulated in HSCs or PBLs. A
Hematopoietic progenitor cells
Mature cells
B A Hematopoietic progenitor cells
Mature cells Hematopoietic progenitor cells Mature cells B B (
)
p
p
p
(
)
p
p
y
(
)
g
MicroRNA (miRNA) expression profiles of hematopoietic stem cells (HSCs) and peripheral blood leukocytes
(PBLs). RNA was isolated from T cells, B cells, monocytes and NK cells from 7 subjects, G-CSF-mobilized CD34+ cells from
5 subjects, AMD3100 (A)-mobilized CD133+ cells from 4 subjects, and AMD3100 plus G-CSF (A+G)-mobilized CD133+ cells
from 4 subjects and miRNA expression was analyzed using an expression array with 457 human miRNAs. A) Unsupervised
hierarchical clustering of Eisen was used to analyze the 148 miRNAs that remained after filtering (miRNA expressed in ≥ 80%
of samples). B) Signature miRNAs whose expression was markedly up-regulated in HSCs or PBLs. Page 4 of 12
(page number not for citation purposes) http://www.translational-medicine.com/content/6/1/39 http://www.translational-medicine.com/content/6/1/39 http://www.translational-medicine.com/content/6/1/39 Journal of Translational Medicine 2008, 6:39 tained a mixture of B cells (n = 7), monocytes (n = 7), T
cells (n = 2) and NK cells (n = 4) (Figure 1A). Within the
HSC group the 4 AMD3100-mobilized and the 4
AMD3100 plus G-CSF-mobilized CD133+ cells clustered
together, but separate from the G-CSF-mobilized CD34+
cells. Comparison of miRNA expression among hematopoietic
progenitor cells and peripheral blood leukocytes The 8
signature HSC miRNAs whose expression was increased
included miR-19a, -19b, -20a, and 20b, which are part of
the polycistronic cancer-associated miR-17-92 cluster. The
3 signature miRNAs whose expression was greatest in the
PBL samples were miR-142-3p, miR-21, and miR-142-5p. Both probes for miR-20b were up-regulated in HSCs and
both for miR-142-3p were up-regulated in PBLs. Among the 148 miRNAs 47 were differentially expressed
between AMD3100-mobilized CD133+ cells and the G-
CSF-mobilized CD34+ cells (t-tests, p < 0.005). The
expression of 17 was increased in the AMD3100 group
and 30 were increased in the G-CSF group (data not
shown). To identify the entire set of miRNAs whose expression dif-
fered between HSCs and PBLs, the expression of miRNAs
by all 13 HSC samples was compared with those by all 28
PBL samples (t-test, p < 0.005). The expression of 35 miR-
NAs differed between the two types of cells: the expression
of 13 miRNAs were increased in HSCs and 22 miRNAs
were increased in PBLs (Table 1). The expression of two
miRNAs, miR-126 and miR-10a, were markedly increased The expression of 4 miRNAs were markedly increased in
PBL: miR-142-3p, miR-218, miR-21, and miR-379. In Page 5 of 12
Table 1: MicroRNAs (miRs) whose expression differed among 13 hematopoietic stem cell
miRs whose expression was
increased in HSCs
Fold-increase
miRs whose expression
was increased in PBLs
Fold-increase
hsa-miR-126
14.43
hsa-miR-142-3p
15.66
hsa-miR-10a
13.46
hsa-miR-218
11.07
hsa-miR-19a
3.89
hsa-miR-21
8.46
hsa-miR-19b
3.11
hsa-miR-379
7.63
hsa-mir-595
2.98
hsa-miR-381
4.61
hsa-miR-146a
2.72
hsa-miR-29b
3.83
hsa-miR-93
2.56
hsa-miR-26b
3.54
hsa-miR-221
2.38
hsa-miR-30c
3.12
hsa-miR-20b
2.35
hsa-miR-142-5p
2.51
hsa-miR-130a
2.25
hsa-miR-29a
2.49
hsa-miR-34a
2.12
hsa-let-7g
2.49
hsa-miR-363
1.92
hsa-let-7i
2.42
hsa-miR-17-5p
1.89
hsa-miR-191
2.3
hsa-miR-30b
2.03
hsa-let-7b
1.88
hsa-miR-26a
1.85
hsa-miR-16
1.78
hsa-let-7c
1.75
hsa-miR-30a-5p
1.73
hsa-miR-373
1.62
hsa-mir-594
1.6
hsa-mir-610
1.34
(HSC) and 28 peripheral blood leukocyte (PBL) samples*
* The expression of miRs between the two groups were compared with t-tests (p < 0.005) Table 1: MicroRNAs (miRs) whose expression differed among 13 hematopoietic stem cell http://www.translational-medicine.com/content/6/1/39 Journal of Translational Medicine 2008, 6:39 addition to these 4 miRNAs, expression of miR-16 which
belongs to the leukemia associated cluster miR-15a-16
was also increased. To identify miRNAs uniquely expressed among PBLs, 5-
way ANOVA was performed to compare the expression of
the 148 miRNAs among T cell, B cell, monocyte, NK cell
and the HSC samples. Discussion In this study we discovered that miRNA expressed by HSC
differed from those expressed by PBLs. We also found that
miRNA profiling and cDNA expression analysis are poten-
tially useful tools for the analysis of clinical HSCs derived
by different mobilization and isolation methods which
may yield functionally diverse products. Among the 13 miRNAs increased in HSCs miR-10a, -17-
5p, -19a, -19b, -20b, -93, -126, -130a, and -221; have pre-
viously been found to be expressed by HSCs [33-36]. miR-
126 and miR-10a have been found to be down-regulated
in erythrocyte and megakaryocyte precursors [33,36]. miR-221 has previously been shown to be important in
erythropoiesis; the expression of miR-221 and -222
inhibit erythropoiesis [37]. The expression of miR-221
has been reported to be down regulated in CD34+ cells
during erythropoesis [33,37]. The expression of miR-221
is also increased in papillary thyroid carincomas [38] and
is involved with the pathogenesis of hepatocelluar carin-
coma [39] and prostate cancer [40]. addition to these 4 miRNAs, expression of miR-16 which
belongs to the leukemia associated cluster miR-15a-16
was also increased. addition to these 4 miRNAs, expression of miR-16 which
belongs to the leukemia associated cluster miR-15a-16
was also increased. To identify miRNAs uniquely expressed among PBLs, 5-
way ANOVA was performed to compare the expression of
the 148 miRNAs among T cell, B cell, monocyte, NK cell
and the HSC samples. T cells were characterized by
increased expression of miR-146a, -146b, -29a, and -29b
and B cells were characterized by the increased expression
of miR-29a -29b, and -29c (Table 3). The expression of
several miRNAs including miR-223 and miR-21 were
increased in monocytes, but none were increased NK cells
(Table 3). Polycistronically transcribed miRNA clusters, so called
onco-miR clusters, were also highly expressed by HSCs
compared to PBLs. These findings are consistent with the
theory that stem cells are important in cancer. The miR-
17-92 cluster has been reported to be up-regulated in dif-
fuse large B cell lymphoma (DLBCL), lung, breast, pros-
tate, and colon cancer [41-43]. Venurini and colleagues
found that the miR-17-92 cluster was up-regulated in
CD34+ cells from healthy subjects and those with early- Comparison of miRNA expression among hematopoietic
progenitor cells and peripheral blood leukocytes T cells were characterized by
increased expression of miR-146a, -146b, -29a, and -29b
and B cells were characterized by the increased expression
of miR-29a -29b, and -29c (Table 3). The expression of
several miRNAs including miR-223 and miR-21 were
increased in monocytes, but none were increased NK cells
(Table 3). cDNA expression profiling
Table 2: Cancer-associated microRNAs (miRNAs) whose
expression was up- and down-regulated in hematopoietic stem
cells (HSCs)
Cluster miR-17-92
miRNA
Fold-increase in HSCs
P
17-p5
1.89
0.004
17-p3
NC
NS
18a
1.31
0.018
19a
3.89
4 × 10-7
20a
1.55
0.015
19b-1
3.1
1.3 × 10-6
92-1
NC
NS
Cluster miR-106-363
miRNA
Fold-increase in HSCs
P
106a
1.55
0.016
18b
NC
NS
20b
2.34
2.9 × 10-05
19b-2
3.1
1.3 × 10-06
92-2
NC
NS
363
NC
NS
Cluster miR-15-16
miRNA
Fold-decrease in HSCs
P
15a
3.33
0.007466
16
1.78
9.93 × 10-4
NC = no change
NS = not significant Table 2: Cancer-associated microRNAs (miRNAs) whose
expression was up- and down-regulated in hematopoietic stem
cells (HSCs) contained 17,088 genes. Unsupervised hierarchical clus-
tering of the 11,023 genes that were expressed in ≥ 80% of
the samples separated the HPC and PBL samples into two
distinct groups (Figure 2). All of the HSC samples clus-
tered separately from mature PBL subsets. The AMD3100-
mobilized CD133+ cells and the AMD3100 plus G-CSF-
mobilized CD133+ cells clustered together and again were
considered as one group, the AMD3100 group. The two
different types of HSCs, AMD3100-mobilized CD133+
cells and G-CSF-mobilized CD34+ cells clustered sepa-
rately (Figure 2). In addition, T cell, B cell, monocyte, and
NK cell samples clustered into separate groups (Figure 2). A comparison of genes expressed by HSCs and PBLs
revealed that 5,392 genes were differentially expressed
among the two types of cells (t-tests, p < 0.001). The 30
genes whose expression increased the greatest in HSCs
included several transcription factors and oncogenes such
as GATA2 and N-myc, while the 30 genes whose expres-
sion was increased the most in PBLs were enriched for
genes known to be expressed by circulating leukocytes
such as Fc-γ receptor III (CD16), integrin α M subunit and
IL-10 receptor α (Table 4). Page 6 of 12
(page number not for citation purposes) cDNA expression profiling Analysis of miRNA expression revealed that HSCs and
PBLs had unique miRNA expression profiles. To investi-
gate this further, gene expression profiles were also com-
pared using cDNA expression microarrays which Page 6 of 12
(page number not for citation purposes) Page 6 of 12
(page number not for citation purposes) Journal of Translational Medicine 2008, 6:39 http://www.translational-medicine.com/content/6/1/39 Table 3: MicoRNAs (miRNAs) characteristic of each type of peripheral blood leukocyte*
T cells
NK cells
Increased
FC
Decreased
FC
Increased
FC
Decreased
FC
miR-146b
4.05
miR-223
16.8
None
NA
miR-19a
3.54
miR-29b
3.86
miR-181b
2.41
miR-19b
3.06
miR-146a
3.45
miR-93
2.32
miR-92
1.85
miR-29a
3.38
miR-20b
2.19
miR-17-5p
2.01
miR-20a
1.88
miR-106a
1.86
Monocytes
B cells
Increased
FC
Decreased
FC
Increased
FC
Decreased
FC
miR-223
21.0
miR-146b
5.85
miR-29b
4.53
miR-22
7.62
miR-21
10.1
miR-146a
5.71
miR-29a
3.66
miR-23a
6.15
miR-424
5.74
miR-29c
2.92
miR-29c
3.05
miR-24
5.87
miR-365
3.28
miR-146b
3.33
miR-191
3.28
miR-27a
3.29
miR-103
3.19
miR-23b
2.42
miR-23a
3.1
miR-27a
2.85
miR-15a
2.77
miR-374
2.74
miR-107
2.58
miR-106b
2.33
miR-16
2.1
miR-422b
2.01
miR-23b
1.78
miR-185
1.62
*The groups of cells were compared using F-tests (p < 0.005)
FC = fold change Table 3: MicoRNAs (miRNAs) characteristic of each type of peripheral blood leukocyte* *The groups of cells were compared using F-tests (p < 0.005)
FC = fold change chronic phase chronic myelogenous leukemia, but was
not up-regulated in CD34+ cells from subjects with CML
blast crisis [44]. In addition, 3 of 6 members of the cancer-
associated miR-106-363 cluster were also up-regulated in
HSCs. Elevated expression of this cluster has been found
in T cell leukemia [45] and T cell lymphoma [46]. chronic phase chronic myelogenous leukemia, but was
not up-regulated in CD34+ cells from subjects with CML
blast crisis [44]. In addition, 3 of 6 members of the cancer-
associated miR-106-363 cluster were also up-regulated in
HSCs. Elevated expression of this cluster has been found
in T cell leukemia [45] and T cell lymphoma [46]. At the transcription level we found that the expression of
a number of oncogenes and genes related to transcription
was greater in HSCs than in PBLs. The expression of
GATA2, a transcription factor important in hematopoietic
stem cell and endothelial cell differentiation [57,58] was
137-fold greater in HSCs and that of N-myc was 97-fold
greater. cDNA expression profiling Computational gene target prediction http://
www.targetscan.org/ indicates that GATA2 is targeted by
miR-27a whose expression was 2-fold less in HSCs than
PBLs (p = 0.0069). miR-21 was increased in PBLs. miR-21 has also been
found to be increased in chronic lymphocytic leukemia
[47], B cell lymphomas [48], breast cancer [49], pancreatic
cancer [50], head and neck cancer cell lines [51]. In addi-
tion, the expression of miR-16 was greater in PBLs, espe-
cially in monocytes, than HSCs. miR-15 and miR-16 are
cancer-associated miRNA that are down-regulated in B-
cell chronic lymphocyte leukemia [52,53] and pituitary
adenomas [54] but upregulated in acute promyelocytic
leukemia [55]. We also found that T cells were character-
ized by the upregulation of miR-29a and -29b and B cells
by miR-29a, -29b, and -29c. These miRNA are down-regu-
lated in aggressive B cell lymphoma [56]. An interesting finding of this study resides in the differ-
ences in both miRNA and gene expression found between
G-CSF-mobilized CD34+ cells and AMD3100-mobilized
CD133+ cells. While both miRNA expression profiling
and DNA expression profiling differentiated the two dif-
ferent types of HSCs, these two populations were
obtained using different HSC mobilization and isolation
procedures. It is not certain if the differences in the two
stem cell types were due to the different mobilizing agents Page 7 of 12
(page number not for citation purposes) Page 7 of 12
(page number not for citation purposes) http://www.translational-medicine.com/content/6/1/39 Journal of Translational Medicine 2008, 6:39 Gene expression profiles of hematopoietic stem cells (HSCs) and peripheral blood leukocytes (PBLs)
Figure 2
Gene expression profiles of hematopoietic stem cells (HSCs) and peripheral blood leukocytes (PBLs). cDNA
was isolated from T cells, B cells, monocytes and NK cells from 7 subjects, G-CSF (G)-mobilized CD34+ cells from 5 subjects
AMD3100 (A)-mobilized CD133+ cells from 4 subjects and AMD3100 plus G-CSF (A+G)-mobilized CD133+ cells from 4 sub
ects. cDNA expression was analyzed using an expression microarray with 17,500 cDNA. Unsupervised hierarchical clustering
of Eisen was used to analyze the 11,023 genes that remained after filtering (cDNA expressed in ≥ 80% of samples). Gene expression profiles of hematopoietic stem cells (HSCs) and peripheral blood leukocytes (PBLs)
Figure 2
Gene expression profiles of hematopoietic stem cells (HSCs) and peripheral blood leukocytes (PBLs). cDNA expression profiling cDNA
was isolated from T cells, B cells, monocytes and NK cells from 7 subjects, G-CSF (G)-mobilized CD34+ cells from 5 subjects
AMD3100 (A)-mobilized CD133+ cells from 4 subjects and AMD3100 plus G-CSF (A+G)-mobilized CD133+ cells from 4 su
jects. cDNA expression was analyzed using an expression microarray with 17,500 cDNA. Unsupervised hierarchical clusterin
of Eisen was used to analyze the 11,023 genes that remained after filtering (cDNA expressed in ≥ 80% of samples). Gene expression profiles of hematopoietic stem cells (HSCs) and peripheral blood leukocytes (PBLs)
Figure 2
Gene expression profiles of hematopoietic stem cells (HSCs) and peripheral blood leukocytes (PBLs). cDNA
was isolated from T cells, B cells, monocytes and NK cells from 7 subjects, G-CSF (G)-mobilized CD34+ cells from 5 subjects,
AMD3100 (A)-mobilized CD133+ cells from 4 subjects and AMD3100 plus G-CSF (A+G)-mobilized CD133+ cells from 4 sub-
jects. cDNA expression was analyzed using an expression microarray with 17,500 cDNA. Unsupervised hierarchical clustering
of Eisen was used to analyze the 11,023 genes that remained after filtering (cDNA expressed in ≥ 80% of samples). or isolation antibodies. We suspect that both the mobiliz-
ing agents and antibodies contributed to the differences. In addition, the G-CSF-mobilized CD34+ cells underwent
an additional freeze-thaw cycle. This was not expected to
affect the HSCs, but we can not exclude this possibility. Further studies are needed which compare HSCs mobi-
lized with AMD3100 alone and with G-CSF alone and
with both types of stem cells isolated with the same mon-
oclonal antibody. Since both CD34 and CD133 are being
used to isolate stem cells for clinical applications, HSCs
mobilized with one agent and isolated with each antibody
should also be compared. When considering new or different mobilization or isola-
tion protocols, changes in both the quantity and quality
of HSCs should be considered. The quantitation of
CD34+ cells has been the gold standard for assessing the
potency of HSCs for clinical therapies. When comparing
HSCs mobilized with G-CSF from different subjects, dif-
ferent G-CSF mobilization protocols, or different HSC
collection protocols, measuring CD34+ cells is a good
indicator of the potency of the HSCs. However, if stem
cells mobilized with G-CSF are to be compared with those
mobilized with AMD3100 or AMD3100 plus G-CSF,
measuring only CD34+ or CD133+ cells may not com-
pletely reflect the differences among these types of cells. *The groups were compared using t-tests (p < 0.001). Acknowledgements We thank the Staff of the Dowling Clinic, DTM, CC, NIH for collecting the
cells and the staff of the Cell Processing Laboratory, DTM, CC, NIH for iso-
lating the CD34+ and CD133+ cells. The study was funded by the NIH,
Bethesda, Maryland, USA. 16. Aiuti A, Webb IJ, Bleul C, Springer T, Gutierrez-Ramos JC: The
chemokine SDF-1 is a chemoattractant for human CD34+
hematopoietic progenitor cells and provides a new mecha-
nism to explain the mobilization of CD34+ progenitors to
peripheral blood. J Exp Med 1997, 185:111-120. p
p
J
p
17. Kim CH, Broxmeyer HE: In vitro behavior of hematopoietic
progenitor cells under the influence of chemoattractants:
stromal cell-derived factor-1, steel factor, and the bone mar-
row environment. Blood 1998, 91:100-110. References 1. Gilboa E: DC-based cancer vaccines. J Clin Invest 2007,
117:1195-1203. 2. Wrzesinski C, Paulos CM, Gattinoni L, Palmer DC, Kaiser A, Yu Z,
Rosenberg SA, Restifo NP: Hematopoietic stem cells promote
the expansion and function of adoptively transferred antitu-
mor CD8 T cells. J Clin Invest 2007, 117:492-501. 18. Liles WC, Broxmeyer HE, Rodger E, Wood B, Hubel K, Cooper S,
Hangoc G, Bridger GJ, Henson GW, Calandra G, Dale DC: Mobili-
zation of hematopoietic progenitor cells in healthy volun-
teers by AMD3100, a CXCR4 antagonist. Blood 2003,
102:2728-2730. J
3. Losordo DW, Schatz RA, White CJ, Udelson JE, Veereshwarayya V,
Durgin M, Poh KK, Weinstein R, Kearney M, Chaudhry M, Burg A,
Eaton L, Heyd L, Thorne T, Shturman L, Hoffmeister P, Story K, Zak
V, Dowling D, Traverse JH, Olson RE, Flanagan J, Sodano D,
Murayama T, Kawamoto A, Kusano KF, Wollins J, Welt F, Shah P,
Soukas P, Asahara T, Henry TD: Intramyocardial transplantation
of autologous CD34+ stem cells for intractable angina: a
phase I/IIa double-blind, randomized controlled trial. Circula-
tion 2007, 115:3165-3172. 19. Devine SM, Flomenberg N, Vesole DH, Liesveld J, Weisdorf D, Badel
K, Calandra G, Dipersio JF: Rapid mobilization of CD34+ cells
following
administration
of
the
CXCR4
antagonist
AMD3100 to patients with multiple myeloma and non-Hodg-
kin's lymphoma. J Clin Oncol 2004, 22:1095-1102. 20. Broxmeyer HE, Orschell CM, Clapp DW, Hangoc G, Cooper S, Plett
PA, Liles WC, Li X, Graham-Evans B, Campbell TB, Calandra G,
Bridger G, Dale DC, Srour EF: Rapid mobilization of murine and
human hematopoietic stem and progenitor cells with
AMD3100, a CXCR4 antagonist. J Exp Med 2005,
201:1307-1318. 4. Kurdi M, Booz GW: G-CSF-based stem cell therapy for the
heart-unresolved issues part A: Paracrine actions, mobiliza-
tion, and delivery. Congest Heart Fail 2007, 13:221-227. tion, and delivery. Congest Heart Fail 2007, 13:221 227. 5. Broxmeyer HE, Hangoc G, Cooper S, Ribeiro RC, Graves V, Yoder
M, Wagner J, Vadhan-Raj S, Benninger L, Rubinstein P, .: Growth
characteristics and expansion of human umbilical cord blood
and estimation of its potential for transplantation in adults. Proc Natl Acad Sci U S A 1992, 89:4109-4113. 21. Nervi B, Link DC, Dipersio JF: Cytokines and hematopoietic
stem cell mobilization. J Cell Biochem 2006, 99:690-705. 21. Nervi B, Link DC, Dipersio JF: Cytokines and hematopoietic
stem cell mobilization. J Cell Biochem 2006, 99:690-705. 22. Authors' contributions 13. Nagasawa T, Hirota S, Tachibana K, Takakura N, Nishikawa S, Kita-
mura Y, Yoshida N, Kikutani H, Kishimoto T: Defects of B-cell lym-
phopoiesis and bone-marrow myelopoiesis in mice lacking
the CXC chemokine PBSF/SDF-1. Nature 1996, 382:635-638. All of the authors read and approved the final manuscript. The studies were designed by PJ and DS and were pre-
formed by PJ, JR, and JWS. Stem cell mobilization and iso-
lation was performed under the direction of RC and HK. The data was analyzed by PJ, EW, DS and FM. PJ and DS
wrote the manuscript with help from RC and EW. 14. Deichmann M, Kronenwett R, Haas R: Expression of the human
immunodeficiency virus type-1 coreceptors CXCR-4 (fusin,
LESTR) and CKR-5 in CD34+ hematopoietic progenitor
cells. Blood 1997, 89:3522-3528. 15. 15. Mohle R, Bautz F, Rafii S, Moore MA, Brugger W, Kanz L: The chem-
okine receptor CXCR-4 is expressed on CD34+ hematopoi-
etic
progenitors
and
leukemic
cells
and
mediates
transendothelial migration induced by stromal cell-derived
factor-1. Blood 1998, 91:4523-4530. Competing interests 12. Larochelle A, Krouse A, Metzger M, Orlic D, Donahue RE, Fricker S,
Bridger G, Dunbar CE, Hematti P: AMD3100 mobilizes hemat-
opoietic stem cells with long-term repopulating capacity in
nonhuman primates. Blood 2006, 107:3772-3778. The authors declare that they have no competing interests. cDNA expression profiling Page 8 of 12
(page number not for citation purposes) http://www.translational-medicine.com/content/6/1/39 Journal of Translational Medicine 2008, 6:39 Table 4: Genes differentially expressed between hematopoietic stem cells (HSCs) and peripheral blood leukocytes (PBLs)
Expression Increased in HSCs
Expression Increased in PBLs
Gene Symbol
Description
Fold
Change
Gene
Symbol
Description
Fold Change
GATA2
GATA-binding protein 2
237.82
CARD14
Caspase recruitment domain protein 14
25.59
MYCN
N-myc
97.75
LTBR
Lymphotoxin-Beta receptor precursor =
Tumor necrosis factor receptor 2 related
protein = Tumor necrosis factor C
receptor
18.83
CRHBP
CRF-BP = corticotropin-releasing factor
binding protein
65.52
GPR65
TDAG8 = putative G protein-coupled
receptor induced during activation-
induced apoptosis of T cells = G protein-
coupled receptor 65
18.33
FHL1
Four and a half LIM domains 1
58.2
FCGR3A
CD16 = Fcgamma receptor IIIa
18.15
ERG
V-ets erythroblastosis virus E26 oncogene
homolog (avian)
43.57
ITGAM
Integrin, alpha M
(complement component 3 receptor 3
subunit)
16.96
NPR3
Natriuretic peptide receptor C/guanylate
cyclase C
(atrionatriuretic peptide receptor C)
42.04
SGSH
N-sulfoglucosamine sulfohydrolase
(sulfamidase)
16.64
SCHIP1
Schwannomin interacting protein 1
41.77
ITGB7
Integrin, beta 7
16.39
MSRB3
Methionine sulfoxide reductase B3
35.29
GNLY
Granulysin
15.21
MYB
V-myb myeloblastosis viral oncogene
homolog (avian)
32.99
ALOX5AP
Arachidonate 5-lipoxygenase-activating
protein
15.11
DEPDC6
DEP domain containing 6
28.85
CD48
CD48 = BLAST-1
14.81
EPDR1
Ependymin related protein 1 (zebrafish)
27.88
IL10RA
Interleukin 10 receptor, alpha
13.71
TRH
Thyrotropin-releasing hormone
26.63
CX3CR1
Chemokine (C-X3-C motif) receptor 1
13.64
NGFRAP1
Nerve growth factor receptor (TNFRSF16)
associated protein 1
25.86
COTL1
Coactosin-like 1 (Dictyostelium)
13.4
SERPING1
Serpin peptidase inhibitor, clade G (C1
inhibitor), member 1, (angioedema,
hereditary)
25.83
ADAM19
ADAM metallopeptidase domain 19
(meltrin beta)
13.24
MAP7
Microtubule-associated protein 7
25.11
IL2RB
IL-2 receptor beta chain
13
SOCS2
Suppressor of cytokine signaling 2
24.44
ITK
IL2-inducible T-cell kinase
11.91
TSC22D1
TSC22 domain family, member 1
24.12
KLRC4
Killer cell lectin-like receptor subfamily
C, member 4
11.36
H1F0
H1 histone family, member 0
22.71
CCL4
MIP-1 beta
11.22
RBPMS
RNA binding protein with multiple splicing
22.67
CTSH
Cathepsin H
11.18
CTDSPL
CTD (carboxy-terminal domain, RNA
polymerase II, polypeptide A) small
phosphatase-like
22.64
EBI2
Epstein-Barr virus induced gene 2
(lymphocyte-specific G protein-coupled
receptor)
10.69
CDCA7
Cell division cycle associated 7
22.49
GIMAP4
GTPase, IMAP family member 4
10.28
CYTL1
Cytokine-like 1
20.9
POU2F2
POU domain, class 2, transcription factor
2
10.22
FLT3
Fms-related tyrosine kinase 3
20.45
CXCR4
CXCR4 = CXC chemokine receptor 4
10.11
PRKAR2B
Protein kinase, cAMP-dependent,
regulatory, type II, beta
20.09
CYP4A11
Cytochrome P450, family 4, subfamily A,
polypeptide 11
10.1
TRIM58
Tripartite motif-containing 58
19.18
LGALS3
Lectin, galactoside-binding, soluble, 3
(galectin 3)
9.61
FSCN1
Fascin homolog 1, actin-bundling protein
(Strongylocentrotus purpuratus)
18.91
SNX27
Sorting nexin family member 27
9.44
C1orf150
Chromosome 1 open reading frame 150
18.83
TRIM26
Tripartite motif-containing 26
9.23
TRH
Thyrotropin-releasing hormone
17.56
SNX27
Sorting nexin family member 27
9.05
KIT
V-kit Hardy-Zuckerman 4 feline sarcoma
viral oncogene homolog
17.27
CSPG2
Chondroitin sulfate proteoglycan 2
(versican)
8.89
WASF1
WAS protein family, member 1
16.75
IL32
Interleukin 32
8.76 xpressed between hematopoietic stem cells (HSCs) and peripheral blood leukocytes (PBLs) Page 9 of 12
(page number not for citation purposes) http://www.translational-medicine.com/content/6/1/39 http://www.translational-medicine.com/content/6/1/39 Journal of Translational Medicine 2008, 6:39 http://www.translational-medicine.com/content/6/1/39 Measures in addition of CD34 and CD133 are needed to
more completely characterize HSCs. cDNA expression profiling blood progenitor cells for allogeneic transplantation: safety,
kinetics of mobilization, and composition of the graft. Br J
Haematol 1994, 87:609-613. 7. Stroncek DF, Clay ME, Herr G, Smith J, Jaszcz WB, Ilstrup S, McCul-
lough J: The kinetics of G-CSF mobilization of CD34+ cells in
healthy people. Transfus Med 1997, 7:19-24. Our results show that the global miRNA and cDNA
expression assessment can distinguish different types of
HSCs. Both types of global gene expression arrays were
able to distinguish the two types of HSCs that we studied
and both should be useful for potency testing of clinical
HPC components. miRNA and cDNA microarray assays
with several thousand probes usually require several days
to complete, but it would be possible to more rapidly ana-
lyze the expression of a smaller selected group of miRNA
or genes. 8. Korbling M, Huh YO, Durett A, Mirza N, Miller P, Engel H, Anderlini
P, van Besien K, Andreeff M, Przepiorka D, Deisseroth AB, Champlin P, van Besien K, Andreeff M, Przepiorka D, Deisseroth AB, Champlin
RE: Allogeneic blood stem cell transplantation: peripherali-
zation and yield of donor-derived primitive hematopoietic
progenitor cells (CD34+ Thy-1dim) and lymphoid subsets,
and possible predictors of engraftment and graft-versus-host
disease. Blood 1995, 86:2842-2848. 9. Prosper F, Stroncek D, Verfaillie CM: Phenotypic and functional
characterization of long-term culture-initiating cells present
in peripheral blood progenitor collections of normal donors
treated with granulocyte colony-stimulating factor. Blood
1996, 88:2033-2042. 10. Flomenberg N, Devine SM, Dipersio JF, Liesveld JL, McCarty JM, Row-
ley SD, Vesole DH, Badel K, Calandra G: The use of AMD3100
plus G-CSF for autologous hematopoietic progenitor cell
mobilization is superior to G-CSF alone. Blood 2005,
106:1867-1874. In conclusion, HSCs and PBLs have unique miRNA
expression profiles and many cancer-associated miRNA
are expressed by HSCs. miRNA and gene expression
microarrays maybe useful for assessing differences in
HSCs. 11. 11. Burroughs L, Mielcarek M, Little MT, Bridger G, Macfarland R, Fricker
S, LaBrecque J, Sandmaier BM, Storb R: Durable engraftment of
AMD3100-mobilized autologous and allogeneic peripheral-
blood mononuclear cells in a canine transplantation model. Blood 2005, 106:4002-4008. References Lang P, Schumm M, Greil J, Bader P, Klingebiel T, Muller I, Feuchtinger
T, Pfeiffer M, Schlegel PG, Niethammer D, Handgretinger R: A com-
parison between three graft manipulation methods for hap-
loidentical stem cell transplantation in pediatric patients:
preliminary results of a pilot study. Klin Padiatr 2005,
217:334-338. 42. He L, Thomson JM, Hemann MT, Hernando-Monge E, Mu D, Good-
son S, Powers S, Cordon-Cardo C, Lowe SW, Hannon GJ, Hammond
SM: A microRNA polycistron as a potential human oncogene. Nature 2005, 435:828-833. 43. 43. Hayashita Y, Osada H, Tatematsu Y, Yamada H, Yanagisawa K, Tom-
ida S, Yatabe Y, Kawahara K, Sekido Y, Takahashi T: A polycistronic
microRNA cluster, miR-17-92, is overexpressed in human
lung cancers and enhances cell proliferation. Cancer Res 2005,
65:9628-9632. 26. Bitan M, Shapira MY, Resnick IB, Zilberman I, Miron S, Samuel S, Ack-
erstein A, Elad S, Israel S, Amar A, Fibach E, Or R, Slavin S: Success-
ful
transplantation
of
haploidentically
mismatched
peripheral blood stem cells using CD133+-purified stem
cells. Exp Hematol 2005, 33:713-718. 44. Venturini L, Battmer K, Castoldi M, Schultheis B, Hochhaus A, Muck-
enthaler MU, Ganser A, Eder M, Scherr M: Expression of the miR-
17-92 polycistron in chronic myeloid leukemia (CML) CD34+
cells. Blood 2007, 109:4399-4405. 27. Isidori A, Motta MR, Tani M, Terragna C, Zinzani P, Curti A, Rizzi S,
Taioli S, Giudice V, D'Addio A, Gugliotta G, Conte R, Baccarani M,
Lemoli RM: Positive selection and transplantation of autolo-
gous highly purified CD133(+) stem cells in resistant/
relapsed chronic lymphocytic leukemia patients results in
rapid hematopoietic reconstitution without an adequate
leukemic cell purging. Biol Blood Marrow Transplant 2007,
13:1224-1232. 45. Landais S, Landry S, Legault P, Rassart E: Oncogenic potential of
the miR-106-363 cluster and its implication in human T-cell
leukemia. Cancer Res 2007, 67:5699-5707. 46. Lum AM, Wang BB, Li L, Channa N, Bartha G, Wabl M: Retroviral
activation of the mir-106a microRNA cistron in T lym-
phoma. Retrovirology 2007, 4:5. 28. Tura O, Barclay GT, Roddie H, Davies J, Turner ML: Absence of a
relationship between immunophenotypic and colony enu-
meration analysis of endothelial progenitor cells in clinical
haematopoietic cell sources. J Transl Med 2007 in press. p
gy
47. References Hess DA, Bonde J, Craft TP, Wirthlin L, Hohm S, Lahey R, Todt LM,
Dipersio JF, Devine SM, Nolta JA: Human progenitor cells rapidly
mobilized by AMD3100 repopulate NOD/SCID mice with
increased frequency in comparison to cells from the same 6. Dreger P, Haferlach T, Eckstein V, Jacobs S, Suttorp M, Loffler H,
Muller-Ruchholtz W, Schmitz N: G-CSF-mobilized peripheral Page 10 of 12
(page number not for citation purposes) Page 10 of 12
(page number not for citation purposes) Journal of Translational Medicine 2008, 6:39 http://www.translational-medicine.com/content/6/1/39 http://www.translational-medicine.com/content/6/1/39 39. donor mobilized by granulocyte colony stimulating factor. Biol
Blood
Marrow
Transplant
2007,
13:398-411. 39. Gramantieri L, Ferracin M, Fornari F, Veronese A, Sabbioni S, Liu CG,
Calin GA, Giovannini C, Ferrazzi E, Grazi GL, Croce CM, Bolondi L,
Negrini M: Cyclin G1 is a target of miR-122a, a microRNA fre-
quently down-regulated in human hepatocellular carcinoma. Cancer Res 2007, 67:6092-6099. donor mobilized by granulocyte colony stimulating factor. Biol
Blood
Marrow
Transplant
2007,
13:398-411. p
23. Fruehauf S, Seeger T, Maier P, Li L, Weinhardt S, Laufs S, Wagner W,
Eckstein V, Bridger G, Calandra G, Wenz F, Zeller WJ, Goldschmidt
H, Ho AD: The CXCR4 antagonist AMD3100 releases a sub-
set of G-CSF-primed peripheral blood progenitor cells with
specific gene expression characteristics. Exp Hematol 2006,
34:1052-1059. 40. 40. Galardi S, Mercatelli N, Giorda E, Massalini S, Frajese GV, Ciafre SA,
Farace MG: miR-221 and miR-222 expression affects the pro-
liferation potential of human prostate carcinoma cell lines by
targeting p27kip1. J Biol Chem 2007. 24. Lang P, Bader P, Schumm M, Feuchtinger T, Einsele H, Fuhrer M,
Weinstock C, Handgretinger R, Kuci S, Martin D, Niethammer D,
Greil J: Transplantation of a combination of CD133+ and
CD34+ selected progenitor cells from alternative donors. Br
J Haematol 2004, 124:72-79. 41. Volinia S, Calin GA, Liu CG, Ambs S, Cimmino A, Petrocca F, Visone
R, Iorio M, Roldo C, Ferracin M, Prueitt RL, Yanaihara N, Lanza G,
Scarpa A, Vecchione A, Negrini M, Harris CC, Croce CM: A micro-
RNA expression signature of human solid tumors defines
cancer gene targets. Proc Natl Acad Sci U S A 2006, 103:2257-2261. J
25. References Fulci V, Chiaretti S, Goldoni M, Azzalin G, Carucci N, Tavolaro S, Cas-
tellano L, Magrelli A, Citarella F, Messina M, Maggio R, Peragine N,
Santangelo S, Mauro FR, Landgraf P, Tuschl T, Weir DB, Chien M,
Russo JJ, Ju J, Sheridan R, Sander C, Zavolan M, Guarini A, Foa R, Mac-
ino G: Quantitative technologies establish a novel microRNA
profile of chronic lymphocytic leukemia. Blood 2007,
109:4944-4951. p
J
p
29. Wang E, Miller LD, Ohnmacht GA, Liu ET, Marincola FM: High-fidel-
ity mRNA amplification for gene profiling. Nat Biotechnol 2000,
18:457-459. 30. Wang E, Miller LD, Ohnmacht GA, Mocellin S, Perez-Diez A,
Petersen D, Zhao Y, Simon R, Powell JI, Asaki E, Alexander HR, Duray
PH, Herlyn M, Restifo NP, Liu ET, Rosenberg SA, Marincola FM: Pro-
spective molecular profiling of melanoma metastases sug-
gests classifiers of immune responsiveness. Cancer Res 2002,
62:3581-3586. 48. Lawrie CH, Soneji S, Marafioti T, Cooper CD, Palazzo S, Paterson JC,
Cattan H, Enver T, Mager R, Boultwood J, Wainscoat JS, Hatton CS:
Microrna expression distinguishes between germinal center
B cell-like and activated B cell-like subtypes of diffuse large B
cell lymphoma. Int J Cancer 2007, 121:1156-1161. y
p
J
,
49. Si ML, Zhu S, Wu H, Lu Z, Wu F, Mo YY: miR-21-mediated tumor
growth. Oncogene 2007, 26:2799-2803. 31. Eisen MB, Spellman PT, Brown PO, Botstein D: Cluster analysis
and display of genome-wide expression patterns. Proc Natl
Acad Sci U S A 1998, 95:14863-14868. g
g
50. Lee EJ, Gusev Y, Jiang J, Nuovo GJ, Lerner MR, Frankel WL, Morgan
DL, Postier RG, Brackett DJ, Schmittgen TD: Expression profiling
identifies microRNA signature in pancreatic cancer. Int J Can-
cer 2007, 120:1046-1054. 32. Dennis G Jr., Sherman BT, Hosack DA, Yang J, Gao W, Lane HC,
Lempicki RA: DAVID: Database for Annotation, Visualization,
and Integrated Discovery. Genome Biol 2003, 4:3. 51. Tran N, McLean T, Zhang X, Zhao CJ, Thomson JM, O'Brien C, Rose
B: MicroRNA expression profiles in head and neck cancer cell
lines. Biochem Biophys Res Commun 2007, 358:12-17. g
y
33. Choong ML, Yang HH, McNiece I: MicroRNA expression profil-
ing during human cord blood-derived CD34 cell erythropoi-
esis. Exp Hematol 2007, 35:551-564. 34. Georgantas RW III, Hildreth R, Morisot S, Alder J, Liu CG, Heimfeld
S, Calin GA, Croce CM, Civin CI: CD34+ hematopoietic stem-
progenitor cell microRNA expression and function: A circuit
diagram of differentiation control. References Proc Natl Acad Sci U S A 2007. 52. Calin GA, Dumitru CD, Shimizu M, Bichi R, Zupo S, Noch E, Aldler
H, Rattan S, Keating M, Rai K, Rassenti L, Kipps T, Negrini M, Bullrich
F, Croce CM: Frequent deletions and down-regulation of
micro- RNA genes miR15 and miR16 at 13q14 in chronic
lymphocytic leukemia. Proc Natl Acad Sci U S A 2002,
99:15524-15529. g
35. Liao R, Sun J, Zhang L, Lou G, Chen M, Zhou D, Chen Z, Zhang S:
MicroRNAs play a role in the development of human hemat-
opoietic stem cells. J Cell Biochem 2008, 104(3):805-817. 53. 53. Calin GA, Liu CG, Sevignani C, Ferracin M, Felli N, Dumitru CD,
Shimizu M, Cimmino A, Zupo S, Dono M, Dell'Aquila ML, Alder H,
Rassenti L, Kipps TJ, Bullrich F, Negrini M, Croce CM: MicroRNA
profiling reveals distinct signatures in B cell chronic lym-
phocytic leukemias. Proc Natl Acad Sci U S A 2004,
101:11755-11760. 36. Garzon R, Pichiorri F, Palumbo T, Iuliano R, Cimmino A, Aqeilan R,
Volinia S, Bhatt D, Alder H, Marcucci G, Calin GA, Liu CG, Bloomfield
CD, Andreeff M, Croce CM: MicroRNA fingerprints during
human megakaryocytopoiesis. Proc Natl Acad Sci U S A 2006,
103:5078-5083. 54. Bottoni A, Piccin D, Tagliati F, Luchin A, Zatelli MC, degli Uberti EC:
miR-15a and miR-16-1 down-regulation in pituitary adeno-
mas. J Cell Physiol 2005, 204:280-285. 37. Felli N, Fontana L, Pelosi E, Botta R, Bonci D, Facchiano F, Liuzzi F,
Lulli V, Morsilli O, Santoro S, Valtieri M, Calin GA, Liu CG, Sorrentino
A, Croce CM, Peschle C: MicroRNAs 221 and 222 inhibit nor-
mal erythropoiesis and erythroleukemic cell growth via kit
receptor down-modulation. Proc Natl Acad Sci U S A 2005,
102:18081-18086. J
y
55. Garzon R, Pichiorri F, Palumbo T, Visentini M, Aqeilan R, Cimmino A,
Wang H, Sun H, Volinia S, Alder H, Calin GA, Liu CG, Andreeff M,
Croce CM: MicroRNA gene expression during retinoic acid-
induced differentiation of human acute promyelocytic leuke-
mia. Oncogene 2007, 26:4148-4157. 38. He H, Jazdzewski K, Li W, Liyanarachchi S, Nagy R, Volinia S, Calin
GA, Liu CG, Franssila K, Suster S, Kloos RT, Croce CM, de la CA:
The role of microRNA genes in papillary thyroid carcinoma. Proc Natl Acad Sci U S A 2005, 102:19075-19080. 56. 57.
Khandekar M, Brandt W, Zhou Y, Dagenais S, Glover TW, Suzuki N,
Shimizu R, Yamamoto M, Lim KC, Engel JD: A Gata2 intronic
enhancer confers its pan-endothelia-specific regulation.
Development 2007, 134:1703-1712. Kipps T, Croce CM: Tcl1 expression in chronic lymphocytic
leukemia is regulated by miR-29 and miR-181. Cancer Res
2006, 66:11590-11593. p
58.
Lugus JJ, Chung YS, Mills JC, Kim SI, Grass J, Kyba M, Doherty JM,
Bresnick EH, Choi K: GATA2 functions at multiple steps in
hemangioblast development and differentiation. Development
2007, 134:393-405. Kipps T, Croce CM: Tcl1 expression in chronic lymphocytic
leukemia is regulated by miR-29 and miR-181. Cancer Res
2006, 66:11590-11593. References Pekarsky Y, Santanam U, Cimmino A, Palamarchuk A, Efanov A, Maxi-
mov V, Volinia S, Alder H, Liu CG, Rassenti L, Calin GA, Hagan JP, Page 11 of 12
(page number not for citation purposes) Page 11 of 12
(page number not for citation purposes) http://www.translational-medicine.com/content/6/1/39 http://www.translational-medicine.com/content/6/1/39 http://www.translational-medicine.com/content/6/1/39 Journal of Translational Medicine 2008, 6:39 57. Khandekar M, Brandt W, Zhou Y, Dagenais S, Glover TW, Suzuki N,
Shimizu R, Yamamoto M, Lim KC, Engel JD: A Gata2 intronic
enhancer confers its pan-endothelia-specific regulation. Development 2007, 134:1703-1712. p
58. Lugus JJ, Chung YS, Mills JC, Kim SI, Grass J, Kyba M, Doherty JM,
Bresnick EH, Choi K: GATA2 functions at multiple steps in
hemangioblast development and differentiation. Development
2007, 134:393-405. Publish with BioMed Central and every
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Teachers' self-efficacy in dealing with students' online learning difficulties: A study of psychopragmatics in Indonesian language learning
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Teachers' self-efficacy in dealing with students'
online learning difficulties: A study of
psychopragmatics in Indonesian language learning Submitted: June 4, 2022; Accepted: February 22, 2023; Published: February 27, 2023 * Corresponding author: mamad_r76@staff.uns.ac.id efikasi,
kesulitan
pembelajar,
psikopragmatik,
kelas daring KEYWORDS efficacy,
learning
difficulties,
psychopragmatics,
online class Efficacy is teachers’ ability to strengthen their self-motivation to help solve learning prob-
lems faced by their students. In the learning process, psychopragmatic mastery is expected
to strengthen the psychological condition of teachers and students through informative,
manipulative, and persuasive sentences so that each student can optimize learning tools
to help them achieve learning goals. During the pandemic, it is important to examine
teachers’ self-efficacy and psychopragmatics mastery to support students in addressing
their online learning difficulties. A qualitative study to investigate these issues was there-
fore conducted. Participants of this study were teachers from all over Indonesia, consisting
of 350 middle and high school teachers. Data were collected through (1) a survey of teacher
efficacy during online learning, (2) observations of the forms of efficacy built by the teach-
ers during learning activities and outside the classroom, (3) interviews with the teachers
to identify forms of efficacy used to shape students' learning fundamentals. It was found
that the teachers tended to increase their efficacy through strong lesson planning. In ad-
dition, they often modified learning tools so that the multimedia used could foster enthu-
siasm and learning motivation for both themselves and the students. The teacher's efficacy
in teaching is the key to analysis in psychopragmatic studies because data in the form of
snippets of teacher speech can influence student motivation. Psychopragmatics in the
teaching and learning process is not only considered the psychology of language that is
practiced in learning the Indonesian language, but teachers also have internalized psycho-
logical reinforcement in the learning activity that has been planned, implemented, and
assessed Available online at
http://journal2.um.ac.id/index.php/jbs
P-ISSN: 0854-8277, E-ISSN: 2550-0635
Volume 51, Number 1, February 2023, 13–26
Teachers' self-efficacy in dealing with students'
online learning difficulties: A study of
psychopragmatics in Indonesian language learning
Efikasi diri guru dalam menghadapi kesulitan
pembelajaran daring siswa: Sebuah studi
psikopragmatik dalam pembelajaran bahasa
Indonesia
Muhammad Rohmadi a *
, Memet Sudaryanto b
a Universitas Sebelas Maret, Indonesia
b Universitas Jenderal Soedirman, Indonesia
Submitted: June 4, 2022; Accepted: February 22, 2023; Published: February 27, 2023 Available online at
http://journal2.um.ac.id/index.php/jbs
P-ISSN: 0854-8277, E-ISSN: 2550-0635
Volume 51, Number 1, February 2023, 13–26
Teachers' self-efficacy in dealing with students'
online learning difficulties: A study of
psychopragmatics in Indonesian language learning
Efikasi diri guru dalam menghadapi kesulitan
pembelajaran daring siswa: Sebuah studi
psikopragmatik dalam pembelajaran bahasa
Indonesia
Muhammad Rohmadi a *
, Memet Sudaryanto b
a Universitas Sebelas Maret, Indonesia
b Universitas Jenderal Soedirman, Indonesia
Submitted: June 4, 2022; Accepted: February 22, 2023; Published: February 27, 2023 Available online at
http://journal2.um.ac.id/index.php/jbs
P-ISSN: 0854-8277, E-ISSN: 2550-0635
Volume 51, Number 1, February 2023, 13–26 Available online at
http://journal2.um.ac.id/index.php/jbs
P-ISSN: 0854-8277, E-ISSN: 2550-0635
Volume 51, Number 1, February 2023, 13–26 Teachers' self-efficacy in dealing with students'
online learning difficulties: A study of
psychopragmatics in Indonesian language learning Introduction The COVID-19 pandemic has a systemic and prolonged impact. For the past three
years, learning activity has been conducted online, and the policies taken by the central
government have continued to change following the COVID-19 trend in Indonesia. Every element of education needs to continue improving and updating the learning of
soft and hard skills to keep up with the changing times (Alqahtani, 2015; Nystrand &
Gamoran, 1991; Utomo et al., 2020). Every policy change taken by the regional govern-
ment is a derivative of central government policies, but most teachers are also confused
about keeping up with these policy changes to be applied as standard processes and
expected learning outcomes. Each policy made involves complexity and demands. Teachers with several cyclical and endless tasks must simultaneously convert every
emotion inside them into energy to improve the quality of learning and motivate them-
selves into forming positive affect that students will feel directly or indirectly. It reflects
handling a sense of crisis during this pandemic, including how to learn. Teachers pre-
vent the stress and fatigue of the students from having to move from school to their
homes (Klapproth et al., 2020). In addition, teachers should not experience stress and
fatigue during learning because it can make the teaching and learning process less ef-
fective. The teacher's psychological needs are one of the supports for achieving learning in-
dicators. These demands and needs add to the teacher's central function, especially in
determining learning objectives following the implementation of learning. The de-
mands and challenges faced by teachers during online learning continue to increase
and become more complex. As part of teaching competency standards, teachers are re-
quired to formulate both curricular and extracurricular learning objectives which are
supposed to be achieved by the students. The demands faced by teachers are increasingly complex, followed by increasingly
limited learning facilities, especially online. Students with increasingly diverse charac-
teristics expect teachers to be more creative and innovative in utilizing every learning
media they use (Carmody, 2005). Various forms of improvised learning development
that can be done include (1) modifying, replacing, and improving learning media that
have been used, (2) searching for the latest learning resources that can be accessed by
students anywhere and anytime, (3) applying various multimedia-based learning de-
vices, and (4) increasing the central role of students as learning actors in the learning
activity. How to cite this article: How to cite this article:
Rohmadi, M. & Sudaryanto, M. (2023). Teachers 'self-efficacy in dealing with students' online learning difficulties:
A study of psychopragmatics in Indonesian language learning. Bahasa dan Seni: Jurnal Bahasa, Sastra, Seni, dan
Pengajarannya, 51(1), 13–26. https://dx.doi.org/10.17977/um015v51i12023p13 KATA KUNCI efikasi,
kesulitan
pembelajar,
psikopragmatik,
kelas daring Efikasi adalah kemampuan guru untuk memperteguh motivasi diri sendiri dalam mem-
bantu memecahkan masalah belajar yang dihadapi siswanya. Dalam proses pembelaja-
ran, penguasaan psikopragmatik diharapkan dapat memperkuat kondisi psikologis guru
dan siswa melalui kalimat-kalimat yang informatif, manipulatif, dan persuasif sehingga
setiap siswa dapat mengoptimalkan perangkat pembelajaran untuk membantu mereka
mencapai tujuan pembelajaran. Di masa pandemi, penting untuk mengkaji efikasi diri
guru dan penguasaan psikopragmatik untuk mendukung siswa dalam mengatasi
kesulitan belajar daring mereka. Oleh karena itu, sebuah studi kualitatif untuk menyeli-
diki masalah ini dilakukan. Partisipan penelitian ini adalah para guru yang tersebar di
seluruh Indonesia, terdiri atas 350 guru SMP dan SMA. Pengumpulan data dilakukan
melalui (1) survei efikasi guru selama pembelajaran daring, (2) observasi terhadap bentuk
efikasi yang dibangun oleh guru selama kegiatan pembelajaran dan di luar kelas, (3) wa-
wancara dengan guru untuk mengetahui bentuk efikasi yang digunakan. untuk memben-
tuk dasar belajar siswa. Berdasarkan data ditemukan bahwa guru cenderung meningkat-
kan keefektifannya melalui perencanaan pembelajaran yang kuat. Selain itu, mereka 14 Rohmadi, Sudaryanto, Teachers ' self-efficacy in dealing with students' … sering memodifikasi perangkat pembelajaran agar multimedia yang digunakan dapat me-
numbuhkan semangat dan motivasi belajar baik bagi diri sendiri maupun bagi siswa. Ef-
ikasi guru dalam mengajar menjadi kunci analisis dalam kajian psikopragmatik karena
data berupa potongan tuturan guru dapat mempengaruhi motivasi belajar siswa. Psiko-
pragmatik dalam proses belajar mengajar tidak hanya mempertimbangkan psikologi ba-
hasa yang dipraktikkan dalam pembelajaran bahasa Indonesia, tetapi keterampilan guru
dalam menginternalisasi penguatan psikologis pada kegiatan pembelajaran yang telah
direncanakan, dilaksanakan, dan dinilai. Introduction On the other hand, this condition continues to burden the teachers. It is be-
cause they also have additional burden of administrative responsibilities. 15 Bahasa dan Seni: Jurnal Bahasa, Sastra, Seni, dan Pengajarannya, 51(1), (2023), 13–26 Online learning can be interpreted as limiting access to education. Education that
usually occurs with direct interaction between different elements (teachers, adminis-
trators, and students) turns into learning with indirect interaction (Kansanen, 1999;
Maeda et al., 2014). Restrictions on direct interaction in the education field sometimes
occur in specific situations but not in the context of social restrictions, such as what
people do to prevent the spread of the virus. Conventionally, offline learning usually
uses worksheets, printed teaching materials, and learning media on television and ra-
dio to help the activities. Offline learning can be understood as media that do not use
the Internet network. This media can be alternative learning media during a pandemic
due to inadequate online learning conditions (Picard & Velautham, 2016). At the same
time, the online learning does not involve face-to-face interaction but uses the Internet
network, the activities of which use devices or gadgets such as laptops, computers, or
smartphones. Online learning also uses platforms that have been provided, for exam-
ple, through online learning applications (Sudaryanto et al., 2020; Utomo et al., 2020). All forms of the instructional materials are delivered online, and communication is car-
ried out online. Even the test is also done online without having to do face-to-face meet-
ings. In other words, both teachers and students need extra motivation and support for
the success of learning. The requirements that teachers have to meet, if not followed by efforts to increase
competence, can lead to depression. Furthermore, there are added complexities in a
way that (1) students are increasingly diverse in their cognitive, psychomotor, and af-
fective abilities, (2) learning materials designed by the ministry are increasingly com-
plex, (3) learning process standards are continuously being improved. (4) demands of
the students' thinking skills are higher, and (5) online learning demands more effort,
technology mastery, and learning designs that are interesting and dynamic. Introduction In the education system in Indonesia, there is a term known as PKB (Pengembangan
Keprofesian Berkelanjutan or Continuous Professional Development), which is a work-
ing system in which teachers are required to continue to productively improve their
competence according to their profession in writing academic papers and conducting
classroom action research and other forms of development. This indicates that the
teacher must continue to be active in (1) understanding the characteristics of students
in terms of physical, social, cultural, emotional, intellectual aspects, (2) understanding
the students' backgrounds, (3) continuously updating the way teachers teach to identify
student learning difficulties, (4) facilitating the development of the potential of stu-
dents, and (5) evaluating the process and learning outcomes to improve the curriculum
in their schools. In terms of conducting online learning, teachers are ultimately re-
quired to convert learning materials and media into digital forms and use technology
in the teaching and learning process. With the emergence of the pandemic situation currently, teachers' workload is in-
creasing. The teaching and learning process is still focused on completing the curricu-
lum, but the teaching duration is reduced and teachers have limited capacity in using
modules that are interesting and fun for students. With all the requirements to make
innovations, teachers' work hours are longer than before the pandemic, from eight
hours to 12 hours every day after the pandemic with the online learning (Kolb & Kolb,
2009). There is also another limitation regarding parents’ support in accompanying Rohmadi, Sudaryanto, Teachers ' self-efficacy in dealing with students' … 16 their children because they have to work. Teachers are much pressured by the demands
of the students' parents, curriculum demands, and the moral burden of a less than the
optimal learning process. Many teachers at higher-grade levels feel that students do
not have sufficient provisions, including attitudes that do not represent a form of active
learning (Ismaili, 2021). The demands from different parties certainly make teachers
experience a crisis of confidence and guilt. The lack of teachers' creativity will undoubtedly affect the direction and objectives
of the learning process. Less consistent learning directions can result in the low
achievement of learning indicators (Fuadiah et al., 2019; Koh, 2019; Williams, 2004). One of the subjects that still requires much improvement is the Indonesian language. Introduction There are some complaints from the students about the learning activities of the Indo-
nesian language subject, such as: Material
The instructional material used is less contextual
and too theoretical, so the students lack practice
in the four language skills. In literature material,
students tend to be only given the task to read and
write, but they do not really discuss and review
literary works, which is more interesting to study. Media Innovation
Most of the learning media used are power points
so that students are required to keep reading the
material while the teacher explains it during the
class. Learning Interaction
Teachers still position themselves as the center of
the learning activity so that students are consid-
ered objects that must listen and become passive,
which in the end, the basic language skills are not
measured accurately and adequately. Classroom Management
Classroom management, such as the selection of
methods, the interaction between students to stu-
dents and students to teachers, and students with
the learning environment, is still very minimal. There is a lack of learning environment optimi-
zation that represents language learning materi-
als. In the efficacy studies, teachers are expected to deal with and solve problems effec-
tively. As part of resilience, efficacy is a psychological study that can effectively improve
self-confidence and increase control over oneself. Resilience is a person's capacity to
accept, face, and transform the problems that s/he faces. Resilience can help someone
deal with and overcome difficult situations and can be used to maintain their life's qual-
ity (Niiya et al., 2004; Sterling, 2010). The seven components of resilience are emotional
regulation, impulse control, optimism, problem-solving ability, empathy, self-efficacy,
and achievement (Semeraro et al., 2019; William, 2013). Resilience is influenced by in-
ternal factors, including cognitive abilities, gender, attachment to culture, and external
factors from family and community. When someone is resilient, that person can control
their emotions, behavior, and attention in dealing with problems. This study does not only focus on locutionary speech acts, which play a role in con-
veying information as the teacher's main speech activity. In addition, this research will
also identify the teacher's speech in preparation for learning and the possibilities it will Bahasa dan Seni: Jurnal Bahasa, Sastra, Seni, dan Pengajarannya, 51(1), (2023), 13–26 17 take when implementing the lesson. Introduction Therefore, the purpose of this study is (1) to de-
scribe the forms of speech that occur in the learning process, (2) to describe the distri-
bution of speech acts in learning, and (3) to describe the speech related to teachers’
efficacy in learning. Research conducted by Insani and Sabardila (2016) shows that perlocutionary
speech acts carried out by teachers in classroom learning can impact student psychol-
ogy during or even after the learning process. Meanwhile, Rahmat et al. (2019) stated
that the psychopragmatics of oral speech could affect the expected derivative results in
carrying out the language process. This study complements the two studies. One nov-
elty aspect of this research is to analyze teachers' efficacy in the teaching and learning
process during the pandemic. This psychopragmatic study manifests in teacher efficacy
in classroom management. The utterances produced by the teacher during learning
show motivational words and things that affect the teacher's and students' psychologi-
cal condition. The form of efficacy produced by the teacher is likely to cause the teacher's
speech to be conveyed more easily to students. Method This study used a qualitative approach. Research instruments were in the form of
(1) a survey of teacher efficacy during online learning, (2) observations of the forms of
efficacy built by the teachers during learning activities and outside the classroom, and
(3) interviews with the teachers to identify forms of efficacy used to shape students'
learning fundamentals. The subjects in this study were Indonesian language teachers
at the secondary school level, namely SMP (Junior High School), SMA (Senior High
School), and SMK (Vocational High School). A total of 350 teachers took part in the
survey, and eight teachers were interviewed about their forms of self-efficacy. The data were in the form of transcripts of the interviews and field notes of the ob-
servations as well as the teacher behavior survey results. Triangulation of sources and
methods to validate the data was carried out to produce reliable data. The data were
analyzed using interactive data analysis techniques by Miles and Huberman namely
data collection, analysis, reduction, and concluding (Miles & Huberman, 1992). Teacher
efficacy instrument data were analyzed using a qualitative survey approach: percep-
tions, interpretations, generalizations, and conclusions. Furthermore, through pattern
diagrams, the tendency of the teacher's answers is mapped regarding the choice of
words, the use of greetings, and personal approaches during learning. Teacher efficacy
instrument data were analyzed using a qualitative survey approach: perceptions, inter-
pretations, generalizations, and conclusions. Furthermore, through pattern diagrams,
the tendency of the teacher's answers is mapped regarding the choice of words, the use
of greetings, and personal approaches during online classes. Learning observation data
consists of lesson plans, video recordings, and student learning experiences outside
online classes. Recordings made via zoom media were transcribed and analyzed start-
ing from the communication built by the teacher, student responses, and the expres-
sions both raised. The results of this analysis reflect the observation instruments con-
structed through a psychopragmatic perspective. After reviewing the results of the ef-
ficacy survey and mapping the forms of efficacy in the learning process, interviews were Rohmadi, Sudaryanto, Teachers ' self-efficacy in dealing with students' … 18 conducted with teachers to review, confirm and ensure various forms of strengthening
efficacy carried out by teachers. conducted with teachers to review, confirm and ensure various forms of strengthening
efficacy carried out by teachers. Results The results of the survey filled out by the teachers indicated a decrease in the teach-
er's motivation to teach. The teacher changed the definition of learning, which initially
increased attitudes, knowledge, and skills to [deliver teaching material only]. Survey
results are supported by learning planning in online classes. The teacher keeps the RPP
(lesson plan), which should adapt to the hybrid learning objectives. According to the
interviews with teachers, teachers do not make lesson plans because of their anxiety
about the pressure students would suffer. In addition, based on the teacher's speech in
class, many words refer to negative charisma. At the beginning of learning, the teacher
does not motivate students; instead, the teacher allows students to ask questions in
other classes or read books themselves. Based on the teacher's utterances, working sen-
tences in lesson plans, and imperative sentences delivered in online classes, some data
refers to the trends below. Table 1. The tendency of weak teacher motivation
No. Aspect
Indicators
1. Teachers experience bore-
dom in designing Indone-
sian language learning
that they will teach. a. Emotional exhaustion: teachers are easily distracted
emotionally
[kenapa saya harus bikin RPP baru; toh anak-anak
juga tidak belajar] (en: [why do I have to make a new
lesson plan; students don't learn anyway]
[tidak perlu muluk-muluk, siswa bisa baca buku
sendiri] (en: Students can read the book by themselve
[Santai saja, siswa pasti diajari orang tuanya.] (en:
Take it easy, students must be taught by their par-
ents.)
b. Excessive fear when learning does not run as it should
Learning objectives should be readjusted, but teachers
don't know how to adjust them either. c. Worry if the indicator is not reached
Teachers reduce learning indicators. In addition, teach
ers often feel let down by students. At the pandemic's
start, teachers prepared lessons as they should, but
students were busy with themselves. Finally, the
teacher did not make any lesson planning. 2. Teachers experience cog-
nitive fatigue in develop-
ing learning tools
a. Feeling hopeless for suboptimal learning
[Saya sudah pasrah] (en: I've given up)
[Saya merasa siswa sudah tidak ingin belajar]
(en: I feel that students don't want to study anymore)
[Saya juga fokus pada keselamatan diri saya sendiri]
(en: I also focus on my own safety)
b. 2.
Teachers experience cog-
nitive fatigue in develop-
ing learning tools Results Feeling hopeless for suboptimal learning
[Saya sudah pasrah] (en: I've given up)
[Saya merasa siswa sudah tidak ingin belajar]
(en: I feel that students don't want to study anymore)
[Saya juga fokus pada keselamatan diri saya sendiri]
(en: I also focus on my own safety) Bahasa dan Seni: Jurnal Bahasa, Sastra, Seni, dan Pengajarannya, 51(1), (2023), 13–26 19 Table 1. The tendency of weak teacher motivation (Continuation)
No. Aspect
Indicators
c. Feeling frustrated when developing material
Based on the results of instrument analysis, in the les-
son planning sub-chapter, it was found (1) the teacher
was confused with the map of learning material; (2) the
teacher does not know the extent of students' under-
standing of the teaching material; (3) the teacher wants
to provide simple material but is afraid that students
will take it for granted; and (4) the teacher finally cop-
ies and cuts material from the internet. 3. Teachers lose motivation
in starting learning
a. Teachers do not get support from the learning environ-
ment so that learning does not achieve the goals accord-
ing to the plan that has been designed
b. Teachers are bored, so they cannot explore internal
solid motivation to start learning activity Table 1. The tendency of weak teacher motivation (Continuation) Results Feeling hopeless for suboptimal learning
[Saya sudah pasrah] (en: I've given up)
[Saya merasa siswa sudah tidak ingin belajar]
(en: I feel that students don't want to study anymore)
[Saya juga fokus pada keselamatan diri saya sendiri]
(en: I also focus on my own safety) Table 1. The tendency of weak teacher motivation
No. Aspect
Indi
1. Teachers experience bore-
dom in designing Indone-
sian language learning
that they will teach. a. Emotional exhaustion: tea
emotionally
[kenapa saya harus bikin
juga tidak belajar] (en: [w
lesson plan; students don'
[tidak perlu muluk-muluk
sendiri] (en: Students can
[Santai saja, siswa pasti d
Take it easy, students mu
ents.)
b. Excessive fear when learn
Learning objectives shoul
don't know how to adjust
c. Worry if the indicator is no
Teachers reduce learning
ers often feel let down by
start, teachers prepared le
students were busy with t
teacher did not make any
2. Teachers experience cog-
nitive fatigue in develop-
ing learning tools
a. Feeling hopeless for subop
[Saya sudah pasrah] (en:
[Saya merasa siswa sudah
(en: I feel that students do
[Saya juga fokus pada kes
(en: I also focus on my ow
b. Feeling hopeless for subop
[Saya sudah pasrah] (en:
[Saya merasa siswa sudah
(en: I feel that students do
[Saya juga fokus pada kes
(en: I also focus on my ow Table 1. The tendency of weak teacher motivation b. Excessive fear when learning does not run as it should
Learning objectives should be readjusted, but teachers
don't know how to adjust them either. c. Worry if the indicator is not reached
Teachers reduce learning indicators. In addition, teach-
ers often feel let down by students. At the pandemic's
start, teachers prepared lessons as they should, but
students were busy with themselves. Finally, the
teacher did not make any lesson planning. a. Feeling hopeless for suboptimal learning
[Saya sudah pasrah] (en: I've given up)
[Saya merasa siswa sudah tidak ingin belajar]
(en: I feel that students don't want to study anymore)
[Saya juga fokus pada keselamatan diri saya sendiri]
(en: I also focus on my own safety) b. Indicators Based on Table 1, teachers need to be more interested in online learning. However,
from the results of interviews, teachers sometimes still hope for students majoring in
science. Teachers' efficacy strategies to develop fun learning include being more respon-
sive to the development of Science, Technology, and the Arts, which are internalized in
the Indonesian language and literature learning. Finally, curriculum adaptation is de-
veloped based on realizing that science, technology, and the arts are developing dynam-
ically. Therefore, during the pandemic, the spirit and the curriculum provide a learning
experience for students to follow and take advantage of scientific developments through
technology. The Indonesian language learning substances include all dimensions of competence,
scientific study field, and subjects that are planned and presented on an ongoing basis
between all levels of education and adapt to the pandemic conditions. Learning activity
is done face-to-face, limited face-to-face, and/or distance learning online and offline
(Amirian et al., 2008; Dubey et al., 2020). These activities are carried out to provide a
meaningful learning experience for students without being burdened with the demands
of completing all curriculum achievements for grade promotion and graduation. Learn-
ing activity is developed creatively and innovatively in optimizing the growth of stu-
dents' critical, creative, communicative, and collaborative skill. The teachers prioritize
the essential subject matter in the learning activity, while students can study other
materials independently. Learning materials are taken, collected, and developed from
(1) sourcebooks such as student books, teacher manuals, and other books or literature
related to the appropriate and correct scope. The books and teaching materials are
packaged in a language that is motivating, full of language power, and able to demon-
strate personal excellence without discriminating against students; (2) things related
to life and/or related to contextual social phenomena, for example, related to the Covid-
19 pandemic or other things that are happening around students but the topics chosen
should have contexts such as healing status, and news about the pandemic, vaccina-
tions, and other good news. Teachers minimize boredom by developing learning models and prioritizing lan-
guage skills: collaborating oral and written skills. In oral skills such as listening and
speaking, students must be more active in conveying their ideas and attainments Rohmadi, Sudaryanto, Teachers ' self-efficacy in dealing with students' … 20 (Sudaryanto et al., 2019; Suwandi & Sudaryanto, 2021; Zhang, 2018). Indicators Learning design
in improving productive skills is conducted using a scientific-based approach in the
form of learning models, such as the discovery learning model, the inquiry learning
model, the inquiry-based learning model, the project-based learning model, problem-
based learning, and other learning models that allow students to learn actively and
creatively. Grotberg (2003) explains that three aspects of resilience include [i have], [i
am], and [i can]. Aspects of internal support owned by the teacher can be a solid impetus
to solve problems. In addition, assistance from outside sources can be a strength for
teachers. Therefore, social competence can be realized by teachers through strong [i
can] aspects. The study results of the three aspects of resilience can be grouped in Table
2. Table 2. Dimensions and teacher efficacy in learning activity
Factor
Dimension
Teachers' Efficacy in Learning Activity
[I am]
Strengths that are
built from within
the teachers such
as behavior,
feelings, and beliefs
related to the
learning process
a. They felt proud when planning, implementing, and
assessing the teaching and learning process carried
out by students and other teachers. Teachers can
identify themselves through lesson development
activities. b. Feeling loved and having an attractive attitude so
students and teachers can feel comfortable during
learning. The role of the teacher is increasingly
visible in attitude management when facing different
responses during interaction with students and with
other teachers
c. Love, empathy and altruism was shown by the
teacher by caring to others, students, and fellow
teachers. Teachers can show they care about
everything that happens in a comfortable learning
environment. d. Teachers can show a sense of independence and
responsibility for the learning process and outside the
classroom. [I have]
External factors
that influence
teachers' attitudes
a. The structure and rules of the school both during
learning and outside of learning activities are
available. Teachers have rules that students must
obey, as well as penalties and warnings if these rules
are not implemented. b. Role Models are people who can show what teachers
should do, such as information on something, and
encourage students to follow it
c. Having relationships, besides support from the closest
people, is also to build good relationships between
teachers, between students, and students and
teachers. [I can]
teachers' social and
interpersonal
competence
a. Teachers' Efficacy in Learning Activity c. Teachers can assess communication skills and
understand feelings. Directly or indirectly, teachers
need to get a sense of security in communicating with
students and building good trust from various parties. Using motivational sentences can show the teacher's ability to construct self-confi-
dence. The teacher uses favorable terms, so students feel free from the subject, even in
class. Teachers use positive affirmations to build courage in planning lessons. In lesson
planning, teachers need to be bolder in giving confidence to students to manage them-
selves. Therefore, using [i am, I can, and I have] increasingly shows the relationship
between students and teachers. Therefore, the turning point of a teacher's efficacy is
using language in planning motivational learning for both himself and his students. On
the other hand, the teachers choose a method that allows learning objectives in pan-
demic conditions using 'magical' words to convey material during the learning process. In receptive skills, teachers creatively develop active learning methods adapted to the
characteristics of the material/theme and the characteristics of the situation in pan-
demic conditions. Learning activities and tasks during the learning period from home
are carried out in various ways between students, according to their respective interests
and conditions, including considering the gap in access/availability of learning facilities
at home. The provision of learning assignments is carried out by considering the concept
of learning from home, namely as an effort to break the chain of the spread of Covid-
19, it is inevitable that the workload assigned to students can be completed without
leaving the house and maintaining health, as well as sufficient resting time to support
students' immunity. Indicators Managing various feelings and stimuli from both
other teachers and students so that the teachers
indirectly and continuously recognize stimuli and all
kinds of emotions then show them in the form of
words or behavior and actions. b. Students are looking for a trustworthy relationship so
that teachers can gain social and interpersonal
competencies that have started to fade and disappear. Table 2. Dimensions and teacher efficacy in learning activity 21 Bahasa dan Seni: Jurnal Bahasa, Sastra, Seni, dan Pengajarannya, 51(1), (2023), 13–26 Table 2. Dimensions and teacher efficacy in learning activity (Continuation)
Factor
Dimension
Teachers' Efficacy in Learning Activity
c. Teachers can assess communication skills and
understand feelings. Directly or indirectly, teachers
need to get a sense of security in communicating with
students and building good trust from various parties. d. Measuring the temperament of oneself and others
during learning takes place with various
measurements that have been made by the teacher
both formally and informally. The teachers'
sensitivity in assessing the measurement results can
develop their self-efficacy. e. Through good feeling management, the teacher can
develop the ability to solve problems that other
teachers and students will face both in class and
outside the classroom. Teachers need to communicate,
measure the surrounding environment, and translate
the measurement results into a psycholinguistic
assessment based on the actual situation. Discussion Effective learning involves teachers and students providing mutual support so that
the achievement of learning indicators can be measured and achieved. In developing
learning efficacy, the teacher is directed to focus on the students’ potential, develop-
ment, needs, and interests, and their environment. In online learning, teachers adapt
the curriculum developed based on the principle that students have a central position
to develop their competencies to become human beings who are faithful and pious, no-
ble, healthy, knowledgeable, capable, creative, independent, democratic, and responsi-
ble citizens (Habe & Ahiruddin, 2017). The scope of learning a language is a medium of Rohmadi, Sudaryanto, Teachers ' self-efficacy in dealing with students' … 22 communication that builds teacher perceptions, whether intentional or unintentional. In learning, the teacher's function is increasingly important, even though, on the other
hand, the teacher feels demotivated during the pandemic. Every teacher has control to plan learning activities to act as an educator. Based on
the data, changes in the tendency for teacher self-confidence can also be seen from the
learning motivation map. Although on the other hand, the teacher realizes that using
words that motivate learning can increase his confidence. Teachers must understand
the different psychological characteristics of their students. It often happens in the
classroom that symptoms of attention disorders are psychological factors experienced
by students in class; this must be recognized and understood by teachers as the educa-
tors in the classroom to prevent and overcome learning difficulties faced by students in
the process of participating in the classroom. Psycho-pragmatics in education is not only considered the psychology of language
practiced in the learning activity. Educational psychology is the knowledge that has the
right to live on its own because certain aspects of educational psychology are philosoph-
ical (Courage & Richards, 2008). However, as a science, educational psychology has its
structure and principles or fundamental truths, objective facts, and valuable techniques
for research. To support the achievement of these objectives, the development of student
competencies is adjusted to the potential, development, needs, interests of students,
and demands from society. Having a central position means that learning activities are
student-centered. Indonesian language teachers have a role in building strong self-confidence through
language in the classroom. In addition, teachers must provide efforts to prevent and
overcome problems faced by their students and themselves during the planning and
implementation of classroom learning (Leech, 2014; Pianta & Hamre, 2009). Discussion Teachers
should use learning methods and strategies that can attract the learning attention so
that teachers themselves can feel very comfortable from the beginning to the end of the
class. Online learning creates new learning patterns that also come with new obstacles in
communication, limited space, students' passive attitudes, lack of students' observa-
tion, and less particular learning object that creates an impossibility to study without
media, remote learning places, and others. In psychopragmatic studies, words have a
magical power to evoke learning interactions (Hodges, 2018; Reyes, 1990). In order to
make the teaching and learning process work well, students should be invited to use all
their senses. When words are presented as narrative, auditory channels can be used for
word processing. At the same time, visual channels can be used for processing images. In this way, the load is balanced between the two lines so that no one line is over-
loaded. Psychopragmatics can bridge constructive thinking processes through forms of
learning designed by the teacher in the classroom (Rahardi, 2017; Rohmadi et al.,
2021). Psychopragmatic studies in learning activity can describe learning needs, such
as showing an imbalance between the learning needs of every member in the learning
environment, the encouragement needed through constructive words, and goals that
describe the form of mentality in learning that is adaptive but can support the learning
indicators formulated by the teacher. 23 Bahasa dan Seni: Jurnal Bahasa, Sastra, Seni, dan Pengajarannya, 51(1), (2023), 13–26 In psychopragmatics, every action taken, including learning, is caused by an im-
pulse. The impulse comes from within the individual to achieve a goal called motivation. Someone's motivation depends on the strength of his motives, which he wants to
achieve (Ball, 2010; Draper, 2014). Here, the motives are the individual's need, desire,
impulse, or urge. In other words, motivation moves someone to act in a certain way, or
at least develop certain things. In efficacy, language that appears and arises in learning
is a form of self-statement that can sometimes be expressed or cannot be expressed. Research data on the distribution of speech acts in learning shows that locutionary
speech acts are more common than illocutionary and perlocutionary speech acts in the
learning implementation process. Based on the analysis results, it can be understood
that the teacher does more direct speech or there is no hidden meaning behind the
speech. Discussion Speech acts are undoubtedly different from illocutions which emphasize indi-
rectly that the speech partner does something according to the intent conveyed by the
speaker. This can happen because the teacher in the online learning process reveals
more/conveys a piece of information which, in the process, the information conveyed
can become new knowledge. Based on mapping the speech acts used by the teacher,
they use three of five forms of speech acts in class: statements, interrogative, and im-
perative (Searle 1979; assertive, directive, commissive, expressive, declarative). The
teacher uses these three speech acts to strengthen students' desires, namely (1) state-
ment (declarative), which serves to say something to others to pay attention, and (2)
statement (interrogative), which functions to ask something so that the listener an-
swers the question asked, and (3) the command (imperative), which aims to get the
listener to respond to the action or action requested. Similar to the statement above, using speech acts to motivate students is expressed
using locutions. Often, the statements used by the teacher are a collection of words that
directly impact the psychology of himself and his students. Locutions in psychoprag-
matic studies in Indonesian language learning refer to an informative action that de-
scribes the ideal and desired learning process by the teachers following the learning
atmosphere in online classes but still prioritizes the achievement of student competen-
cies (Abbot-Smith et al., 2020; Ryabova, 2015). Every learning activity that refers to
the illocutionary act is behind the locutionary action. In learning Indonesian, the
teacher shows the perlocutionary form in a speech that intends to work according to the
hidden intent of an utterance inserted by the teacher so that students act as expected
or there is an effect from the speech. The data explains that the strength of speech acts
uttered by the teacher can affect students' effectiveness: locutionary speech acts are
widely used to provide psychological solid injections. The research data shows that the distribution of perlocutionary speech acts is more
prevalent in instructional planning. This can happen because teachers tend to develop
better lesson plans so that the learning process can run smoothly. These expectations
are implemented in speech acts contained in the implementation of learning so that
they can emphasize the speech effects that will occur (Rohmadi, 2014). Discussion In addition, this
can be understood from the perspective of learning design that teachers have rarely
faced, namely digital learning design. The process of change has an impact on teachers
in planning learning. Learning activity that is carried out by teachers directly can make
(1) learning as a process of encouraging students to act by moving motor skills and Rohmadi, Sudaryanto, Teachers ' self-efficacy in dealing with students' … 24 building energy, (2) determining teachers' actions through constructive, active learning
forms, (3) selecting good points and not good in the learning process so that every atti-
tude in the class can be built through the formation of active learning according to
learning indicators (Sudaryanto, et al: 2020). These forms form the contextuality of ef-
ficacy carried out by teachers during learning both inside and outside the classroom. Conclusions In this study, the teachers chose the essential subjects to be prioritized in language
learning to optimize the form of speech acts to support the mentality of both teachers
and students. Based on the research data and analysis results, it can be understood
that teachers experience (1) cognitive fatigue in developing learning tools, (2) boredom
in designing Indonesian language learning to be taught, and (3) loss of motivation to
start learning. This can be overcome with teacher efficacy strategies to develop fun
learning and adapt to science, technology, and art development. In addition, the results
show that (1) the distribution of speech acts in the lesson planning carried out by the
teacher uses more perlocutionary speech acts, (2) the distribution of speech acts in the
implementation of learning uses more locutionary speech acts, and (3) the distribution
of speech acts in the assessment process uses more locutionary speech acts. The use of
locutionary speech acts is more than perlocutionary and illocutionary acts. It is caused
by the learning process, which conveys information that can become new knowledge
because teachers tend to choose direct speech or speech which does not contain hidden
meanings. Our study has also found that teacher's efficacy in teaching is the key to
analysis in psychopragmatic studies because data in the form of snippets of teacher
speech can influence student motivation. We have shown that psychopragmatics in the
teaching and learning process is not only considered the psychology of language that is
practiced in learning the Indonesian language, but teachers also have internalized psy-
chological reinforcement in the learning activity that has been planned, implemented,
and assessed. References Abbot-Smith, K., Williams, D., & Matthews, D. (2020). ‘What’s new for you?’: Interlocutor-spe-
cific perspective-taking and language interpretation in autistic and neuro-typical children. Research
in
Autism
Spectrum
Disorders,
70,
101465. https://doi.org/10.1016/j.rasd.2019.101465 Alqahtani, M. (2015). The importance of vocabulary in language learning and how to be taught. International
Journal
of
Teaching
and
Education,
III(3),
21-34
https://doi.org/10.20472/te.2015.3.3.002 Amirian, Z., Kassaian, Z., & Tavakoli, M. (2008). Genre analysis: An investigation of the discus-
sion sections of applied linguistics research articles. The Asian ESP Journal, 4(1), 39-63. Ball, J. (2010). An analysis of teacher self-efficacy, teacher trust, and collective efficacy in a south-
west Texas school district. Texas A&M University. Carmody, D. P. (2005). Psychometric characteristics of the Beck Depression Inventory-II with
college students of diverse ethnicity. International Journal of Psychiatry in Clinical Practice,
9(1), 22–28. Courage, M. L., & Richards, J. E. (2008). Attention. In M. M. Haith & J. B. Benson (Eds.), En-
cyclopedia
of
infant
and
early
childhood
development
(pp. 106-117). https://doi.org/10.1016/B978-012370877-9.00013-X 25 Bahasa dan Seni: Jurnal Bahasa, Sastra, Seni, dan Pengajarannya, 51(1), (2023), 13–26 de Beaugrande, R. (2006). Critical discourse analysis: History, ideology, methodology. Stu
in Language and Capitalism, 1, 29-56. Draper, J. (2014). Teacher self-efficacy: Internalized understandings of competence. In S. N. Phillipson, K. Y. L. Ku, & S. Phillipson (Eds.), Constructing educational achievement (pp. 84–
98). Routledge. Dubey, S., Biswas, P., Ghosh, R., Chatterjee, S., Dubey, M. J., Chatterjee, S., Lahiri, D., & Lavie,
C. J. (2020). Psychosocial impact of COVID-19. Diabetes and Metabolic Syndrome: Clinical
Research and Reviews, 4(5), 779–788. https://doi.org/10.1016/j.dsx.2020.05.035 Forrest-Lawrence, P. (2019). Case study research. In P. Liamputtong (Ed.), Handbook of re-
search
methods
in
health
social
sciences
(pp. 317-331). Springer
Singapore. https://doi.org/10.1007/978-981-10-5251-4_67 Fuadiah, N. F., Suryadi, D., & Turmudi. (2019). Teaching and learning activities in classroom
and their impact on student misunderstanding: A case study on negative integers. Interna-
tional Journal of Instruction, 12(1), 407–424. Grotberg, E. H. (Ed.). (2003). Resilience for today: Gaining strength from adversity. Greenwood
Publishing Group. Habe, H., & Ahiruddin, A. (2017). Sistem Pendidikan Nasional. Ekombis Sains: Jurnal
Ekonomi, Keuangan Dan Bisnis, 2(1), 39-45. Hodges, C. B. (2018). Self-efficacy in instructional technology contexts. Springer. Ismaili, Y. (2021). Evaluation of students’ attitude toward distance learning during the pan-
demic (Covid-19): A case study of ELTE university. On the Horizon, 29(1),17-30. http://dx.doi.org/10.1108/OTH-09-2020-0032 Kansanen, P. (1999). Teaching as teaching‐studying‐learning interaction. Scandinavian Jour-
nal of Educational Research, 43(1), 81–89. References Klapproth, F., Federkeil, L., Heinschke, F., & Jungmann, T. (2020). Teachers’ experiences of
stress and their coping strategies during COVID-19 induced distance teaching. Journal of
Pedagogical Research, 4(4), 444–452. Koh, J. H. L. (2019). Four pedagogical dimensions for understanding flipped classroom practices
in higher education: A systematic review. Educational Sciences: Theory & Practice, 19(4), 14–
33. Kolb, A. Y., & Kolb, D. A. (2009). Experiential learning theory: A dynamic, holistic approach to
management learning, education and development. In S. J. Armstrong & C. V. Fukami (Eds.),
The SAGE handbook of management learning, education and development (pp. 42-68). Sage. https://dx.doi.org/10.4135/9780857021038 Leech,
G. (2014). The
pragmatics
of
politeness. Oxford
University
Press. https://doi.org/10.1093/acprof:oso/9780195341386.001.0001 Maeda, G., Ewerton, M., Lioutikov, R., Amor, H. Ben, Peters, J., & Neumann, G. (2014). Learn-
ing interaction for collaborative tasks with probabilistic movement primitives. 2014 IEEE-
RAS International Conference on Humanoid Robots, 527–534. https://doi.org.10.1109/HU-
MANOIDS.2014.7041413 Miles, M. B., & Huberman, M. A. (1992). Analisis data kualitatif: Buku sumber tentang metode-
metode baru. In Universitas Indonesia_UI Press. Ed: Rohendi & Mulyarto. Niiya, Y., Crocker, J., & Bartmess, E. N. (2004). From vulnerability to resilience: Learning ori-
entations buffer contingent self-esteem from failure. Psychological Science, 15(12), 801–805. Nystrand, M., & Gamoran, A. (1991). Instructional discourse, student engagement, and litera-
ture achievement. Research in the Teaching of English, 25(3), 261-290. Pianta, R. C., & Hamre, B. K. (2009). Conceptualization, measurement, and improvement of
classroom processes: Standardized observation can leverage capacity. Educational Re-
searcher, 38(2), 109-119. https://doi.org/10.3102/0013189X09332374 Picard, M., & Velautham, L. (2016). Developing independent listening skills for English as an
additional language students. International Journal of Teaching and Learning in Higher Ed-
ucation, 28(1), 52–65. Rohmadi, Sudaryanto, Teachers ' self-efficacy in dealing with students' … 26 ahardi, K. (2017). Linguistic impoliteness in the sociopragmatic perspective. Humanior
29(3), 309-315. https://doi.org/10.22146/jh.v29i3.24954 Reyes, P. (1990). Teachers and their workplace: Commitment, performance, and productivity. Sage Publications. Rohmadi, M., Sadhhono, K., & Sudaryanto, M. (2021). A creative library management in digital
era in Indonesia from psychopragmatic perspective. Webology, 18(Special Issue on Infor-
mation Retrieval and Web Search), 332–343. Rohmadi, M. (2014). Kajian pragmatik percakapan guru dan siswa dalam pembelajaran bahasa
Indonesia. Paedagogia, 17(1), 53-61. Ryabova, M. (2015). Politeness strategy in everyday communication. Procedia - Social and Be-
havioral Sciences, 206, 90–95. https://doi.org/10.1016/j.sbspro.2015.10.033 Searle, R. J. (1979). Expression and meaning: Studies in the theory of speech acts. Cambridge
University Press. References Semeraro, C., Coppola, G., Cassibba, R., & Lucangeli, D. (2019). Teaching of cursive writing in
the first year of primary school: Effect on reading and writing skills. PLoS ONE, 14(2),
e0209978. https://doi.org/10.1371/journal.pone.0209978 Sterling, S. (2010). Learning for resilience, or the resilient learner? Towards a necessary recon-
ciliation in a paradigm of sustainable education. Environmental Education Research, 16(5-
6), 511–528. Sudaryanto, M., Mardapi, D., & Hadi, S. (2019). Multimedia-based online test on Indonesian
language receptive skills development. Journal of Physics: Conference Series, 1339(1). https://doi.org/10.1088/1742-6596/1339/1/012120 Sudaryanto, Memet, Ulya, C., Rohmadi, M., & Kuhafeesah, K. (2020). Inter-rater assessment
on listening media for foreign language speakers. Proceedings of the 2nd Konferensi BIPA
Tahunan by Postgraduate Program of Javanese Literature and Language Education in Col-
laboration with Association of Indonesian Language and Literature Lecturers, KEBIPAAN,
9 November, 2019, Surakarta, Central Java, Indonesia. https://doi.org/10.4108/eai.9-11-
2019.2295064 Sudaryanto, M., Ulya, C., Rohmadi, M., & Wulansari, K. (2020, May). Learning trajectory: Re-
actions of prospective teachers looking for solutions to learning problems. In B-SPACE 2019:
Proceedings of the First Brawijaya International Conference on Social and Political Sciences,
BSPACE, 26-28 November, 2019, Malang, East Java, Indonesia (p. 7). European Alliance for
Innovation. Suwandi, S., & Sudaryanto, M. (2021). Benefits and challenges of learning Indonesian language
with an environmental system: An action research at high school in Surakarta. Psychology
and Education Journal, 58(2), 4403–4413. Utomo, M. N. Y., Sudayanto, M., & Saddhono, K. (2020). Tools and strategy for distance learning
to respond COVID-19 pandemic in Indonesia. Ingénierie Des Systèmes d’Information, 25(3),
383–390. William, D. (2013). Assessment : The bridge between teaching and learning. Voices from the
Middle, 21(2), 15-20. Williams, B. (2004). Self direction in a problem based learning program. Nurse Education Today,
24(4), 277–285. https://doi.org/10.1016/j.nedt.2004.01.008 Zhang, L. (2018). Reading and writing connections. In The TESOL Encyclopedia of English Lan-
guage Teaching. https://doi.org/10.1002/9781118784235.eelt0479
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Mapping citizens’ emotions: participatory planning support system in Olomouc, Czech Republic
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Journal of maps
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THE EMOTIONAL MAP OF OLOMOUC íjna
Pekaská
Kozí
Panská
Mahlerova
Lafayettova
9védská
Opletalova
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Hynaisova
Palackého
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Ol
Olomouc-Nová Ulice
17°18' E
17°17' E
17°16' E
17°15' E
17°14' E
17°13' E
17°12' E
49°37' N
49°36' N
49°35' N
49°34' N
17°18' E
17°17' E
17°16' E
17°15' E
17°14' E
17°13' E
17°12' E
49°37' N
49°36' N
49°35' N
49°34' N
49°37' N
49°36' N
49°35' N
49°34' N
49°37' N
49°36' N
49°35' N
49°34' N
European Union
European social fund
Location of The Czech Republic within Europe
Location of Olomouc within The Czech
15° E
15° E
13° E
13° E
51° N
50° N
49° N
48° N
30° E
20° E
10° E
0°
60° N
60° N
50° N
50° N
40° N
40° N
0°
20° W
20° E
40° E
10° W
10° E
30° E
50° E
train station
bus station
tram stop
tram rail
railway
highway
I. class road
II. class road
III. class road
street
service road
walkways
city border
protected area
streems
rivers and lakes
forest
buildings
cemetery
army area
Base map
train station
bus station
tram stop
tram rail
railway
highway
I. class road
II. class road
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street
service road
walkways
city border
protected area
streems
rivers and lakes
forest
buildings
cemetery
army area
extreme
high value
middle value
low value
Pleasant area
W
Where do you feel unsafe? Which areas should be improved re
Which is a pleasant public space? Which places should be improved
© Journal of Maps, 2018
Which is a pleasant public space? Which areas should be improved regarding public transport? Which places should be improved regarding walkability? Where do you feel unsafe? Which areas should be improved regarding public transport? Which is a pleasant public space? Which is a pleasant public space? Gorazdovo nám. Polská
t. Svobody
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8. THE EMOTIONAL MAP OF OLOMOUC THE EMOTIONAL MAP OF OLOMOUC Participatory mapping of citizens´ perceptions in urban environment (Olomouc in 2016)
Jií PÁNEK Jií PÁNEK
Department of Development and Environmental Studies, Faculty of Science, Palacký University Olomouc, Czech Republic
© Journal of Maps 2018 55
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Pasteurova
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Ka!parova
Tylova
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Karolíny Sv*tlé
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Reme!ova
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R3eny Svobodové
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nám. VídeDská
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Tr
Kapucínská
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Havelkova
Mlýnská
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Wellnerova
Kollárovo nám. Dobrovs
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Ka!parova
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Tylova
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9vermova
Slovenská
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Zámenická
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Kaení
Hanáckého pluku
Helceletova
Pekární
U botanické zahrady
Reme!ova
Dánská
Vanurova
Mariánská
Komenského
Bezruova
Franklinova
R3eny Svobodové
Erbenova
U výpadu
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Barvíská
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Mlochova
Broíkova
Blahoslavova
Divadelní
Boeny N*mcové
Pöttingova
Dómská
Marie Pospí!ilové
Var!avské nám. Aksamitova
Ne!verova
Krakovská
Aksamitova
Akademická
Legionáská
Wurmova
Polská
Blahoslavova
Ztracená
Riegrova
Kivá
Kateinská
Ostrunická
Denisova
28. íjna
Pekaská
Kozí
Panská
Hrníská
Mahlerova
Lafayettova
9védská
Opletalova
9kolní
Pavelákova
Uhelná
Univerzitní
Purkrabská
Koeluská
Trnice
Kapucínská
8. kv*tna
Michalská
9emberova
Kerotínovo n. Na hrad*
Havelkova
Mlýnská
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Výstavit Flora
Námstí
Republiky
Olomouc-Smetanovy sady
Olomouc-Nová Ulice
Which areas should be improved regarding public transport? Gorazdovo nám. Polská
t. Svobody
t. Kosmonaut3
17. listopadu
9títného
Masarykova tída
Sokolovská
Holická
Havlíkova
Husova
Vejdovského
Wolkerova
Legionáská
17. listopadu
t. Svobody
Tovární
Vejdovského
Wittgensteinova
Biskupské nám. Horní
nám*stí
Dolní
nám*stí
Palachovo
nám. VídeDská
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Sokolská
1. máje
t. Spojenc3
9meralova
Wellnerova
Nábeí
Dukelská
8. kv*tna
Boivojova
Nezvalova
Mlýnská
Boleslavova
Praskova
U místní dráhy
Babíkova
Koeluská
U stadiónu
9antova
Ka!parova
Domovina
Kavalerist3
Tylova
U reálky
Zamenhofova
Kateinská
Javoíská
Charkovská
Kubíkova
Karolíny Sv*tlé
Kosinova
Wanklova
Grégrova
Kíkovského
Jiího z Pod*brad
U soutoku
9vermova
Slovenská
Lolkova
Zámenická
Lafayettova
náb. Pemyslovc3
Vodární
Nerudova
Pasteurova
Myslbekova
Kaení
Hanáckého pluku
Helceletova
Pekární
U botanické zahrady
Reme!ova
Dánská
Vanurova
Mariánská
Komenského
Bezruova
Franklinova
R3eny Svobodové
Erbenova
U výpadu
U Husova sboru
Barvíská
U hradeb
Mlochova
Broíkova
Blahoslavova
Divadelní
Boeny N*mcové
Pöttingova
Dómská
Marie Pospí!ilové
Var!avské nám. Aksamitova
Ne!verova
Krakovská
Aksamitova
Akademická
Legionáská
Wurmova
Polská
Blahoslavova
Ztracená
Riegrova
Kivá
Kateinská
Ostrunická
Denisova
28. íjna
Pekaská
Kozí
Panská
Hrníská
Mahlerova
Lafayettova
9védská
Opletalova
9kolní
Pavelákova
Uhelná
Univerzitní
Purkrabská
Koeluská
Trnice
Kapucínská
8. kv*tna
Michalská
9emberova
Kerotínovo n. Na hrad*
Havelkova
Mlýnská
Ka!parova
Smetanovy sady
Na stelnici
Wellnerova
Dobrovského
Kollárovo nám. Václavské nám. Dobrovského
Studentská
Hynaisova
Palackého
Komenského
Galerie antovka
)i kova kasárna
Kasárna 9. THE EMOTIONAL MAP OF OLOMOUC kvtna
Teplárna
Smetanovy sady
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Vejdovského
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Okresní soud
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Msto
Námstí
Hrdin:
)i kovo námstí
Výstavit Flora
Námstí
Republiky
Olomouc-Smetanovy sady
Olomouc-Nová Ulice
Which areas should be improved regarding public transport? Gorazdovo nám. Polská
t. Svobody
t. Kosmonaut3
17. listopadu
9títného
Masarykova tída
Sokolovská
Holická
Havlíkova
Husova
Vejdovského
Wolkerova
Legionáská
17. listopadu
t. Svobody
Tovární
Vejdovského
Wittgensteinova
Biskupské nám. Horní
nám*stí
Dolní
nám*stí
Palachovo
nám. VídeDská
Krapkova
Sokolská
1. máje
t. Spojenc3
9meralova
Wellnerova
Nábeí
Dukelská
8. kv*tna
Boivojova
Nezvalova
Mlýnská
Boleslavova
Praskova
U místní dráhy
Babíkova
Koeluská
U stadiónu
9antova
Ka!parova
Domovina
Kavalerist3
Tylova
U reálky
Zamenhofova
Kateinská
Javoíská
Charkovská
Kubíkova
Karolíny Sv*tlé
Kosinova
Wanklova
Grégrova
Kíkovského
Jiího z Pod*brad
U soutoku
9vermova
Slovenská
Lolkova
Zámenická
Lafayettova
náb. Pemyslovc3
Vodární
Nerudova
Pasteurova
Myslbekova
Kaení
Hanáckého pluku
Helceletova
Pekární
U botanické zahrady
Reme!ova
Dánská
Vanurova
Mariánská
Komenského
Bezruova
Franklinova
R3eny Svobodové
Erbenova
U výpadu
U Husova sboru
Barvíská
U hradeb
Mlochova
Broíkova
Blahoslavova
Divadelní
Boeny N*mcové
Pöttingova
Dómská
Marie Pospí!ilové
Var!avské nám. Aksamitova
Ne!verova
Krakovská
Aksamitova
Akademická
Legionáská
Wurmova
Polská
Blahoslavova
Ztracená
Riegrova
Kivá
Kateinská
Ostrunická
Denisova
28. íjna
Pekaská
Kozí
Panská
Hrníská
Mahlerova
Lafayettova
9védská
Opletalova
9kolní
Pavelákova
Uhelná
Univerzitní
Purkrabská
Koeluská
Trnice
Kapucínská
8. kv*tna
Michalská
9emberova
Kerotínovo n. Na hrad*
Havelkova
Mlýnská
Ka!parova
Smetanovy sady
Na stelnici
Wellnerova
Dobrovského
Kollárovo nám. Václavské nám. Dobrovského
Studentská
Hynaisova
Palackého
Komenského
Galerie antovka
)i kova kasárna
Kasárna 9. kvtna
Teplárna
Smetanovy sady
"echovy sady
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U Dómu
Tr nice
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antovka
Palackého
Wolkerova
U Byst7i$ky
Vejdovského
U Sv. Mo7ice
U Sv. Mo7ice
Okresní soud
Nádra í
Msto
Námstí
Hrdin:
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Výstavit Flora
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Republiky
Olomouc-Smetanovy sady
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p
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p Which places should be improved regarding walkability? Which places should be improved regarding walkability?
|
https://openalex.org/W1677864759
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https://parasitesandvectors.biomedcentral.com/track/pdf/10.1186/s13071-015-1076-y
|
English
| null |
Baseline susceptibility to alpha-cypermethrin in Lutzomyia longipalpis (Lutz & Neiva, 1912) from Lapinha Cave (Brazil)
|
Parasites & vectors
| 2,015
|
cc-by
| 4,543
|
© 2015 Pessoa et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Given the increase in cases of visceral leishmaniasis in recent years, associated with the socio-economic
impact of this disease, as well as the wide distribution of Lutzomyia longipalpis in Brazil and the likelihood that this
vector may develop resistance to insecticides used for control, the Ministry of Health considers as crucial the creation
of a network in order to study and monitor the resistance of this vector to insecticides used for control. In this sense,
this study aimed: 1) to characterize the susceptibility of L. longipalpis from Lapinha Cave (Lagoa Santa, MG - Brazil) to
Alfateck SC200 in field bioassays, and 2) to define the susceptibility baseline to alpha-cypermethrin in laboratory
bioassays, checking the possibility of using it as susceptibility reference lineage (SRL). Findings: The field bioassays revealed that the tested population was highly susceptible to alpha-cypermethrin in all
time periods with high mortality (~100 %) in all treated surfaces before six months after spraying. In the laboratory
bioassays, the studied population presented LD50, LD95 and LD99 to 0.78013, 10.5580 and 31.067 mg/m2, respectively. The slope was 1.454121. Conclusions: The studied population of L. longipalpis was considered as adequate for SRL according criterion
recommended by Pan-American Health Organization and has proven susceptibility to tested insecticide in the field. One cannot rule out the possibility of finding populations of L. longipalpis more susceptible to alpha-cypermethrin;
therefore, further research is necessary on other populations with potential use as a SRL. rds: Lutzomyia longipalpis, Insecticide resistance, Alpha-cypermethrin, Susceptibility reference lineage There is increasing evidence for the importance of
vector control against leishmaniasis, especially for re-
ducing risks of transmission of VL [4–7]; recommended
measures include spraying households with pyrethroid in-
secticides [8]. However, these efforts have not been able to
prevent the geographical expansion and the rise in the
incidence and lethality of VL in Brazil. Furthermore,
although most sand fly species remain susceptible to all of
the major classes of insecticides in the world, there is in-
creasing evidence that some phlebotomine sand flies may
be developing insecticide resistance [9]. In many vector
species such as Neotropical phlebotomine sand flies, the
occurrence of insecticide resistance is still poorly studied. Insecticide resistance has not yet been demonstrated
in L. longipalpis but there are some indications of its
occurrence in this specie [10, 11]. Baseline susceptibility to alpha-cypermethrin
in Lutzomyia longipalpis (Lutz & Neiva, 1912)
from Lapinha Cave (Brazil) Grasielle Caldas DÁvila Pessoa1, Josiane Valadão Lopes2, Marília Fonseca Rocha3, Letícia C. Pinheiro1,
Aline Cristine Luiz Rosa1, Érika Monteiro Michalsky2 and Edelberto Santos Dias2* Pessoa et al. Parasites & Vectors (2015) 8:469
DOI 10.1186/s13071-015-1076-y Pessoa et al. Parasites & Vectors (2015) 8:469
DOI 10.1186/s13071-015-1076-y * Correspondence: edel@cpqrr.fiocruz.br
2Laboratório de Leishmanioses, Centro de Pesquisas René Rachou, Fundação
Oswaldo Cruz, Av. Augusto de Lima 1715, Barro Preto, 29190-002 Belo
Horizonte, MG, Brazil
Full list of author information is available at the end of the article Findings The slope and the lethal doses
required to kill 50 % (LD50), 95 % (LD95) and 99 %
(LD99) of treated individuals were estimated. During
the test, the temperature and humidity were con-
trolled (25 ºC ± 1 °C , 60 % ± 10 % RU). mortality was recovered 24 h after phlebotomine sand flies
exposition. A specimen was classified as dead if it was im-
mobile or unable to stand or fly in a coordinated way [14]. y
y [
]
In the laboratory, the insects were subjected to im-
pregnated paper with ten different alpha-cypermethrin
(BASF, Brazil) concentrations (0.1 to7.5 mg i.a./m2),
for one hour. For control tests, insects were exposed
to impregnated paper prepared only with vaseline oil
and chloroform [15]. At least three replicates of 30
females were tested per concentration. The mortality
was recovered 24 h after insects’ exposition. A speci-
men was classified as dead if it was immobile or un-
able to stand or fly in a coordinated way [13]. Data
from dose–response tests were analyzed using the
PROBIT program [16]. The slope and the lethal doses
required to kill 50 % (LD50), 95 % (LD95) and 99 %
(LD99) of treated individuals were estimated. During
the test, the temperature and humidity were con-
trolled (25 ºC ± 1 °C , 60 % ± 10 % RU). The phlebotomine sand flies were collected from
Lapinha cave (licence n° SISBIO 18728–1). HP traps
[13] were used for overnight indoor insect collection. After collection, the insects were allowed to settle for
a few hours for conditioning during which time they
were supplied with 10 % sucrose solution. In this sense,
we identify 30 % of the specimens per capture, as an in-
ternal control. The species identifications adopted in our
laboratory takes into account the taxonomic keys available
in Young and Ducan [1], as well as comparison with speci-
mens deposited at the Phlebotomine Sand fly Reference
Collection at CPqRR/Fiocruz. Results obtained in the field bioassays revealed that
the tested population was highly susceptible to alpha-
cypermethrin during all the time period. The mortality
was close to 100 % during the six months (Table 1). The
comparison of mortality observed in control and ex-
perimental DUs by the Kruskal-Wallis test revealed a
significant difference between at least two of the four
DUs (p = 2.2 × 10−16). Findings The Mann–Whitney post-test
showed a difference only between the control DU and
experimental DUs, proving that the mortality of phle-
botomine specimen was related to the presence of the
insecticide on the timeline, in the intradomicile and
peridomicile, in all types of surface tested. There was
no statistical difference in the mortality rate observed in
the intradomicile and peridomicile between different
surfaces and between different experimental DUs. Field bioassays were carried out in Montes Claros,
MG. The residents voluntarily signed a Statement of
Informed Consent. A sample was formed with three
domestic units (DUs), comparable in terms of type of
surface (adobe with plaster, adobe without plaster, wood)
and environmental characteristics, treated with alpha-
cypermethrin 20 % -Alfatek ® SC’(Rogama, Brazil). This is
the insecticide currently used by the Ministry of Health of
Brazil for phlebotomine sand fly control, with residual
effect expected for up to 6 months. Spraying was carried
out by agents of endemic diseases in the region, as recom-
mended by the Ministry of Health. A Guarani hand
sprayer was used attached to a Tee-jet 8002 nozzle. Testing was performed from July to December, 2014. At 1, 30, 60, 90, 120 and 150 days after spraying the
DUs, 30 females (fasting; parental generation) were
submitted to wall surface tests in plastic cones for
1 h. Then, the phlebotomine sand flies were trans-
ferred to insecticide-free bottles and kept in an insectary
with temperature and humidity controlled (25 ºC ± 1 ºC,
60 % ± 10 % RU). Three cones were exposed inside the
intradomicile and another three cones around in the peri-
domicile, of each surface type, per treated DU (experimen-
tal DUs – number 1,2,3). The insects of the control group
were exposed to insecticide-free surfaces of the intradomi-
cile and peridomicile (control DU – number 4). The The choice of the susceptibility reference lineage is a crit-
ical and fundamental step that precedes bioassays that
check insect resistance to insecticides. The Pan-American
Health Organization (PAHO) [17] defined a SRL as a strain
with more than five generations in the laboratory (without
contact with insecticides and inclusion of external mater-
ial), and/or one collected from an area that has never been
treated with insecticides. The phlebotomine sand flies from
Lapinha Cave have met this criterion, because this region
has had little anthropic interference; thus, they had not
been subjected to chemical control. Findings Lutzomyia
longipalpis
(Diptera,
Psychodidae)
has
a
broad distribution in Brazil and has been recorded in
North, Central and South America, from Mexico to
Argentina [1]. This phlebotomine sand fly is consi-
dered a vector for visceral leishmaniasis (VL), caused
by Leishmania (Leishmania) infantum. During the
urbanization of visceral leishmaniasis, from 1980 to
2005, 59,129 new cases of VL have been reported in
Brazil [2]. In 2013, 3117 new cases of VL were reported in
the whole national territory [3]. * Correspondence: edel@cpqrr.fiocruz.br
2Laboratório de Leishmanioses, Centro de Pesquisas René Rachou, Fundação
Oswaldo Cruz, Av. Augusto de Lima 1715, Barro Preto, 29190-002 Belo
Horizonte, MG, Brazil
Full list of author information is available at the end of the article Page 2 of 6 Pessoa et al. Parasites & Vectors (2015) 8:469 Therefore, considering the increase in VL cases in re-
cent years in Brazil and the consequent socio-economic
impact caused by this disease, the wide distribution of
L. longipalpis in the country and the likelihood that
this vector may develop resistance to insecticides used
for control, the Ministry of Health considers as cru-
cial the creation of a network in order to study and
monitor the resistance of this vector to insecticides [12]. In response to this need, this study aimed to characterize
the susceptibility of L. longipalpis from Lapinha Cave
(Lagoa Santa, MG - Brazil) within a timeline, to the in-
secticide currently being used for control of phlebotomine
sand flies in the country through field bioassays, as well as
define the susceptibility baseline of alpha-cypermethrin in
laboratory bioassays; checking the possibility of using it as
a susceptibility reference lineage (SRL). mortality was recovered 24 h after phlebotomine sand flies
exposition. A specimen was classified as dead if it was im-
mobile or unable to stand or fly in a coordinated way [14]. In the laboratory, the insects were subjected to im-
pregnated paper with ten different alpha-cypermethrin
(BASF, Brazil) concentrations (0.1 to7.5 mg i.a./m2),
for one hour. For control tests, insects were exposed
to impregnated paper prepared only with vaseline oil
and chloroform [15]. At least three replicates of 30
females were tested per concentration. The mortality
was recovered 24 h after insects’ exposition. A speci-
men was classified as dead if it was immobile or un-
able to stand or fly in a coordinated way [13]. Data
from dose–response tests were analyzed using the
PROBIT program [16]. Findings The high susceptibility
of this population of L. longipalpis to alpha-cypermethrin
was demonstrated in field bioassays in which high mortal-
ity was observed throughout the monitoring period. In the laboratory bioassays, the studied population
presented LD50, LD95 and LD99 to 0.78013, 10.5580 and
31.067 mg/m2, respectively. The slope was 1.454121. Such data is unprecedented in the literature and lethal
doses were defined for the first time in studies with Table 1 Mortality of L. longipalpis in response to exposure on different surfaces impregnated with alpha-cypermethrin 20 %
Domestic
Surfaces
1 day after spraying
30 days after spraying
60 days after spraying
Unit (DU)
Treated
Intradomicile
Peridomicile
Intradomicile
Peridomicile
Intradomicile
Peridomicile
Tested
Dead (%)
Tested
Dead (%)
Tested
Dead (%)
Tested
Dead (%)
Tested
Dead (%)
Tested
Dead (%)
DU 1a
Wood
30
28 (93)
30
23 (77)
30
30 (100)
30
30 (100)
30
30 (100)
30
30 (100)
Adobe with plaster
30
30 (100)
30
30 (100)
30
30 (100)
30
30 (100)
30
30 (100)
30
30 (100)
Adobe without plaster
30
30 (100)
30
29 (97)
30
30 (100)
30
30 (100)
30
30 (100)
30
27 (90)
Wood
30
29 (97)
30
29 (97)
30
30 (100)
30
30 (100)
30
30 (100)
30
30 (100)
DU 2a
Adobe with plaster
30
30 (100)
30
30 (100)
30
30 (100)
30
30 (100)
30
30 (100)
30
30 (100)
Adobe without plaster
30
30 (100)
30
30 (100)
30
29 (97)
30
27 (90)
30
28 (93)
30
30 (100)
Wood
30
30 (100)
30
30 (100)
30
30 (100)
30
30 (100)
30
30 (100)
30
30 (100)
DU 3a
Adobe with plaster
30
30 (100)
30
30 (100)
30
30 (100)
30
30 (100)
30
30 (100)
30
30 (100)
Adobe without plaster
30
30 (100)
30
30 (100)
30
30 (100)
30
25 (83)
30
30 (100)
30
27 (90)
DU 4b
Wood
30
1 (3)
30
0 (0)
30
0 (0)
30
0 (0)
30
0 (0)
30
0 (0)
Adobe with plaster
30
0 (0)
30
0 (0)
30
0 (0)
30
0 (0)
30
0 (0)
30
0 (0)
Adobe without plaster
30
0 (0)
30
0 (0)
30
0 (0)
30
0 (0)
30
0 (0)
30
0 (0) Pessoa et al. Parasites & Vectors (2015) 8:469 Table 1 Mortality of L. Comparison of mortality in the four domestic unit ; p = 2.2 × 10−16, Kruskal-Wallis test; p(DU1, DU2) = 0.77, p(DU
4) = 2.8 × 10−16, Kruskal-Wallis post-hoc tests (Mann–Whitney tests considering correction for multiple tests) Findings longipalpis in response to exposure on different surfaces impregnated with alpha-cypermethrin 20 %
Domestic
90 days after spraying
120 days after spraying
150 days after spraying
Unit (DU)
Intradomicile
Peridomicile
Intradomicile
Peridomicile
Intradomicile
Peridomicile
Tested
Dead (%)
Tested
Dead (%)
Tested
Dead (%)
Tested
Dead (%)
Tested
Dead (%)
Tested
Dead (%)
DU 1a
30
30 (100)
30
29 (97)
30
30 (100)
30
29 (97)
30
30 (100)
30
29 (97)
30
30 (100)
30
28 (93)
30
30 (100)
30
27 (90)
30
30 (100)
30
29 (97)
30
28 (93)
30
25 (83)
30
30 (100)
30
29 (97)
30
30 (100)
30
30 (100)
30
29 (97)
30
29 (97)
30
29 (97)
30
30 (100)
30
30 (100)
30
28 (93)
DU 2a
30
30 (100)
30
30 (100)
30
30 (100)
30
28 (93)
30
30 (100)
30
30 (100)
30
29 (97)
30
29 (97)
30
30 (100)
30
10 (33)
30
28 (93)
30
28 (93)
30
30 (100)
30
29 (97)
30
30 (100)
30
29 (97)
30
30 (100)
30
29 (97)
DU 3a
30
30 (100)
30
29 (97)
30
29 (97)
30
29 (97)
30
30 (100)
30
30 (100)
30
29 (97)
30
30 (100)
30
30 (100)
30
30 (100)
30
30 (100)
30
30 (100)
DU 4b
30
0 (0)
30
0 (0)
30
0 (0)
30
0 (0)
30
0 (0)
30
0 (0)
30
0 (0)
30
0 (0)
30
0 (0)
30
0 (0)
30
0 (0)
30
0 (0)
30
0 (0)
30
0 (0)
30
0 (0)
30
1 (3)
30
0 (0)
30
0 (0)
Comparison of mortality in the four domestic unit ; p = 2.2 × 10−16, Kruskal-Wallis test; p(DU1, DU2) = 0.77, p(DU 1, DU 3) = 0.45, p(DU 2, DU 3) = 0.32, p(DU 1, DU 4) = 4.8 × 10-16, p(DU 2, DU 4) = 4.9 × 10-16, p(DU3, DU
4) = 2.8 × 10−16, Kruskal-Wallis post-hoc tests (Mann–Whitney tests considering correction for multiple tests)
aExperimental group
bControl group of L. longipalpis in response to exposure on different surfaces impregnated with alpha-cypermethrin 20 % Pessoa et al. Parasites & Vectors (2015) 8:469 Page 5 of 6 Page 5 of 6 phlebotomine sand flies. Findings The LDs defined for the SRL
have a direct and immediate use if this population is used
as SRL for studies with L. longipalpis. The LD99 of
SRL is fundamental for defining a diagnostic dose (DD) -
a tool that allows the distinction of the field population
vigor against the SRL, killing the most susceptible speci-
mens and the minimally resistant specimens. In studies
with triatomines, the WHO [18] recommends the use of
DD = 1×LD99 and, for mosquitoes, 2×LD99 [15]. Because
research on phlebotomine sand flies is still incipient, fur-
ther studies are required in order to adapt this method-
ology to biological/physiological characteristics of L. longipalpis, thus defining cutoff points that are used to
foster understanding of susceptibility and resistance to
insecticides. LPC analyzed the data. GCDP and ESD wrote the manuscript. All authors
read and approved the final manuscript. LPC analyzed the data. GCDP and ESD wrote the manuscript. All authors
read and approved the final manuscript. Received: 18 June 2015 Accepted: 8 September 2015 Received: 18 June 2015 Accepted: 8 September 2015 Acknowledgements
Th
d This study was supported by the Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq), Centro de Pesquisa Rene Rachou (CPqRR),
Fundação Oswaldo Cruz (FIOCRUZ), Secretaria de Vigilância em Saúde (SVS)
and Ministério da Saúde (MS) - Brazil. We would also like to thank to BASF
Brazil for providing the insecticide used in laboratory bioassays and Msc. Newmar Pinto Marliéri for the support provided in the process of paper
sheet impregnation. Special thanks to Dra. Liléia Diotaiuti for permission to
perform bioassays in Laboratório de Referência em Triatomíneos e
Epidemiologia da Doença de Chagas – CPqRR/FIOCRUZ Minas, also for
valuable suggestions and comments. Competing interests
Th
h
d
l
h 12. Brasil. Ministério da Saúde. I Oficina da implantação da “Rede de
Monitoramento de Flebotomíneos a Inseticidas”. Resumo executivo,
Ministério da Saúde, Secretaria de Vigilância em Saúde, Brazilian Ministry of
Health. 2012. p. 4. 12. Brasil. Ministério da Saúde. I Oficina da implantação da “Rede de
Monitoramento de Flebotomíneos a Inseticidas”. Resumo executivo,
Ministério da Saúde, Secretaria de Vigilância em Saúde, Brazilian Ministry of
Health. 2012. p. 4. Author details
1 1Laboratório de Referência em Triatomíneos e Epidemiologia da Doença de
Chagas, Centro de Pesquisas René Rachou, Fundação Oswaldo Cruz, Belo
Horizonte, MG, Brazil. 2Laboratório de Leishmanioses, Centro de Pesquisas
René Rachou, Fundação Oswaldo Cruz, Av. Augusto de Lima 1715, Barro
Preto, 29190-002 Belo Horizonte, MG, Brazil. 3Centro de Controle de
Zoonoses de Montes Claros, Montes Claros, MG, Brazil. The other LDs characterized for SRL (e.g. LD50 and
LD95) may be used in the initial design of the dose–re-
sponse bioassays to study field populations of L. longipal-
pis. In addition, knowledge of the LD50 for SRL is
fundamental for calculation of the resistance ratio of 50 %
(RR50). This indicator, calculated by dividing the LD50 of
each field population by the LD50 value of the SRL, allows
the susceptibility status classification of the study field
population. Based on this classification, actions can be
taken for vector control strategies in the field. According
to PAHO [17] Triatominae populations with RR >5 are
considered resistant to the insecticide tested, whereas
populations of Aedes aegypti with RR >3 are considered
resistant [19]. For phlebotomine sand flies, this informa-
tion does not exist, which justifies the need for a screening
of various populations of L. longipalpis in the laboratory
and in the field bioassays, simultaneously, to define such
cut-off points. Zoonoses de Montes Claros, Montes Claros, MG, Brazil. Received: 18 June 2015 Accepted: 8 September 2015 References 1. Young DG, Duncan MA. Guide to the identification and geographic
distribution of Lutzomyia sandflies in Mexico, the West Indies, Central and
South America (Diptera: Psychodidae). Mem Am Entomol Inst. 1994;54(1):881. 2. Maia-Elkloruty ANS, Alves WA, Sousa-Gomes ML, Sena JM, Luna EA. Visceral
leishmaniasis in Brazil: trends and challenges. Cad Saude Publ, Rio de
Janeiro. 2008;24(12):2941–7. 2. Maia-Elkloruty ANS, Alves WA, Sousa-Gomes ML, Sena JM, Luna EA. Visceral
leishmaniasis in Brazil: trends and challenges. Cad Saude Publ, Rio de
Janeiro. 2008;24(12):2941–7. 3. Brasil. Ministério da Saúde. Sistema de Informação de Agravos de
Notificação. Leishmaniose visceral: casos notificados. http://
dtr2004.saude.gov.br/sinanweb Accessed 13 Sept 2015. 4. Alexander B, Usma MC, Cadena H, Quesada BL, Solarte Y, Roa W, et al. Evaluation of deltamethrin-impregnated bednets and curtains against
phlebotomine sandflies in Valle de la Cauca, Colombia. Med Vet Entomol. 1995;9:279–83. 5. Nadin A, Motabar M, Houshmand B, Keyghobady K, Aflatonian MR. Evaluation of pyrethroid impregnated bednets for control of anthroponotic
cutaneous leishmaniasis in Bam (Islamic Republic of Iran). WHO documents
WHO/LEISH/95.37. WHO, Geneva; 1995. The population from Lapinha Cave has been characte-
rized as one of the putative sibling species of L. longipalpis
[20], and it was considered as appropriate as a SRL for
laboratory bioassays because: 1) it meets the criteria for
SRL, recommended by PAHO and 2) it shows insecticide
susceptibility in the field on the timeline confirmed in
laboratory bioassays. However, one cannot rule out the
possibility of finding more susceptible populations of
L. longipalpis. This has already been verified with tria-
tomine studies [21]. This fact reinforces the need for
further research on other populations and putative sibling
species of L. longipalpis with potential to be used as SRL,
such as those from Jacobina (Bahia, Brazil) and Espírito
Santo do Pinhal (São Paulo, Brazil) [12, 20]. 6. Tayeh A, Jalouk L, Al-Khiami AM. A cutaneous leishmaniasis control trial
using pyrethroid-impregnated bednets in villages near Aleppo, Syria. WHO
document WHO/LEISH/97.41. WHO, Geneva; 1997. 7. Reyburn H, Ashford R, Mohsen M, Hewitt S, Rowland M. Randomized
controlled Trial insecticide-treated bednets and chaddars or top sheets and
residual spraying of interior rooms for the prevention of cutaneous
leishmaniasis in Kabul, Afghanistan. Trans R Soc Trop Med Hyg. 2000;94:361–6. 8. Brasil. Ministério da Saúde. Secretaria de Vigilância em Saúde. Departamento
de Vigilância Epidemiológica. Manual de controle da leishmaniose visceral:
normas e manuais técnicos. Brasília: Ministério da Saúde; 2006. 8. Brasil. Ministério da Saúde. 13.
Pugedo H, Barata RA, França-Silva JC, Silva JC, Dias ES. HP: um
modeloaprimorado de armadilha luminosa de sucção para a captura de
pequenos insetos. Rev Soc Bras Med Trop. 2005;38(1):70–2. References Secretaria de Vigilância em Saúde. Departamento
de Vigilância Epidemiológica. Manual de controle da leishmaniose visceral:
normas e manuais técnicos. Brasília: Ministério da Saúde; 2006. 8. Brasil. Ministério da Saúde. Secretaria de Vigilância em Saúde. Departamento
de Vigilância Epidemiológica. Manual de controle da leishmaniose visceral:
normas e manuais técnicos. Brasília: Ministério da Saúde; 2006. 9. Alexander B, Maroli M. Control of Phlebotomine sandflies. Med Vet Entomol. 2003;17:1–18. 9. Alexander B, Maroli M. Control of Phlebotomine sandflies. Med Vet Entomol. 2003;17:1–18. 10. Falcão AR, Pinto CT, Ferreira Gontijo CM. Susceptibility of Lutzomyia
longipalpis to deltamethrin. Mem Inst Oswaldo Cruz. 1988;83:395–6. 10. Falcão AR, Pinto CT, Ferreira Gontijo CM. Susceptibility of Lutzomyia
longipalpis to deltamethrin. Mem Inst Oswaldo Cruz. 1988;83:395–6. 11. Mazzarri MB, Feliciangeli MD, Maroli M, Hernandez A, Bravo A. Susceptibility
of Lutzomyia longipalpis (Diptera: Psychodidae) to selected insecticides in na
endemic focus of visceral leismaniasis in Venezuela. J American Mosquito
Control Association. 1997;13:335–41. 11. Mazzarri MB, Feliciangeli MD, Maroli M, Hernandez A, Bravo A. Susceptibility
of Lutzomyia longipalpis (Diptera: Psychodidae) to selected insecticides in na
endemic focus of visceral leismaniasis in Venezuela. J American Mosquito
Control Association. 1997;13:335–41. Competing interests
The authors declare that they have no competing interests. Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
All authors have contributed substantially to this study. GCDP and ESD
conceived and designed the experiments. MR contributed biologic material.
GCDP, JLV, EMM, ACLR, MFR and ESD performed the experiments. GCDP and 9.
Alexander B, Maroli M. Control of Phlebotomine sandflies. Med Vet Entomol.
2003;17:1–18. Pessoa et al. Parasites & Vectors (2015) 8:469 Pessoa et al. Parasites & Vectors (2015) 8:469 14. WHO. Instructions for determining the susceptibility or resistance of
blackflies, sandflies and biting midges to insecticides. WHO/VBC/81.810
1981; p.1-6. WHO, Geneva. 14. WHO. Instructions for determining the susceptibility or resistance of
blackflies, sandflies and biting midges to insecticides. WHO/VBC/81.810
1981; p.1-6. WHO, Geneva. blackflies, sandflies and biting midges to insecticides. WHO/VBC/81.810
1981; p.1-6. WHO, Geneva. 15. World Health Organization. Criteria and meaning of testes for determining
the susceptibility or resistance of insects to insecticides. VBC/81.6. 1981. 16. Finney JD. Probit analysis: statistical treatment of the sigmoid response
curve. Cambridge: Cambridge University Press; 1971. 17. Organización Panamericana de la Salud (PAHO). II Reunion técnica
latinoamericana de monitoreo de resistência a insecticidas em triatominos
vectores de Chagas, OPS. Panamá: PAHO; 2005. 18. World Health Organization (WHO). Taller sobre la evaluación de efecto
insecticida sobre triatominos. Workshop on the insecticide effect evaluation
in triatominos. Acta Toxicol Argentina. 1994;2:29–33. 19. Brasil. Ministério da Saúde. Secretaria de Vigilância em Saúde. Coordenação
geral do Programa Nacional do Controle de Dengue. Reunião Técnica para
discutir o status de resistência de Aedes aegypti aos inseticidas. Brasília:
Ministério da Saúde; 2006. p. 2. 20. Lins RM, Souza NA, Brazil RP, Maingon RD, Peixoto AA. Fixed differences in
the paralytic gene define two lineages within the Lutzomyia longipalpis
complex producing different types of courtship songs. PLoS One. 2012;7:e44323. 21. Pessoa GCD, Dias LS, Diotaiuti L. Deltamethrin pyrethroid susceptibility
characterization of Triatoma sordida Stal, 1859 (Hemiptera:Reduviidae)
populations in the Northern Region of Minas Gerais, Brasil. Rev Soc Bras
Med Trop. 2014;47(4):426–9. 17. Organización Panamericana de la Salud (PAHO). II Reunion técnica
latinoamericana de monitoreo de resistência a insecticidas em triatominos
vectores de Chagas, OPS. Panamá: PAHO; 2005. 17. Organización Panamericana de la Salud (PAHO). II Reunion técnica
latinoamericana de monitoreo de resistência a insecticidas em triatominos
vectores de Chagas, OPS. Panamá: PAHO; 2005. g
18. World Health Organization (WHO). Taller sobre la evaluación de efecto
insecticida sobre triatominos. Workshop on the insecticide effect evaluation
in triatominos. Acta Toxicol Argentina. 1994;2:29–33. 18. World Health Organization (WHO). Taller sobre la evaluación de efecto
insecticida sobre triatominos. Workshop on the insecticide effect evaluation
in triatominos. Acta Toxicol Argentina. 1994;2:29–33. 19. Brasil. Ministério da Saúde. Secretaria de Vigilância em Saúde. Coordenação
geral do Programa Nacional do Controle de Dengue. Reunião Técnica para
discutir o status de resistência de Aedes aegypti aos inseticidas. Authors’ contributions
All
h
h
ib All authors have contributed substantially to this study. GCDP and ESD
conceived and designed the experiments. MR contributed biologic material. GCDP, JLV, EMM, ACLR, MFR and ESD performed the experiments. GCDP and 13. Pugedo H, Barata RA, França-Silva JC, Silva JC, Dias ES. HP: um
modeloaprimorado de armadilha luminosa de sucção para a captura de
pequenos insetos. Rev Soc Bras Med Trop. 2005;38(1):70–2. 13. Pugedo H, Barata RA, França-Silva JC, Silva JC, Dias ES. HP: um
modeloaprimorado de armadilha luminosa de sucção para a captura de
pequenos insetos. Rev Soc Bras Med Trop. 2005;38(1):70–2. Page 6 of 6 Page 6 of 6 Pessoa et al. Parasites & Vectors (2015) 8:469 Brasília:
Ministério da Saúde; 2006. p. 2. 20. Lins RM, Souza NA, Brazil RP, Maingon RD, Peixoto AA. Fixed differences in
the paralytic gene define two lineages within the Lutzomyia longipalpis
complex producing different types of courtship songs. PLoS One. 2012;7:e44323. 21. Pessoa GCD, Dias LS, Diotaiuti L. Deltamethrin pyrethroid susceptibility
characterization of Triatoma sordida Stal, 1859 (Hemiptera:Reduviidae)
populations in the Northern Region of Minas Gerais, Brasil. Rev Soc Bras
Med Trop. 2014;47(4):426–9. Submit your next manuscript to BioMed Central
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English
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The Vacuolar-Type<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mrow><mml:msup><mml:mtext>H</mml:mtext><mml:mtext>+</mml:mtext></mml:msup></mml:mrow></mml:math>-ATPase in Ovine Rumen Epithelium is Regulated by Metabolic Signals
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Hindawi Publishing Corporation
Journal of Biomedicine and Biotechnology
Volume 2010, Article ID 525034, 12 pages
doi:10.1155/2010/525034 Hindawi Publishing Corporation
Journal of Biomedicine and Biotechnology
Volume 2010, Article ID 525034, 12 pages
doi:10.1155/2010/525034 Judith Kuzinski,1 Rudolf Zitnan,2 Christina Warnke-Gurgel,3 and Monika Schweigel1
1Research Unit Nutritional Physiology “Oskar Kellner”, Research Institute for the Biology of Farm Animals (FBN),
Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany g
g
1Research Unit Nutritional Physiology “Oskar Kellner”, Research Institute for the Biology of Farm Animals (FBN),
Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany 1Research Unit Nutritional Physiology “Oskar Kellner”, Research Institute for the Biology of Farm Animals (FBN)
Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany 2Research Institute of Animal Production, Slovak Agricultural Research Centre, Nitra, Komenskeho 73, 041 81 Kosice, Slovakia
3Institute of Farm Animal Sciences and Technology, University of Rostock, Justus-Von Liebig-Weg 8, 18059 Rostock, Germany Correspondence should be addressed to Monika Schweigel, mschweigel@fbn-dummerstorf.de Correspondence should be addressed to Monika Schweigel, mschweigel@fbn-dummerstorf.de Received 1 July 2009; Accepted 13 October 2009 Academic Editor: Kenichiro Kitamura Copyright © 2010 Judith Kuzinski et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited. In this study, the effect of metabolic inhibition (MI) by glucose substitution with 2-deoxyglucose (2-DOG) and/or application
of antimycin A on ovine rumen epithelial cells (REC) vacuolar-type H+-ATPase (vH+-ATPase) activity was investigated. Using
fluorescent spectroscopy, basal pHi of REC was measured to be 7.3 ± 0.1 in HCO3
−-free, glucose-containing NaCl medium. MI
induced a strong pHi reduction (−0.44 ± 0.04 pH units) with a more pronounced effect of 2-DOG compared to antimycin A
(−0.30 ± 0.03 versus −0.21 ± 0.03 pH units). Treatment with foliomycin, a specific vH+-ATPase inhibitor, decreased REC pHi by
0.21±0.05 pH units. After MI induction, this effect was nearly abolished (−0.03±0.02 pH units). In addition, membrane-associated
localization of vH+-ATPase B subunit disappeared. Metabolic control of vH+-ATPase involving regulation of its assembly state by
elements of the glycolytic pathway could provide a means to adapt REC ATP consumption according to energy availability. Journal of Biomedicine and Biotechnology 2 Journal of Biomedicine and Biotechnology 2 (stratum spinosum, stratum granulosum) of the rumen
epithelium has been observed. We speculate that this flexible
location could reflect reversible recycling of ruminal vH+-
ATPase between the plasma membrane and a pool of cyto-
plasmic vesicles and/or dissociation of V1 catalytic complex
from membrane-bound VO domains. In various epithelia
and other cell types, such mechanisms are known to be
involved in the regulation vH+-ATPase activity [12–15, 30–
32]. Regulatory factors in ruminal vH+-ATPase recycling are
unknown but for yeasts [33–36] and renal epithelia [37];
metabolic control has been demonstrated. Physiological
signals that modulate vH+-localization and activity include
pHi, HCO3
−, pCO2, and glucose [14, 15, 18, 37, 38], all
related to cell metabolism. papillae were removed by scissors and washed three times in
antibiotic-containing PBS and ones in antibiotic-free PBS. Then, primary cultures of ruminal epithelial cells (REC)
were prepared as described by Galfiet al. [39]. Briefly,
REC were isolated by fractional trypsination and those cell
fractions containing mostly cells of the strata spinosum
and basale were grown in Medium 199 containing 15%
FCS, 1.36 mM glutamine, 20 mM HEPES, and antibiotics
(50 mg/L gentamycin, 100 mg/L kanamycin, 2.4 × 105 U/L
nystatin) in an atmosphere of humidified air-5% CO2 at
38◦C. From day 2 of culture, the medium was nystatin-
free and contains 10% FCS only. The experiments were
performed 5-6 days after seeding. The present study was designed to investigate a pos-
sible modulation of ruminal vH+-ATPase activity by sub-
strate/energy availability. To do this, we used fluorescent
spectroscopic pHi measurements to study the effects of
glucose removal and/or reduction of the cellular ATP
concentration ([ATP]) on vH+-ATPase functional activity. In addition, Western blot and immunocytochemistry are
used to analyze if changes of vH+-ATPase expression and
localization play a role in adaptation of the pump activ-
ity. 2.4. Solutions for pHi Measurements. Control experiments
were performed in HCO3
−-free, HEPES-buffered measuring
solution (in mM): 125 NaCl, 5 KCl, 1 CaCl2, 2 MgCl2, 5
glucose, 10 HEPES, pH 7.1. Journal of Biomedicine and Biotechnology In experiments designed to
reduce the energy metabolism of REC (metabolic inhi-
bition): (1) glucose was removed from the medium and
antimycin A (5 μM), a known inhibitor of Complex III of
the mitochondrial respiratory chain, was added; (2) glucose
was replaced with 20 mM 2-DOG to inhibit the glycolytic
pathway; (3) antimycin A (5 μM) and 2-DOG (20 mM) were
added with concomitant glucose removal. The osmolarity
of all solutions was adjusted to 280 mOsmol/kg using D-
mannit. All experiments were performed in the nominal
absence of CO2/HCO3
−, to suppress Na+-HCO3
−symporter
related pHi regulation [40] and to enable comparability with
results of our previous studies [10, 11]. Amiloride (250 μM)
and foliomycin (2 μM), known as specific inhibitors of
Na+/H+ exchanger (NHE) and vH+-ATPase, respectively,
were used to differentiate Na+- and pump dependent H+-
secretion. 2. Material and Methods 2.1. Materials. Medium 199, trypsin, glutamine, antibiotics
(gentamycin, nystatin, kanamycin, penicillin-streptomycin),
fetal calf serum (FCS), and Dulbecco’s phosphate-buffered
saline (DPBS) were purchased from PAN Biotech (Aiden-
bach, Germany). HyQTase was obtained from Thermo Fisher
Scientific (Bonn, Germany). BCECF-AM and pluronic acid
were from Molecular Probes Inc. (Eugene, OR). Foliomycin,
amiloride, antimycin A, and 2-deoxyglucose (2-DOG) were
from Sigma Aldrich (Munich, Germany). All chemicals
for Western blot analysis were purchased from Carl Roth
(Karlsruhe, Germany). 2.5. Measurement of pHi by Spectrofluorometry. For the
determination of pHi, cells were loaded with 1 μM BCECF-
AM for 30 minutes and subsequently washed twice in
DPBS. REC were incubated for a further 30 minutes to
allow complete de-esterification and washed twice before
measurement of fluorescence. Intracellular pH was detected
by measuring the fluorescence of the probe-loaded REC
in a spectrofluorometer (LS-50 B, Perkin-Elmer) equipped
with a fast-filter accessory that allowed fluorescence to be
measured at 20-ms intervals with excitation for BCECF at
440 and 480 nm and emission at 515 nm. All measurements
were made at 37◦C in a 3-ml cuvette containing 2 ml cell
suspension (10% cytocrit) under stirring. BCECF signals
were calibrated to pH by placing the cells in medium con-
taining 135 mM KCl and the ionophore nigericin (10 μM)
to equilibrate intra- and extracellular [H+]. The procedure
was repeated for various pH values between 6.0 and 8.0. For data evaluation, 10-s data sets were each averaged at
the beginning of the measurement and then after 50, 100,
150, 200, 250, 300, 350, 400, 450, 500, and 550 s. The final
pHi was determined as the mean pHi of the last 10 s of
the measurement. Thus, for the calculation of any given pHi
value, 500 data points were used. 2.2. Antibodies. The monoclonal mouse antibodies used in
this study were specific for 60-kDa subunit of the yeast vH+-
ATPase (13D11-B2, Molecular Probes) and the α subunit of
the sheep Na+/K+-ATPase (M7-PB-E9, Affinity Bioreagents). Both antibodies have been shown to detect the sheep
proteins specifically [10, 11]. Relevant secondary antibodies
conjugated to Alexa fluor 488 (Invitrogen) were used for
immunocytochemistry. For Western blotting, a horseradish-
peroxidase (HRP)-conjugated antibody (ECL Anti-mouse
IgG) obtained from Amersham Bioscience was used. 2.2. Antibodies. The monoclonal mouse antibodies used in
this study were specific for 60-kDa subunit of the yeast vH+-
ATPase (13D11-B2, Molecular Probes) and the α subunit of
the sheep Na+/K+-ATPase (M7-PB-E9, Affinity Bioreagents). 1. Introduction cell membrane and ion transport and/or the regulation of
cytosolic pH has been found in osteoclasts [18], macrophages
[19], and various epithelia, for example, frog and toad skin,
mammalian renal collecting duct, endolymphatic sac of the
inner ear, and epididymis [20–25]. Resulting from its considerable role in the absorption of
nutrients, mainly of short chain fatty acids (SCFAs) and of
electrolytes [1–3], the rumen epithelium ranks among the
tissues with high metabolic rates [4, 5]. A main proportion
of the rumen ATP utilization is related to activity of a
Na+/K+-ATPase that has been shown to be expressed at
high levels [6–8] in the cell membrane of rumen epithelial
cells (REC) [9, 10]. In addition, functional vacuolar-type
H+ pumps (vH+-ATPase) are existent in REC [10, 11]. The
vH+-ATPase is well known as being present in intracellular
membrane components such as endosomes, lysosomes,
clathrin-coated vesicles, and the Golgi complex [12–15]. The
pump-mediated acidification of such cell compartments is
required for a variety of processes, including transcytosis of
receptor-ligand complexes and other molecules, for example,
NH3/NH+
4, coupled transport of neurotransmitters and
protein breakdown [16, 17]. In addition, a link between
electrogenic H+ secretion by vH+-ATPases localized on the The existence of the vH+-ATPase as an active transport
mechanism in addition to the Na+/K+-ATPase implies a
special functional role of the protein in the rumen. We
have shown that the pump plays a considerable role in REC
pHi regulation being responsible for about 30% of total H+
release [11]. Moreover, indirect evidence for the involvement
of vH+-ATPase in ruminal transport processes comes from
experiments showing that mucosal nitrate, known to inhibit
vH+-ATPase activity [20], reduced propionate and Cl−
absorption markedly [26, 27]. Foliomycin, a specific vH+-
ATPase blocker [28], has been found to inhibit the uptake
of Mg2+ into REC [29]. In our previous study [10], a variable subcellular
distribution of vH+-ATPase in cell membranes and/or
cytosolic pools of the more luminally oriented cell layers 2. Material and Methods After three changes of PBS (5 minutes each),
nuclei were counterstained with 300 nM of 4,6-diamidino-
2-phenylindole (DAPI) in S-buffer (containing: 75 mM KCl,
3 mM MgSO4·7H2O, 1 mM EGTA, 0.2 mM dithiothreitole,
10 mM imidazol, 1 μg/mL aprotinin, 0.1 mM phenylmethane
sulfonyl-fluoride). Cover slips were then mounted with 30 μL
mounting medium (Dianova, Hamburg, Germany). Digital
images were acquired by using a fluorescence microscope
Olympus IX50 (Hamburg, Germany) and MetaMorph ver-
sion 7.5.2.0 and AutoDeblur version 1.4.1 software (Visitron
Systems GmbH, Puchheim, Germany). 2.9. Light Microscopy and Morphometry of Rumen Papillae. Samples of the rumen wall (1 cm2 surface) were obtained
from identical sites of the atrium ruminis and fixed in 4%
neutral formaldehyde solution for morphometric investi-
gations. After rinsing with water, the rumen wall tissues
were dehydrated in a graded series of ethanol (30%, 50%,
70%, 90%, and absolute ethanol), cleared with benzene,
saturated with and embedded in paraffin. At each sampling,
sections of 5 μm thickness were made of 30 papillae and
stained with haematoxylin/eosin. The length and width of
papillae were determined by the computer-operated Image C
picture analysis system (Intronic GmbH, Berlin, Germany)
and the IMES analysis program, by using a color video
camera (SONY 3 CCD) and a light microscope (Axiolab,
Carl Zeiss Jena, Germany). The number of papillae per cm2
mucosa was estimated by using a video camera equipped
with a picture analysis system. According to Hofmann and
Schnorr [42], the total surface of papillae per cm2 mucosa
was determined as length × width × 2, multiplied by the
number of papillae/cm2. 2.10. Statistical Analysis. If not otherwise stated, data are
presented as means ± standard error (SE). Significance
was determined by Student’s t-test or the paired t-test as
appropriate. P < .05 was considered to be significant. All
statistical calculations were performed by using SigmaStat
(Jandel Scientific). 2.7. Western Blot Analysis. For Western blots, total protein
from washed REC was extracted by use of the M-PER
Mammalien Protein Extraction Reagent (Pierce), comple-
mented with Halt protease inhibitor cocktail (Pierce). The
protein concentration was determined by using the Bradford
assay (Bio-Rad, Munich, Germany). Protein samples (10–
115 μg) were separated by SDS (12.5%)-polyacrylamide gel
electrophoresis and subsequently transferred to polyvinyli-
dene fluoride (PVDF) membrane (GE Healthcare). 2. Material and Methods After
transfer, membranes were blocked with 3% non-fat dry
milk in PBS (pH 7.5) containing 0.05% Tween 20 (PBS-T)
for 2 hours and washed two times for 5 minutes in PBS-
T. Thereafter, membranes were incubated at 4◦C with the
primary antibodies (anti-vH+-ATPase: 1 : 5,000 dilution;
anti Na+/K+-ATPase: 1 : 1,000 dilution) overnight, washed
three times (1 × 15 minutes, 2 × 5 minutes) with PBS-T,
and incubated for 1 hour with HRP-conjugated secondary
antibodies (1 : 10,000 dilution). Then, after three washings (1
× 10 minutes and 2 × 5 minutes) in PBS-T, membranes were
developed with ECL Western Blotting Substrate (Pierce). 2. Material and Methods Both antibodies have been shown to detect the sheep
proteins specifically [10, 11]. Relevant secondary antibodies
conjugated to Alexa fluor 488 (Invitrogen) were used for
immunocytochemistry. For Western blotting, a horseradish-
peroxidase (HRP)-conjugated antibody (ECL Anti-mouse
IgG) obtained from Amersham Bioscience was used. 2.3. Tissue Preparation and Cell Culture. The ruminal tissues
were obtained from a local slaughter house. Samples were
excised from the forestomachs of sheep within 10 minutes of
slaughter. Two pieces of rumen tissue, each about 100 cm2,
were taken from the Atrium ruminis, washed at least
three times in ice-cold phosphate-buffered saline (PBS)
containing penicillin-streptomycin, and then transported
to the laboratory in the same solution. There, rumen 3 Journal of Biomedicine and Biotechnology fluid and 2 mL iso-capronic acid (internal standard) was
centrifuged at 3000× g at 4◦C for 20 minutes. The filtered
(0.22 μm pore size) supernatant was used to measure the
SCFA concentration by gas chromatography (Shimadzu GC-
14A, Shimadzu Corporation, Kyoto, Japan) on a capillary
column (Free Fatty Acid Phase, 25 m × 0.25 mm, Machery-
Nagel GmbH & Co. KG, D¨uren, Germany) according to the
method of Geissler et al. [41]. 2.6. Immunocytochemistry. REC (2 × 106 cells/ml) were
grown on sterile glass cover slips (Neolab, Germany) for 24
to 48 hours. Thereafter, a metabolic inhibition (20 minutes)
was performed as described above. After being rinsed twice
with PBS, REC were fixed in methanol (10 minutes at
−20◦C). If not otherwise stated, all the following steps were
carried out at room temperature. After two PBS washes,
cells were permeabilized in 0.25% Triton X-100 for vH+-
ATPase or in 100 μM digitonin for Na+/K+-ATPase for 10
minutes and again rinsed three times with PBS. Nonspecific
binding of IgG was suppressed by incubation of specimens
with 7% goat serum in PBS for 20 minutes. Subsequently,
cells were rinsed with PBS (three times for 5 minutes) and
then incubated overnight at 4◦C with the primary anti-vH+-
ATPase antibody (13 μg/mL) or the primary anti-Na+/K+-
ATPase antibody (15 μg/mL) solved in PBS with 1% BSA;
PBS-BSA. After being rinsed three times with PBS, cells
were incubated for 2 hours with the secondary, Alexa Fluor-
488-labeled goat anti-mouse IgG1(γ1) antibody (1 : 200 in
PBS-BSA). 3. Results −0.6
−0.5
−0.4
−0.3
−0.1
−0.2
Decrease of pHi (pH units)
2.5 μM/10 mM
5 μM/20 mM
10 μM/40 mM
Concentration of
antimycin A/2-DOG
Antimycin A
2-DOG
Antimycin A
(5 μM)
+2-DOG
2-DOG
(20 mM)
+antimycin A
a a
a a
a
a
Figure 1: Results of dose-response experiments with antimycin A
and/or 2-DOG. The pHi of ruminal epithelial cells was measured
in either HEPES-buffered HCO3
−-free Na-medium containing
5 mM glucose (control) or in glucose-free media with blockers as
indicated. The mean pHi reduction from the pHi measured in
control medium with glucose is given. Values are means ± SE from
3 single measurements; aP < .05 versus the lowest dosage used. 6.9
7
7.1
7.2
7.3
7.4
pHi
0
100
Control
Antimycin A
0 3
−0.2
−0.1
Hi (pH units)
Antimycin
∗ −0.6
−0.5
−0.4
−0.3
−0.1
−0.2
Decrease of pHi (pH units)
2.5 μM/10 mM
5 μM/20 mM
10 μM/40 mM
Concentration of
antimycin A/2-DOG
Antimycin A
2-DOG
Antimycin A
(5 μM)
+2-DOG
2-DOG
(20 mM)
+antimycin A
a a
a a
a
a
Figure 1: Results of dose-response experiments with antimycin A
and/or 2-DOG. The pHi of ruminal epithelial cells was measured
in either HEPES-buffered HCO3
−-free Na-medium containing
5 mM glucose (control) or in glucose-free media with blockers as
indicated. The mean pHi reduction from the pHi measured in
control medium with glucose is given. Values are means ± SE from
3 single measurements; aP < .05 versus the lowest dosage used. −0.6
−0.5
−0.4
−0.3
−0.1
−0.2
Decrease of pHi (pH units)
Antimycin A
2-DOG
Antimycin A
(5 μM)
+2-DOG
2-DOG
(20 mM)
+antimycin A
a a
a a
a
a 6.9
7
7.1
7.2
7.3
7.4
pHi
0
100
200
300
400
500
600
Ti
( ) Decrease of pHi (pH units) ( )
Control
Antimycin A
2-DOG
Antimycin A + 2-DOG
(a) Figure 1: Results of dose-response experiments with antimycin A
and/or 2-DOG. The pHi of ruminal epithelial cells was measured
in either HEPES-buffered HCO3
−-free Na-medium containing
5 mM glucose (control) or in glucose-free media with blockers as
indicated. The mean pHi reduction from the pHi measured in
control medium with glucose is given. Values are means ± SE from
3 single measurements; aP < .05 versus the lowest dosage used. 3. Results −0.5
−0.4
−0.3
−0.2
−0.1
Decrease of pHi (pH units)
Antimycin A
2-DOG
Antimycin A + 2-DOG
∗
∗
∗
(b) by 0.21 ± 0.03, 0.35 ± 0.04, and 0.44 ± 0.04 pH units,
respectively. With antimycin A or both inhibitors in the
medium, this pHi reduction was stable in most cases. However, if 2-DOG only was present, the effect was slightly
diminished and the observed pHi decrease amounted to 0.30
± 0.03 pH units at the end of the measuring period. Figure 2: Effect of glucose replacement by 2-DOG and/or of
antimycin A application on pHi of ruminal epithelial cells. Mea-
surements were made in either HEPES-buffered HCO3
−-free Na-
medium containing 5 mM glucose (control) or in glucose-free
media with blockers as indicated. (a): The figure shows original
traces from one experiment. (b): To summarize the inhibitor effects,
the mean pHi reduction from the pHi measured in control medium
with glucose and without metabolic inhibitors has been calculated
and is shown for each condition. Inhibitor concentrations are 5 μM
and 20 mM for antimycin A and 2-DOG, respectively. Values are
means ± SE from 8 to 9 single measurements;
∗P < .05 versus
control. 3.2. Effect of Foliomycin on the pHi of REC before and after
Metabolic Inhibition with Glucose Substitution by 2-DOG
and Antimycin A Application. Next, we investigated whether
a decreased vH+-ATPase activity was responsible for the
pHi reducing effect of metabolic inhibition (MI). Therefore,
BCECF-loaded REC were suspended in foliomycin (2 μM)-
containing HEPES-buffered Na-medium or glucose-free,
HEPES-buffered Na-medium with antimycin A (5 μM), 2-
DOG (20 mM), and foliomycin (2 μM). Then, the pHi
was measured continuously over a 10-min period. Control
measurements were performed with cells handled in the
same way, but without foliomycin present in the solutions. In
comparison to the pHi of control cells measured in HEPES-
buffered Na-medium, foliomycin-treated REC showed a
decreased pHi (Figures 3(a) and 3(b)). The mean pHi
reduction observed in foliomycin-treated REC amounted to
0.21 ± 0.05 pH units (Figure 3(b)). As shown in Figures 3(a)
and 3(B), the foliomycin effect was nearly abolished when
REC were incubated in antimycin A- and 2-DOG-containing
glucose-free media. Under such conditions, the pHi dif-
ference between control (HEPES-buffered, glucose-free Na-
medium with antimycin A and 2-DOG) and foliomycin-
treated REC amounted to 0.03 ± 0.02 pH units only (Figure 3(b)). 3. Results 3.1. Basal Intracellular pH of REC and Effects of Glucose
Removal, 2-DOG, and Antimycin A on pHi. BCECF-loaded
REC were suspended in either HEPES-buffered Na-medium
(control) or HEPES-buffered, glucose-free Na-medium with
2-DOG and/or antimycin A, and pHi was measured over
a 10-min period. The appropriate concentrations of 2-
DOG (20 mM) and antimycin A (5 μM) were chosen from
dose-response experiments (Figure 1). Characteristic orig-
inal traces showing the time course of the REC pHi for
all four conditions are shown in Figure 2(a). Data on the
pHi reduction induced by either antimycin A or 2-DOG
alone or by a combination of both metabolic inhibitors
are summarized in Figure 2(b). The initial and final pHi of
control REC incubated in HEPES-buffered NaCl-medium
were 7.2 ± 0.1 and 7.3 ± 0.1, respectively. The presence of
antimycin A and/or 2-DOG in the media led to a reduction
of pHi (Figures 2(a) and 2(b)). In REC exposed to antimycin
A, 2-DOG or antimycin A and 2-DOG, the pHi measured
at the beginning of the measuring period was reduced 2.8. Rumen Fluid Analysis. Rumen fluid was taken from the
perforated rumen immediately after slaughtering. Its pH was
measured directly after sampling with a glass electrode (N
1042A, pH meter CG 841, Schott, Mainz, Germany). Then,
the rumen fluid was strained through 4 layers of gauze and
prepared for SCFA analysis. First, a mixture of 5 mL rumen Journal of Biomedicine and Biotechnology 4 6.9
7
7.1
7.2
7.3
7.4
pHi
0
100
200
300
400
500
600
Time (s)
Control
Antimycin A
2-DOG
Antimycin A + 2-DOG
(a)
−0.5
−0.4
−0.3
−0.2
−0.1
Decrease of pHi (pH units)
Antimycin A
2-DOG
Antimycin A + 2-DOG
∗
∗
∗
(b)
Figure 2: Effect of glucose replacement by 2-DOG and/or of
antimycin A application on pHi of ruminal epithelial cells. Mea-
surements were made in either HEPES-buffered HCO3
−-free Na-
medium containing 5 mM glucose (control) or in glucose-free
media with blockers as indicated. (a): The figure shows original
traces from one experiment. (b): To summarize the inhibitor effects,
the mean pHi reduction from the pHi measured in control medium
with glucose and without metabolic inhibitors has been calculated
and is shown for each condition. Inhibitor concentrations are 5 μM
and 20 mM for antimycin A and 2-DOG, respectively. Values are
means ± SE from 8 to 9 single measurements;
∗P < .05 versus
control. 3. Results These results point to a fast deactivation of vH+-
ATPase activity after MI with antimycin A and 2-DOG. Reg-
ulatory mechanisms responsible for such adaptive response
could be reversible disassembly of the VO and V1 subunits
of the pump [30, 33] and/or a translocation between the cell
membrane and intracellular compartments [43]. Thus, in a next step we tested whether MI affect the
distribution of vH+-ATPase and performed an immunoflu-
orescence study using an antibody directed against the 60-
kDa subunit of the yeast vH+-ATPase which has been shown
to detect vH+-ATPase in ovine rumen epithelium [10]. Journal of Biomedicine and Biotechnology 5 6.4
6.6
6.8
7
7.2
7.4
7.6
pHi
0
100
200
300
400
500
600
Time (s)
Control
Foliomycin
Antimycin A + 2-DOG
Antimycin A + 2-DOG + foliomycin
(a)
−0.25
−0.2
−0.15
−0.1
−0.05
0
Decrease of pHi (pH units)
Foliomycin
Antimycin A + 2-DOG
+ foliomycin
∗
(b)
Figure 3: Effect of the vH+-ATPase inhibitor foliomycin before and
after metabolic inhibition (MI) induced by glucose replacement
with 2-DOG and application of antimycin A on pHi of ruminal
epithelial cells (REC). Foliomycin was added to REC suspended in
either HEPES-buffered HCO3
−-free Na-medium containing 5 mM
glucose (control; n = 6) or in glucose-free media with 2-DOG
and antimycin A (n = 7). A: The figure shows original traces
from one experiment. B: To summarize the foliomycin effects, the
mean pHi reduction from the pHi measured in respective control
medium without inhibitor has been calculated and is shown for
both conditions. Values are means ± SE; ∗P < .05 versus control. 6.4
6.6
6.8
7
7.2
7.4
7.6
pHi
0
100
200
300
400
500
600
Time (s) In comparison with the Na+/K+-ATPase, the vH+-ATPase
distribution showed a much higher variability and depen-
dence on the substrate availability. Examples of characteristic
staining patterns are given in figure 4(B). When incubated
in glucose-containing standard medium, REC vH+-ATPase
appeared in the cell membrane and/or perimembrane area
as well as in the cytoplasma of REC (Figure 4(B), a). The cytosolic vH+-ATPase staining is clearly defined and
either distributed throughout the cytoplasm or concentrated
around the nucleus. After glucose deprivation and applica-
tion of 2-DOG (Figure 4(B), b), no membrane staining can
be found and typically the vH+-ATPase staining is clustered
in the perinuclear area of the cells. 3. Results Compared to control con-
ditions, glucose deprivation and combined application of 2-
DOG and antimycin A (Figure 4(B), c) induced appearance
of diffuse, cytosolic vH+-ATPase staining. 6.4
6.6
6.8
7
7.2
7.4
7.6
pHi
0
100
200
300
400
500
600
Time (s)
Control
Foliomycin
Antimycin A + 2-DOG
Antimycin A + 2-DOG + foliomycin
(a)
−0.25
−0.2
−0.15
−0.1
−0.05
0
Decrease of pHi (pH units)
Foliomycin
Antimycin A + 2-DOG
+ foliomycin
∗
(b) 3.4. Effect of Amiloride on the pHi of REC before and after
Metabolic Inhibition with Glucose Substitution by 2-DOG and
Antimycin A Application. As in our previous investigation
with ovine and bovine REC [10, 11], a more or less expressed
compensatory pHi increase was seen after application of
foliomycin. In Na+-containing media, an NHE is known
to be responsible for this effect [40] as application of the
NHE inhibitor amiloride reduced REC pHi by 65% [11]. Therefore, we repeated the above described experiments to
investigate the NHE activity before and after MI with 2-DOG
and antimycin A. Experiments were performed as described
for the first series, however, in addition to foliomycin (2 μM),
the NHE inhibitor amiloride (250 μM) was used alone or in
combination with the vH+-ATPase blocker. −0.25
−0.2
−0.15
−0.1
−0.05
0
Decrease of pHi (pH units)
Foliomycin
Antimycin A + 2-DOG
+ foliomycin
∗
(b) Surprisingly, the results differ from that seen before. MI induced a pHi decrease of 0.10 ± 0.03 pH units only
(Figure 5), which corresponds to a 75% reduction of the
effect compared with the results of the first experiment. In addition, the mean inhibitory effect of foliomycin was
strongly diminished (−0.05 ± 0.03 pH units; Figure 5). Interestingly, there was a coupling between MI and the
foliomycin effect. Figure 5 shows that REC responding to
substrate deprivation and application of metabolic inhibitors
were also foliomycin-sensitive. In such cells pHi was reduced
by 0.14 ± 0.03 or 0.10 ± 0.05 pH units after MI induction
or exposure to foliomycin. In contrast, REC that did not
respond to MI were foliomycin-insensitive (Figure 5). (b) Figure 3: Effect of the vH+-ATPase inhibitor foliomycin before and
after metabolic inhibition (MI) induced by glucose replacement
with 2-DOG and application of antimycin A on pHi of ruminal
epithelial cells (REC). 3.3. Vacuolar H+-ATPase Localization before and after
Metabolic Inhibition. As expected, the α subunit-specific
anti-Na+/K+-ATPase antibody identified the protein in the
cytoplasmic membrane of REC and its localization was not
changed by a 20-min incubation in glucose-free, antimycin
A- and/or 2-DOG-containing media (Figure 4(A), a–c). 3. Results Foliomycin was added to REC suspended in
either HEPES-buffered HCO3
−-free Na-medium containing 5 mM
glucose (control; n = 6) or in glucose-free media with 2-DOG
and antimycin A (n = 7). A: The figure shows original traces
from one experiment. B: To summarize the foliomycin effects, the
mean pHi reduction from the pHi measured in respective control
medium without inhibitor has been calculated and is shown for
both conditions. Values are means ± SE; ∗P < .05 versus control. In Figure 6, the inhibitory effects of foliomycin and/or
amiloride before and after MI are summarized. Only results
from measurements with REC responding to metabolic
inhibition are included and for comparison data from the
first series of experiments are also shown. Figure 6 shows that
compared to series 1 (a) the foliomycin effect was strongly
reduced (−0.10 ± 0.02 pH units versus −0.27 pH units)
and b) the residual foliomycin effect (−0.08 ± 0.03 pH
units) was not influenced by MI. As in series 1, foliomycin
application led to a compensatory pHi increase, an effect
that was reduced after MI. A strong amiloride-sensitive
component was observed. At the beginning and end of the
measurement, the pHi of amiloride-treated REC was reduced
by 0.83 ± 0.05 and 0.89 ± 0.06 pH-units, respectively. As a control, the Na+/K+-ATPase which is constantly found
in the cell membrane of REC [10] has been investigated using
an α subunit-specific anti-Na+/K+-ATPase antibody. As a control, the Na+/K+-ATPase which is constantly found
in the cell membrane of REC [10] has been investigated using
an α subunit-specific anti-Na+/K+-ATPase antibody. 3.3. Vacuolar H+-ATPase Localization before and after
Metabolic Inhibition. As expected, the α subunit-specific
anti-Na+/K+-ATPase antibody identified the protein in the
cytoplasmic membrane of REC and its localization was not
changed by a 20-min incubation in glucose-free, antimycin
A- and/or 2-DOG-containing media (Figure 4(A), a–c). 6 Journal of Biomedicine and Biotechnology 6 a
b
c
(A)
a
b
c
(B)
Figure 4: Immunolocalization of Na+/K+-ATPase α-subunit (A, green) and of vH+-ATPase B subunit (B, green) in ovine REC. Monoclonal
mouse antibodies against sheep Na+/K+-ATPase α-subunit or yeast vH+-ATPase 60 kDa subunit, and an Alexa fluor 488 conjugated
secondary antibody were used. The nuclei are counterstained with DAPI (blue). 3. Results Compared to control conditions (Ba) glucose substitution
with 2-DOG (Bb) and complete MI (glucose substitution with 2-DOG and antimycin A application; Bc) induced loss of membrane staining
and appearance of diffuse, cytosolic staining of vH+-ATPase B subunits. In contrast, the Na+/K+-ATPase was always localized to the cell
membrane and not influenced by metabolic inhibition (A a–c). b
(A) c b
(A) a b (A)
b
(B) c a (B) Figure 4: Immunolocalization of Na+/K+-ATPase α-subunit (A, green) and of vH+-ATPase B subunit (B, green) in ovine REC. Monoclonal
mouse antibodies against sheep Na+/K+-ATPase α-subunit or yeast vH+-ATPase 60 kDa subunit, and an Alexa fluor 488 conjugated
secondary antibody were used. The nuclei are counterstained with DAPI (blue). Compared to control conditions (Ba) glucose substitution
with 2-DOG (Bb) and complete MI (glucose substitution with 2-DOG and antimycin A application; Bc) induced loss of membrane staining
and appearance of diffuse, cytosolic staining of vH+-ATPase B subunits. In contrast, the Na+/K+-ATPase was always localized to the cell
membrane and not influenced by metabolic inhibition (A a–c). 3.6. Ruminal Fluid Analysis and Morphometry. We speculate
that varying feeding conditions of sheep could be responsible
for the different results seen in both series. To get some
information on prior slaughter nutrition, we started rumen
fluid analysis (SCFA concentration, pH) and morphometric
measurements of the rumen papillae at the end of the second
series of experiments. Figure 8 shows measured rumen fluid
pH values and the accompanying SCFA concentrations
determined from 22 sheep. As expected, an inverse rela-
tionship (y = −57,7x + 462.6; r2 = 0.61) between ruminal
fluid pH and the amount of SCFA was observed (Figure 8). The mean pH value amounted to 7.1 ± 0.1 with minimum
and maximum values ranging from 6.4 to 7.9. The mean
concentration of SCFA was 51.6 ± 5.7 mM/l and minimum
to maximum levels ranged from 11.7 to 115.7 mM/l. For
comparison, data from a preliminary feeding experiment
with sheep fed hay ad libitum are also given in Figure 8. 4. Discussion rate of butyrate oxidation to CO2 and increases the rate
of ketogenesis [49]. Besides glutamine, glucose is the main
metabolic fuel in cultured REC and known to be primarily
metabolized through glycolysis [53]. In this study, inhibition
of the glycolytic pathway by substitution of glucose with 2-
DOG induced a significant reduction of pHi (−0.35± 0.04
pH units) in REC showing that their ability to regulate
pHi, at least partly, depends on this metabolic pathway. Like
glucose, its analogue 2-DOG is transported into the cells
and is phosphorylated into 2-DOG-6-phosphate. However,
then it is not further metabolized and accumulates inside
the cells causing energy deprivation. Even in the absence of
glucose, antimycin A application reduced the REC pHi to a
lesser extent (−0.21± 0.03 pH units) than 2-DOG showing
that an intact glycolytic pathway is more important than
mitochondrial ATP production for H+ secretion under our
experimental conditions. This is in accordance with our data
showing that the combination of glucose substitution with
2-DOG and antimycin A led to a stable pHi decrease by 0.44
± 0.04 pH units. To date, the functional role and the regulation of the
recently described ruminal vH+-ATPase [10, 11] is not well
understood. Based on our own work [10] and that of other
investigators [15, 37, 44] showing metabolic regulation of
the pump, we assume that the availability of substrates
and of energy are main regulatory factors to adapt ruminal
vH+-ATPase activity. To prove this hypothesis, we here
investigated the effect of MI induced by glucose removal and
application of inhibitors of the glycolytic pathway (2-DOG)
and/or of mitochondrial ATP production (antimycin A) on
vH+-ATPase activity. 4.1. Basal pHi of REC. The pHi (7.2 to 7.3 ± 0.1) of REC
suspended in control solution (HEPES-buffered, HCO3
−-
free Na+-media with 5 mM glucose) was in the range of 7.1 to
7.5 reported for REC [11, 40, 45] and other cell types [44, 46–
48] under HCO3
−-free conditions. 4.2. Effect of Metabolic Inhibition on pHi of REC. Although
oxidative metabolism of SCFA, mainly of butyrate and
propionate, is the main energy source for metabolically
matured REC [2, 49–51], glucose has also been shown to
be used at a basal rate in vivo and in vitro [5, 51–54]. Journal of Biomedicine and Biotechnology Experiments
were performed as described in Figure 3, however, in addition
to foliomycin (2 μM), amiloride (250 μM) was used alone or in
combination with the vH+-ATPase blocker. For comparison, the
foliomycin effects observed in series 1 of our experiments are also
shown. Values are means ± SE; ∗P < .05. low-pH group when compared to the high-pH group (797.7
± 27.1 mm2/cm2). Journal of Biomedicine and Biotechnology Journal of Biomedicine and Biotechnology Journal of Biomedicine and Biotechnology 7 −0.2
−0.1
0
0.1
0.2
Change of pHi (pH units)
All; n = 12
Responders; n = 9
Non-responders; n = 3
All; n = 12
Responders; n = 9
Non-responders; n = 3
Antimycin A + 2-DOG
Foliomycin
Figure 5: Effect of metabolic inhibition (MI) and of the vH+-
ATPase inhibitor foliomycin in the second series of experiments. Experiments were performed as described in Figures 2 and 3. Note
the coupling between MI and foliomycin sensitivity. Values are
means ± SE; n = number of single experiments = number of
animals. −1.2
−1
−0.8
−0.6
−0.4
−0.2
0
0.2
Change of pHi (pH units)
∗
∗
∗
∗
∗
Series 2
Control
Series 1
Foliomycin
Amiloride
+foliomycin
+foliomycin
Foliomycin
Metabolic inhibition
Series 1
Series 2
Amiloride
Start
End
Figure 6: Effect of vH+-ATPase and Na+/H+ exchanger inhibitors
on REC pHi before and after metabolic inhibition. Experiments
were performed as described in Figure 3, however, in addition
to foliomycin (2 μM), amiloride (250 μM) was used alone or in
combination with the vH+-ATPase blocker. For comparison, the
foliomycin effects observed in series 1 of our experiments are also
shown. Values are means ± SE; ∗P < .05. 1 2
−1
−0.8
−0.6
−0.4
−0.2
0
0.2
Change of pHi (pH units)
∗
∗
∗
∗
∗
Series 2
Control
Series 1
Foliomycin
Amiloride
+foliomycin
+foliomycin
Foliomycin
Metabolic inhibition
Series 1
Series 2
Amiloride All; n = 12
Responders; n = 9
Non-responders; n = 3
All; n = 12
Responders; n = 9
Non-responders; n = 3 Figure 5: Effect of metabolic inhibition (MI) and of the vH+-
ATPase inhibitor foliomycin in the second series of experiments. Experiments were performed as described in Figures 2 and 3. Note
the coupling between MI and foliomycin sensitivity. Values are
means ± SE; n = number of single experiments = number of
animals. Figure 5: Effect of metabolic inhibition (MI) and of the vH+-
ATPase inhibitor foliomycin in the second series of experiments. Experiments were performed as described in Figures 2 and 3. Note
the coupling between MI and foliomycin sensitivity. Values are
means ± SE; n = number of single experiments = number of
animals. Figure 6: Effect of vH+-ATPase and Na+/H+ exchanger inhibitors
on REC pHi before and after metabolic inhibition. 3. Results Although morphometric data showed no significant differ-
ences in rumen papillae surface from sheep with ruminal
fluid pH values <6.9 (n = 6) or >6.9 (n = 16), there was a
tendency to higher surfaces (872.0 ± 47.8 mm2/cm2) in the The foliomycin- and amiloride-sensitive components were
additive under control conditions and the observed pHi
decrease amounted to 0.93 ± 0.05 at the start and to 0.95 ±
0.08 at the end of the measuring period. MI led to a reduction
of the amiloride-sensitive, NHE-related component. At the
end of the measurement the pHi decrease was only 0.69 ±
0.06 pH units. The foliomycin- and amiloride-sensitive components were
additive under control conditions and the observed pHi
decrease amounted to 0.93 ± 0.05 at the start and to 0.95 ±
0.08 at the end of the measuring period. MI led to a reduction
of the amiloride-sensitive, NHE-related component. At the
end of the measurement the pHi decrease was only 0.69 ±
0.06 pH units. 3.5. Western Analysis. Using Western analysis, we investi-
gated the expression of vH+-ATPase B subunit and Na+/K+-
ATPase α subunit protein expression in REC protein extracts
from sheep slaughtered during the first or second series of
experiments. Characteristic examples of immunoblots are
given in Figure 7. Only with protein samples from series one,
a clear 60-kDa band representing the vH+-ATPase B subunit
was seen (Figure 7, lane 1). However, with protein extracts
obtained during the second experimental series, the 60-kDa
band was very weak or completely absent (Figure 7, lane 2-3). In contrast, a distinct 110-kDa band was constantly detected
showing the presence of Na+/K+-ATPase α subunit (Figure 7,
lane 4–6) in all the samples. 4. Discussion The
metabolism of SCFA has been shown to be supported by
glucose [55] and particularly it is known to exert a positive
effect on ruminal butyrate metabolism [49]. Addition of
glucose to butyrate in the incubation medium decreases the 4.3. Effect of Foliomycin on the pHi of REC before and after
Glucose Removal and Metabolic Inhibition with Antimycin A
and/or 2-DOG. In yeast [34, 35, 56], various mammary cell
types [18, 37, 46], and turtle urinary bladder [57], a coupling
between vH+-ATPase activity and cellular energetic processes
has been demonstrated. To evaluate the role of metabolic
regulation on ruminal vH+-ATPase activity, we investigated
the effect of its specific inhibitor foliomycin before and Journal of Biomedicine and Biotechnology 8 8 15
20
25
37
50
75
100
150
250
MW
(kDa)
1
2
3
4
5
6
vH+-ATPase
Na+/K+-ATPase
Figure 7: Immunoblot of the vH+-ATPase subunit B (≈60 kDa) and the Na+/K+-ATPase α subunit (≈110 kDa) in ovine REC. Protein
extracts were prepared from REC isolated during series 1 (lane 1 and 4) or series 2 (lane 2, 3, 5 and 6) of the experiments. For the Na+/K+-
ATPase α subunit, a band at the expected size of approximately 110 kDa was always detected (lane 4–6). In contrast, a clear 60 kDa-band
for H+-ATPase subunit B protein was found with protein extracts from series 1 only (lane 1). With protein extracts from REC with low
metabolic activity, staining was very weak (lane 2) or completely absent (lane 3). Figure 7: Immunoblot of the vH+-ATPase subunit B (≈60 kDa) and the Na+/K+-ATPase α subunit (≈110 kDa) in ovine REC. Protein
extracts were prepared from REC isolated during series 1 (lane 1 and 4) or series 2 (lane 2, 3, 5 and 6) of the experiments. For the Na+/K+-
ATPase α subunit, a band at the expected size of approximately 110 kDa was always detected (lane 4–6). In contrast, a clear 60 kDa-band
for H+-ATPase subunit B protein was found with protein extracts from series 1 only (lane 1). With protein extracts from REC with low
metabolic activity, staining was very weak (lane 2) or completely absent (lane 3). 0
20
40
60
80
100
120
6
6.5
7
7.5
8
pH of ruminal fluid
[SCFA] (mM/L)
Slaughter house
Hay ad libitum
Figure 8: Ruminal fluid concentrations of short chain fatty acids
and accompanying pH values. 4. Discussion Ruminal fluid were obtained from
sheep (n = 22) slaughtered during the second experimental period. For comparison, data from sheep fed hay ad libitum are also given. Note the high proportion of samples showing high pH values and
compared to hay fed sheep low SCFA concentrations indicative of
low feed intake. 0
20
40
60
80
100
120
6
6.5
7
7.5
8
pH of ruminal fluid
[SCFA] (mM/L)
Slaughter house
H
d libit units) have been reported in studies with cells of the human
eccrine sweat duct [58], cultured rabbit non-pigmented
ciliary epithelium [59] and alveolar macrophages [19]. Thus,
a remarkable part of the REC H+ secretion resulted from
vH+-ATPase activity. y
The vH+-ATPase-related pHi component (0.21 ± 0.05
pH units) was nearly abolished (0.03 ± 0.02 pH units) after
substitution of glucose by the glycolytic inhibitor 2-DOG
and treatment with the mitochondrial electron transport
inhibitor antimycin A. This result clearly shows that the pHi
reduction observed after MI mainly results from deactivation
of REC vH+-ATPase. As 2-DOG exerts stronger effects
on REC pHi than treatment with antimycin A, it can be
assumed that ruminal vH+-ATPase activity mainly depends
on an intact glycolytic pathway. Glucose deprivation and/or
inhibition of glycolysis has also been shown to reduce
or prevent vH+-ATPase activity in cells of the medullary
collecting duct [44, 60], the porcine kidney proximal tubule
cell line LLC-PK1 [15, 37], and yeast cells [35, 56]. In
addition, glucose-induced activation of vH+-ATPase activity
has been shown [18, 37]. A coupling between the ATP-
generating glycolytic pathway and vH+-ATPase-mediated
H+ secretion is supported by the findings that iodoacetate,
an inhibitor of glyceralaldehyde-3-phosphate dehydrogenase
(GAPDH), reduced vH+-ATPase activity [46] and that the
enzyme coimmunoprecipitates with the pump [35]. Also,
the rate limiting enzyme of glycolysis, phosphofructokinase-
1 interacts with vH+-ATPase, a subunit which has been
suggested to play a role crucial for proton translocation [61]. A direct physical interaction between the vH+-ATPase E,
B and a subunits, and aldolase, an enzyme responsible for
cleavage of fructose-6-phosphate in the glycolytic pathway,
has been demonstrated [34, 35]. With glucose present, this
interaction increased dramatically leading to the suggestion Figure 8: Ruminal fluid concentrations of short chain fatty acids
and accompanying pH values. Ruminal fluid were obtained from
sheep (n = 22) slaughtered during the second experimental period. For comparison, data from sheep fed hay ad libitum are also given. 4. Discussion Note the high proportion of samples showing high pH values and
compared to hay fed sheep low SCFA concentrations indicative of
low feed intake. after MI with 2-DOG and antimycin A in glucose-free
media. Under control conditions, the foliomycin-induced
pHi decrease amounted to 0.21 ± 0.05 pH units at the end
of the measurements, which is in agreement with results
(−0.18 ± 0.07 pH units) seen in a previous study with sheep
REC [11]. Very similar effects of vH+-ATPase inhibition
with bafilomycin A1 or foliomycin (−0.16 to −0.26 pH 9 Journal of Biomedicine and Biotechnology that aldolase acts as a glucose sensor and regulates vH+-
ATPase assembly, expression, and activity via direct physical
association and by providing ATP for H+ extrusion from
cytosol [35, 36]. regulation under HCO3
−-free conditions responsible for
about 70% of proton secretion and the remaining proton
secretion was related to vH+-ATPase [11]. Therefore, our
second series of experiments was originally designed to
investigate the role of NHE before and after MI. REC conversion of glucose to pyruvate and CO2 is rather
small in proportion to conversion to lactate [53] and thus,
an increased lactate production is indicative of higher rates
of glycolysis [62]. Interestingly, it was found, that at pHe =
7.4, REC converted 3-fold more D-glucose than n-butyrate
and produced about two-fold more lactate than at pHe =
6.2 [62]. Since glycolysis generates protons, its activation at
higher pHe will stimulate vH+-ATPase-mediated H+ efflux
and thereby, will help to maintain SCFA absorption under
such conditions. Provision of extracellular protons is critical
for diffusion-mediated uptake of SCFA in their undissociated
form [3, 50]. Unexpectedly however, the effect of MI was decreased
by 75% in our secondary series of experiments and this
was accompanied by a strongly reduced (−81%) foliomycin
effect. These results point to an impairment of the glycolytic
pathway in those REC and corroborate with tight coupling
between glycolysis and/or components of the glycolytic
pathway and vH+-ATPase activity. Because REC used in
this study were isolated from rumen tissue obtained from a
local slaughter house, we can only speculate on the reasons
for the very different metabolic properties of the cells. However, the latter has been shown to depend on the level
of metabolizable energy (ME) intake and on the type of
nutrition [51, 68]. 4. Discussion Ruminal fluid analysis showed a high
proportion of samples (16 out of 22) showing pH values >6.9
and [SCFA] <60 mM/l (Figure 8) indicating decreased ME
intake [51]. In a preliminary feeding experiment with sheep
fed hay ad libitum (Figure 8) lower ruminal pH values (6.7
± 0.2) and higher [SCFA] (81.7 ± 15.5 mM/l) were observed
than in this study (7.1 ± 0.1; 51.6 ± 5.7 mM/l). In this study, deactivation of vH+-ATPase activity
induced by metabolic inhibition has been shown to be a fast
process appearing within a few minutes. A general mech-
anism responsible for such response to energy deprivation
could be reversible disassembly of the catalytic VO and the
proton-translocating V1 domains of the pump [30, 33, 56]. In yeast cells, glucose deprivation induced a disassembly of
75% of the assembled vH+-ATPase complexes in as little as
5 minutes [63]. In accordance with our previous study with
bovine rumen epithelium [10], ovine REC vH+-ATPase B
subunit was found in close vicinity of the cell membrane
as well as in the cytosolic compartment under control
conditions (glucose-containing NaCl-medium). However,
while Na+/K+-ATPase used as control protein was always
membrane-bound, we here showed for the first time that
the B subunit associated with the REC vH+-ATPase V1
domain shows diffuse cytosolic distribution after MI. The
data provide first evidence that the reduction of vH+-
ATPase-mediated transmembrane proton efflux observed
after substrate and energy deprivation of REC could result
from a higher proportion of disassembled V1 and VO sectors. Since vH+-ATPases are major cellular proteins that can
consume significant amounts of total cellular ATP, their
graduated disassembly could help to conserve energy under
such conditions. However, with the method used in this
study, we were not able to exclude endocytosis of vH+-
ATPase-bearing vesicles from the cell membrane into the
cytosolic compartment. Endo- and exocytotic translocations
have been demonstrated in epithelia of kidney, pancreas,
and placenta [13–15, 64] as an additional mechanism for
regulating vH+-ATPase activity. y
A tendency for a reduced capacity to oxidize glucose
in REC isolated from low-intake sheep had been observed
[51]. Different feeding conditions are also known to induce
changes of the chemical composition of the ruminal fluid. As shown by Kauffold et al. [68], such changes are most
strongly expressed between rations consisting of fresh green
feed or maize silage and such consisting of concentrate or
dried green feeds. References [1] J. Sehested, L. Diernaes, P. D. Moller, and E. Skadhauge,
“Transport of sodium across the isolated bovine rumen
epithelium: interaction with short-chain fatty acids, chloride
and bicarbonate,” Experimental Physiology, vol. 81, no. 1, pp. 79–94, 1996. [16] N. Nelson, “Structure and function of V-ATPases in endocytic
and secretory organelles,” Journal of Experimental Biology, vol. 172, pp. 149–153, 1992. [2] J. Sehested, L. Diernaes, P. D. Moller, et al., “Ruminal
transport and metabolism of short-chain fatty acids (SCFA)
in vitro: effect of SCFA chain length and pH,” Comparative
Biochemistry and Physiology A, vol. 123, no. 4, pp. 359–368,
1999. [17] D. Weihrauch, A. Ziegler, D. Siebers, and D. W. Towle, “Active
ammonia excretion across the gills of the green shore crab
Carcinus maenas: participation of Na+/K+-ATPase, V-type H+-
ATPase and functional microtubules,” Journal of Experimental
Biology, vol. 205, no. 18, pp. 2765–2775, 2002. [3] G. G¨abel, J. R. Aschenbach, and F. M¨uller, “Transfer of energy
substrates across the ruminal epithelium: implications and
limitations,” Animal Health Research Reviews, vol. 3, no. 1, pp. 15–30, 2002. [18] K. I. Larsen, M. L. Falany, L. V. Ponomareva, W. Wang, and J. P. Williams, “Glucose-dependent regulation of osteoclast H+-
ATPase expression: potential role of p38 MAP-kinase,” Journal
of Cellular Biochemistry, vol. 87, no. 1, pp. 75–84, 2002. [4] C. K. Reynolds and G. B. Huntington, “Partition of portal-
drained visceral net flux in beef steers. 1. Blood flow and
net flux of oxygen, glucose and nitrogenous compounds
across stomach and post-stomach tissues,” British Journal of
Nutrition, vol. 60, no. 3, pp. 539–551, 1988. [19] T. A. Heming and A. Bidani, “Plasmalemmal H+ extruders in
mammalian alveolar macrophages,” Comparative Biochemistry
and Physiology A, vol. 133, no. 1, pp. 143–150, 2002. [20] S. L. Gluck, R. D. Nelson, B. S. Lee, et al., “Biochemistry of the
renal V-ATPase,” Journal of Experimental Biology, vol. 172, pp. 219–229, 1992. [5] D. Remond, I. Ortigues, and J. P. Jouany, “Energy substrates
for the rumen epithelium,” Proceedings of the Nutrition Society,
vol. 54, no. 1, pp. 95–105, 1995. [21] E. H. Larsen, N. J. Willumsen, and B. C. Christoffersen, “Role
of proton pump of mitochondria-rich cells for active transport
of chloride ions in toad skin epithelium,” Journal of Physiology,
vol. 450, pp. 203–216, 1992. [6] J. M. Kelly, B. W. McBride, and L. P. Acknowledgments The authors gratefully acknowledge the excellent technical
assistance of R. Brose, H. Pr¨ohl (FBN Dummerstorf). They
thank M. Althaus (FBN Dummerstorf) for GC analysis and
R. Zitnan (Research Institute of animal Production, Kosice,
Slovakia) for morphometric analysis of rumen papillae. They
also thank Dr. Theresa Jones for linguistic corrections. This
study was supported by the DFG (M. Schweigel, SCHW
652). [13] E. Roussa, S. L. Alper, and F. Thevenod, “Immunolocalization
of anion exchanger AE2, Na+/H+ exchangers NHE1 and
NHE4, and vacuolar type H+-ATPase in rat pancreas,” Journal
of Histochemistry and Cytochemistry, vol. 49, no. 4, pp. 463–
474, 2001. [14] N. Pastor-Soler, V. Beaulieu, T. N. Litvin, et al., “Bicarbonate-
regulated adenylyl cyclase (sAC) is a sensor that regulates
pH-dependent V-ATPase recycling,” The Journal of Biological
Chemistry, vol. 278, no. 49, pp. 49523–49529, 2003. [15] Y. Y. Sautin, M. Lu, A. Gaugler, L. Zhang, and S. L. Gluck,
“Phosphatidylinositol 3-kinase-mediated effects of glucose on
vacuolar H+-ATPase assembly, translocation, and acidification
of intracellular compartments in renal epithelial cells,” Molec-
ular and Cellular Biology, vol. 25, no. 2, pp. 575–589, 2005. Journal of Biomedicine and Biotechnology 10 An explanation is a reduced ability to maintain low intra-
cellular [Na+] by Na+/K+-ATPase leading to slow dissipation
of the transmembrane Na+ gradient [74]. Inhibition of REC
Na+/K+-ATPase has been shown to reduce pHi by lowering
NHE activity [45]. [7] N. B. Kristensen, O. Hansen, and T. Clausen, “Measurement of
the total concentration of functional Na+,K+-pumps in rumen
epithelium,” Acta Physiologica Scandinavica, vol. 155, no. 1, pp. 67–76, 1995. [8] O. Hansen, “Isoform of Na+, K+-atpase from rumen epithe-
lium identified and quantified by immunochemical methods,”
Acta Physiologica Scandinavica, vol. 163, no. 2, pp. 201–208,
1998. In conclusion, our results demonstrate metabolic regu-
lation of ruminal vH+-ATPase activity. A fast reduction of
vH+-ATPase mediated proton extrusion occurs few minutes
after initiation of MI by glucose substitution with 2-DOG
and application of antimycin A. An intact glycolytic path-
way seems to be more important for vH+-ATPase activity
regulation than mitochondrial ATP production. The very
fast response possibly results from disassembly of the V1
and VO domains of the pump. Prolonged energy deficiency
may result in a higher level of disassembled, inactive vH+-
ATPase complexes but also led to a reduced expression of the
essential B subunit of the pump. This may provide a means to
control ruminal epithelial ATP consumption in dependence
of substrate and energy availability. [9] C. Graham and N. L. Simmons, “Functional organization of
the bovine rumen epithelium,” American Journal of Physiology,
vol. 288, no. 1, pp. R173–R181, 2005. [10] E. Albrecht, M. Kolisek, T. Viergutz, R. Zitnan, and M. Schweigel, “Molecular identification, immunolocalization,
and functional activity of a vacuolar-type H+-ATPase in
bovine rumen epithelium,” Journal of Comparative Physiology
B, vol. 178, no. 3, pp. 285–295, 2008. [11] B. Etschmann, K. S. Heipertz, A. von der Schulenburg, and
M. Schweigel, “A vH+-ATPase is present in cultured sheep
ruminal epithelial cells,” American Journal of Physiology, vol. 291, no. 6, pp. G1171–G1179, 2006. [12] A. Nanda, J. H. Brumell, T. Nordstr¨om, et al., “Activation of
proton pumping in human neutrophils occurs by exocytosis
of vesicles bearing vacuolar-type H+-ATPases,” Journal of
Biological Chemistry, vol. 271, no. 27, pp. 15963–15970, 1996. 4. Discussion The former feeding conditions led to a
fast decrease of metabolic activity and protein synthesis in
the germinative layers of the rumen epithelium character-
ized by low O2-consumption and REC nucleus diameter
[68]. It seems therefore possible that adaptation to low energy
availability that could include reduced proton production
from glycolysis [69, 70] gives an explanation for the low
or absent vH+-ATPase activity. This hypothesis is supported
by our finding that the expression of vH+-ATPase B sub-
unit protein was drastically reduced or absent in protein
extracts from REC used in the second series of experiments. The B subunit is essentially involved in the regulation of
normal trafficking, assembly, and activity of vH+-ATPase
[36, 71]. Interestingly, REC identified to have low metabolic
and vH+-ATPase activity were characterized by a very
high amiloride-sensitive component of pHi reflecting NHE
activity. The amiloride-induced reduction of pHi (−0.89 ±
0.06 pH-units) was as high as that from butyrate-stimulated
REC (−1.00 ± 0.25 pH units) [72]. An increased activity of
NHE has also been shown to occur during early stages of
apoptosis in response to growth factor withdrawal [73]. 4.4. A High NHE Activity Was Observed in REC with Low
Glycolytic and vH+-ATPase Activity. REC belong to cells
that have a specific requirement for high levels of proton
transport and possess Na+/H exchangers of subtype 1 to 3
[40, 45, 65] and monocarboxylate transporter 1 (MCT1)
[66, 67] in addition to the vH+-ATPase. The partial recovery
of pHi from the acidosis induced by MI or foliomycin
application may result from compensatory activation of
one or both of these H+-secreting transport proteins. The
NHE has been shown to be most important for REC pHi- Elevation of NHE activity may compensate for the
loss of vH+-ATPase-related H+ extrusion thereby improving
pHi homeostasis in energy-deprived REC. However, after
induction of MI, NHE activity decreases slowly resulting
in a reduction of the amiloride effect to 0.69 pH units. Journal of Biomedicine and Biotechnology Journal of Biomedicine and Biotechnology 11 [23] J. Ehrenfeld and U. Klein, “The key role of the H+ V-ATPase
in acid-base balance and Na+ transport processes in frog skin,”
Journal of Experimental Biology, vol. 200, no. 2, pp. 247–256,
1997. [39] P. Galfi, S. Neogrady, and F. Kutas, “Culture of epithelial
cells from bovine ruminal mucosa,” Veterinary Research
Communications, vol. 4, no. 4, pp. 295–300, 1981. [40] F. M¨uller, J. R. Aschenbach, and G. G¨abel, “Role of Na+/H+
exchange and HCO−
3 transport in pHi recovery from intra-
cellular acid load in cultured epithelial cells of sheep rumen,”
Journal of Comparative Physiology B, vol. 170, no. 4, pp. 337–
343, 2000. [24] L. J. Jensen, J. N. Sorensen, E. H. Larsen, and N. J. Willumsen,
“Proton pump activity of mitochondria-rich cells: the inter-
pretation of external proton-concentration gradients,” Journal
of General Physiology, vol. 109, no. 1, pp. 73–91, 1997. [25] H. Dou, J. Xu, Z. Wang, et al., “Co-expression of pendrin,
vacuolar H+-ATPase α4-subunit and carbonic anhydrase II in
epithelial cells of the murine endolymphatic sac,” Journal of
Histochemistry and Cytochemistry, vol. 52, no. 10, pp. 1377–
1384, 2004. [41] Ch. Geissler, M. Hoffmann, and B. Hickel, “Determination of
volatile fatty acids by gas chromatography,” Archives of Animal
Nutrition, vol. 26, pp. 123–129, 1976 (German). [42] R. R. Hofmann and B. Schnorr, The Functional Morphology of
the Forestomachs, Ferdinand Enke, Stuttgart, Germany, 1982. [26] R. W¨urmli, S. Wolffram, and E. Scharrer, “Inhibition of
chloride absorption from the sheep rumen by nitrate,” Journal
of Veterinary Medical A, vol. 34, no. 6, pp. 476–479, 1987. [43] D. Brown and S. Breton, “H+V-ATPase-dependent lumi-
nal acidification in the kidney collecting duct and the
epididymis/vas deferens: vesicle recycling and transcytotic
pathways,” Journal of Experimental Biology, vol. 203, no. 1, pp. 137–145, 2000. [27] T. Kramer, T. Michelberger, H. G¨urtler, and G. G¨abel, “Absorp-
tion of short-chain fatty acids across ruminal epithelium of
sheep,” Journal of Comparative Physiology B, vol. 166, no. 4,
pp. 262–269, 1996. [44] M. L. Zeidel, P. Silva, and J. L. Seifter, “Intracellular pH
regulation and proton transport by rabbit renal medullary
collecting duct cells. Role of plasma membrane proton
adenosine triphosphatase,” Journal of Clinical Investigation,
vol. 77, no. 1, pp. 113–120, 1986. [28] S. Drose, K. U. Bindseil, E. J. Bowman, A. Siebers, A. Zeeck,
and K. Journal of Biomedicine and Biotechnology Altendorf, “Inhibitory effect of modified bafilomycins
and concanamycins on P- and V-type adenosinetriphos-
phatases,” Biochemistry, vol. 32, no. 15, pp. 3902–3906, 1993. [45] M. Schweigel, M. Freyer, S. Leclercq, et al., “Luminal hyperos-
molarity decreases Na transport and impairs barrier function
of sheep rumen epithelium,” Journal of Comparative Physiology
B, vol. 175, no. 8, pp. 575–591, 2005. [29] M. Schweigel and H. Martens, “Anion-dependent Mg2+ influx
and a role for a vacuolar H+-ATPase in sheep ruminal
epithelial cells,” American Journal of Physiology, vol. 285, no. 1, pp. G45–G53, 2003. [46] I. Kurtz, “Apical Na+/H+ antiporter and glycolysis-dependent
H+-ATPase regulate intracellular pH in the rabbit S3 proximal
tubule,” Journal of Clinical Investigation, vol. 80, no. 4, pp. 928–
935, 1987. [30] H. Merzendorfer, R. Gr¨af, M. Huss, W. R. Harvey, and H. Wieczorek, “Regulation of proton-translocating V-ATPases,”
Journal of Experimental Biology, vol. 200, no. 2, pp. 225–235,
1997. [31] M. A. Skinner, L. A. MacLaren, and A. G. Wildeman, “Stage-
dependent redistribution of the V-ATPase during bovine
implantation,” Journal of Histochemistry and Cytochemistry,
vol. 47, no. 10, pp. 1247–1254, 1999. [47] L. C. McKinney and A. Moran, “Regulation of intracellular
pH in J774 murine macrophage cells: H+ extrusion processes,”
American Journal of Physiology, vol. 268, no. 1, pp. C210–C217,
1995. [32] C. Winter, N. Schulz, G. Giebisch, J. P. Geibel, and C. A. Wagner, “Nongenomic stimulation of valcuolar H+-ATPases
in intercalated renal tubule cells by aldosterone,” Proceedings
of the National Academy of Sciences of the United States of
America, vol. 101, no. 8, pp. 2636–2641, 2004. [48] R. Fern´andez and G. Malnic, “H+ ATPase and Cl−Interaction
in regulation of MDCK cell pH,” Journal of Membrane Biology,
vol. 163, no. 2, pp. 137–145, 1998. [49] U. Beck, B. Emmanuel, and D. Giesecke, “The ketogenic effect
of glucose in rumen epithelium of ovine (Ovis aries) and
bovine (Bos taurus) origin,” Comparative Biochemistry and
Physiology B, vol. 77, no. 3, pp. 517–521, 1984. [33] P. M. Kane and K. J. Parra, “Assembly and regulation of the
yeast vacuolar H+-ATPase,” Journal of Experimental Biology,
vol. 203, no. 1, pp. 81–87, 2000. [50] M. Bugaut, “Occurrence, absorption and metabolism of
short chain fatty acids in the digestive tract of mammals,”
Comparative Biochemistry and Physiology B, vol. 86, no. 3, pp. 439–472, 1987. [34] M. Lu, L. S. Holliday, L. Zhang, W. A. Dunn Jr., and S. L. References Milligan, “In vitro
ouabain-sensitive respiration and protein synthesis in ruminal
epithelial papillae of Hereford steers fed either alfalfa or
bromegrass hay once daily,” Journal of Animal Science, vol. 71,
no. 10, pp. 2799–2808, 1993. [22] D. Brown and S. Breton, “Mitochondria-rich, proton-
secreting epithelial cells,” Journal of Experimental Biology, vol. 199, no. 11, pp. 2345–2358, 1996. Journal of Biomedicine and Biotechnology 12 [70] C. P. de Albuquerque, G. Gerstenblith, and R. G. Weiss,
“Importance of metabolic inhibition and cellular pH in
mediating preconditioning contractile and metabolic effects
in rat hearts,” Circulation Research, vol. 74, no. 1, pp. 139–150,
1994. [55] P. Kauffold, B. Piatkowski, and J. Voigt, “Studies on the effect
of nutritional factors on the ruminal mucosa. 4. The effect of
plant proteins and urea on mucosal structure and function,”
Archives of Animal Nutrition, vol. 27, no. 6, pp. 379–391, 1977. [56] K. J. Parra and P. M. Kane, “Reversible association between
the V1 and V0 domains of yeast vacuolar H+-ATPase is
an unconventional glucose-induced effect,” Molecular and
Cellular Biology, vol. 18, no. 12, pp. 7064–7074, 1998. [71] Q. Yang, G. Li, S. K. Singh, E. A. Alexander, and J. H. Schwartz, “Vacuolar H+-ATPase B1 subunit mutations that
cause inherited distal renal tubular acidosis affect proton
pump assembly and trafficking in inner medullary collecting
duct cells,” Journal of the American Society of Nephrology, vol. 17, no. 7, pp. 1858–1866, 2006. [57] P. R. Steinmetz, R. F. Husted, A. Mueller, and R. Beauwens,
“Coupling between H+ transport and anaerobic glycolysis in
turtle urinary bladder: effects of inhibitors of H+ ATPase,”
Journal of Membrane Biology, vol. 59, no. 1, pp. 27–34, 1981. [72] K. S. Heipertz, Die vH+-ATPase und ihre Bedeutung f¨ur die pH-
Regulation bei ovinen Pansenepithelzellen, Promotion A, Free
University of Berlin, Berlin, Germany, 2006. [58] D. Granger, M. Marsolais, J. Burry, and R. Laprade, “V-type
H+-ATPase in the human eccrine sweat duct: immunolocal-
ization and functional demonstration,” American Journal of
Physiology, vol. 282, no. 6, pp. C1454–C1460, 2002. [73] A. L. Grenier, K. Abu-Ihweij, G. Zhang, et al., “Apoptosis-
induced alkalinization by the Na+/H+ exchanger isoform 1 is
mediated through phosphorylation of amino acids Ser726 and
Ser729,” American Journal of Physiology, vol. 295, no. 4, pp. C883–C896, 2008. [59] Y. Hou and N. A. Delamere, “Studies on H+-ATPase in
cultured rabbit nonpigmented ciliary epithelium,” Journal of
Membrane Biology, vol. 173, no. 1, pp. 67–72, 2000. [74] C. R. Rose, S. G. Waxman, and B. R. Ransom, “Effects
of glucose deprivation, chemical hypoxia, and simulated
ischemia on Na+ homeostasis in rat spinal cord astrocytes,”
Journal of Neuroscience, vol. 18, no. 10, pp. 3554–3562, 1998. [60] R. C. Stanton, D. C. Boxer, and J. L. Journal of Biomedicine and Biotechnology Gluck, “Interaction between aldolase and vacuolar H+-
ATPase,” Journal of Biological Chemistry, vol. 276, no. 32, pp. 30407–30413, 2001. [51] R. L. Baldwin VI and K. R. McLeod, “Effects of diet forage:
concentrate ratio and metabolizable energy intake on isolated
rumen epithelial cell metabolism in vitro,” Journal of Animal
Science, vol. 78, no. 3, pp. 771–783, 2000. [35] M. Lu, Y. Y. Sautin, L. S. Holliday, and S. L. Gluck, “The
glycolytic enzyme aldolase mediates assembly, expression,
and activity of vacuolar H+-ATPase,” Journal of Biological
Chemistry, vol. 279, no. 10, pp. 8732–8739, 2004. [52] R. A. Prins, F. van Vugt, A. Lankhorst, and J. Akkermans-
Kruyswijk, “Metabolism of lactate isomers by rumen mucosa,”
Journal of Dairy Science, vol. 57, no. 5, pp. 556–561, 1974. [36] M. Lu, D. Ammar, H. Ives, F. Albrecht, and S. L. Gluck,
“Physical interaction between aldolase and vacuolar H+-
ATPase is essential for the assembly and activity of the proton
pump,” Journal of Biological Chemistry, vol. 282, no. 34, pp. 24495–24503, 2007. [53] B. Emmanuel, “Further metabolic studies in the rumen
epithelium of camel (Camelus dromedarius) and sheep (Ovis
aries),” Comparative Biochemistry and Physiology B, vol. 68, no. 1, pp. 155–158, 1981. [37] S. Nakamura, “Glucose activates H+-ATPase in kidney epithe-
lial cells,” American Journal of Physiology, vol. 287, no. 1, pp. C97–C105, 2004. [54] M. A. Lane, R. L. Baldwin VI, and B. W. Jesse, “Sheep
rumen metabolic development in response to age and dietary
treatments,” Journal of Animal Science, vol. 78, no. 7, pp. 1990–
1996, 2000. [38] J. van Adelsberg and Q. Al-Awqati, “Regulation of cell pH
by Ca2+-mediated exocytotic insertion of H+-ATPases,” The
Journal of Cell Biology, vol. 102, pp. 1638–1645, 1986. Journal of Biomedicine and Biotechnology Seifter, “Expression of
Na+-H+ exchange and ATP-dependent proton extrusion in
growing rat IMCD cells,” American Journal of Physiology, vol. 258, no. 3, pp. C416–C420, 1990. [61] Y. Su, A. Zhou, R. S. Al-Lamki, and F. E. Karet, “The a-subunit
of the V-type H+-ATPase interacts with phosphofructokinase-
1 in humans,” Journal of Biological Chemistry, vol. 278, no. 22,
pp. 20013–20018, 2003. [62] P. Galfiand S. Neogrady, “The pH-dependent inhibitory
action of n-butyrate on gastrointestinal epithelial cell divi-
sion,” Food Research International, vol. 34, no. 7, pp. 581–586,
2001. [63] P. M. Kane, “Disassembly and reassembly of the yeast vacuolar
H+-ATPase in vivo,” Journal of Biological Chemistry, vol. 270,
no. 28, pp. 17025–17032, 1995. [64] A. Banerjee, G. Li, E. A. Alexander, and J. H. Schwartz,
“Role of SNAP-23 in trafficking of H+-ATPase in cultured
inner medullary collecting duct cells,” American Journal of
Physiology, vol. 280, no. 4, pp. C775–C781, 2001. [65] C. Graham, I. Gatherar, I. Haslam, M. Glanville, and N. L. Simmons, “Expression and localization of monocarboxylate
transporters and sodium/proton exchangers in bovine rumen
epithelium,” American Journal of Physiology, vol. 292, no. 2, pp. R997–R1007, 2007. [66] F. M¨uller, K. Huber, H. Pfannkuche, J. R. Aschenbach, G. Breves, and G. G¨abel, “Transport of ketone bodies and
lactate in the sheep ruminal epithelium by monocarboxylate
transporter 1,” American Journal of Physiology, vol. 283, no. 5,
pp. G1139–G1146, 2002. [67] D. Kirat, H. Inoue, H. Iwano, et al., “Monocarboxylate
transporter 1 gene expression in the ovine gastrointestinal
tract,” Veterinary Journal, vol. 171, no. 3, pp. 462–467, 2006. [68] P. Kauffold, J. Voigt, and B. Piatkowski, “Studies of the
influence of nutritional factors on the ruminal mucosa. 1. Structure and functional state of the ruminal mucosa after
feeding of extreme rations and abrupt change in nutrition,”
Archives of Animal Nutrition, vol. 25, no. 4, pp. 247–256, 1975. [69] C. E. Murry, V. J. Richard, K. A. Reimer, and R. B. Jennings,
“Ischemic preconditioning slows energy metabolism and
delays ultrastructural damage during a sustained ischemic
episode,” Circulation Research, vol. 66, no. 4, pp. 913–931,
1990.
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Integrating Task Specific Information into Pretrained Language Models for Low Resource Fine Tuning
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cc-by
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*These authors contributed equally to this work Rui Wang1*
Shijing Si1*
Guoyin Wang1,2
Lei Zhang3
Lawrence Carin1
1Duke University
2Amazon Alexa AI
3Fidelity Investments
rui.wang16@duke.edu Ricardo Henao1 Abstract A clear-cut solution to this problem is to focus
more on samples that are more relevant to the tar-
get task during pretraining. However, this requires
a task-specific pretraining, which in most cases
is computational or time prohibitive. Another ap-
proach is to pretrain on an auxiliary dataset before
fine-tuning on the target task (Phang et al., 2018). Such method requires the availability of an appro-
priate auxiliary datasets. Unfortunately, in some
cases it may negatively impact the downstream
transfer (Wang et al., 2018a). Label embeddings
(Akata et al., 2015) can be regarded as a feature-
based definition of a classification task, in which
detailed information of the task is encoded. One
natural question is whether we can combine the
general knowledge in a PLM and the task-specific
characterization contained within label embeddings
for better fine-tuning on low-resource tasks. Pretrained Language Models (PLMs) have im-
proved the performance of natural language
understanding in recent years. Such mod-
els are pretrained on large corpora, which
encode the general prior knowledge of natu-
ral languages but are agnostic to information
characteristic of downstream tasks. This of-
ten results in overfitting when fine-tuned with
low resource datasets where task-specific in-
formation is limited. In this paper, we inte-
grate label information as a task-specific prior
into the self-attention component of pretrained
BERT models. Experiments on several bench-
marks and real-word datasets suggest that the
proposed approach can largely improve the
performance of pretrained models when fine-
tuning with small datasets. The code repos-
itory is released in https://github.com/
RayWangWR/BERT_label_embedding. In this paper, we propose to utilize the label em-
beddings as a task-specific prior, complementary to
the general prior already encoded during pretrain-
ing. We learn and integrate these label embeddings
into BERT models (Devlin et al., 2018) to regular-
ize its self-attention modules, so the task-irrelevant
tokens or patterns can be readily filtered out, while
the task-specific information can be enhanced dur-
ing fine-tuning. Such a modification is compatible
with any PLM built upon self-attention and will not
degrade the original pretrained structure. Findings of the Association for Computational Linguistics: EMNLP 2020, pages 3181–3186
November 16 - 20, 2020. c⃝2020 Association for Computational Linguistics Integrating Task Specific Information into Pretrained Language Models
for Low Resource Fine Tuning Rui Wang1*
Shijing Si1*
Guoyin Wang1,2
Lei Zhang3
Lawrence Carin1
1Duke University
2Amazon Alexa AI
3Fidelity Investments
rui.wang16@duke.edu 3.1
The BERT Model The encoder of BERT and other popular PLMs
are built upon the transformer architecture, which
is composed of multiple layers of multi-head self-
attention and position-wise feed-forward layers. Multi-head Self-attention The multi-head self-
attention is an ensemble of multiple single-head
self-attention modules. Let X ∈RL×D be the em-
bedding matrix of the input sequence with length
L. For each single head, the input sequence is
first mapped into the key, query and value triplet,
denoted as, K = XWK, Q = XWQ, V = XWV ,
(1) 2
Related Work Positional-wise Feed Forward Layer After self-
attention, a fully connected network is applied on
each token representation x using Label embeddings have been previously leveraged
for image classification (Akata et al., 2015), multi-
modal learning between images and text (Kiros
et al., 2014), text recognition in images (Rodriguez-
Serrano and Perronnin, 2015), zero-shot learning
(Li et al., 2015; Ma et al., 2016) and text classifica-
tion (Zhang et al., 2017). Notably, LEAM (Wang
et al., 2018b) jointly embeds words (tokens) and
labels in a common latent space as a means to im-
prove the performance on general text classification
tasks. Further, Moreo et al. (2019) concatenates
label embedding with word embeddings. However,
this approach cannot be directly implemented into
PLMs since the new (concatenated) embedding is
not compatible with the pretrained parameters. We
integrate label embeddings into the self-attention of
BERT models, so the attention can be regularized
to better focus on task-relevant information. which consists of two linear transformations and
ReLU activations. In BERT, the input sequence starts with a [CLS]
token, whose hidden state will be extracted as
the sequence representation for classification. Let
CE(·, ·) be the cross-entropy loss, C(·) be the final
classifier and enc(·) be the encoder consisting of a
stack of transformer layers. The classification loss
can be written as, Lc = E(X,y)∼D[CE(C(enc(X)[CLS]), y)]
(4) where enc(X)[CLS] is the representation of [CLS]
after encoding, y is the classification label and D
is a dataset. In the context of graph embeddings (Kipf and
Welling, 2016), the [CLS] token acts as a super
node that connects to all other tokens (nodes) and
aggregates global information during self-attention
(convolution). After training, the embedding of
the [CLS] token should contain the task-specific
information, so that it can mostly attend to task rel-
evant information in self-attention during inference. However, embeddings of the PLMs are pretrained
agnostic to downstream tasks. When fine-tuning
with low-resource datasets where label informa-
tion is scarce, a single [CLS] token may not cap-
ture enough task specific information, resulting in
model overfitting to task irrelevant tokens or pat-
terns in the input sequences. 1
Introduction Recently, Pretrained Language Models (PLMs)
(Devlin et al., 2018; Radford et al., 2019) have yield
significant progress on various natural language
processing (NLP) tasks, e.g., neural language un-
derstanding, text generation, etc. Existing PLMs
are usually pretrained in a task-agnostic manner, in
which the model is expected to capture the general
knowledge of natural language from a large corpus,
independent of downstream-specific information. This is not a problem when data is abundant in the
downstream dataset, in which case, the model can
effectively extract task-specific information during
fine-tuning. However, in real scenarios, data may
be difficult to collect and labeling is usually expen-
sive. We show that PLMs pretrained with general
knowledge can overfit without enough guidance
from the task-specific information, resulting in de-
graded performance during testing. Recently, Pretrained Language Models (PLMs)
(Devlin et al., 2018; Radford et al., 2019) have yield
significant progress on various natural language
processing (NLP) tasks, e.g., neural language un-
derstanding, text generation, etc. Existing PLMs
are usually pretrained in a task-agnostic manner, in
which the model is expected to capture the general
knowledge of natural language from a large corpus,
independent of downstream-specific information. In order to validate the performance of our ap-
proach in a real-world setting, we collected two text
classification datasets from the online patient portal
of a large academic health system, each with a few
thousand sequences. These are the first datasets
for automatic patient message triage, which consti-
tute an important problem in the field of clinical
data analysis. Experimental results show that our
approach significantly improves the performance
of fine-tuning on low-resource datasets, e.g., those
consisting of only several thousand data samples. *These authors contributed equally to this work 3181 3.2
Integrating Label Embedding into
Self-Attention where {WK, WQ, WV } ∈RD×d are projection
matrices. The self-attention can be formalized as In this paper, we propose to leverage label embed-
dings to optimize the self-attention modules, so the
model can better focus on task-relevant information
when fine-tuned with small datasets. A = QKT
√
d
∈RL×L,
(2)
Hi = softmax(A)V ∈RL×D,
(3) (2) We reformulate the representations in (1) as
{Kw, Qw, Vw} by replacing X with block ma-
trix Xw = [XCLS, X], where XCLS ∈R1×D
and X ∈R(L−1)×D represent the embeddings of We reformulate the representations in (1) as
{Kw, Qw, Vw} by replacing X with block ma-
trix Xw = [XCLS, X], where XCLS ∈R1×D
and X ∈R(L−1)×D represent the embeddings of
[CLS] and the other tokens in the sequence, respec-
tively. The attention score matrix can be rewritten
as, √
d
Hi = softmax(A)V ∈RL×D, (3) where i = 1, . . . , h, h is the number of heads,
softmax(·) is the row-wise softmax function and
d is the head dimension. A is the attention score
matrix representing the compatibility between Q
and K. The multi-head self-attention is defined by
concatenating and projecting {Hi}h
i=1, the repre-
sentation of each head, into ˆH ∈RL×D. where i = 1, . . . , h, h is the number of heads,
softmax(·) is the row-wise softmax function and
d is the head dimension. A is the attention score
matrix representing the compatibility between Q
and K. The multi-head self-attention is defined by
concatenating and projecting {Hi}h
i=1, the repre-
sentation of each head, into ˆH ∈RL×D. [CLS] and the other tokens in the sequence, respec-
tively. The attention score matrix can be rewritten
as, [CLS] and the other tokens in the sequence, respec-
tively. The attention score matrix can be rewritten
as, as, A =
1
√
d
"
Q[CLS]KT
[CLS]
Q[CLS]KT
QKT
[CLS]
QKT
#
. (5) (5) 3182 (a)
(b)
Figure 1: (a) Incorporating label embeddings into multi-head self-attention. C(·) is the classifier for the BERT
model. (b) Modifying self-attention scores with label embeddings. L indicates row concatenation. (a) (b) (b) (a) Figure 1: (a) Incorporating label embeddings into multi-head self-attention. C(·) is the classifier for the BERT
model. (b) Modifying self-attention scores with label embeddings. L indicates row concatenation. be classified into their corresponding classes. 3.2
Integrating Label Embedding into
Self-Attention The
final loss for classification is then We denote the cross-attention between the [CLS]
token and all the other input tokens as S
≜
QT
[CLS]KT ∈R1×(L−1). Let Xl ∈RM×D be the
label embedding matrix, where M is the number
of classes. We first compute the cross attention
between Xl and X as Llabel =
M
X
i=1
CE(C(Xi
l ), i),
(9)
Lfinal = Lc + λLlabel. (10) (9) (10) Al = QlKT
√
d
, Ql = XlWQ,
(6) (6) where Xi
l is the i-th label embedding, λ is a trade-
off parameter between the regularization on label
embeddings and the original classification loss. where Xl is encoded in to Ql with the same map-
ping matrix WQ as in (1). Then, we compute a
modified cross-attention row vector S′ by concate-
nating S and Al by row and keeping the maximum
value of each column, The label embeddings can be initialized ran-
domly or by the pretrained embeddings of rele-
vant keywords. When the label is not identified by
keywords, e.g., in sentence entailment tasks, their
embeddings can be initialized with the represen-
tations of [CLS], averaged over samples from the
same class. All other parameters can be initialized
from the pretrained BERT. This modification can
be adapted to any PLM with self-attention modules. S′ = max([S; Al]) ∈R1×L. (7) (7) As a result, S′ represents the maximum attention
score of a input token with both [CLS] and the
label embeddings. A new attention score matrix A′
can be obtained by replacing S with S′ in (5), 4
Experiments A′ =
1
√
d
"
Q[CLS]KT
[CLS]
S′
QKT
[CLS]
QKT
#
. (8) (8) We focus on fine-tuning with small datasets. We
integrate label embeddings into the pretrained
(Bio)BERT models, and fine-tune on various clas-
sification benchmarks as well as two real-world
clinical datasets that we collected from the online
patient portal of a large academic health system. In (8), when a token is highly relevant to one of the
labels, it will result in a larger attention score in
S′, thus the [CLS] embedding will be less affected
by irrelevant information in the sequence, unlike
(2) where only attention from the current [CLS]
embedding is considered. The proposed attention
layer is shown in Figure 1(b). The attention score
matrix A in (2) is replaced as A′ in (8). All other
components are the same as the original layers in
BERT as in (1)–(3.1). 4.2
Patient Message Triage (b) Attention from our method. (b) Attention from our method. We further evaluate the proposed approach in real-
world scenarios of patient message classification. This is a task motivated by the increasing popular-
ity of online patient portals. Most of the patient
messages generated from the portal are non-urgent,
while the doctors are expected to focus on the ur-
gent requests, which amount to only a small por-
tion (about 10%) of all messages. As a result, the
heath providers will have to spend considerable
time just identifying urgent messages, thus being
less efficient at emergency responses. We obtain
two healthcare datasets –Message-urgency and Ac-
knowledgment– from a large academic health sys-
tem online portal. Detailed description of these two
datasets can be found in Appendix A. Figure 2: Examples of the attention from the [CLS]
token in the final attention layer. The sequences are
sampled from the Message-urgency dataset. Red color
indicates higher attention score. It can be shown that
our method can better focus on keywords, e.g., ’chest’,
’bad’ and ’stairs’, which are more likely to ocurr on
urgent requests. Alternatively, BioBERT fine-tuned on
such a small dataset tends to overfit to task-irrelevant
words, such as ’holiday’, ’school’, ’tests’, etc. to overfit when there is limited task-specific infor-
mation during fine-tuning. However, our method
produces comparable results on larger datasets such
as MNLI and QQP. This is consistent with the study
in Lazar (2003) where additional priors are less use-
ful when the size of dataset grows larger. These
results suggest that our method is more suitable
for fine-tuning with smaller amounts of data, and
that our approach to injecting the label informa-
tion is at least not detrimental to the original pre-
trained model. This supports the intuition of com-
bining the pretrained general knowledge and the
task-specific information for better fine-tuning with
small datasets. We employ our method on the BioBERT pre-
trained model (Lee et al., 2020), which has the
same architecture as BERT but further pretrained
on the clinical corpora. Results are shown in Ta-
ble 2. Our model improves on all the baselines in
terms of F1 score, which validates the usefulness of
the proposed method for low-resource fine-tuning
in the real scenarios. 4.1
Public Benchmarks Table 1 shows the results of integrating label em-
bedding into the pretrained bert-based-uncased
model on 9 public classification benchmarks of var-
ious sizes. We find that our method improves the
results from BERT on small datasets, e.g, WNLI,
MRPC, CoLA, etc, which typically have only sev-
eral thousand data samples available for fine-tuning. This shows that the BERT model, which is pre-
trained with task-agnostic objectives, is more likely We share the same label embedding Xl for all
the layers. The label embedding is adapted on each
layer via WQ in the multi-head attention module. As shown in Figure 1a, we also feed Xl into the
final classifier C(·), so the label embeddings can 3183 Table 1: Results on public benchmarks. Method
TREC
(5.5k)
WNLI
(0.6k)
RTE
(2.5k)
MRPC
(3.7k)
CoLA
(8.5k)
IMDB
(25k)
SST-2
(67k)
MNLI-M/MM
(393k)
QQP
(364k)
Avg
BERT (Devlin et al., 2018)
97.00
55.11
63.90
87.29
54.47
92.36
92.32
84.38/ 84.87
87.53
79.92
Our Method
97.40
57.75
66.43
89.48
56.26
92.43
92.58
84.12/ 84.62
87.84
80.89 Table 1: Results on public benchmarks. (a) Attention from the BioBERT. (b) Attention from our method. (a) Attention from the BioBERT. (b) Attention from our method. Table 2: Results on our healthcare datasets. Values are
shown as F1/Precision/Recall. Table 2: Results on our healthcare datasets. Values are
shown as F1/Precision/Recall. Dataset
Message-urgency
(1.7k)
Acknowledgment
(1.6k)
BERT
(Devlin et al., 2018)
0.761/0.762/0.761
0.980/0.976/0.984
BioBERT
(Lee et al., 2020)
0.764/0.774/0.758
0.985/0.990/0.980
Our Method
0.789/0.784/0.797
0.990/0.993/0.987 Table 2: Results on our healthcare datasets. Values are
shown as F1/Precision/Recall. that can be emphasized by the attention from label
embeddings. References Yukun Ma, Erik Cambria, and Sa Gao. 2016. Label
embedding for zero-shot fine-grained named entity
typing. In Proceedings of COLING 2016, the 26th
International Conference on Computational Linguis-
tics: Technical Papers, pages 171–180. Zeynep Akata, Florent Perronnin, Zaid Harchaoui, and
Cordelia Schmid. 2015. Label-embedding for image
classification. IEEE transactions on pattern analy-
sis and machine intelligence, 38(7):1425–1438. Alejandro Moreo, Andrea Esuli, and Fabrizio Se-
bastiani. 2019. Word-class
embeddings
for
multiclass text classification. arXiv preprint
arXiv:1911.11506. Jimmy Lei Ba, Jamie Ryan Kiros, and Geoffrey E Hin-
ton. 2016. Layer normalization. arXiv preprint
arXiv:1607.06450. Jason Phang, Thibault F´evry, and Samuel R Bowman. 2018. Sentence encoders on stilts: Supplementary
training on intermediate labeled-data tasks. arXiv
preprint arXiv:1811.01088. L´eon Bottou. 2010. Large-scale machine learning
with stochastic gradient descent. In Proceedings of
COMPSTAT’2010, pages 177–186. Springer. Kevin Clark, Minh-Thang Luong, Quoc V Le, and
Christopher D Manning. 2020. Electra: Pre-training
text encoders as discriminators rather than genera-
tors. arXiv preprint arXiv:2003.10555. Alec Radford, Jeff Wu, Rewon Child, David Luan,
Dario Amodei, and Ilya Sutskever. 2019. Language
models are unsupervised multitask learners. Jose Antonio Rodriguez-Serrano and Florent C Per-
ronnin. 2015. Label-embedding for text recognition. US Patent 9,008,429. Jacob Devlin, Ming-Wei Chang, Kenton Lee, and
Kristina Toutanova. 2018. Bert: Pre-training of deep
bidirectional transformers for language understand-
ing. arXiv preprint arXiv:1810.04805. Alex Wang, Jan Hula, Patrick Xia, Raghavendra Pap-
pagari, R Thomas McCoy, Roma Patel, Najoung
Kim, Ian Tenney, Yinghui Huang, Katherin Yu, et al. 2018a. Can you tell me how to get past sesame
street? sentence-level pretraining beyond language
modeling. arXiv preprint arXiv:1812.10860. Kaiming He, Xiangyu Zhang, Shaoqing Ren, and Jian
Sun. 2015. Delving deep into rectifiers: Surpassing
human-level performance on imagenet classification. In Proceedings of the IEEE international conference
on computer vision, pages 1026–1034. Guoyin Wang, Chunyuan Li, Wenlin Wang, Yizhe
Zhang, Dinghan Shen, Xinyuan Zhang, Ricardo
Henao, and Lawrence Carin. 2018b. Joint embed-
ding of words and labels for text classification. In
ACL. Kexin Huang, Jaan Altosaar, and Rajesh Ranganath. 2019. Clinicalbert: Modeling clinical notes and
predicting hospital readmission. arXiv preprint
arXiv:1904.05342. Zhilin Yang, Zihang Dai, Yiming Yang, Jaime Car-
bonell, Russ R Salakhutdinov, and Quoc V Le. 2019. Xlnet: Generalized autoregressive pretraining for
language understanding. In Advances in neural in-
formation processing systems, pages 5754–5764. Diederik P Kingma and Max Welling. 2013. Auto-
encoding
variational
bayes. arXiv
preprint
arXiv:1312.6114. Thomas N Kipf and Max Welling. 2016. 5
Conclusion We propose to integrate task specific information
into PLMs that are pretrained with task-agnostic ob-
jectives. To do this, we leverage label embeddings
to regularize the self-attention in PLMs. Results on
public benchmarks and real-world datasets suggest
that our method can effectively improve the results
for low resource fine-tuning. We note that label information can improve the
results on many tasks of neural language inference,
e.g., WMLI and QQP, where classes are not iden-
tified by keywords, but rather certain patterns in
the input sentence pair. This may be because the
self-attention will encode these input patterns into
intermediate tokens, which act as pseudo keywords 3184 References Semi-
supervised classification with graph convolutional
networks. arXiv preprint arXiv:1609.02907. Honglun
Zhang,
Liqiang
Xiao,
Wenqing
Chen,
Yongkun Wang, and Yaohui Jin. 2017. Multi-task la-
bel embedding for text classification. arXiv preprint
arXiv:1710.07210. Ryan Kiros, Ruslan Salakhutdinov, and Rich Zemel. 2014. Multimodal neural language models. In In-
ternational conference on machine learning, pages
595–603. A
Description of healthcare datasets Label
Count
Typical Example
1
1123
Thank you. Have a
good day. 0
566
I have continued hav-
ing chest pain short-
ness of breath since
waking. Please let me
know what to do. Label
Count
Typical Example
1
1123
Thank you. Have a
good day. 0
566
I have continued hav-
ing chest pain short-
ness of breath since
waking. Please let me
know what to do. cant portion of these messages is purely acknowl-
edgment, like ’Thank you’. It would be helpful if
this type of messages can be filtered out, so that
hospital staff can focus on non-trivial messages. A doctor and a nurse labelled and validated this
dataset. A.1
Message-urgency dataset In message-urgency dataset, portal messages were
manually labeled by experienced sub-specialty (car-
diology) clinicians into three levels of priority:
non-urgent, medium and urgent. Non-urgent la-
bels include notes of appreciation (e.g., thank you). The Medium urgency class contains messages that
could be reasonably responded to in 1-3 days. Ur-
gent messages are those requiring an immediate
phone call to the patient by the clinician. Condi-
tions suggesting acute myocardial infarction, ex-
acerbation of heart failure respiratory distress or
possible stroke were labeled as urgent and would
be inappropriate for an asynchronous patient portal. B
Implementation Details For all the experiments, we use finetune the pre-
trained model for 3 epoches with learning rate 2e-5
and batch size 32. We use the Adam training al-
gorithm. λ is generally set to 3. We set warm up
steps as 10 percent of the total training steps. We
do not apply weight decay and the norm of all the
gradients are clipped by 1. Experiments on the
public benchmarks are run on a TITAN X (Pascal)
1080 gpu. The healthcare experiment are run on
the CPU in a secured virtual machine system. Table 4:
Typical examples of patient messages to
providers. Label 1 for messages being pure acknowl-
edgment, while 0 for non-trivial messages. Table 4:
Typical examples of patient messages to
providers. Label 1 for messages being pure acknowl-
edgment, while 0 for non-trivial messages. Table 4:
Typical examples of patient messages to
providers. Label 1 for messages being pure acknowl-
edgment, while 0 for non-trivial messages. protected health information. These messages in-
cluded free, unstructured plain text sent by patients
to their healthcare team. Responses and messages
sent from the clinician or health system to the pa-
tient were excluded from the analysis. A
Description of healthcare datasets Nicole A Lazar. 2003. Bayesian empirical likelihood. Biometrika, 90(2):319–326. In this work, we utilized 1,756 web portal mes-
sages generated from 10/2014 to 08/2018 by adult
patients (> 18 years old) of a large academic med-
ical center. The Electronic Health Record (EHR)
system (Epic Verona, WI, USA) with associated pa-
tient portal (MyChart) was the source of all patient
messages. A custom-built Application Program-
ming Interface (API) securely made available the
portal messages from the EHR enterprise data ware-
house into a highly protected virtual network space
offered by the medical center. Approved users
were allowed access to work with the identifiable Jinhyuk
Lee,
Wonjin
Yoon,
Sungdong
Kim,
Donghyeon Kim, Sunkyu Kim, Chan Ho So, and
Jaewoo Kang. 2020. Biobert: a pre-trained biomed-
ical language representation model for biomedical
text mining. Bioinformatics, 36(4):1234–1240. Xirong Li, Shuai Liao, Weiyu Lan, Xiaoyong Du, and
Gang Yang. 2015. Zero-shot image tagging by hi-
erarchical semantic embedding. In Proceedings of
the 38th International ACM SIGIR Conference on
Research and Development in Information Retrieval,
pages 879–882. 3185 Label
Count
Typical Example
Non-urgent
631
That would be awesome... thank you. Medium
955
Dr. [name]. All seems well now. I am at home resting. My wife and I have a trip planned to Maryland this week
beginning on Wednesday. We can fly, drive or stay home
if I should not travel. Are there any reasons that I should
not fly. Urgent
170
I have continued having chest pain shortness of breath
since waking. Please tell me what to do. I have tried in
hailers am going to try nebulizers. I just feel extremely
tight in my chest. Table 3: Typical examples of patient messages to providers grouped by urgency. These are examples of the me
urgency dataset used in the experiments. Table 3: Typical examples of patient messages to providers grouped by urgency. These are examples of the message
urgency dataset used in the experiments. Table 3: Typical examples of patient messages to providers grouped by urgency. These are examples of the message
urgency dataset used in the experiments. Label
Count
Typical Example
1
1123
Thank you. Have a
good day. 0
566
I have continued hav-
ing chest pain short-
ness of breath since
waking. Please let me
know what to do. Table 4:
Typical examples of patient messages t
providers. Label 1 for messages being pure acknow
edgment, while 0 for non-trivial messages. A.2
Acknowledgment dataset This acknowledgment dataset is randomly selected
from patient’s responses to the hospital. A signifi- 3186
|
https://openalex.org/W1986081946
|
https://zenodo.org/records/2508234/files/article.pdf
|
English
| null |
Discussion on ``Disruptive Strength with Transient Voltages'', ``The Electric Strength of Air'' and ``Dielectric Strength of Oil''. Jefferson, N. H., June 29, 1910
|
Transactions of the American Institute of Electrical Engineers
| 1,910
|
public-domain
| 14,465
|
DIELECTRIC STRENGTH DIELECTRIC STRENGTH [June 29 [June 29 1208 DISCUSSION
ON
"DISRUPTIVE
STRENGTH
WITH
TRANSIENT
VOLTAGES ", "THE ELECTRIC STRENGTH
OF AIR " AND
"DIELECTRIC STRENGTH
OF OIL
. JEFFERSON, N. H.,
JUNE 29, 1910. D. B. Rushmore:
Mr. Whitehead in his paper says something
which is perhaps more true than many people realize:
" As
a result of the increase in values of transmission voltage, how-
ever, and of improvements in high voltage apparatus and line
insulators, the electric strength of air has become a limiting
factor in the long distance transmission of power."
It is only
of a very recent period that this has been true. It is, however,
of great interest and import. In the somewhat old problem of
transmitting
power,
one
obstacle
after
another
has
been
overcome until at present we are confronted with the effect of
the insulating properties of air, and what action will be taken
with iegard to overcoming the obstacles presented by it is a
question of much importance at the present time. Until very
recently, line insulators were the limiting feature, and at 60,000
volts the standard form of insulator was operating near its
limitations. With the new suspension insulators, however, the
line can be insulated far beyond the point at which the dielectric
strength of air will stand. The question of altitude in trans-
mission lines is also becoming important, because of the lower
dielectric strength of air at the higher altitudes. The physical
phenomena which take place when corona
has
formed
are
extremely interesting, and a considerable number of points arise
in connection with it. For instance, it is not quite clear without
fuller understanding, why corona should cease to be formed
by the lower voltages than that at which it begins. y
g
g
The phenomena described in the paper are those which are of
immediate interest to many members of the Institute. To take
an example, a line which was to operate at 60,000 volts had
the design of the insulators carefully worked out so that they
would always flash over before puncturing. A number of times,
however, lightning punctured these insulators and the dielectric
time-lag of air was used to explain the occurrence. Also many
operating engineers are familiar with the slight blackening of the
bright copper wires in their stations, due to the effect of corona
formation. DIELECTRIC STRENGTH The problem of how best to perform experiments of this
kind is a difficult one. We all like to see the conditions sur-
rounding such investigations as nearly as possible like those
under which the application of the results will be made, such as
the interesting work carried on by Mr. Mershon some years
ago on outside commercial
lines. It
is, however, extremely
difficult to obtain exact data in this way, and it would seem that
the basis for our engineering recommendations must to a large
extent be the result of experiments carried on in the Laboratory. 1910] DISCUSSION AT JEFFERSON 1209 The relation of power or energy to breakdown or ionization
is not clear, and the effect of frequency on such dielectric failure
still remains a subject for discussion. The point most deserving
of attention is that the future progress of the art depends very
largely on the results of the study of the properties of dielectrics. The characteristics of this class of material bring about the
limiting features in high voltage power transmission. V. Karapetoff:
Those familiar with the history of the theory
of elasticity and of the mechanical strength of materials will
find it quite similar to the history of practical
electro-statics. During the early stages of the development of civil engineering
the engineers who needed to know the strength of columns,
bridges, etc., tested them in their actual complicated shapes. The result, therefore, did not characterize the materials, but
only certain objects of definite dimensions made out of these
materials. Knowing the strength of a column or of a beam of
a certain size, it was not possible to predict the strength of a
column or of a beam of a different cross-section and of a dif-
ferent length. Then, gradually, a theory of resistance of mater-
ials was developed and formulae established into which the
elastic coefficient of a material, the dimensions of an object made
out of this material, and the given forces entered as independent
factors. The next stage in the theory was to combine various
stresses and to combine the resultant strains. In most struc-
tures and parts of machines there is usually more than one set
of stresses (for instance a normal stress and a shear), and un-
fortunately stresses are combined according to a different law
than the resulting strains. DIELECTRIC STRENGTH Finally the fact became clear that
it is the resulting strain and not the stress that limits the safe load
of a piece of material-I am speaking of mechanical strength
now-no matter by what combination of stresses the strain is
produced. As the last step, the theory was extended to include
the action of dynamic loads or what we call in electrical engi-
neering transient loads. In bridges, in reciprocating engines,
and in the many other cases loads are not steady, they are inter-
mittent; kinetic energy and inertia enter therefore as disturbing
factors. A similar development took place in applied electrostatics. Our predecessors started with a theory of charges acting at a
distance. Naturally, with such a wrong assumption, not much
progress was made, because the phenomena take place in the
dielectric and not on metallic surfaces. f
d
d
ll After Faraday and Maxwell had cleared the way for a correct
interpretation of electrostatic phenomena, and after it had be-
come necessary for engineers to take into account the dielectric
strength of materials, a new impetus was given to electrostatic
experiments. But as in the case of early experiments on columns,
beams, etc., "practical " electrical engineers still use electrodes
of all kinds of fancy shapes, from which the dielectric strength
of dielectrics proper cannot be conveniently calculated. Know- 1210 DIELECTRIC STRENGTH [June 29 [June 29 [June 29 ing, for instance, the striking distance between two pointed
electrodes,
it is impossible to calculate the striking distance
between electrodes of a different shape, or to determnine the
critical voltage with the same electrodes at a different distance. The question is, however, being gradually put on a more ra-
tional basis; it is at present clearly understood by physicists
and by many engineers that the striking
distance
per centi-
meter of air-gap between needle points really does not mean
much, as regards the dielectric strength of air. Experiments
must be preferably made on large (practically infinite) parallel
plates, in which case the stress in the dielectric is the same at all
points. Or,
else,
electrodes must be used
of such a shape
that the true strains (critical
dielectric flux density)
can be
calculated from the
striking
distance. Concentric
cylinders
are convenient for the purpose. p
p
I wish to refer to the sketches Figs. 1, 2 and 3 of Mr. Tobey's
paper, which illustrate my point. DIELECTRIC STRENGTH The sketches show the dif-
ference between a uniform dielectric field in the case of parallel
plates, and non-uniform fields in the case of sharp points on
spheres. p
In a non-uniform electrostatic field it is necessary to consider
the actual stresses from point to point, because the air is not
broken down simultaneously at all points. It is possible to
break dowrn certain portions of the air nearest to the electrodes,
to ionize it, to make it a conductor, without producing a jump-
spark. The air so ionized becomes a part of the electrode, so
that the break-down finally occurs between new shapes of elec-
trodes. This leads to the consideration of the quantity usually
called the electrostatic capacity between two electrodes, but
which ought to be more properly called the permittance
of the
dielectric. By using proper values of permittance and " per-
mittivity " of the dielectric, calculations of the dielectric strength
of insulation are put on the same scientific bases as calculations
of conductors of non-cylindrical shapes. y
p
At present we are not yet advanced far enough to be able to
calculate the distribution of electrostatic stresses in all cases,
but in the simplest cases, for instance, such as is presented by
Dr. Whitehead, or in the case of two spheres, it is possible to
calculate the actual stresses and their distribution. It is not
the average strength of the air, considered as a mass, that in-
terests the engineer, but the weakest point at which the air
breaks down. The safety of insulation must be considered with
regard to this weakest point, the same as the civil engineer
figures out the factor of safety of the weakest part in his bridge. And fortunately the electrical problem is simpler than the cor-
responding problem in the theory of elasticity, because there
we have two kinds of stresses, normal stress and shear, while in
dielectrics there is but one kind of stress. I do not know what
kind it is; perhaps it is some kind of shear between positive and
negative electricity; at any rate we have to consider only one
kind of stress. 1211 1910J DISCUSSION AT JEFFERSON Now we come to the last stage, the same as civil engineers
had to face, namely, the effect of dynamic loads, of transient
voltages. The
paper by
Mr. Hayden
and
Dr. DIELECTRIC STRENGTH Steinmetz
represents the results of a pioneer investigation in this field. Let us consider their experiments in the light of a mechanical
analogy, that of an elastic rod or a column suddenly subjected
to a normal stress by a released weight. Is it correct to say
that the weight is instantly balanced to its full extent by elastic
forces in the rod? To me such an assumption contradicts the
laws of mechanics of elastic bodies. In order to produce an elastic reaction a strain or a deforma-
tion must first occur in the rod. The rod can resist only (1)
as the result of such a strain (elasticity), or (2) by opposing an
acceleration
of
its parts
(inertia). In the
case under con-
sideration there is no initial strain in the rod, and in order to
produce it parts of the rod must be accelerated layer by layer. At the very first instant the load is balanced by a very high
acceleration of a very small part of the mass of the rod ad-
jacent to it; then the next layers are accelerated so that we get
the effect of a travelling compression wave. Only after a certain
displacement has taken place the load is balanced in part or in
full by the elastic forces. Hence this paradox that with a very
short application of the weight, that corresponds to our transient
voltage, the column or the rod can seemingly support without
being destroyed a heavier load than with a load applied gradu-
ally. Is it not more logical to suppose that the molecular strain,
the actual strain at which the material breaks down, is the same
in both cases? Only in the case of a transient application a part
of the load is balanced by the acceleration of the mass and has
no time to produce elastic stresses, before the load is removed. p
It seems to me that the results obtained by Mr. Hayden and
Dr. Steinmetz must be interpreted in the same light. I cannot
conceive how a voltage of say 100 kilovolts can be applied sud-
denly to a neutral dielectric, that is to say to a dielectric which
is not strained. The action must be equal to the reaction, and
a dielectric which is not strained can produce no electrostatic
reaction. DIELECTRIC STRENGTH We must remember that the rush of current at the first instant
is tremendous, in order to produce the required displacement in
the dielectric. There could have been also a considerable mag-
netic field in the dielectric proper. We have in reality a much
more complicated phenomenon, electromagnetic as well as electro-
static, than it appears on the surface. It is hardly scientific
to say that we simply have 100 kilovolts momentarily applied
to air or oil, and that momentarily these dielectrics can support
more than with a steady application of the voltage. y
pp
g
We must be particularly careful now, when we just begin to
understand the full meaning of these phenomena, not to describe
them in a " short-hand " way, but to specify carefully the
actual physical relations. Without this precaution, busy practi-
tioners are only too prone to misunderstand the results, and to
apply them in cases for which they were not intended at all. hi
h
d pp y
y
Percy H. Thomas:
Professor Whitehead's paper suggests
many questions that I presume he is not in position to answer
as
yet,
but
there are a few
points
which
I
think
would
clear up some matters. p
Professor Whitehead has apparently made a very desirable
advance in having devised a method for the study of corona
and allied phenomena in which he can eliminate all variables
which he does not control. His tests, being laboratory tests,
and made under somewhat limited conditions, do not tell us
everything about corona, but apparently he has succeeded in con-
trolling all the variables actually present in his tests, so that he
can absolutely reproduce his results on different days and under
quite different circumstances. With such a result realized, it
is possible to add another known variable, and get its effect,
and so on step by step. p
y
p
I ask Dr. Whitehead
if he will tell us something concerning
the electrical conditions at the ends of his tube. i
i
i
i
i I would ask Dr. Whitehead if it is possible that the peculiar
action of small wires
in giving the apparently high dielectric
strength for air could be due to the fact that near the surface of the
small wire the strain is changing rapidly. DIELECTRIC STRENGTH It can react only electromagnetically, by means of
displacement currents in it, or in the other parts of the circuit. I am willing to grant that the voltage calculated by the authors
was actually induced in the high-tension windings of the trans-
former, but it does not follow from this that at the very instant
of the closure of the primary switch this full voltage acted across
the air-gap, producing static stresses in the dielectric. The elec-
tromagnetic inertia effects are predominant at the first instant. h
l
f
h
l
i
i
i It seems to me that the results obtained by Mr. Hayden and
Dr. Steinmetz must be interpreted in the same light. I cannot
conceive how a voltage of say 100 kilovolts can be applied sud-
denly to a neutral dielectric, that is to say to a dielectric which
is not strained. The action must be equal to the reaction, and
a dielectric which is not strained can produce no electrostatic
reaction. It can react only electromagnetically, by means of
displacement currents in it, or in the other parts of the circuit. I am willing to grant that the voltage calculated by the authors
was actually induced in the high-tension windings of the trans-
former, but it does not follow from this that at the very instant
of the closure of the primary switch this full voltage acted across
the air-gap, producing static stresses in the dielectric. The elec-
tromagnetic inertia effects are predominant at the first instant. The slope of the voltage wave is very steep at the first instant;
therefore, even a very small magnetic leakage between the two
windings of the transformer, a leakage negligible with ordinary
frequencies, must have produced an enormous counter-electro-
motive force. I am inclined to think that at the first instant
the voltage between the electrodes was practically equal to g
p
The slope of the voltage wave is very steep at the first instant;
therefore, even a very small magnetic leakage between the two
windings of the transformer, a leakage negligible with ordinary
frequencies, must have produced an enormous counter-electro-
motive force. I am inclined to think that at the first instant
the voltage between the electrodes was practically equal to 1212 Uune 29 DIELECTRIC STRENGTH zero. The unstrained dielectric acted as a short-circuit, and the
applied voltage was balanced by the reactance counter e.m.f. DIELECTRIC STRENGTH That is, the volume
of air that is subjected to the maximum stress is very much
more limited than where the wire is larger; the potential gradient
being much sharper in the case of the small rods. i
i g
p
I would ask whether the air which passes through the tube
coming from the wire at the time of the formation of corona is
ionized in the sense that there are positive and negative charges
therein equally balanced, or whether it comes away with a
predominant positive or with a predominiant negative charge,
and if so, which? Also whether he has tried any other gases
than air. i
i
i
i
h
h Another point, in the visual observation of the corona through
oscillographs, has he observed both positive and negative waves, DISCUSSION AT JEFFERSON 1910] 1213 direction being taken with reference to the test wire-that is,
were observations made with the positive potential on the inner
wire or the negative potential on the inner wire, or with both? If so did any difference appear? i One other question-I would ask how the actual arc, that
is, the complete break down, follows after the ionization first
begins, that is how wide a range of voltage, for example, exists
between the point where you can first observe a rapid dropping
of the electroscope, and formation of the actual arc. p ,
A. E. Kennelly
In regard to one particular point in these very
interesting papers, Dr. Whitehead emphasizes the fact the elec-
tric intensity at which disruption appears is greater for small
wires than for large wires. A suggestion for the reason of this
remarkable phenomenon may be offered tentatively. We know,
referring to the properties of the magnetic field for a moment,
that a small magnetically polarized iron particle, or iron filing,
is not acted upon by any magnetic force of translation-any
bodily moving force-when subjected to a uniform magnetic
field, or a magnetic flux distribution acting in parallel straight
lines. There are forces of rotation,
couples,
or torques, but
no forces of translation. When, however, the magnetic field
is not uniform but divergent, the iron filings are subjected to
translatory forces as well as couples, and are pulled bodily
towards the denser parts of the field. DIELECTRIC STRENGTH This effect may roughly
be described by saying that the magnetic pull, in the denser
part of the field, on the attracted pole, is less than the opposing
push, in the weaker part, of the field on the repelled pole. In
fact, we know that the pull of a divergent field 3C upon a spherical
iron particle or spherical iron filing depends upon the product Jc
. , where
-
is the greatest space rate of change of the
field. Now returning to the electric field, if we may assume that a
molecule of gas subjected to an electric field is polarized thereby,
then the molecule will be subjected to a couple or aligning force;
but if the electric field is uniform, there will be no pull, or force
of translation, on the molecule. If, however, the field is con-
vergent, a force of translation will exist as well as the couple,
and the molecule will be pulled towards the denser part of the
field. In the case of an electrified round wire, the field will be
radially divergent from its surface, and the divergence will be
numerically
greater,
the
smaller
the
wire's
diameter. We
should, therefore, expect air-molecules to be drawn in towards
the surface of the wire, and to crowd together near that surface,
just as though the atmospheric pressure were locally increased
in this vicinity. If such a local increase of air-density occurred
around and near the surface of a wire, the electric intensity
required to produce disruption should be increased, as it in-
creases with the atmospheric pressure, and the increase should DIELECTRIC STRENGTH 1214 [June 29 be greater the smaller the wire. I tried the experiment recently,
with Dr. G. W. Pierce, since reading Dr. Whitehead's paper, of
electrifying a two-millimeter wire running down the axis of a
glass tube and observing whether the pressure of the air, within
the tube as a whole, became thereby altered with respect to the
pressure of the air outside the tube. We were unable to detect
any such effect. Consequently, although the effect above sug-
gested might occur, and might yet be masked in this experi-
ment by opposing actions, yet it must be admitted that thus
far, the suggestion is not supported experimentally. ,
gg
pp
p
y
W. H. DIELECTRIC STRENGTH Pratt:
There is one suggestion which comes to me in
connection with the second paper, which has read. The results
quoted with moist air are apparently in disagreement with cer-
tain other measurements which have been made. May it not
be that moist air, as we find it in the open, at times contains elec-
tric nucleii due to the partial re-evaporation of condensations that
have occurred around these electric nucleii, and hence the moist
air, at such times, is really somewhat conducting, whereas in the
experiments that were outlined, the moisture was added to the
air in such a way that it certainly would not be expected to be
conducting. g
E. E. F. Creighton:
Two of the big problems in the trans-
mission and distribution of electrical energy are the lightning
protection of the transmission circuit and the suppression of
troubles from internal surges of electrical energy. The dielectric
spark lag and the spark energy enter into both problems and most
vitally in the latter. Speaking of internal surges only, it has been
noted here and there all over the country that transformers and
generators would fail at some internal part of the winding. On,
any particular system these failures are rather infrequent, but
the aggregate is considerable. Some time ago this problem of
internal surges was confused by the presence of end turn effects. Modern designers have eliminated the end turn failures by heavily
reinforced insulation. This leaves the problem of caring for
internal surges distinct and prominent. The conditions of cost
are not such that a designer may place everywhere in the wind-
ings, as he does on the end turning, an insulation that will stand
a test pressure of 6,000 to 20,000 volts when the dynamic voltage
normally present is only ten to twenty volts. The amount of
energy in these internal surges is usually small. The excellence
of a design taking these factors into consideration would never
be casually apparent, yet they would be vital. With these
practical features in view, the theoretical and experimental stud-
ies made by the authors assume a great importance. Careful
measurements are the life of further progress. A simple meth94
of test is given which can be extended easily to further studies. DIELECTRIC STRENGTH The speaker has had occasion to study a particular feature of
the dielectric spark lag of protective apparatus, with the object
in view of getting rid of it. We were surprised to find
lags
extending
to
several
seconds. To the speaker's knowledge E. E. F. Creighton:
Two of the big problems in the trans-
mission and distribution of electrical energy are the lightning
protection of the transmission circuit and the suppression of
troubles from internal surges of electrical energy. The dielectric
spark lag and the spark energy enter into both problems and most
vitally in the latter. Speaking of internal surges only, it has been
noted here and there all over the country that transformers and
generators would fail at some internal part of the winding. On,
any particular system these failures are rather infrequent, but
the aggregate is considerable. Some time ago this problem of
internal surges was confused by the presence of end turn effects. Modern designers have eliminated the end turn failures by heavily
reinforced insulation. This leaves the problem of caring for
internal surges distinct and prominent. The conditions of cost
are not such that a designer may place everywhere in the wind-
ings, as he does on the end turning, an insulation that will stand
a test pressure of 6,000 to 20,000 volts when the dynamic voltage
normally present is only ten to twenty volts. The amount of
energy in these internal surges is usually small. The excellence
of a design taking these factors into consideration would never
be casually apparent, yet they would be vital. With these
practical features in view, the theoretical and experimental stud-
ies made by the authors assume a great importance. Careful
measurements are the life of further progress. A simple meth94
of test is given which can be extended easily to further studies. The speaker has had occasion to study a particular feature of
the dielectric spark lag of protective apparatus, with the object
in view of getting rid of it. We were surprised to find
lags
extending
to
several
seconds. To the speaker's knowledge DISCUSSION A T JEFFERSON 1910] 1215 this long lag has never been observed before. These data have
a strong bearing on the subject of the paper. DIELECTRIC STRENGTH But the points at con-
siderable super-spark potential are somewhat variable, showing
an erratic behavior not understood at present. At only 35-volt
super-spark potential, the lag is 200 milli-seconds. At 250-volt
super-spark potential, it is 20 milliseconds. At 300-volt super-
spark potential it is 6 milliseconds. STAlIC MACHINE
_ _ _
I_ _ _t
_
_ _t_.,__. _GAP
I0
LEYDEN
200C
--1_ __
JARS
1lta
X
_ _ 1+ __ _ __
_ __PROTECTOR
100
L
~~~~~~~~~~~000000
SML
; X
o
i
i
i
t
t
L o
°SINDUCTANCE
23
IJ
-
-
J
50
100
150
20
TIME IN MILLNSECONDS
INSULATION
FIG. 1. -Sup3r-spark pot?ntial curve. The points
F
are
tLken by measurement and
the curve is the
'IG. 2
hyperbola x (y -23) = 1780
case x (y - 23) = 1780. (See Fig. 1). But the points at con-
siderable super-spark potential are somewhat variable, showing
an erratic behavior not understood at present. At only 35-volt
super-spark potential, the lag is 200 milli-seconds. At 250-volt
super-spark potential, it is 20 milliseconds. At 300-volt super-
spark potential it is 6 milliseconds. The asymtote to the curve is 23 volts above the spark potential
at an infinite time of application. This may have been due to
difference in conditions of tests as this value was taken some time
previous to the other readings. The dielectric-spark-lag was demonstrated by another test
using
alternating
potential. The
spark-potential
for
con-
tinuous application was less than the peak value of the alter-
nating potential, yet under these conditions it was not possible
to spark across the gap. Successive alternations apparently
deionized the gap. At any rate the time of application of super- STAlIC MACHINE
LEYDEN
JARS
OTECTOR
~~~~~~~~~~~000000
SML
t
L o
°SINDUCTANCE
INSULATION
'IG. 2 FIG. 1. -Sup3r-spark pot?ntial curve. The points
are
tLken by measurement and
the curve is the
hyperbola x (y -23) = 1780 'IG. 2 case x (y - 23) = 1780. (See Fig. 1). But the points at con-
siderable super-spark potential are somewhat variable, showing
an erratic behavior not understood at present. At only 35-volt
super-spark potential, the lag is 200 milli-seconds. At 250-volt
super-spark potential, it is 20 milliseconds. At 300-volt super-
spark potential it is 6 milliseconds. h
h
i
i p
p
The asymtote to the curve is 23 volts above the spark potential
at an infinite time of application. DIELECTRIC STRENGTH Among several
other things, the dielectric spark lag depends on the excess
voltage above the voltage which would cause a spark to pass if
applied for a long time, theoretically for an infinite time. For
brevity, call this, excess voltage the super-spark potential. i y
g
p
p
p
As would naturally be expected, the relation between the
suiper-spark potential and the time,
is hyperbolic. For very
great superspark potentials the time interval before the spark
passes becomes only a few milliseconds, or may even go down
into the microseconds. In the curve herewith reproduced the measurements were
taken by the simple method of the oscillograph. Direct current
potential was applied to a gap and the time interval measured
between the application of the potential and the beginning of the
current in the spark. The points of slight super-spark potential
deviate very little from hyperbolic curve-in this particular _ _ _
I_ _ _t
_
_ _t_.,__. _GAP
I0
200C
--1_ __
1lta
X
_ _ 1+ __ _ __
_ __P
100
L
; X
o
i
i
i
23
IJ
-
-
J
50
100
150
20
TIME IN MILLNSECONDS
FIG. 1. -Sup3r-spark pot?ntial curve. The points
are
tLken by measurement and
the curve is the
hyperbola x (y -23) = 1780 STAlIC MACHINE
_ _ _
I_ _ _t
_
_ _t_.,__. _GAP
I0
LEYDEN
200C
--1_ __
JARS
1lta
X
_ _ 1+ __ _ __
_ __PROTECTOR
100
L
~~~~~~~~~~~000000
SML
; X
o
i
i
i
t
t
L o
°SINDUCTANCE
23
IJ
-
-
J
50
100
150
20
TIME IN MILLNSECONDS
INSULATION
FIG. 1. -Sup3r-spark pot?ntial curve. The points
F
are
tLken by measurement and
the curve is the
'IG. 2
hyperbola x (y -23) = 1780 STAlIC MACHINE
_ _ _
I_ _ _t
_
_ _t_.,__. _GAP
I0
LEYDEN
200C
--1_ __
JARS
1lta
X
_ _ 1+ __ _ __
_ __PROTECTOR
100
L
~~~~~~~~~~~000000
SML
; X
o
i
i
i
t
t
L o
°SINDUCTANCE
23
IJ
-
-
J
50
100
150
20
TIME IN MILLNSECONDS
INSULATION
FIG. 1. -Sup3r-spark pot?ntial curve. The points
F
are
tLken by measurement and
the curve is the
'IG. 2
hyperbola x (y -23) = 1780
case x (y - 23) = 1780. (See Fig. 1). DIELECTRIC STRENGTH This may have been due to
difference in conditions of tests as this value was taken some time
previous to the other readings. h
di l
i
k l
d
d b g
The dielectric-spark-lag was demonstrated by another test
using
alternating
potential. The
spark-potential
for
con-
tinuous application was less than the peak value of the alter-
nating potential, yet under these conditions it was not possible
to spark across the gap. Successive alternations apparently
deionized the gap. At any rate the time of application of super- DIELECTRIC STRENGTH 1216 Uune 29 spark-potential was not sufficient to cause a spark and the suc-
cessive impulses were not cumulative. p
The obnoxious effect of the spark lag on the operation of light-
ning arresters was noticed first, years ago, in taking equivalent
needle-gaps and other direct measurements of the value of light-
ning arresters. g
The gap at G of a static machine requires a certain pressure to
spark. (Fig. 2). This determines the value of pressure that
will appear at the protector. At the terminals of the protector is
attached the insulation to be protected. Between the protector
or lightning arrester and the insulation a small amount of in-
ductance
is placed to imitate circuit conditions. When the
pressure at G was as high as 100,000 volts, the insulation was not
injured as the spark took place quickly at the protector. When,
however, the electric pressure at the gap (G) was relatively
small, in one case about 3,000 volts, the insulation was punctured. The greater time lag of the protector allowed the charge to pass
beyond into the insulation and although the spark sometimes
took place in the protector, it occurred too late to give protection. d
hi
h
d
i h
h
i
h p
p
,
g
p
In regard to Dr. Whitehead's paper, I wish to say that in the
study of the dielectric spark-gap, we came across the same thing
that he has studied in what he has named dirty wires, or the
oxidation of the wire. We had ionization taking place on wires
which allowed a spark to pass at less than fifty per cent of the
normal spark potential, and I felt that it could not be explained
by any roughness of the surface. DIELECTRIC STRENGTH Steinmetz, a laboratory paper; next is the Telluride test
by Mr. Mershon, which was a field test; next is the paper by
Professor Ryan, a laboratory test; next follows a Mershon paper,
giving field tests, and now the Whitehead paper, a laboratory
paper follows. The next thing on the regular schedule would
be to have another paper from Mershon. p p
Harris J. Ryan:
Dr. Whitehead's paper is a most valuable
contribution to the subject. h
i
i h
h
h
h
d
i
i
i contribution to the subject. The writer agrees with the author that " secondary ionization
is the cause of initial breakdown and formation of corona."
For out-door conditions he prefers the terms " native " ioniza-
tion to " antecedent " ionization as used in the paper. He
differs from the author in the conclusion that native ionization
enlarges the effective terminal until the stress (intensity)
at
its surface falls to the ionizing value. Native ionization strength-
ens the stress near the terminal and diminishes the distancetothe
ionizing zone. This distance is required to accomplish the process
of general ionization by collision, i.e. the formation of corona. At
a stress of 76 kilovolt-in., or 30 kilovolt-cm., ions travel nearly one
inch in 0.0002 second. The region about the electrode enclosed
by the zone in which the corona starts contains mostly native
ions of opposite polarity. Such ions will strengthen the stress
and lessen the striking distance to the corona forming zone. g
g
Recent studies of the normal in-door air show that the value
of J. J. Thomson and the one in which the author of this paper
expressed confidence, a year ago, is correct, namely, 76 kilovolt-
in. There is much evidence that 40 kilovolt-cm. as given now
by the author is too high for normal in-door air. y
g
The rapid outward spread of the stress from the surface of
a small conductor and the greater concentration of native ions
combine to make the striking' distance to the initial corona
zone shorter. Since the initial corona zone always forms in
the normal air at 76 kilovolt-in., the net result is that the surface
stress of the conductor rises as the initial corona striking distance
falls. DIELECTRIC STRENGTH It seemed evident from the
tests we made, that the ionization was due to the oxide on the
surface, in some inexplicable manner. In other words, the oxide
apparently causes a considerable degree of auto-ionization. After
we removed the oxide it disappeared, although the surface was
equally rough. The tests were made in hydrogen vapor, and
consequently the oxide on the copper was reduced by the
passage of an arc over the surface. These results are suggestions
although the phenomenon may be different from the effect of
duty copper wires in air. y
pp
J. C. Lincoln:
There is one question I would ask Dr. White-
head. In the diagram of his apparatus, he shows a wire in the
medium which
is controllable, and
I take
it from the
dia-
gram
that
there
is
passed
through
the medium an
alter-
nating voltage, and therefore that the ionization, such as there
would be, would tend to produce positive and negative ions in
the tube, and in that way the ions are passed across the indicating
instrument and some kind
of an indication
is
given. Ap-
parently either the negative or the positive ions are in pre-
ponderance, and I would like to know which did preponderate. p
,
p
p
Charles F. Scott:
I want to say a word in appreciation of
the three papers which have been presented, and the work they
represent, and the valuable part which these papers will play
in the progress of the art. i
i
fi ld lik
hi
hi h
i
i p
p
p
Charles F. Scott:
I want to say a word in appreciation of
the three papers which have been presented, and the work they
represent, and the valuable part which these papers will play
in the progress of the art. p
g
In going into a new field like this high voltage field, there is DISCUSSION AT JEFFERSON 19101 1217 good deal of pioneer work to be done of two kinds-work in the
laboratory and work in the field, theoretical work and practical
work. Very often these alternate. First we take one kind of
step and then the other. Dr. Whitehead enumerated several
papers which have been presented to the Institute, which are
preliminary to his, and form a series. In running over these
papers, I note the first one is on the dielectric strength of air by
Dr. DIELECTRIC STRENGTH In regard to the statement " In view of these discrepancies
one naturally turns to Mershons' method for measuring the loss
as a possible source of error."
A careful study of the methods
used at Niagara and recent laboratory tests have convinced the
writef that Mershon's methods and results are correct. There
are no real discrepancies. The surprises are due mainly to the
larger native ionization at the Falls. Mershon's Niagara corona
zone
stresses go below 76
kilovolt-in.,
because
of irregular [June 29 DIELECTRIIC STRENGTH 1218 [June 29 distribution of stress about parallel conductors worked with
alternating high-tension; this irregularity is produced and en-
hanced by the larger supply of native ions occurring at Niagara
Falls. The vapor-product
is an
indirect
evidence
of
their
quantitative variation. q
R. D. Mershon:
Referring to the paper by Mlr. Hayden and
Dr. Steinmetz, I would ask them to make clear how they de-
termined the energy available at the spark gap; how they found
out what energy was effective. i
i
i
i
i gy
C. P. Steinmetz:
We did not determine it, do not know it. R
D
M
h
Th
it
t
it i
tt h
d t
i R. D. Mershon:
Then it seems to me it is pretty hard to arrive
at any definite conclusion. y
C. P. Steinmetz:
The only conclusion
is the
total energy
supplied to the apparatus, hence the energy at the spark gap
must be less than that. We give only the energy limit. g
y
gy
R. D. Mershon:
The question of energy is related to that of
voltage. I do not see how you can have any idea of the voltage
finally impressed upon the dielectric. y
p
p
C. P. Steinmetz:
I will answer that in the final discuss
R
D
M
h
R f
i
t
D
Whit h
d' y
p
p
C. P. Steinmetz:
I will answer that in the final discussion. R
D
M
h
R f
i
t
D
Whit h
d'
th R. D. Mershon:
Referring to
Dr. Whitehead's paper, the
questions and doubts he raises in regard to the accuracy of my
high-voltage measurements at Niagara are all of them questions
and doubts which I myself raised at the time the results were
obtained, and which I feel were resolved by the careful tests
made of the accuracy of measurement. DIELECTRIC STRENGTH Before we dis-
covered the relation between loss and vapor product, we would
sometimes get consistent results over a period of several days,
and then the results of the next day would be all out of line. It
even happened at times that of a set of observations all taken on
the same day the results would be erratic and entirely incon-
sistent. But all these readings, when interpreted by means of
the vapor product, came together and were consistent; the re-
sults of different days came together; discrepancies occurring
on the same day were reconciled. So I cannot conceive of the
relation referred to as being in any way due to errors in either
measurements or apparatus. In this connection I would call your attention to the facts
brought out in one of the other papers here, relative to the
very small amount of moisture necessary in oil in order to enor-
mously change its dielectric qualities. In what condition does
that moisture exist in the oil? Is it water, or vapor? It cer-
tainly must be very finely divided, at any rate, for it is uni-
formly distributed through the
oil. This may have a con-
siderable bearing upon the loss in air due to moisture; a loss
entirely aside from the so-called ionization loss. y
The only error that I can conceive of as possible, due to the
method of measurement, would arise from phase displacement
in the measuring apparatus
itself. The
tests made
of the
apparatus, as detailed in my paper, seem to me to pretty clearly
show that there was no such error, or at any rate it was not
great enough to account for the discrepancies, if you may call
them such, between my results and those of Professor Ryan and
Dr. Whitehead. I would call your attention to the fact that the results ob-
tained by Dr. Whitehead, and to a considerable extent, the
results obtained by Professor Ryan, are not necessarily com-
parable with my results. I did not investigate the matter of
ionization. I investigated the point at which the loss between
outdoor transmission
line conductors begins to rise rapidly,
which point I called the critical point. I do not think this loss
is necessarily all ionization loss. It seems to me it may include
losses other than those due to ionization. The results of Pro-
fessor Ryan and Dr. DIELECTRIC STRENGTH We tested out the
method of measurement in every way possible and in consequence
of these tests I am well satisfied that the method of measure-
ment is a reliable one, and that the results obtained are sub-
stantially correct. I feel confident the accuracy of the measure-
ment is within 5 per cent, and probably much closer than that. Reference to my paper will show the kind of tests made, and
the method of making them. g
The apparatus used at Niagara was not that used at Telluride. It was of the same kind, but better designed and executed. At
Telluride I had a 1,000,000-ohm resistance with which to test
out the accuracy of the measuring apparatus. At Niagara no
such resistance was available, but in view of the closely checked
results obtained at Telluride making use of the resistance, to-
gether with the fact that the Niagara apparatus was an im-
provement on that at Telluride, and in view of the results of
such check-readings as could be taken at Niagara, I have no
suspicion whatever of the accuracy of the Niagara apparatus
and the results obtained with it. Dr. Whitehead says: Dr. Whitehead says: y
" Since Mershon's observations appear to have been taken
in
all
seasons, it may be stated that a range of temperature of 35 deg. cent. and
a pressure of 25 mm. of mercury, and an error of 5 per cent in thecritical
point as taken from the curves, would give a range of critical voltage as
wide as that attributed by him to moisture ". y
I judge from this that Dr. Whitehead thinks the relation I
found between critical point and vapor product may be the re- DISCUSSION AT JEFFERSON 1910] 1219 sult of a series of errors. If this is the case, it is curious that
these errors should have occurred so consistently over a year and
a half, during which time measurements were taken almost
every day. y
y
I do not see how it is possible that any errors in our observa-
tions or in our method of measurement could account for the
variation
of
critical
point with vapor product. The
effect
was entirely too marked to be so accounted for. DIELECTRIC STRENGTH Whitehead were obtained under laboratory
conditions. My
results
were
obtained
under
engineering
conditions. I do not believe we are safe in applying the results
of laboratory investigation to the engineering problem, unless
we can check up these results under engineering conditions. DIELECTRIC STRENGTII 1220 [une 29 There are too many other elements which may enter to modify
laboratory results as applied ir- practice. y
pp
p
What I have said applies to the conclusions. Conclusion 5
says, " Water vapor or moisture has no influence on the electric
strength of air. Increasing moisture content probably lessens
the loss above the critical voltage ". That depends on what
you mean by the critical point. If you mean the point at which
ionization begins, the statement is possibly true, but if you mean
the point at which the loss between the wires of a transmission
line begins to rapidly ascend, I do not agree with that at all. Similarly with regard to Conclusion 6-" There is no loss through
air until the critical voltage accompanied by ionization and
corona is reached ". I believe there may be a loss below the
critical point, as I have just defined it, and that this critical
point does not necessarily correspond to the inception of ioniza-
tion. Now, as regards Conclusion No. 10: " The corona has high
conductivity, and most of the loss takes place beyond
it ". I do not remember anything in the paper which would appear
to justify this conclusion. If there is, I shall be glad if Dr. Whitehead will bring it out more strongly. j
y
,
Whitehead will bring it out more strongly. i
i g
g y
Referring to Mr. Tobey's paper, Fig. 7 would be much more
interesting if the melting point of the oil were shown, and it is
to be hoped that in closing Mr. Tobey will give us the melting
point. This curve presents some facts in regard to which
I
have often wondered. I have been afraid sometimes that if the
oil in transformers was allowed to solidify, due to cold, we might
get into trouble, but according to this curve we are safer with the
oil solidified than with it in its normal condition. C. P. DIELECTRIC STRENGTH Steinmetz:
First, to answer some questions regarding
our paper-the energy values, are, as stated in the paper, not
the energy appearing at the spark-gap, but are the total amount
of energy supplied to the system through the transformer, and,
as stated in the paper, the actual energy supplied to the spark-
gap probably is only a small part thereof, so that these values
merely mean the upper limit of energy, which is sufficient to
break down the gap, and probably much less energy will be
enough. g
As regards the question whether the voltage was actually at
the spark gap or not, we carefully looked into the matter and
came to the conclusion that this voltage is there, at least approx-
imately
this voltage,
as
near
as
it was possible
to judge. Mathematically, I gave the reasoning in the appendix, which
shows how we
calculated
the
voltage
which
appears
at
the secondary terminals and thereby at the spark-gap. We
know if there is one thing where experiment and calculation
agrees, it is in transient phenomena, provided you consider all
the factors involved in the problem. p
There are two factors which have not been included in that
mathematical
calculation. One
is the
variation
of the
in- DISCUSSION AT JEFFERSON 1910] 1221 ductance; the second is the capacity of the secondary circuit. It can easily be seen that the variation of the inductance with
the voltage has no effect on the voltage, but merely modifies
the shape of the voltage curve of the secondary. There re-
mains then the consideration of the secondary distributed ca-
pacity. From the constants of the transformer, its estimated
resistance, inductance and capacity, it appears obvious, that the
effect of capacity could result only in an oscillation, not in a
steady change, that is, that at the beginning of the impulse
there might appear and probably does appear an oscillation
superimposed on the steady discharge curve, calculated in the
appendix. Such oscillation may be either limited to the high
voltage circuit, and then be of very short duration or small
energy, or it may extend into the primary circuit. gy
y
p
y
In the paper we gave evidence that such oscillation did not
extend into the primary circuit, by changing the resistance of the
primary circuit, increasing the non-inductive resistance of the
primary supply circuit nearly a hundred-fold, and getting the
same result. DIELECTRIC STRENGTH This phenomenon of the great increase of
disruptive strength of air close to the solid terminals I observed
and gave the explanation, that it is due to the condensed layer
of air, some years ago, I believe in the paper which I read on
disruptive strength before the Institute in 1893. There I found
that in testing very small air gaps the disruptive strength very
greatly increased, beyond that observed in the case of larger
air-gaps between parallel plates. g p
p
p
As we know, Professor Ryan has very carefully looked into
this effect, and in his paper of 1904 gave it a numerical value. I believe it would be very interesting, first, to take his n.umerical
value of the layer of condensed air, and reduce Dr. Whitehead's
tests by this assumption, thereby seeing whet;her we do not
get a greater constancy, and then, inversely, from the data of this
test, calculate how thick a layer of condensed air we have to
assume to get constant break-down strength of the air. It is
reasonable to assume that the layer of air is not the same, but
decreases with decreasing diameter of the wire, due to the effect
of divergence. g
If there exists such a layer of condensed air, the result would
be that the initial break-down occurs, not at the surface of the
conductor, but at a small, though finite distance from the con-
ductor surface, and with the increasing voltage the break-down
extends outwardly, but also
extends
inwardly
towards
the
conductor, and at a second higher voltage reaches the conductor. g
g
This gives a second point in the curve where a change of the
phenomenon might be expected to occur. It is very interesting
to realize that with our increasing knowledge of the phenomena
of disruptive strength, we are more and more impressed with the
similarity of the electro-static strength, and its laws and phe-
nomena, with the laws and phenomena existing in the case of
mechanical forces, as pointed out also by Dr. Karepetoff. DIELECTRIC STRENGTH It is obvious if there is any oscillation taking effect
in the primary, that by increasing the resistance of the oscil-
lating circuit nearly one hundred-fold, from about
3 ohm to
26
ohm, the
oscillation
must
be
enormously
reduced and
damped out, and we found the same transient discharge. I
believe, however, there was in the high potential secondary
circuit an oscillation superimposed on the exponential curve of
the calculated voltage ratio, but of extremely short duration,
and as the entire effect is a transient, you see that an oscillation
of still much shorter duration at the beginning of the effect,
could not well be expected to have an appreciable effect. It is,
however, desirable, and we hope some time to be able to carry
out corresponding investigations by some other method of pro-
ducing the transient voltage, and thereby being able to de-
termine exactly the amount of energy existing at the spark-gap. i
i
i y
gy
g
p
g p
A most interesting paper we have in the next paper, that by
Dr. Whitehead, which I consider as extremely valuable and im-
portant at present. I may say, indeed, that we had intended
to make the same investigation, even assembling apparatus of
the same kind, a conductor in the center of a tube, and measuring
the beginning of the breakdown by an electroscope. We ex-
pected to carry that investigation a little further, that is, to
lower air pressure, so as to study more carefully the effect of
higher altitudes, since we know that transmission lines at the
present time extend to altitudes where the air pressure is ap-
preciably reduced. i p
y
There is only one point in Dr. Whitehead's paper, concerning
which I do not agree with him, and that is his opinion that the
explanation of the apparently greater breakdown strength
of
air with small wires cannot well be given by the assumption of
the zone of condensed air around that wire. We know, and it
can easily be shown physically, that all solids are surrounded DIELECTRIC STRENGTH [June 29 [June 29 [June 29 1222 by a zone of condensed air, and a rough calculation will show
that the densities of this air at the immediate neighborhood of the
conductor, by gravitational or molecular attraction, may reach
considerable values. DIELECTRIC STRENGTH Only
I do not agree that the electrostatic phenomena are simpler than
the mechanical, due to the absence of shear, but there is very
good evidence which makes it probable that electrostatic shear
also exists, because at the edge of the electrostatic field, where
the electrostatic field very abruptlv changes, for instance, at
the edge of two parallel plates, there is a very much greater
disruptive force; although the potential gradient would be the
same, and there
is some evidence
qualitatively, though not
sufficient quantitatively, that not only the disruptive potential
gradient, but also the abrupt variation of the potential gradient
causes a breakdown. This was discovered
first in the old
Ferranti system in London, England, in bygone ages, when the
cables had the unhappy habit of breaking down at the edge of
the insulation-where there is an abrupt change in the electro-
static field, that is an electrostatic shear, and it is so well known DISCUSSION AT JEFFERSON 1910] 1223 in the industry that we always carefully avoid abrupt changes
of the electrostatic field, for instance, at the end of lead covered
cables taper the field, etc. Experience has shown that if we
allow too sudden variations of the static field, we break the insu-
lation down, although the insulation is just as thick as where
the field is uniform. Originally when electrostatic phenomena were first investi-
gated, they did not appear to agree with a constant breakdown
strength of air, but gradually the phenomena lined themselves
up in the direction of law and order, pointing towards a definite
strain which air will stand,; and beyond that will break down,
and what we here are mainly interested in is the range beyond
the break down, that is the phenomena beyond the electrostatic
elastic limit of insulating material, where the phenomena have
ceased to be reversible and become irreversible. It is the same
thing in mechanical engineering, and we shall listen still to a
paper dealing with an analogous problem in the mechanical force. DIELECTRIC STRENGTH That
means the distribution of the lines of force changes and the
entire field changes and the problem is to find that equi-gradient
surface which at the same time is an equi-potential surface, and
has the particular numerical value of the break down strength of
air, and that is rather a more difficult problem. h
d
h
i
h
l
f
d
d
i
hi h p
The second phenomenon is the layer of condensed air which
surrounds the solid material. The result is that the disruptive
strengtk at the surface of the terminal is different from what it
is in the space at some distance. At atmospheric pressure it is
very much higher. The result of that would be, as I stated,
that the initial break-down occurs at a voltage corresponding
to, not the break-down gradient at the conductor surface, but at
the space some distance away, or, in other words, it occurs when
the ratio of the electrostatic gradient to the density of the air
has reached the maximum at some point at some distance from
the conductor. This also requires further theoretical investiga-
tion, which probably can be done, because we know the law of
gravitation, and the gas laws, and the layer of condensed air
could be calculated. When you have electrostatic forces acting, the phenomena
may be expected to be complicated still further, as Dr. Kennelly
pointed out, by the electrostatic attraction, which changes the
density of the layer of air and its volume. y
y
Lastly, and one feature which we usually overlook, is the
resistance of the conducting space of the effective terminals. It is not correct to say that the space within which the air is
broken down and which forms the effective terminal, is a perfect
conductor, but it has some resistance, and therefore quite con-
siderably modifies the phenomena. To illustrate
it by the
discharge between needle points-we have a discharge occurring
between these effective conductors, spherical spaces of ionized
air, but these spaces are not perfect conductors-they have a
certain resistance, and the resistance has the characteristic of gas
resistance, that is, it increases enormously with decreasing current
density. DIELECTRIC STRENGTH This is an extremely interesting field, but the difficulty of ex-
ploring it is the same as in the case of almost all phenomena-
you never find in nature a clear cut case of the action of one
phase or one form, you always have it complicated by numerous
other conditions, forces or phenomena which are existing as
secondary actions, and practically most of the work of the elec-
trical engineer and other engineers consists of a very simple cal-
culation of the theoretical condition, and then he must calculate
and estimate the secondary actions which modify, and some-
times entirely obscure the primary action of the phantom ap-
paratus under phantom conditions. p
p
The most important secondary actions in electrostatic phe-
nomena are:
i
i
i First, the phenomena of the effective terminal. That
is,
when we have electrostatic fields, the terminals of the field are
not the solid terminals, the needle points or spheres, but are
made up of that space surrounding the solid terminals within
which the air or space is broken down, has passed beyond its
elastic limit and is more or less conductive. Between needle
points, even at extremely low voltages, the break down strength
is excedeed at the points, nevertheless the spark discharge does
not occur until much higher voltages are reached, and when it
occurs it is not a discharge between needle points, but between
these effective terminals, these approximately spherical spaces
of conducting or ionized air. g
The study
of that phenomenon mathematically
is rather
difficult; is not as simple as it appears. We can easily consider
the electrostatic field with its force lines and equi-potential sur-
faces between needle points or spheres, and then see at a given
voltage what is the equi-gradient surface which corresponds to
the break-down of air, and all the air space inside of this surface
would become conductive. But as soon as the space becomes
conductive up to the equi-gradient surface, the equi-gradient DIELECTRIC STRENGTH Uune 29 Uune 29 Uune 29 1224 surface becomes an equi-potential surface, while it is not an
equi-potential surface in the original electrostatic field. DIELECTRIC STRENGTH The
cause of this deviation from the true theoretical curve seems to
be the resistance of the effective terminals which, within that
range, are still so small that the current consumed by them is
sufficiently low to give a resistance, consuming a voltage com-
parable to the voltage between the needle points. At higher
voltage the space ionized is so large and the current absorbed
is so great that the effect has disappeared. That is another
secondary phenomenon which requires consideration. y p
q
Again, we really do not observe the break down voltage, or
the elastic limit of air directly, but always indirectly by dif-
ferent methods, and there is no evidence that the point we
observe by different methods is the same point. We get it by
the disruptive discharge. We get it by observing the beginning
of ionization, as described in Dr. Whitehead's paper. We get
it by the corona. We get it by measuring the energy loss in the
conductor as in Mr. Mershon's paper or Mr. Ryan's paper. Then we get it by the increase of capacity of the conductor,
at the voltages where the corona spreads out and increases the
effective
conductor. There
is no reason why the
different
methods should really give you the same point. As soon as the
air begins to break down anywhere in space, an irreversible
process occurs. The energy consumed by it is not returned. It means at that point the energy loss in the electric system must
appear. If we could measure the beginning of the energy loss
exactly, which is an extremely difficult problem, we could show
the beginning of the energy loss, the beginning of the break down. i g
g
gy
g
g
As there is a layer of condensed air, that initial break down oc-
curs at some distance from the conductor, and the conductor,
therefore, is surrounded by a zone of conducting air which is
separated from the conductor by a layer of compressed and
insulating
air. There
is no conduction of current from the
conductor into the ionized air region until a higher voltage is
reached, when the zone of disrupted air spreads to the conductor
and current begins to flow into the zone. DIELECTRIC STRENGTH Now, the current density depends on the size of the
conducting terminals, the volume which has to be ionized, and
also on the frequency, and you see, then, within this range,
until you have reached so large conducting spheres, effective
terminals, that the amount of current required for ionization
is large enough to give a negligible resistance, the frequency as
well as other features, have no effect. That is very markedly
shown in the striking distance curve between needle points. Theoretically, we can calculate
it, allowing for the effective
terminals, the ionized air space, and no matter what allowance
we make we can easily show that the striking distance between
needle points should be a straight line going through the origin. Now, the actual curve, as we found it by test, is given in the
paper in the 1898 TRANSACTIONS, at 125 cycles; it differs from the DISCUSSION AT JEFFERSON 1225 1910] straight line at lower voltages. More recent tests at 60 cycles
seem to show a curve in which the deviation from a straight line
extends to somewhat higher voltages, and there seems to be
some evidence which makes it probable that at extremely high
frequencies, 100,000 cycles, the curve remains straight practi-
cally down to zero, that is, is the true theoretical curve. The
cause of this deviation from the true theoretical curve seems to
be the resistance of the effective terminals which, within that
range, are still so small that the current consumed by them is
sufficiently low to give a resistance, consuming a voltage com-
parable to the voltage between the needle points. At higher
voltage the space ionized is so large and the current absorbed
is so great that the effect has disappeared. That is another
secondary phenomenon which requires consideration. straight line at lower voltages. More recent tests at 60 cycles
seem to show a curve in which the deviation from a straight line
extends to somewhat higher voltages, and there seems to be
some evidence which makes it probable that at extremely high
frequencies, 100,000 cycles, the curve remains straight practi-
cally down to zero, that is, is the true theoretical curve. DIELECTRIC STRENGTH It appears possible
that the point where the capacity of the conductor increases is
not the point where the initial break down occurs, but is the point
where the zone of broken down air gas spread up to the conductor
and reached it. It is curious to note that there are also two visible steps in the
luminosity which are quite sharply marked, and are also ap-
parently sharply distinguished by their chemical action. First
appears the glow, the noiseless or silent discharge. Perhaps this
is the break down at some distance from the conductor, not at the
conductor-while the air at the conductor still is insulating, but DIELECTRIC STRENGTH rJune 29 rJune 29 1226 rJune 29 at some distance therefrom has broken down. This does not
appear at needle points, where the break down must occur at the
beginning, but
it appears verv markedly
at plane surfaces. Then at higher voltage, the blue glow is superseded by violet
streamers, which are noisy and are interrupted, intermittent,
and appear to represent conduction of current, when that ionized
layer of air has reached the conductor. When conduction occurs
into the space, that conduction, following the gas laws, has the
effect that as soon as current begins to pass locally the resistance
at the space falls, a discharge occurs, the voltage disappears and
the current disappears, so by the laws of gas conduction the
current must be intermittent and must always be a localized
expenditure of energy as a spark or streamer. At this point,
apparently, the chemical action changes. Where we have the
blue glow, the energy seems to be sufficient to dissociate the
oxygen molecules, but not the nitrogen molecules, and ozone
formation takes place. As soon as you see these violet streamers,
not only the oxygen molecules, but also the nitrogen molecules
split, and you get nitric-oxide, but less ozone, because at the
high temperature of that streamer discharge the ozone is broken
up, so that there seems to be a sharp dividing line. Below that
the blue discharge produces ozone, above it the streaming dis-
charge or brush is intermittent, and of different color, noisy,
and seems able to fix nitrogen. h
h l
bj
i
l
i
i g
p
John B. DIELECTRIC STRENGTH While there are still many phenomena which have not yet been
brought into perfect accord, those which have been studied
most widely show conclusively that for a proper understanding
of the conditions governing the break-down of insulation whether
gaseous, liquid or solid, the way must be through the knowledge
of the properties of ions which has been already gained. These
comments are suggested- by the mode of interpretation
used
by Messrs. Hayden and Steinmetz in discussing their results. We are to be congratulated upon the presentation of so careful
and accurate a set of experiments in a field so little understood. The conclusions drawn from the experiments by the authors,
however, invite some comment. ,
co
e t. Disruptive discharge is said to require a definite minimum
amount of energy. The laws of spark discharge in air which
may be said to be firmly established, explain the phenomena
in terms of motion of gaseous ions, i.e., charged particles, acted
on by electric force. These ions have mass, and therefore ac-
celeration and momentum when in an electric field. The laws
of motion and impact of the simple mechanical system thus
presented lead in many cases to complete explanation. Spark
discharge is the result of secondary ionization or the collision
of a charged particle moving under electric force with a neutral
molecule, thus breaking up the latter into new ions and so fur-
nishing the conductors for the discharge. Since the free ions
have mass they require time to attain speed. If the electric
force is transient and of sufficiently short duration the ion may
remain practically unaffected. The study then of the phe-
nomena of transient voltages should be extended to that of the
shape and duration of the voltage impulse, i.e., the integrated
product of intensity and time in relation to the known properties
of gaseous ions. Even granting that the initial discharge voltage
of the experiments is properly deduced from the ratio of turns,
this value cannot exist for more than an infinitesimal time and
the appendix concludes that the shortest and longest time dura-
tions of a pulse are in the ratio 1 to 100. It is concluded that
the discharge does not instantly follow the application of voltage,
although the observations leading to this conclusion are not
described. DIELECTRIC STRENGTH Whitehead:
Before answering the questions which
have been asked in connection with my paper, I want to say a
few words in general comment on the three papers which have
been presented at the morning session. p
g
These three papers all deal with the effects of high voltage on
insulation. Each of the papers presents a set of experimental
results on this perhaps most important of all electrical questions. All of them fail, however, to explain the observations in terms
of simpler phenomena or physical laws at present understood. Nor do the few suggestions of explanations which are offered
coincide
either in point
of view or language of expression. There is here then a sharp indication of present uncertainty
surrounding these highly interesting phenomena. The uncer-
tainty, however, is by no means as great as may appear from
this confusion. The phenomenon of spark discharge in all its
manifestations has been a subject of study by experimental
physicists for many years. As a result of this study the ionic
theory has not only paved the way for most fruitful investiga-
tion, but has been finally established as a medium through which DISCUSSION AT JEFFERSON 1910] 1227 many electrical manifestations now receive their explanations
in terms of the laws of simple mechanics. For no set of phe-
nomena is this more true than for the spark discharge in gases. While there are still many phenomena which have not yet been
brought into perfect accord, those which have been studied
most widely show conclusively that for a proper understanding
of the conditions governing the break-down of insulation whether
gaseous, liquid or solid, the way must be through the knowledge
of the properties of ions which has been already gained. These
comments are suggested- by the mode of interpretation
used
by Messrs. Hayden and Steinmetz in discussing their results. We are to be congratulated upon the presentation of so careful
and accurate a set of experiments in a field so little understood. The conclusions drawn from the experiments by the authors,
however, invite some comment. many electrical manifestations now receive their explanations
in terms of the laws of simple mechanics. For no set of phe-
nomena is this more true than for the spark discharge in gases. DIELECTRIC STRENGTH It is also stated that during the interval energy is
being supplied to the dielectric, and relations are deduced be-
tween energy and sparking distance for air and oil, as given on
page 770. Although the observed points in the two cases fall on
curves of different shape they are assumed to have the same
equations, and although the energy is measured at the trans-
former primary, it is assumed to be a basis of comparison for
the secondary spark-gaps. It would be interesting to know
what part of the energy delivered to the magnetic circuit is
regained upon opening the primary circuit. It would also be DIELECTRIC STRENGTH 1228 [June 29 [June 29 [June 29 interesting if the authors would present a conception of the
mechanism by which a dielectric accumulates energy leading
up to disruptive discharge. p
p
g
Taking up the points that were brought out in connection
with my own paper, the first question was that of Mr. Mershon
as to the point on the voltage wave at which the corona ceases
in relation to the point at which it starts, and he raised the
question as to just how you could expect the corona to stop at a
lower voltage than that at which it started. The indication is
that there is only a small lag, if any, in the point of disappearance
of the corona. The corona, however, is a source of heat in the
gas, and the raising of temperature has the effect of lowering
the corona voltage. g
Regarding Mr. Thomas' questions-first, the conditions at the
ends of the tube: The wire is carried through a cylindrical metal
bushing set in a glass receptacle of the shape very much as
indicated in the drawing on page 1063. There is no observa-
tion of spark discharge, no corona nor anything else indicating
ionization at this point, occurring before that on the length of
wire within the tube. If you carried the wire through a rubber
or other insulating bushing, ionization would first set in at that
point, and would spread to some extent along the wire inside
of the tube. The variation of electric field at the end of the
tube is quite marked, of course, outside the tube, but does not
extend to the region inside for any considerable distance. DIELECTRIC STRENGTH These
differences are to be expected in view of the known fact of the
difference in the rates at which the positive and negative ions
move in the gas and the different rates at which they diffuse. The point of break-down can be observed with either kind of ion. k d
i h
h
h
i p
I have not worked with any other gas than air. h
i
i i
i
i y
g
The question as to the positive and negative corona is taken
up in the paper, rather briefly, and I have pointed out that
there is no apparent difference in the positive and negative corona
unless
it be possibly the somewhat greater brightness and
sharper definition of one. I am not prepared to say that there
was any noticeable difference. y
As to the relation of initial ionization to the following arc,
the corona starts evenly, as you are, of course, aware. As you
go on increasing the voltage you will finally get to either a
spark or an arc. Now then, just what this range of voltage is
will depend on the relative diameters of the inner wire and the
tube. The potential gradient between a tube and the central
wire depends upon the diameter of the wire in such way that
under some circumstances an increase in diameter will mean
a lowering of the electric intensity and under other conditions
it will mean an increase in the electric intensity. With the
increase in corona diameter, resulting from increased voltage,
when the corona reaches a point so that any further increase
means an increase of intensity the spark or arc follows. y
p
Dr. Kennelly and Dr. Steinmetz have each suggested that an
increase due to pressure in the neighborhood of a small wire
may offer a means of explaining the increase of electric intensity
of corona and break-down. I am not quite sure that I grasp
Dr. Steinmetz's idea, in that I cannot see why you should expect
any difference between the case of small and large wires. Pro-
fessor Kennelly's idea is, of course, a possible one, and there is
no difficulty about imagining such a thing to occur. DIELECTRIC STRENGTH As
soon as corona forms, it is very evident that no error is intro-
duced on this account. The whole apparatus was, of course,
enclosed with the idea of conducting any ionization that was
generated out through the gap leading to the electrode. I think
there was no error on this score. With reference to the question as to whether in a small wire
rapid change of gradient at the surface would explain any of
these phenomena: The potential gradient does change very
rapidly; however the distances within which the potential gra-
dient varies widely are very much greater than mean free paths
of the molecules and ions. P. H. Thomas:
You did not catch my point-the question
is whether the volume of air which was subjected to something
like the critical strain was not greater in the case of the large
wire, not that the mean free path was involved, but the sur-
rounding presence of supporting ionized air might prevent the
overwhelming of the very narrow zone there, very narrow parti-
cles, with reference to the free paths. ,
p
J. B. Whitehead:
I am not sure that I get the idea. Ioniza-
tion in this case is a sharply cumulative effect. As soon as you
get a region where there
is this secondary ionization, when
this second ionization starts, it is a continuous thing, and if
you attempt to explain it by any relation between the mean
free paths of the ion or the molecule, and the variation in diameter
of the wire, there is the widest kind of discrepancy. I have DISCUSSION AT JEFFERSON 1910] 1229 given the figure 105 in the paper showing that the two quantities
are of different orders of magnitude. g
Referring to the question as to the state of the air as it leaves
the wire, whether it has positive or negative ions-I think Mr. Lincoln asked a similar question-a gas ionized in this way has
both positive and negative charges-the point of initial break-
down will be observed by charging the electroscope either posi-
tively or negatively. The only differences which come in are
in the shape of the discharge curve of the electroscope. DIELECTRIC STRENGTH I would
say, however, that I have made efforts to observe any increase
in pressure in the neighborhood of these wires by optical means
-the increased density of this air in its relation to the neighbor-
ing portions, permits the use of a very simple optical method
of observation. Such experiments were negative in my case,
although I cannot say I think that is any conclusive proof that
some such increase of pressure may not explain the observed
facts. Since we are dealing with extremely small distances, and
I am not sure that even with the optical method I have used the Ujune 29 Ujune 29 Ujune 29 DLELECTRIC STRENGTH 1230 layer would be sufficiently thick to manifest itself in this way. The test is known to physicists as the " Schlieren " method. p y
Professor Creighton made some comments as to the auto-
ionization of the air and the necessity of removing ions. I do
not know the word auto-ionization. Ions may be generated
by secondary ionization,
as in coronae, and recombine
very
promptly; the air is then to all appearances in quite the same
state as it was before secondary ionization or coronae set
in. This is shown very well by the sharpness with which the corona
stops and by the absence of any after effects of ionization. There are always a certain number of free ions in the air, and
these ions account for the small conductivity which air has. If
you generate ions by some external means, and put them in the
air, it simply means you can get a greater leakage current, but
the presence of these ions does not in any way hasten or lower
the point at which secondary ionization steps in,
it simply
increases the leakage current. There are numerous ways of
removing free ions from the air; the use of glass wool, or crude
raw cotton and various materials of like nature, through which the
air can be drawn to a perfectly pure state, so far as ionization is
concerned, but it will always have a certain extremely small
number of free ions. The natural conductivity of the air is
entirely explainable on this basis. DIELECTRIC STRENGTH Mershon has pointed out that his critical point is not neces-
sarily the same as my own, but I am unable to see any other
explanation than that of corona and break-down for the sharp,
upward bend of his curves. I am unable to think of any means,
also, by which there is a possible loss through the air below this
point. po
t. It has been suggested by several that laboratory methods
present conditions different from those obtained outside and
that this may be an explanation of some of these differences. I should be interested to hear some one suggest some means
or method by which a loss could take place through air at
voltages below those at which it actually breaks down. i
(
i
i g
y
M. A. de Chatelain (by letter):
The writer and his associate,
Professor V. F. Mitkevitch, were led to the question of corona
formation and the accompanying losses in connection with the
question of possibility of high-tension power transmission in the
vicinity of St. Petersburg, Russia. i
f
d
li d i
l y
g
Laboratory experiments were performed on cylindrical con-
ductors of different diameters, placed at different distances from
each other. In view of discrepancies in the values of the critical
voltage, as given by Messrs. Scott, Mershon, Ryan, Kapp, and
Berg, among themselves and with our results, Professor Mit-
kevitch was led to investigate the matter theoretically. Based
upon J. J. Thomson's researches on conductivity of gases he
has found the following formula for the critical voltage: E
5= H273
. r log10,- (in kilovolts) where H is the barometric pressure in mm. of mercury;
i
i
i
i H is the barometric pressure in mm. of mercury;
i
h
f
i
i
d
i
d p
y
t is the temperature of air in degrees centigrade;
i
h
di
f
h
d
i p
g
r is the radius of the conductor, in cm.;
d i
h
di
b
h
i d is the distance between the centers, in cm. Changes in humidity did not produce any appreciable varia-
tions in results. The values calculated according to the fore-
going formula were checked experimentally in the electrical
laboratory of St. Petersburg Polytechnic Institute, and were
compared to the results given by Professors Ryan and Kapp
for the same conditions. DIELECTRIC STRENGTH For reasons which will be
found in the remarks I have just made, I do not think Professor
Ryan's suggestion, as well as I was able to gather it from hearing
his letter read in the meeting, that there may be a difference
between indoor and outdoor ionization, offers an explanation of
the discrepancies that he is calling attention to. The range of
natural ionization throughout the regions which have been in-
vestigated all over the earth, is about in the ratio of 1 to 4,
and as I have said, the presence of more or less free ions in the
air, simply increases conductivity, but I do not believe under
any circumstances it would hasten the point at which secondary
ionization, spark discharge, or corona would start. h
h
k d
h
ff
f
h
f
h p
g
Mr. Mershon has asked the effect of the wave form on the
observations. The discussion of the wave form is given in the
paper, showing the relation of maximum to effective values, and
also in the reference to the curve Fig. 5. It
is
possible
to
observe the corona very close to the peak of the wave, and it is
so sensitive that it was possible by careful voltage adjustment
to pick up the three little peaks by the stroboscopic method
with intervening dark spaces corresponding to the dimples. i
i
i i g
p
p
g
p
As to Conclusion 10, that the corona has high conductivity
and most of the loss takes place beyond it, that is based on the
results given in the paper, where it is shown that the intensity
at the edge of the corona is the same, practically the same, differ-
ing from it by a small amount, as that of a solid wire of the same
diameter, and if that is the case it is a fairly reasonable as-
sumption that the corona has high conductivity, and marks
the limit at which ionization takes place. 1910] DISCUSSION AT JEFFERSON 1231 In calling attention to Mr. Mershon's method of measurement
I have been led by the process of elimination of all other ex-
planations
of
the
discrepancies
between
our
results. Mr. DIELECTRIC STRENGTH The comparison is given in Tables
I and II. It will be seen that the difference between the volt-
ages at which the corona became visible, and those calculated
according to Professor Mitkevitch's formula, are not large. i
i g
g
It may be appropriate to mention here that Professor Kapp's
adaptation
of Mershon's formula
(Journal
of the
Inst. El. Engs., February, 1910) leads to an odd result, namely, that the
critical voltage, beyond a certain limit, becomes lower with
increasing diameter of conductors (see Table III). 232
DIELECTRIC DIELECTRIC STRENGTH 1232 Uune 29 Uune 29 Mr. Mershon's statement that the losses increase with the
frequency led us to investigate the effect of frequency. Our
results seem to indicate that the loss is practically independent
of the frequency, and rather decreases at higher frequencies. Namely, we measured the losses between two parallel planes,
27 x 27 cm. each, one of which was covered with needles of the
same length. The distance between the points and the opposite
plane was 2 cm. The frequency was varied from 15 to 90 cycles
per second. After having taken into account the necessary
corrections for the capacity, etc., we found the following values
of losses: p
y
g
of losses:
Cycles
Watts
15
56
50
54.5
90
54
TABLE
I
d
Critical kilovolts
2 r (mm.)
mm. Observed
Calculated*
5 (cable)
...... 1000
65
60
11 (cable)
....... ..".120-125
115
11 (tube)
....... .. .125-130
115
14.3 (tube)
...... 145-150
141
*Formula Professor Mitkevitch
TABLE II
CRITICAL KILOVOLTS
d
mm. 2r =lOmm. 2r = 15mm. 2r2 20mm. Ryan
Kapp
Mitke-
Mitke-
Mitke-
vitch
Ryan
Kapp
vitch
Ryan
Kapp i
vitch
500
143
82
98
180
89.5
135
212
92.5
165
1000.......165
94
110
209
104
155
250
109.5
195
2000
l
106
125
236
119.5
177
287
125.5
225
3000 ........ 2
114
135
256
128
190
310
1135
240
TABLE III
d
2 r mm. mm. 5
10
15
20
25
30
50
100
150
200
2000....... 79.3
106
119.5
125.5
128.5
131
130
119.5
10000 ....... 98
136
154
164
171
172
177
169
163
156 50
54.5
90
54
TABLE
I
d
Critical kilovolts
2 r (mm.)
mm. Observed
Calculated*
5 (cable)
...... 1000
65
60
11 (cable)
....... ..".120-125
115
11 (tube)
....... .. .125-130
115
14.3 (tube)
...... 145-150
141
*Formula Professor Mitkevitch
|
https://openalex.org/W3034204874
|
https://link.springer.com/content/pdf/10.1007%2F978-3-030-42274-5_6.pdf
|
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| null |
From Scenarios to Use Cases, Test Cases and Validation Examples
|
Springer eBooks
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|
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From Scenarios to Use Cases, Test Cases
and Validation Examples K. Maki, A. Kulmala, K. Heussen, O. Gehrke, E. Rikos, J. Merino, M. Rossi,
L. Pellegrino, C. Sandroni, M. Z. Degefa, H. Taxt, D. Lagos,
and P. Kotsampopoulos 1
Test Scenario Descriptions In the context of ERIGrid, scenarios are meant to be higher-level circumstance
descriptions which will provide a basis for more detailed use case and test case
definitions. As a term, scenario often refers to visionary descriptions of future devel-
opment and the factors influencing it. Scenarios obviously apply long view perspec-
tives where many uncertainties are present. In the context of ERIGrid, scenarios
reaching to 2050 are of interest. In many cases, scenario work can feed in to political
processes and decision making on different levels. In the course of ERIGrid, generic system configurations have been considered
more useful than traditional high-level scenarios. A system configuration approach
allows including more detailed and quantitative data in the descriptions and providing
a better technical basis for developing the use cases and test cases. Whereas high-level K. Maki (B) · A. Kulmala
VTT Technical Research Centre of Finland, Tampere, Finland
e-mail: kari.maki@vtt.fi
K. Heussen · O. Gehrke
Technical University of Denmark, Roskilde, Denmark
E. Rikos
Centre for Renewable Energy Sources and Saving, Athens, Greece
J. Merino
TECNALIA Research & Innovation, Derio, Spain
M. Rossi · L. Pellegrino · C. Sandroni
RSE Ricerca Sistema Energetico, Milan, Italy
M. Z. Degefa · H. Taxt
SINTEF Energi AS, Trondheim, Norway
D. Lagos · P. Kotsampopoulos
National Technical University of Athens, Athens, Greece
© The Author(s) 2020
T. I. Strasser et al. (eds.), European Guide to Power System Testing,
https://doi.org/10.1007/978-3-030-42274-5_6 K. Heussen · O. Gehrke
Technical University of Denmark, Roskilde, Denmark 87 K. Maki et al. K. Maki et al. 88 scenarios give some qualitative statements about the progress, system configuration
uses quantitative data such as numbers of components, size of the system, etc. At the
same time, the system configuration becomes more complex due to the amount of
data but also more locally due to dimensions and local parameters. The system configurations allow development of use cases, which give a descrip-
tion of a process leading to a specific objective. In other words, use case defines the
actions needed to obtain some goal. Use cases are often described from an external
perspective in a neutral manner, utilizing a formal methodology. Use cases can also
be thought to define the interfaces of the process with its environment, inputs and
eventual outputs. Use cases can be defined from two perspectives: behavioural perspective and
interaction perspective. 1
Test Scenario Descriptions Behavioural perspective is always function-type; it defines
the behaviour of the process internally and towards external stakeholders. In the
interaction perspective, most interest is on interactions between components and
describing them, for instance by means of sequences. Test cases with reference to system configurations require information on system
parameters, ranges of parameters, system functionalities and quantitative measures. They also require information on test procedures and design of experiments. Test
cases define the actual test setup; which are the combinations and series to be tested
and which are the prevailing circumstances in which the tests are performed. Following definitions have been used within ERIGrid [3]: • System defined as a set of interrelated elements considered in a defined context a
a whole and separated from their environment. • System Configuration defined as an assembly of (sub-)systems, components, con-
nections, domains, and attributes relevant to a particular test case. • Scenario defined as a compilation of System Configuration, Use Cases, and Test
Cases in a shared context. • Use Case defined as a specification of a set of actions performed by a system,
which yields an observable result that is, typically, of value for one or more actors
or other stakeholders of the system. • Use Case defined as a specification of a set of actions performed by a system,
which yields an observable result that is, typically, of value for one or more actors
or other stakeholders of the system. • Test Case defined as a set of conditions under which a test can determine whether
or how well a system, component or one of its aspects is working given its expected
function. Distribution Grid System configuration “Distribution grid” considers the electricity distribution sys-
tem at MV and LV voltage levels. The area covered by this configuration starts at the
HV/MV transformer, where also the responsibility area of DSO typically starts. On
the low voltage side, the configuration is limited to the customer interface (metering
point) or at the connection point of each active component or DER unit. However,
the configuration also needs to consider components beyond the network connection
point to the degree they impact on the state of the distribution grid. Hence compo-
nents like control systems for DER units or controllable loads are included in the
configuration. The distribution grid as a domain includes a significant number of control-related
challenges and developments. Communication is also increasingly present for mon-
itoring and control purposes. One issue faced in this work was how to present these
different layers. It could be possible to build up separate layers for the power sys-
tem, communication system and control systems. This would enable a more detailed
presentation of each system and especially of their interfaces. Eventually, control
systems and ICT have been included as separate domains in this configuration. Multi-
domain components are located in domain interfaces, for instance smart meters which
are physically connected to the power domain but also connected to the ICT domain
in terms of data and control. This system configuration includes a long list of traditional power system compo-
nents such as lines, loads, transformers and switches. They all belong to the electrical
power system domain. Some active components such as DER units, storage units,
EV charging stations or intelligent controllers are also present; they are also physi-
cally connected to the electrical power system do-main, but they are also connected
to control and ICT domains via their controllers and com-munications. The system configuration also includes a heat system domain. The purpose of
including a heat system is to be able to represent aspects of cross-impacts between
heat and electricity; for instance, in a Combined Heat and Power (CHP) production,
between heat exchangers, heat pumps, etc. However, heat system parametrisation is
left very generic with the main focus to include connectivity. The control domain includes various controllers connected with components. They have been categorized to central (coordinated) and local control methodologies. ICT domain includes metering systems, communication and data management areas. 2
ERIGrid Generic System Configurations ERIGrid has defined three system configurations addressing key system areas [3]: • Distribution grid • Transmission grid and offshore wind • Vertical integration • Vertical integration From Scenarios to Use Cases, Test Cases and Validation Examples 89 The project has developed system configurations for these dedicated system areas
as well as structures and templates for describing them. The templates apply similar
hierarchy for the structure, starting from domain information and proceeding to
more local information (such as area/level) and finally to individual components. Parameters are defined for each component as well as for the whole system as global
parameters. Transmission Grid and Offshore Wind The offshore wind power plant scenario has been selected because it is a predomi-
nant future scenario with special operation characteristics and impact on transmis-
sion grids. For specifying the system configuration, the following assumptions have
been made: • A meshed HVDC network will be adopted because it seems a cost-effective solu-
tionforhostinghigh-powerwindgenerationand,asatopic,itpresentsanadditional
research interest. • A meshed HVDC network will be adopted because it seems a cost-effective solu-
tionforhostinghigh-powerwindgenerationand,asatopic,itpresentsanadditional
research interest. • AC grid parts are assumed for the connections of the wind power plants to
the HVDC hubs and an aggregated representation for the on-shore substations/
connections. • More than one connection to the shore may be used because it adds extra benefits
in terms of services and allows the wind power plant to participate in various
processes of operation and the energy market. Also, this increases the number of
applicable use cases. • Interconnection with different control areas (different countries) so as to increase
diversity of operating characteristics and processes at the ends of the system. • Simple configuration with the minimum possible number of components that at
the same time satisfy the abovementioned requirements. • Hierarchical control structure based on levels, with each level assigned with spe-
cific roles for the system’s protection, operation and optimisation. • The system is assumed to have specific role(s) in the energy and ancillary ser-
vices market which help to establish concrete interconnections with the ‘Market’
domain. • The interconnection with other physical domains such as weather conditions is
more specific since there is only one RES technology involved. Nevertheless, the
effects of weather conditions are considered only as a boundary of the system and
are not analytically modelled. Based on example scenarios, the system configuration is extended according to the
aforementioned assumptions. To this end, components given in the basic scenario
have been identified followed by components for possible extensions to the basic
scenario. For those components, attributes and domains have been identified as well
as the connections between. One of the most crucial discussion topics was the importance of considering
onshore wind power plants together with the offshore scenario. The former is (and
will be) the predominant wind-production scenario of the future. However, taking
into account only the share of a scenario for selecting it, it means that other large-
scale technologies should also be considered. Distribution Grid Stakeholders and markets have also been presented as separate domains, indicating
different roles and markets within the scope of this system configuration. K. Maki et al. K. Maki et al. 90 Vertical Integration The vertical integration scenario and system configuration provides a possible back-
ground for use cases requiring coordination and integration of transmission and
distribution grid related tasks. In principle, it includes all domains used in other
system configurations; however, in this system configuration often abstractions and
aggregations of usually included components are employed, as the full detail may
overload a given test requirement. Due to its cross-cutting nature, vertical integration system configuration sets a lot
of attention on connectivity of components, their information exchange as well as
on the roles of stakeholders. Transmission Grid and Offshore Wind Thus, only the offshore wind power
plant scenario is considered, not just for its contribution to the RES share but also for
its technical characteristics. Specifically, the incorporation of meshed HVDC grids
is a value added for the selection of the scenario. The topology of the system was also an important discussion topic. Among differ-
ent options such as pure AC, radial DC, and meshed DC configurations, the meshed
scenario has been selected which is technologically the most promising solution for
bulk transmission of offshore wind power. 91 From Scenarios to Use Cases, Test Cases and Validation Examples HVDC onshore fault ride-through protection was also identified as a serious chal-
lenge from an operational standpoint, as well as from testing and simulation perspec-
tives. HVDC onshore fault ride-through protection was also identified as a serious chal-
lenge from an operational standpoint, as well as from testing and simulation perspec-
tives. A third point of discussion was the way of modelling the onshore connection
points and, in general, the overall onshore transmission grid’s behaviour in combina-
tion with the selected scenario. To this end, aggregation of production/consumption
at various grid nodes (at transmission level) and simplified representation of the trans-
mission grid has been agreed. With the use cases in mind (e.g., fault ride-through,
energy balancing, active power control, stability to a lesser extent) this is a plausible
assumption. 3
Focal Use Cases The ERIGrid Focal Use Case Collection has been gathered during the project, based
on existing outputs from earlier projects and networks. Several repositories, for
instance EPRI (The Electric Power Research Institute) and SGCG (Smart Grid Coor-
dination Group) ones have been utilized while building the ERIGrid collection. Focal use cases have been categorised according to the service they provide for
the system [4]: • SS1 Energy balance
• SS2 Energy efficiency
• SS3 Power quality
• SS4 Power system stability
• SS5 Infrastructure integrity, protection and restoration • SS1 Energy balance • SS2 Energy efficiency • SS3 Power quality • SS4 Power system stability • SS5 Infrastructure integrity, protection and restoration The following sub-sections present these services that can be provided at system
level and show exemplary use cases for each of them. These use cases are aggregates
of several use cases within the ERIGrid collection. SS1 Energy Balance The energy balance of a network is a fundamental require-
ment for its operation; in fact, the generation has to constantly follow the demand
curve in order to maintain the system stable. Taking into account the time horizon
for which the ERIGrid system configurations have been developed, scenarios are K. Maki et al. 92 included in which also the demand is controlled in order to match the generation
availability. Here the energy balancing functions are defined as follows: included in which also the demand is controlled in order to match the generation
availability. Here the energy balancing functions are defined as follows: included in which also the demand is controlled in order to match the generation
availability. Here the energy balancing functions are defined as follows: • Functions aimed at guaranteeing the long-term energy balancing and which have
been categorized in SS1 Energy balance in the restoration of the planned power
exchanges with external systems. • Functions aimed at guaranteeing fast and prompt support in the restoration of the
power balancing have been categorized as Focal UC in SS4 Power system stability. 3
Focal Use Cases • Functions aimed at guaranteeing fast and prompt support in the restoration of the
power balancing have been categorized as Focal UC in SS4 Power system stability The focal Use Cases listed below describe the selected functions for the support of
system energy balance: The focal Use Cases listed below describe the selected functions for the support of
system energy balance: • SS1.SC1 Management of Flexible DERs for the Long-term Balancing (Fre-
quency/Voltage Restoration Reserve) of Microgrids in Island-Mode • SS1.SC2 Automatic Frequency Restoration Reserve from VSCs of Large Wind
Farms • SS1.SC3 Automatic Frequency Restoration Reserve from DERs SS2 Energy Efficiency The containment of the losses in energy conversion, trans-
portation and storage has always been one of the main objectives in the design and
operation of power systems. In fact, several use cases can be found or deduced from
literature specifically aimed at enhancing the energy efficiency of systems. Energy
efficiency related use cases have high relevance among the ERIGrid collection: • SS2.SC1 Optimal Distribution Network Control for the Reduction of System
Energy Losses • SS2.SC1 Optimal Distribution Network Control for the Reduction of System
Energy Losses • SS2.SC2 Optimal Transmission Network Management Level for System Energy
Losses Reduction • SS2.SC2 Optimal Transmission Network Management Level for System Energy
Losses Reduction • SS2.SC3 Incentivising Distribution Network Local Balancing to • Minimize Transmission Network Loading SS3 Power Quality The increasing penetration of distributed generation is par-
ticularly challenging from the power quality point of view and, currently, one of the
most relevant limitations in terms of renewable integration are the voltage issues
caused by generation at distribution level. In order to mitigate these effects, potential
solutions have to be developed and most of them require the coordination of more
resources in order to manage the voltage congestions. 3
Focal Use Cases As for other services, use cases are listed,
including also a use case describing functions aimed at guaranteeing ICT integrity,
protection and restoration: • SS5.SC1 Fault Detection and Corrective Management of Distribution Grid Assets
and Energy Resources • SS5.SC1 Fault Detection and Corrective Management of Distribution Grid Assets
and Energy Resources • SS5.SC2 VSCs (of HVDC and Large Windfarms) Support During Transmission
Network Restoration • SS5.SC2 VSCs (of HVDC and Large Windfarms) Support During Transmission
Network Restoration • SS5.SC3 Intentional Islanding of Microgrids During Widespread Disturbances
and Restoration of the Transmission System • SS5.SC3 Intentional Islanding of Microgrids During Widespread Disturbances
and Restoration of the Transmission System • SS5.SC4 Identification of ICT Anomalies and Restoration of the Communication
Links • SS5.SC4 Identification of ICT Anomalies and Restoration of the Communication
Links These listed sixteen focal use cases have been considered representative of most
relevant functions that can be reasonably expected to be operative in the ERIGrid
system configurations. These focal use cases have been designed in order to cover
a large spectrum of system domains and actors, and to comprehend several more
specific functions. Based on this, several test cases can have been designed, taking
advantage of the different domains which can be easily reproduced and/or simulated
within ERIGrid research infrastructures. 3
Focal Use Cases According to this, all the ERI-
Grid system configurations can be considered as proper scenarios in which power
quality functions can be tested and, for each of them, specific focal use cases are
listed: • SS3.SC1 Advanced Voltage Control of Distribution Grids Supported by DERs
Power Interfaces • SS3.SC1 Advanced Voltage Control of Distribution Grids Supported by DERs
Power Interfaces • SS3.SC2 Voltage Quality Support by Onshore and Offshore (VSC-HVDC con-
nected) Wind Power Plants • SS3.SC3 Transmission Network Voltage Quality Support by the Distribution Net-
work (VPP) 93 From Scenarios to Use Cases, Test Cases and Validation Examples SS4 Power System Stability Another particularly challenging aspect in future
power systems is represented by stability. Most of the functions aimed at supporting
thesystemrobustnessarecurrentlyperformedbytraditionalgenerators.Forscenarios
in which their presence is expected to be less predominant, other solutions have to be
exploited. In order to support the power system stability, many actors can be involved
as well as all the domains considered within ERIGrid. Also in this case, on the basis
of ERIGrid System Configurations, a list of use cases is presented: • SS4.SC1 Management of Flexible DERs for the Instantaneous Active/Reactive
Power Balancing of Microgrids in Island-Mode • SS4.SC1 Management of Flexible DERs for the Instantaneous Active/Reactive
Power Balancing of Microgrids in Island-Mode • SS4.SC1 Management of Flexible DERs for the Instantaneous Active/Reactive g
Power Balancing of Microgrids in Island-Mode • SS4.SC2 Large-scale Wind Power Plant (Onshore and Offshore VSC-HVDC Con-
nected) Support in Frequency Containment Control and Power System Inertia • SS4.SC2 Large-scale Wind Power Plant (Onshore and Offshore VSC-HVDC Con-
nected) Support in Frequency Containment Control and Power System Inertia • SS4.SC3 DERs Support in Frequency Containment Control and Power System
Inertia SS5 Infrastructure Integrity, Protection, and Restoration Other functions that
are expected to evolve in the ERIGrid system configurations are represented by the
ones supporting the integrity, protection and restoration of the System Configura-
tions’ infrastructures. In fact, taking into account the high flexibility that energy
players are able to provide at all power system levels, significant benefits can be pro-
vided through theses dedicated use cases. 4
Test Cases ERIGrid covers all testing approaches consisting of virtual-based and/or real-world-
based methods. The test set-ups can be divided into four categories: (i) pure simula- K. Maki et al. K. Maki et al. K. Maki et al. 94 tion (incl. co-simulation), (ii) Controller Hardware-in-the-Loop (CHIL) simulations,
(iii) Hardware (HW) experiments, and (iv) Power Hardware-in-the-Loop (PHIL)
experiments. These testing approaches can be considered to form a structured test-
ing chain consisting of the following steps: • Pure simulation: Virtual-based approaches both offline and in real-time. All aspects
of the System under Test (SuT) are modelled using suitable software(s) and
the accuracy of the results depends on the accuracy of the utilized models. Co-
simulation can be used to combine different simulators that consider each domain-
specific part of the SuT individually. • CHIL experiments: Real control hardware is utilized in a closed-loop simulation of
the system. Virtual-based and real-world-based approaches are combined. CHIL
experiments enable more accurate simulations in case an exact model of the con-
troller is not available. Communication delays, noise, execution time of algorithms
etc. can be taken into account more easily than with a pure simulation approach. CHIL experiments can also be used to verify the correct operation of a specific
control hardware. • Hardware experiments: Open-loop testing of real components. This can be seen
as the conventional part of component testing and the results are mainly related to
component characteristics. • PHILexperiments:Closed-looptestingofrealcomponents.Virtual-basedandreal-
world-based approaches are combined. Interactions between the hardware under
test and the overall system can be studied. The next step after the four testing approaches would be demonstration in a real
operational environment. The test cases are selected to cover all of the four test-
ing approaches. Better RI integration is needed to enable comprehensive testing of
multi-domain systems and also to enhance the already existing single-domain testing
procedures. Better integration can be achieved by at least two means: By simplifying
the process of porting an experiment from one RI to another, e.g., by using standard-
ized interfaces, and by enabling joint use of RIs with different capabilities through a
real-time communication between the RIs. Here the concept of an RI is understood
to include off-line simulation tools as well as physical laboratory infrastructure con-
sisting of real equipment such as generators and virtual equipment such as real-time
simulators. Research questions determine the test objectives for each of the selected
test cases. 4
Test Cases Additionally, previously defined system configurations and focal use cases
as well as capabilities of different RIs are used as inputs for the selection process. The
number of tested use cases is intentionally quite low so that the work can concentrate
on the research questions on infrastructure integration. Same use cases are used for
many different test cases so that the testing approaches can be developed and results
of individual setups can be compared. Selected Test Cases The work concentrated on demonstrating and validating research infrastructure inte-
gration. Two types of test cases have been defined: In single-RI integration test cases,
models, algorithms etc. developed in one RI are used in another RI as a part of a From Scenarios to Use Cases, Test Cases and Validation Examples 95 test but real-time communication between the RIs is not needed. Single-RI test cases
are also used to compare different experiment set-ups to enable performing the same
tests in different facilities. In multi-RIs integration test cases, the interfacing and
real-time communication between the RIs is needed. Technical challenges identified for single-RI test cases include comparison of
different testing approaches and test setups, integrating third-party Software (SW)
as a part of a test case and model transfer between RIs. Four single-RI test cases
have been selected as presented below. The following test cases have been selected
as single-RI test cases [2]: • TC.S.1 Component testing at different RIs with different setup • TC.S.2 Use of SW developed by RI1 in HW RI2 • TC.S.3UseofcomponentmodeldevelopedinRI1toperformmulti-domainsystem
tests in RI2 • TC.S.4 Test of distributed cyber-physical systems in RI1 as a monolithic setup in
RI2 • TC.S.4 Test of distributed cyber-physical systems in RI1 as a monolithic setup in
RI2 Technical challenges identified for multi-RI test cases include integration of
remote software and integration of remote simulators or hardware. The multi-RI
test cases are used both to validate the correct operation of interfaces developed and
to demonstrate the real-time joint operation of research infrastructures for smart grid
testing purposes. Two multi-RI test cases have been selected as presented below. The
following test cases have been selected as multi-RI test cases [2]: • TC.M.1 Integrate control SW running in RI1 with HW RI2 TC.M.2 Extend HW resources of RI1 using resources of other RIs • TC.M.2 Extend HW resources of RI1 using resources of other R More detailed implementation plans for each test case have been developed,
started by constructing test specifications and experiment specifications as well as
experiment setups. Full test case descriptions include system configurations as a
basis and requirements for research infrastructures. System configurations provide
information on the required components and connectivity. 5.1
Analysis of the Centralized Voltage Control for Rhodes
Island This test case aims to demonstrate the “Hardware/Software integration between dif-
ferent Research Infrastructures”. This test case aimed to demonstrate how modern
advanced testing techniques, such as CHIL, can be used to fill the gap and ensure
faster and more secure transition between pure simulations and field implementa-
tions. In this test case, a control hardware in the loop setup was used in ICCS RI
in order to test a DSO’s control algorithm of a Non-Interconnected Islanded Power
System (NIIPS) in realistic conditions. A dynamic model of Rhodes Island system
implemented in ICCS’s real time digital simulator in full detail consisted of: • Synchronous Generators and their control systems (automatic voltage regulators,
governors, secondary frequency controls) of the 2 different power stations in the
island. • The HV network of the Rhodes Island system. • The HV network of the Rhodes Island system. • The 5 controllable WTs that exist in Rhodes network (as average P, Q models). • 5 average P, Q models that represent the demand of the 5 different HV/MV sub
stations of the island. The dynamic model of the island power system was simulated in real time, send-
ing also measurements and receiving setpoints from a controller hosting DSO’s algo-
rithm which operates in a CHIL setup as presented in the next figure. The first control
algorithm measures the production of the synchronous generators (thermal units),
the power produced by the WTs and the available power of the WTs. From those
measurements the total demand is derived. The controller then determines the max-
imum production allowed cumulative by all the WTs according to their maximum
available power, the maximum permitted penetration level that is set for stability
purposes (e.g. 30% of the total demand) and the non-violation of the minimum load-
ing levels of generators. The algorithm then decides how to distribute this available
power according to the nominal rating of each one of the 5 WTs as well as their
respective available power at that moment. Finally, the setpoints for the thermal gen-
erators calculated by trying to reduce the production cost while at the same time
supply the remaining demand and provide the required reserves for the safe opera-
tion of the system. Those setpoints sent alongside with the WTs setpoints back to
the RTDS. 5
System Validation Examples The following sections present two more detailed examples utilizing the procedure. The first one deals with a voltage control application and the second one with the
development of a converter controller. K. Maki et al. K. Maki et al. 96 5.1
Analysis of the Centralized Voltage Control for Rhodes
Island Furthermore, this CHIL setup is ideal to examine the behaviour of the
existing algorithm as well as a possible improvement of it in real time conditions (e.g. noise, time delays) and also in events that can cause stability issues in the system
such us the largest WT disconnection. In order to illustrate this, an improvement
of the DSO’s centralized control tested also in ICCS CHIL testbed and compared
to the existing controller. The proposed control algorithm also tries to reduce the
voltage deviation from the nominal value, similar to the CVC, as well as to ensure
stability due to RES penetration levels through a different approach. The controller
hosting this algorithm receives measurements (WT Power, Thermal Unit Production,
WT available power, active and reactive power demand of the 5 HV/MV substation) From Scenarios to Use Cases, Test Cases and Validation Examples 97 from RTDS, solves an optimization problem and sent back in RTDS the WT active
and reactive power setpoints and the thermal generator setpoints. The optimization
problem tries to minimize the voltage deviation and the operating costs according to
the following objective function: minx
⎧
⎨
⎩wcost ·
∞
i=1
Costi · Pg + wv ·
n
j=1
Vn −Vj
2
⎫
⎬
⎭ Subject to the constraints: Subject to the constraints: Subject to the constraints: • Power Balance equations in each node • Voltage constraints (Vmin ≤Vj ≤Vmax) j
• Angle Constraints (−180◦≤d j ≤180◦) • Thermal Generator Production Limits (Pmin ≤Pi ≤Pmax)
W T • WT power constraint according to the available power (PW T
i
≤PW Tavail
i
) i
i
• WT power factor constraint (QW T
i
≤PW T
i
· tan arccos 0.9) i
i
• Dynamic Frequency Constraints (F(H, Pdis) ≤49.4) The last constraints are a set of linear constraints ensuring that the frequency will
not drop below a frequency level (here 49.4Hz which is the setting of the first acting
Under Frequency Load Shedding (UFLS) relays and is a recorded transient that has
caused black out in the Rhodes NIIPS). This is a different approach compared to the
existing control which enforces a penetration limit to the WT power. Both methods
try to ensure that if a contingency occurs (e.g. the largest WT is disconnected) the
frequency remains within limits and no UFLS relays trip. 5.1
Analysis of the Centralized Voltage Control for Rhodes
Island The CHIL setup in ICCS
infrastructures allows to run in the loop both algorithms and perform at the same
time contingencies in RTDS in order to examine if the existing and the proposed
control algorithm ensure safe operation. In addition, the CHIL allows both control
algorithms to be tested in realistic conditions, ensuring that time delays and noise on
the signals does not affect the stability of the system (e.g. introduce oscillations in
power which also introduce oscillations in frequency). The DSO’s proposed control
achieved better results compared to the existing control in terms of voltage since it
utilizes the ability of the WT’s power electronics to absorb or produce reactive power
in order to mitigate the voltage issues. The voltage profiles of 2 HV/MV substations
in the HV side are presented in Figs.1 and 2. The resulting reactive power profiles of the 5 WTs of the second method are
presented in Fig.3. Furthermore, the proposed control achieved higher penetration levels compared
to the existing control as presented in Fig.4. This was mainly implemented by dispatching differently the WTs, utilizing more
the WTs that have a lower rating if it is not secure to further increase the production
of the WTs that a have higher rating. The profiles of the production of 2 WTs are
presented in Figs.5 and 6. It is observed that the proposed control achieves lower production only in WT2
which has the highest nominal rating and results in higher production levels according K. Maki et al. K. Maki et al. K. Maki et al. 98 to the existing DSO control. The proposed control reduces the power production of
this WT in order to avoid a frequency transient that would result in a frequency nadir
below 49.4Hz. The existing CHIL setup allows also to perform such transient in the
RTDS in order to compare the two algorithms according to the frequency transients
that could occur if any of them is under operation. In Fig.7 the recorded transients
in frequency for the disconnection of the largest producing WT were performed and
recorded in different hours of the day proving the superiority of the proposed method. to the existing DSO control. The proposed control reduces the power production of
this WT in order to avoid a frequency transient that would result in a frequency nadir
below 49.4Hz. 5.1
Analysis of the Centralized Voltage Control for Rhodes
Island 7 Comparison of the frequency transients that could occur under each control algorithm
operation 5.1
Analysis of the Centralized Voltage Control for Rhodes
Island The existing CHIL setup allows also to perform such transient in the
RTDS in order to compare the two algorithms according to the frequency transients
that could occur if any of them is under operation. In Fig.7 the recorded transients
in frequency for the disconnection of the largest producing WT were performed and
recorded in different hours of the day proving the superiority of the proposed method. To sum up, this test case allowed a second party (DSO) to test its control algorithms
in an advanced testing setup of CHIL, not available at the DSO’s premise, provided by
a second party (ICCS). In this setup, the comparison was made in realistic conditions
(noise, time delays) and for complex scenarios (WT outages) that could assist the
DSO to evaluate and compare those 2 methods. y p
g
p
y
p
p
To sum up, this test case allowed a second party (DSO) to test its control algorithms
in an advanced testing setup of CHIL, not available at the DSO’s premise, provided by
a second party (ICCS). In this setup, the comparison was made in realistic conditions
(noise, time delays) and for complex scenarios (WT outages) that could assist the
DSO to evaluate and compare those 2 methods. Fig. 1 Comparison of Ialisos HV profile
Fig. 2 Comparison of Gennadiou HV profile Fig. 1 Comparison of Ialisos HV profile Fig. 1 Comparison of Ialisos HV profile Fig. 2 Comparison of Gennadiou HV profile From Scenarios to Use Cases, Test Cases and Validation Examples
99
Fig. 3 Reactive Power Profiles throughout the day for the proposed HEDNO control
Fig. 4 Comparison of RES penetration levels for both controls
Fig. 5 Comparison of WT1 Power Production profiles From Scenarios to Use Cases, Test Cases and Validation Examples 99 Fig. 3 Reactive Power Profiles throughout the day for the proposed HEDNO control Fig. 4 Comparison of RES penetration levels for both controls Fig. 5 Comparison of WT1 Power Production profiles 100 K. Maki et al. Fig. 6 Comparison of WT2 Power Production profiles
Fig. 7 Comparison of the frequency transients that could occur under each control algorithm
operation
5.2
Converter Controller Development Fig. 6 Comparison of WT2 Power Production profiles
Fig. 7 Comparison of the frequency transients that could occur under each control algorithm
operation Fig. 6 Comparison of WT2 Power Production profiles Fig. 5.2
Converter Controller Development Time of peak (Tp): Time at which the peak value occurs. • Damping factor (DF): θ =
ln
OS
100
π + ln2 OS
100
⎧
⎨
⎩
θ < 1
Under damped
θ = 1 Critically damped
θ > 1
Over damped θ =
ln
OS
100
π + ln2 OS
100
⎧
⎨
⎩
θ < 1
Under damped
θ = 1 Critically damped
θ > 1
Over damped Following a description of the main results achieved with the testing chain. 5.2
Converter Controller Development This test case aims to demonstrate the “Testing Chain” approach. In order to demon-
strate this approach, a characterization of a converter controller followed by a tuning
was done. The test system includes: distribution LV grid, converter controller, PV,
inverter. To characterize the converter controller a pure simulation test was imple-
mented firstly, then after a tuning of the converter controller based on the simula- 101 From Scenarios to Use Cases, Test Cases and Validation Examples Fig. 8 System under test of the testing chain test Fig. 8 System under test of the testing chain test tion results, a CHIL and a PHIL test were performed to validate the improvements. Figure8 shows the system under test taken into account in each step of the testing
chain. tion results, a CHIL and a PHIL test were performed to validate the improvements. Figure8 shows the system under test taken into account in each step of the testing
chain. The selected LV test grid, considered as relevant enough for testing the converte
controller, is based on the CIGRE LV network modified with DER that can be found The selected LV test grid, considered as relevant enough for testing the converter
controller, is based on the CIGRE LV network modified with DER that can be found
in [1]. The object under test is a droop controller that is used to control the PV source
in the grid. In order to compare the test results four KPIs have been defined and
evaluated: controller, is based on the CIGRE LV network modified with DER that can be found
in [1]. The object under test is a droop controller that is used to control the PV source
in the grid. In order to compare the test results four KPIs have been defined and
evaluated: • Settling time (ST): Time elapsed from the application of an instantaneous step
input to the time at which the amplifier output has entered and remained within an
error band of 5%. • Settling time (ST): Time elapsed from the application of an instantaneous step
input to the time at which the amplifier output has entered and remained within an
error band of 5%. • Overshoot (OS): OS(%) = (Vpeak −VSS)/VSS · 100 • Overshoot (OS): OS(%) = (Vpeak −VSS)/VSS · 100 • Overshoot (OS): OS(%) = (Vpeak −VSS)/VSS · 100 • Time of peak (Tp): Time at which the peak value occurs. • CHIL test: Both sets of controller parameters which were discussed in the previous paragraph
have been tested also with a CHIL setup. The results with the original converter
controller are in Table2. With the CHIL experiments, the improved controller shows that the ST of the
system is lower than the original controller, but it has a very oscillatory behaviour. For large steps in active power injection, such as the one required by this scenario,
the controller became unstable. Therefore, it is expected that the second version of
the controller will behave worse also in reality (in terms of oscillatory response) than
the first version of the controller. • PHIL test: The last step of the testing chain is the PHIL experiment. In this case the converter
controller was implemented on a real power converter. Similar to the CHIL experi-
ment, also in this case the results show that the improved converter controller reduces
the TS at the expense of the level of the OS (Table3). • Pure simulation test: To cover a wide spectrum of possible operating conditions, several experiment speci-
fications have been defined, for analysing the converter response in case of generation
and load variations. The experiment results are in Table1. 102 K. Maki et al. Table 1 Pure simulation results
Test type
Action
ST [s]
OS [%]
Tp [s]
DF
Simulation
Step up PV output of 1 p.u. 0.35
4.34
0.22
0.7
Simulation
Step up of the grid load or
generation of 1 p.u. 0.16
0.9
0.15
0.83
Fig. 9 Response of two [Kp, Ki] sets of parameters Table 1 Pure simulation results Simulation
Step up PV output of 1 p.u. 0.35
4.34
0.22
0.7
Simulation
Step up of the grid load or
generation of 1 p.u. 0.16
0.9
0.15
0.83
Fig. 9 Response of two [Kp, Ki] sets of parameters Fig. 9 Response of two [Kp, Ki] sets of parameters Based on these results the parameters of the converter controller were tuned fur-
ther. Regarding the inner loop, a parametric analysis has been carried out to evaluate
the impact of the PID constants in the system response when facing a step in the
solar power generated. In any case, a wide range of values has been tested to find
the better trade-off between the settling time and the overshoot. Two sets of [Kp, Ki]
constants were selected. Figure9 shows the new response of the converter controller
compared to the old values of set [Kp, Ki]. In view of the results a final tuning of Kp = 2 and Ki = 50 has been eventually
selected for the second round of tests. Regarding the outer loop, potential improvements are linked to the adjustment of
the measurement filters in the Id and Iq, and being more concrete, in the adjustment
of the damping ratios (D). The damping ratio is a parameter linked to the quality of
the filter in the way a higher damping ratio means a higher quality. Also, in this case
several values of D have been evaluated in order to establish the best solution. The
experiment’s results show that the most suitable value for the D parameter of the Iq
filter is 0.5 while the recommendation of the D value for the Id filter for the second
round of tests is 0.85. • Pure simulation test: From Scenarios to Use Cases, Test Cases and Validation Examples 103 From Scenarios to Use Cases, Test Cases and Validation Examples
103
Table 2 CHIL results
Test type
Action
ST [s]
OS [%]
Tp [s]
DF
CHIL
Step up PV output of 1 p.u. 0.02
0.73
0.1
0.94
Table 3 PHIL results
Test type
Action
ST [s]
OS [%]
Tp [s]
DF
PHIL
Step up PV output of 1 p.u. with
the original converter controller
0.0084
16
0.0012
0.49
PHIL
Step up PV output of 1 p.u. with the improved converter
controller
0.0062
22
0.0014
0.43 CHIL
Step up PV output of 1 p.u. 0.02
0.73
0.1
0.94
Table 3 PHIL results
Test type
Action
ST [s]
OS [%]
Tp [s]
DF
PHIL
Step up PV output of 1 p.u. with
the original converter controller
0.0084
16
0.0012
0.49
PHIL
Step up PV output of 1 p.u. with the improved converter
controller
0.0062
22
0.0014
0.43 6
Conclusions This chapter described the progress from general scenario thinking towards more
detailed system configurations, use cases and test cases. Validation examples were
presented to demonstrate the usage. The main objective was to access the relevancy
of testing needs for smart grid system development. The work has started with the
development of generic system configurations which provide a high-level context
for ERIGrid testing. The work has proceeded to define focal use cases which cover
the whole range of smart grid activities relevant to ERIGrid. Based on focal use case
collection, most relevant testing scenarios for have been defined on an abstract level, 104 K. Maki et al. K. Maki et al. outlining the most important areas of smart grid testing. The application examples
demonstrate usage of the process within different circumstances. A first example
demonstrates hardware/software integration across research infrastructures. In this
case, CHIL and dynamic modelling were used as testing techniques. The second
example focuses on testing chain approach, progressing from simulation studies to
CHIL and PHIL tests. 4. Rossi, M., Carlini, C., Pala, D., Sandroni, C., et al.: D-JRA1.2 Focal use case collection. Deliv-
erable D7.2, ERIGrid Consortium (2016) 3. Maki, K., Blank, M., Heussen, K., Bondy, E., et al.: D-JRA1.1 ERIGrid scenario descriptions.
Deliverable D7.2, ERIGrid Consortium (2016) References 1. Kotsampopoulos, P., Lagos, D., Hatziargyriou, N., Faruque, M.O., et al.: A benchmark system
for hardware-in-the-loop testing of distributed energy resources. IEEE Power Energy Technol
Syst J 5(3), 94–103 (2018) 2. Kulmala, A., Maki, K., Sandroni, C., Pala, D., et al.: D-JRA1.3 Use case implementation plan
Deliverable D7.3, ERIGrid Consortium (2017) 3. Maki, K., Blank, M., Heussen, K., Bondy, E., et al.: D-JRA1.1 ERIGrid scenario descriptions
Deliverable D7.2, ERIGrid Consortium (2016) 3. Maki, K., Blank, M., Heussen, K., Bondy, E., et al.:
Deliverable D7.2, ERIGrid Consortium (2016) 4. Rossi, M., Carlini, C., Pala, D., Sandroni, C., et al.: D-JRA1.2 Focal use case collection. Deliv-
erable D7.2, ERIGrid Consortium (2016) Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing,
adaptation, distribution and reproduction in any medium or format, as long as you give appropriate
credit to the original author(s) and the source, provide a link to the Creative Commons license and
indicate if changes were made. The images or other third party material in this chapter are included in the chapter’s Creative
Commons license, unless indicated otherwise in a credit line to the material. If material is not
included in the chapter’s Creative Commons license and your intended use is not permitted by
statutory regulation or exceeds the permitted use, you will need to obtain permission directly from
the copyright holder.
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An update of the Worldwide Integrated Assessment (WIA) on systemic insecticides. Part 3: alternatives to systemic insecticides
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Environmental science and pollution research international
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Abstract Over-reliance on pesticides for pest control is inflicting serious damage to the environmental services that underpin agricultural
productivity. The widespread use of systemic insecticides, neonicotinoids, and the phenylpyrazole fipronil in particular is
assessed here in terms of their actual use in pest management, effects on crop yields, and the development of pest resistance
to these compounds in many crops after two decades of usage. Resistance can only be overcome in the longterm by implementing
methods that are not exclusively based on synthetic pesticides. A diverse range of pest management tactics is already available, all
of which can achieve efficient pest control below the economic injury level while maintaining the productivity of the crops. A
novel insurance method against crop failure is shown here as an example of alternative methods that can protect farmer’s crops
and their livelihoods without having to use insecticides. Finally, some concluding remarks about the need for a new framework
for a truly sustainable agriculture that relies mainly on natural ecosystem services instead of chemicals are included; this
reinforcing the previous WIA conclusions (van der Sluijs et al. Environ Sci Pollut Res 22:148-154, 2015). Keywords Systemic insecticides . Neonicotinoids . Fipronil . Mutual funds . Insurance cover . Pest control . Resistance . Biological control . IPM . Review Responsible editor: Philippe Garrigues An update of the Worldwide Integrated Assessment (WIA) on systemic
insecticides. Part 3: alternatives to systemic insecticides Received: 8 August 2017 /Accepted: 13 December 2017
# The Author(s) 2018. This article is an open access publication
/ Published online: 25 February 2018 https://doi.org/10.1007/s11356-017-1052-5
Environmental Science and Pollution Research (2021) 28:11798–11820 https://doi.org/10.1007/s11356-017-1052-5
Environmental Science and Pollution Research (2021) 28:11798–11820 WORLDWIDE INTEGRATED ASSESSMENT OF THE IMPACT OF SYSTEMIC PESTICIDES ON BIODIVERSITY
AND ECOSYSTEMS Introduction Responsible editor: Philippe Garrigues
* Jean-Marc Bonmatin
bonmatin@cnrs-orleans.fr
1
Veneto Agricoltura, Legnaro (PD), Italy
2
Department of Agronomy, Food, Natural Resources, Animals and
Environment, University of Padova, Viale dell’Università 16,
35020 Legnaro (PD), Italy
3
Beekeeping Research and Information Centre, Louvain la
Neuve, Belgium
4
School of Life and Environmental Sciences, The University of
Sydney, 1 Central Avenue, Eveleigh, NSW 2015, Australia
5
Institut Technique de l’Agriculture Biologique (ITAB), 149 Rue de
Bercy, 75595 Paris, France
6
Condifesa Veneto, Associazione regionale dei ccnsorzi di difesa del
Veneto, Via F.S. Orologio 6, 35129 Padova (PD), Italy
7
Task Force on Systemic Pesticides, 46 Pertuis-du-Sault,
2000 Neuchâtel, Switzerland
8
Centre de Biophysique Moléculaire, Centre National de la Recherche
Scientifique (CNRS), Rue Charles Sadron, 45071 Orléans, France * Jean-Marc Bonmatin
bonmatin@cnrs-orleans.fr * Jean-Marc Bonmatin
bonmatin@cnrs-orleans.fr After the publication of the World Integrated Assessment
(WIA) on Systemic Insecticides (Bijleveld van Lexmond
et al. 2015; van der Sluijs et al. 2015), some new research
about neonicotinoids and fipronil have been made available. In this update, we have endeavored to collect all new infor-
mation that has been published since 2014 onwards on the
same topics covered by the WIA. The first review paper of
the updated WIA (Giorio et al. 2017, this special issue) deals
with the mode of action of neonicotinoids and fipronil, their
metabolism, synergies with other pesticides or stressors, deg-
radation products, and the contamination of the environment. The second updated WIA review covers the lethal and suble-
thal effects of neonicotinoids and fipronil on organisms, from
aquatic and terrestrial invertebrates to vertebrates, and their
impacts on ecosystems (Pisa et al. 2017, this special issue). The present review focuses on alternatives to the uses of these
systemic insecticides for annual and perennial crops. Pest re-
sistance to neonicotinoids and fipronil is also reviewed. 1
Veneto Agricoltura, Legnaro (PD), Italy 2
Department of Agronomy, Food, Natural Resources, Animals and
Environment, University of Padova, Viale dell’Università 16,
35020 Legnaro (PD), Italy 3
Beekeeping Research and Information Centre, Louvain la
Neuve, Belgium 4
School of Life and Environmental Sciences, The University of
Sydney, 1 Central Avenue, Eveleigh, NSW 2015, Australia 5
Institut Technique de l’Agriculture Biologique (ITAB), 149 Rue de
Bercy, 75595 Paris, France 6
Condifesa Veneto, Associazione regionale dei ccnsorzi di difesa del
Veneto, Via F.S. * Jean-Marc Bonmatin
bonmatin@cnrs-orleans.fr Introduction Orologio 6, 35129 Padova (PD), Italy 6
Condifesa Veneto, Associazione regionale dei ccnsorzi di difesa del
Veneto, Via F.S. Orologio 6, 35129 Padova (PD), Italy 7
Task Force on Systemic Pesticides, 46 Pertuis-du-Sault,
2000 Neuchâtel, Switzerland 8
Centre de Biophysique Moléculaire, Centre National de la Recherche
Scientifique (CNRS), Rue Charles Sadron, 45071 Orléans, France 11799 Environ Sci Pollut Res (2021) 28:11798–11820 The prophylactic use of neonicotinoids and fipronil in crop
protection is contrasted with integrated pest management
(IPM) for controlling pests (Barzman et al. 2015; Furlan
et al. 2016; Stenberg2017). We have divided this task in ac-
cordance with two main types of crops: (a) annual crops and
(b) perennials (e.g., orchards and vineyards). A new Mutual
Funds (MF) insurance approach that covers risk from IPM
implementation, applied at a large scale for maize in Italy,
showed that it is possible to bring numerous advantages for
farmers and ecosystems when implementing IPM. seed dressing increased. At the same time, the availability of
honey bee colonies with respect to the growing area of crops
benefitting from insect pollination had a linear, significant im-
pact on turnip rapeseed yield trends. Since landscape and num-
bers of honey bee colonies had not changed during the period of
the turnip rapeseed study, the authors of that study indicate that
the only factor that could explain this decline in yields was the
seed-treatment with neonicotinoids in the past 15 years. In the UK, Budge et al. (2015) showed that yields of oil-
seed rape crops are not significantly increased by using
imidacloprid in treated seeds. While the authors reported that
farmers may get better economic returns some years, as they
apply seed coatings which reduced the number of subsequent
applications of foliar insecticide sprays, they also revealed a
correlation over an 11-year-period between honey bee colony
losses and national-scale imidacloprid usage patterns across
England and Wales. These findings on oilseed rape yields are
consistent with previous reports on the non-usefulness of
neonicotinoids in soybean (Seagraves and Lundgren 2012)
and wheat crops (Macfadyen et al. 2014). Abundant information is available about the negative im-
pacts of neonicotinoids and fipronil on the environment,
which are due to the sum of (1) their extreme toxicity to
invertebrates (Pisa et al. 2015, 2017); (2) their high toxicity
to vertebrates (Gibbons et al. 2015; Pisa et al. Introduction 2017); (3) their
high persistence in soils and the contamination of surface wa-
ter, both of which impact ecosystems and the services they
provide (Bonmatin et al. 2015; Chagnon et al. 2015; Giorio
et al. 2017; Pisa et al. 2017); and (4) their large-scale and
widespread usage in all kinds of crops, even in non-
agricultural settings (Simon-Delso et al. 2015; Douglas and
Tooker 2015). However, there is a great deal of reluctance to
reduce or phase out these insecticides because of fears that
crops may experience yield losses and hurt farmer’s econo-
mies. Accurate information on the efficacy of prophylactic
usage and other applications of these systemic insecticides
and the environmental damage they cause should help resolve
this issue in a rational way. Moreover, a suite of alternative
methods already available, in the context of pest resistance to
synthetic insecticides, should provide regulators with more
sustainable possibilities for pest management of crops. In regard to maize, the available literature, mainly about
studies in Italy, shows that the effect of seed-coated
neonicotinoids on grain yield was mainly negligible (Furlan
and Kreutzweiser 2015 citing different papers on field trials
covering a 15-year period). This was mainly due to the fact
that the majority of pest populations were under the economic
injury level. Other studies have shown that neonicotinoid insecticides
can have effects on germination. For instance, Nogueira
Soares et al. (2017) co-published with Syngenta that
thiamethoxam improves physiological performances of melon
and watermelon seeds treated with this neonicotinoid. By con-
trast, Tamindžić et al. (2016) have shown that three commercial
formulations (i.e., Poncho, Gaucho and Cruiser) were harmful
and reduced germination of three inbred maize varieties. The
most harmful treatment was Gaucho (active ingredient (a.i.)
imidacloprid) when compared to Cruiser (a.i. thiamethoxam)
and Poncho (a.i. clothianidin, a derivative of thiamethoxam). Annual crops The use of systemic insecticides against key pests of annual
crops The use of neonicotinoids or fipronil against the main
pests of annual crops was previously described in Furlan and
Kreutzweiser (2015) and Simon-Delso et al. (2015). It was
stressed that the main use of these systemic insecticides is
prophylactic, for instance by means of seed coating tech-
niques, and that this systematic approach is contrary to IPM
principles (Furlan et al. 2016). The results of this work enable mapping of each cultivated
region and high-risk areas to be pinpointed. Mapping the risk
factors found in this survey, and that of Saussure et al. (2015)
outside Italy, may allow us to prove that the cost–benefit of past
soil-insecticide use was extremely negative. The first layer of
the map includes the main soil characteristics (organic-matter
content, texture, pH); the second includes the key agronomic
characteristics (rotation, drainage); and the third the available
entomological information, such as click-beetle population
levels for the main Agriotes species, or wireworm presence/
density assessed with bait traps over the years. A fourth layer
reproduces the effects that occur when existing risk factors
interact. This system enables areas with different risk levels
to be highlighted. Each wireworm-risk category (e.g., low, me-
dium, or high, based on the presence of one or more risk fac-
tors) will have its own IPM strategy, e.g., assessing wireworm
density in high-risk areas or opting not to treat and not to
continue monitoring in low-risk areas. Where risk factors are
present, a precise procedure to spot land with an economic
wireworm population has been described. In this way, control
strategies will be implemented only when and where economic
thresholds for maize are exceeded, and then it will be possible
to avoid expensive soil insecticide use. Note that the risk fac-
tors causing high wireworm populations in maize are the same
as those in non-maize crops. Therefore, they can be used to
implement IPM in all arable crops, with possible adaptations. Alternative methods for pest control in annual crops Some
new IPM strategies that reliably limit the need for treat-
ments involving neonicotinoids were also described in
Furlan and Kreutzweiser (2015). Annual crops The need for more data on
the factors driving the risk of soil pest damage to maize and
other susceptible crops, mainly to wireworm attacks, was em-
phasized in order to improve both the practical approach for
farmers and the setup of specific MF to support IPM imple-
mentation. In practice, pest level evaluation is often not done
mostly because of a lack of non-time-consuming and low cost
methods (essential for low revenue crops) and insurance to
cover for the risk of mistakes in pest population estimations. Some interesting recently published papers contribute to this
issue: after a 29-year long-term study in Italy, the strongest
factors increasing the risk of wireworm damage have been
isolated, making now possible low-cost and reliable predic-
tions to meet crop protection needs (Furlan et al. 2017). A univariate analysis in the risk assessment was applied to
identify the main factors that influence the occurrence of dam-
age. Then, a multifactorial model was applied using the sig-
nificant factors identified in the previous step. This model
allows the strongest factors to be highlighted and to analyze
how the main factors together influence the damage risk. The
strongest factors were Agriotes brevis as the prevalent damag-
ing species; organic matter content > 5%; rotation, including
meadows and double crops; poor soil drainage; and
A. sordidus as the prevalent damaging species. Also, the sur-
rounding landscape with prevalent meadows was an important
risk factor, confirming previous findings by other authors
(Blackshaw and Hicks 2013; Benefer et al. 2012; Hermann
et al. 2013; Saussure et al. 2015). The multifactorial model
also showed how the simultaneous occurrence of two or more
of the aforementioned risk factors can conspicuously increase
the risk of wireworm damage to the maize crop, while the
probability of damage for a field with no risk factors is always
low (< 1%). These results make it possible to prepare risk
maps for any country identifying low-risk and high-risk areas. Choosing fields with no risk factors may reduce the damage
risk for all crops, including sensitive vegetable crops. Assessing
the risk of wireworm damage affords a solid basis for estimating
the amount of farmland that can be left untreated each season,
without any risk of yield reduction. Alternatives to systemic insecticides in agriculture considerable reduction in the use of soil insecticides and the
immediate containment of the environmental impact of agri-
culture with no negative repercussions on farmers’ income. This can be achieved by implementing two phases: (i) Barea-
wide^ risk assessment, including click-beetle population mon-
itoring with pheromone traps (Furlan and Kreutzweiser 2015)
and (ii) complementary field monitoring where risk assess-
ment has identified the presence of risk factors (Furlan et al. 2016). When a harmful population is found, wireworm-
activity-predicting models based on soil humidity and temper-
ature may be useful to assess if the damage is really being
done by larvae (Jung et al. 2014; Milosavljević et al. 2016). Neonicotinoids and crop yields Little information is available about the actual performance of
neonicotinoids on crop production. However, concerns that crop
yields might decrease significantly after the European moratori-
um (EU 2013a, b) of three neonicotinoids (clothianidin,
imidacloprid, and thiamethoxam) and fipronil have been raised
in the media and a few scientific publications (e.g., in
Matyjaszczyket al. 2015 for maize and oilseed rape in Poland),
although they were not supported by reliable data or statistics. Deguines et al. (2014) analyzed a country-wide dataset of
the 54 major crops in France produced over the past two
decades. They found that the benefits of agricultural intensifi-
cation decrease with increasing pollinator dependence, to the
extent that intensification failed to increase the yield of
pollinator-dependent crops and decreased the stability of their
yield over time. The authors concluded that benefits from
agricultural intensification may be offset by reductions in pol-
lination services and support the need for an ecological inten-
sification (reviewed by Kovács-Hostyánszki et al. 2017) of
agriculture through optimization of ecosystem services. In
other words, the prophylactic use of systemic insecticides,
which impacts on both managed and wild pollinators (Pisa
et al. 2015, 2017), is opposed to yield increases for pollinator
dependent crops. In Finland, yields of insect-pollinated crops are variable,
whereas yields for wind-pollinated crops have been increasing
for decades. While analyzing the possible factors related to
yield declines for insect-pollinated crops, a significant linear
correlation was found between the yield trends in rapeseed
and the extent of neonicotinoid seed dressing used in provinces
of that country (Hokkanen et al. 2017). In particular, yield de-
clines in turnip rapeseed decreased as the use of neonicotinoid Environ Sci Pollut Res (2021) 28:11798–11820 11800 Annual crops In Italy, implementing IPM
is likely to result in a maximum of about 4% of maize-cultivated
land being treated with soil insecticides or by using insecticide-
coated seeds (Furlan et al. 2017). This means that 96% of these
fields will not need any insecticide treatment. Precise IPM
thresholds for soil pests in maize could be set everywhere. For
instance, in no-risk areas, soil insecticides or insecticide-coated
seeds may need to be used on no more than 1% of maize-
cultivated land. In areas where organic matter content is over
5%, soil insecticides could be used on about 20% of maize-
cultivated land if the prevalent species is A. sordidus. For large This information may be used to implement IPM and to
tackle soil pests attacking maize in many European regions
(Furlan et al. 2016) and beyond, which may lead to a Environ Sci Pollut Res (2021) 28:11798–11820 11801 areas with scattered-risk situations, IPM thresholds will be a
balanced mean of the damage risk caused by various risk factors
and the surface area of cultivated land where each risk factor
occurs. This could be immediately applied to areas harboring
the species studied therein and to other areas shortly afterwards. In fact, local checks and adaptations should be assessed in re-
gions where other species and/or conspicuous climatic differ-
ences occur, but the aforementioned IPM approach should be
used since it is likely that the same main risk factors would play
a key role. This would allow IPM to be extended wherever the
Agriotes species studied in this work are widespread, and prob-
ably also to wherever other Elateridae species occur, once ac-
curate comparisons have been made. covers also the risk of mistakes in IPM implementation, in-
cluding any underestimation in the size of the area with wire-
worm economic populations (Furlan et al. 2015). Based on
this risk assessment, a specific Maize Mutual Fund was set up. Its main features are summarized in Table 1. In essence,
farmers should use pesticides only when they are really need-
ed, based on IPM procedures suggested by the Annual Crops
Bulletin (accessible at http://www.venetoagricoltura.org/
subindex.php?IDSX=120), while getting insurance cover for
mistakes and unexpected damages. Annual crops The practical implementation of Maize MF in 2015–2016
resulted in the following economic and management effects:
47.558 ha were covered by the Maize MF on average over the
2 years; the cost was 3.3 €/ha (about one tenth the cost for a
soil insecticide); total revenue to cover damage by wireworms,
western corn rootworm, wild fauna, and other minor pitfalls
was 160.335 €, while total damage paid was 83.863 € (~
52%). Therefore, there was a very significant increase of the
MF stock for following years. 1.
MF insurance cover (rotten roots, seedlings)
• Diabrotica (WCR) damage
• Loss of production caused by wild fauna
Cost
€3–5/ha all inclusive (flooding, excessive rain, freezing
cold, drought, pest risk, diseases, and wild fauna)
Compensation • Changing crop for WCR damage (up to €1000/ha)
• Up to €500/ha including the cost of:
○Re-sowing, if stand below 4 PLS/m2 (up to €250/ha)
○Yield reduction because of sowing delay (up to
€250/ha)
PLS pure life seed Table 1
Main features of the Mutual Fund (MF) strategy for maize in
Italy. Participants
Members of the farmer consortia
Obligations
• Contract to be signed within 7 days after sowing
• Implementation of good cultivation practices
• Implementation of Directive 128/2009/EC
• Implementation of suggestions in the BAnnual Crops
Bulletin^
Risks covered
• Insufficient plant density (stand) due to adverse weather
conditions (i.e., drought, flooding, freezing cold)
• Insufficient plant density (stand) due to soil pests
(e.g., wireworms, black cutworms)
• Insufficient plant density (stand) due to diseases such as
Fusarium spp. (rotten roots, seedlings)
• Diabrotica (WCR) damage
• Loss of production caused by wild fauna
Cost
€3–5/ha all inclusive (flooding, excessive rain, freezing
cold, drought, pest risk, diseases, and wild fauna)
Compensation • Changing crop for WCR damage (up to €1000/ha)
• Up to €500/ha including the cost of:
○Re-sowing, if stand below 4 PLS/m2 (up to €250/ha)
○Yield reduction because of sowing delay (up to
€250/ha)
PLS pure life seed Table 1
Main features of the Mutual Fund (MF) strategy for maize in
Italy. Participants
Members of the farmer consortia
Obligations
• Contract to be signed within 7 days after sowing
• Implementation of good cultivation practices
• Implementation of Directive 128/2009/EC
• Implementation of suggestions in the BAnnual Crops
Bulletin^
Risks covered
• Insufficient plant density (stand) due to adverse weather
conditions (i.e., drought, flooding, freezing cold)
• Insufficient plant density (stand) due to soil pests
(e.g., wireworms, black cutworms)
• Insufficient plant density (stand) due to diseases such as
Fusarium spp. 1.
MF insurance cover A low-cost IPM approach for low-risk pests based on risk
factors and limited direct monitoring of fields makes it impor-
tant that farmers get an appropriate compensation for the few
fields that suffer from pest soil damage which differ from IPM
predictions due to natural variability of the phenomena. In this
case, risk insurance coverage may be extremely useful. Insurance cover/MF may be taken out privately by associated
farmers, or with the support of EU regulations (Reg. 1305/2013/EU). With risks below 1%, a few Euros per hectare
(about ten times less than soil-insecticide costs) would be
enough to pay for damaged fields (Ferrari et al. 2015), includ-
ing those damaged despite having been treated with soil in-
secticides, the likelihood of which is high (Saussure et al. 2015). MFs are instruments managed by collectives of farmers
aimed at creating compensation through an interregional dis-
tribution of risks. They are non-profitable and have transpar-
ent rules. Compensation is commensurate with the financial
resources of the Fund. The Fund stock is increased by savings
in forecast costs. They cover risks that private insurance com-
panies currently do not (e.g., climatic adversities such as
flooding, and damage by wild animals and pests, just before
and after the emergence of arable crops). The Italian Case
study for implementation described here covered large-scale
areas (> 47,000 ha) in two regions of high agricultural impor-
tance, Veneto and Friuli-Venezia Giulia. The comparison between the prophylactic approach with
soil insecticides to protect maize seeds/young plants at sowing
and IPM approaches based on MF implementation are showed
in Table 2. Costs of IPM implementation for farmers and of
running MF are detailed below as farmer’s cost and institu-
tion’s cost: Table 1
Main features of the Mutual Fund (MF) strategy for maize in
Italy. Participants
Members of the farmer consortia
Obligations
• Contract to be signed within 7 days after sowing
• Implementation of good cultivation practices
• Implementation of Directive 128/2009/EC
• Implementation of suggestions in the BAnnual Crops
Bulletin^
Risks covered
• Insufficient plant density (stand) due to adverse weather
conditions (i.e., drought, flooding, freezing cold)
• Insufficient plant density (stand) due to soil pests
(e.g., wireworms, black cutworms)
• Insufficient plant density (stand) due to diseases such as
Fusarium spp. PLS pure life seed 1.
MF insurance cover (rotten roots, seedlings)
• Diabrotica (WCR) damage
• Loss of production caused by wild fauna
Cost
€3–5/ha all inclusive (flooding, excessive rain, freezing
cold, drought, pest risk, diseases, and wild fauna)
Compensation • Changing crop for WCR damage (up to €1000/ha)
• Up to €500/ha including the cost of:
○Re-sowing, if stand below 4 PLS/m2 (up to €250/ha)
○Yield reduction because of sowing delay (up to
€250/ha)
PLS pure life seed Table 1
Main features of the Mutual Fund (MF) strategy for maize in
Italy. In the above-cited regions, a long-term research and survey
(> 29 years) demonstrated that economic damage risk by soil
pests is less than 4%, and that a reliable IPM procedure is
available to spot which fields really need protection from
pests. As described above, the absence of risk factors greatly
decreases the chance of economic damage and makes the ap-
plication of soil insecticides in most fields useless. Where risk
factors are present, an appropriate practice is to assess wire-
worm populations with bait traps and to introduce control
strategies only when and where economic thresholds for
maize are exceeded. This is clearly opposite to the systematic
and prophylactic uses of systemic insecticides. Insurance 11802 Environ Sci Pollut Res (2021) 28:11798–11820 . Farmers’ costs of IPM implementation are given in the
fifth column from left in Table 2. The obligation for
farmers is to follow IPM suggestions of the Annual
Crops Bulletin for an actual implementation of IPM prin-
ciples. If just an evaluation of risk factors presence is done
(see the fourth row from above), only 4–5 hours of a
technician are needed (about 100€/100 ha). If a full IPM
implementation is considered (see the third row from
above), the estimated total cost is 1000€/100 ha. It corre-
sponds to 16 ha at risk, as assuming they are monitored
with bait traps (Furlan 2014). It includes 2 h/ha to do the
monitoring—about 40€/ha with a subtotal cost of 640€,
60€ for materials, 100€ for travel costs and overheads, and
about 200€ that might be needed for an accurate risk anal-
ysis with mapping of the cultivated fields and further in-
sight. Note that in Europe, these costs should not be con-
sidered as additional costs since the compliance with IPM
principles and the connection with IPM Bulletins are
compulsory for all the crops in all Member States in
Europe. 1.
MF insurance cover Table 2
Comparisons of four agricultural strategies for maize in Italy
Strategy
Surface of
reference
(ha)
Surface with
insecticide
(ha)
Insecticide
cost (€)
IPM
cost (€)
MF
cost (€)
Strategy
cost (€)
Damage
cost (€)
Total
cost (€)
Differential
costa (€)
Compliance
with
Directive
2009/128/CE
Risks for
environment
and public health
Synthetic
evaluation
scale (1 to
MF (only)
100
0
0
0
500
500
2000
2500
-1500
Yes
None
+++++
IPM based on risk factors +
monitoring + MF
10
400
1000
500
1900
500
2400
-1600
Yes
Low
++++
IPM based on risk factors + MF
20
800
100
500
1400
750
2150
-1850
Partial
Medium
+++
Soil insecticide (prophylactic)
100
4000
0
0
4000
0
4000
0
No
High
+
Data are based on four prudential assumptions for 100 ha of arable crops including 16% of land having the most important risk factors: (1) Mutual fund (MF) cost of €5/ha (worst case), (2) soil insectici
cost of €40/ha (realistic), (3) highest damage cost of €500/ha on 4 ha out of 100 (worst case), and(4) soil insecticide efficacy set at 100% (best case) 2. Institution’s costs are considered in the sixth column from
left in Table 2. They correspond to a total MF cost of 5
€/ha. This includes both 4€/ha of pure premium to cover
the actual damage risk (a prudent figure higher than pre-
cise estimation done), and 1€/ha for specific administra-
tive costs (including fixed costs) and for the costs of dam-
age assessments by experts in fields where farmers ask to
visit. Over the 4 years of practical implementation of MF,
this latter cost per year ranged between 5 and 15% of the
revenue from farmers to cover damage by pitfalls. More
precisely, this maximum cost was 0.6 €/ha but, to follow a
prudential approach, we overestimated this cost at 1 €/ha
in Table 2. 2. Institution’s costs are considered in the sixth column from
left in Table 2. They correspond to a total MF cost of 5
€/ha. This includes both 4€/ha of pure premium to cover
the actual damage risk (a prudent figure higher than pre-
cise estimation done), and 1€/ha for specific administra-
tive costs (including fixed costs) and for the costs of dam-
age assessments by experts in fields where farmers ask to
visit. Perennial crops The use of neonicotinoids against key pests of perennial crops
A large number of arthropod pests of temperate fruits (e.g.,
apple, pear, peach, and cherry) and grapevine have been man-
aged for a long time using synthetic pesticides such as organ-
ophosphates and carbamates. Problems associated with their
wide use, such as pest resistance, pest resurgence, and out-
breaks of secondary pests, as well as concerns about their
toxicity towards beneficial invertebrates and mammals, have
progressively reduced their availability in many developed
countries. Pyrethroids were suggested to replace these pesti-
cides because of their relatively low toxicity towards mam-
mals, but their impact on natural enemies of pests (i.e., pred-
ators and parasitoids) with consequent risks of secondary pest
outbreaks reduced their appeal for growers involved in IPM in
perennial crops (Duso et al. 2014). Additionally, similar risks
for aquatic invertebrates as compared to neonicotinoids have
been reported (Douglas and Tooker 2016). Chitin synthesis
inhibitors were then successfully proposed due to their long
persistence activity on target pests and relatively low acute
toxicity to mammals. Later, their popularity also declined
due to technical (e.g., pest resistance) and environmental is-
sues (risks to aquatic crustaceans) associated with some active
ingredients (Castro et al. 2012; Rebach and French 1996). More recently, neonicotinoids were proposed as a category
of insecticides characterized by reduced risks to human health. A number of active ingredients showed a high efficacy in the
control of sucking insects and other pests, probably because of 1.
MF insurance cover Over the 4 years of practical implementation of MF,
this latter cost per year ranged between 5 and 15% of the
revenue from farmers to cover damage by pitfalls. More
precisely, this maximum cost was 0.6 €/ha but, to follow a
prudential approach, we overestimated this cost at 1 €/ha
in Table 2. Because of this generally low risk level, the crop insurance
program (MF to protect maize at the early stages) proved to be
more convenient than insecticide protection on large scale. Growers may purchase MF cover instead of soil insecticides,
to provide financial compensation when yield losses can be
attributed to pests or adverse weather conditions. In fact, the
total cost of damage to maize (e.g., need for re-sowing and
yield loss due to delayed sowing or reduced stand) plus the
MF cost was much lower than the total cost of the soil insec-
ticide treatments of most fields as the result of prophylactic
protection approach (Table 2), even when all the fields are left
untreated. In the two intermediate IPM scenarios of Table 2, the as-
sumptions were (1) a little bit larger land than that presenting
risk factors is being treated, including all the border line cases,
to minimize the risk of unpredicted damage; (2) since the
efficiency of soil insecticide is set at 100% (an optimistic es-
timate), by enlarging the treated land the probability of finding Environ Sci Pollut Res (2021) 28:11798–11820 11803 a field with an economic damage is very low. In the practical
application of these two IPM scenarios, economic damages
observed in north-east Italy were < 0.1%, which is really neg-
ligible. However, to make the exercise extremely severe, we
have considered in Table 2 a case worse than the worst case
found in practice: a damage of 1 ha out of 100 ha (500€) is
considered as unpredicted for IPM based on a risk factor eval-
uation and a monitoring scenario, whereas an unpredicted
damage of 1.5 ha out of 100 (750€) was considered for the
scenario without monitoring. Nevertheless, a great advantage
of applying IPM instead of a prophylactic approach is clear. In
addition to economic considerations, MFs avoid the environ-
mental side effects of insecticides on beneficial species, biodi-
versity, ecosystems, and human health (Furlan et al. 2015; van
der Sluijs et al. 2015; Cimino et al. 2017; Pisa et al. 2017). 3.
Ecological engineering for pest suppression: habitat ma-
nipulation for pest management and cultural control 3. Ecological engineering for pest suppression: habitat ma-
nipulation for pest management and cultural control 3. Ecological engineering for pest suppression: habitat ma-
nipulation for pest management and cultural control In rice, ecological engineering practices that were first de-
veloped in China (Gurr et al. 2012) have been field tested in
three countries: China, Thailand, and Vietnam, for multiple
years (Gurr et al. 2016; Spangenberg et al. 2015). The results
showed that in rice fields grown with flowers on the bunds,
insecticide use was reduced by 70%, biological control was
increased by 45%, pest populations were decreased by 30%,
and yields were increased by 5%. These ecological engineer-
ing practices are now widespread in Vietnam (Heong et al. 2014) and China (Lu et al. 2015). When risks are low, the insurance approach is thus conve-
nient for farmers and safe for people, biodiversity (including
pollinators), the environment, and ecosystems. An insurance
approach is much more cost-effective than insecticides since
its large-scale and multiannual implementations demonstrated
that MF costs are much cheaper for farmers than insecticide
use. Obviously, the lower the damage risk is, the more effi-
cient an MF becomes, even without any subsidy. The MF
insurance approach can immediately reduce pesticide use
and increase farmers’ net income by replacing pesticides with
a lower cost strategy. Interestingly, MFs allow an increase of
IPM application by making farmers more comfortable with
IPM procedures, since mistakes in IPM implementation are
also covered. 1.
MF insurance cover and-kill strategy also against Agriotes adults using sex phero-
mones (Kabaluk et al. 2015). However, while biocidal plants and meals have become
commercial products available for farmers, the setup of ordi-
nary control tools based on the entomopathogens described
above requires efforts for the future. A careful cost-benefit
analysis is also needed to practically evaluate these alternative
tools. 2.
Biological control and natural derived insecticides A few papers have recently been published on biological
control and natural derived insecticides to control pests in
arable crops. One new suggestion is about attract-and-kill
strategies using biological tools against soil pests. Brandl
et al. (2017) proved this strategy may reduce potato damage
by wireworms in organic potato production systems in Lower
Saxony, Germany. This strategy is based on the attraction of
wireworms towards an artificial carbon dioxide-emitting
source, using baker’s yeast (Saccharomyces cerevisiae) in
combination with Metarhizium brunneum conidia for wire-
worm infection. This strategy offers the potential to promote
biological wireworm control as an alternative to insecticide
use by potentially reducing the inoculum compared to an in-
undate M. brunneum conidia release strategy (Kabaluk et al. 2007). This approach had some practical successful
implementations in corn (Kabaluk and Ericsson 2007), like
the use of biocidal plants and meals as described in Furlan
and Kreutzweiser (2015). Kabaluk (2014) showed that appli-
cations of M. brunneum conidia may cause high mortality to
adult Agriotes obscurus click beetles in field trials. This was
followed by experiments that showed a potential for an attract- Environ Sci Pollut Res (2021) 28:11798–11820 11804 their novel mode of action (MoA) and systemic distribution
within plants (Bonmatin et al. 2015 and Giorio et al. 2017). pathogen BCandidatus^ Liberi bacterasiaticus, the presumptive
causal agent of citrus greening disease (or huanglongbing). Neonicotinoid insecticides appear to be the most valuable op-
tion for containment of this pest (Ichinose et al. 2010). Aphids are key pests in apple and peach orchards. Pre-
blossom pesticide applications are considered essential to re-
duce their damage to fruit production, and neonicotinoids
proved to be effective in keeping these pests below economic
thresholds in fruit orchards (Shearer and Frecon 2002; Beers
et al. 2003; Lowery et al. 2005; Brück et al. 2009). In Europe,
the use of three active ingredients (i.e., imidacloprid,
thiamethoxam, and clothianidin) has been restricted to post-
blossom applications due to their side effects on honeybees
(EU Regulation 485/2013a, b; Pisa et al. 2015, 2017) and
further restrictions have been applied in some countries. In
some areas, the elimination of a number of broad-spectrum
insecticides has been associated with an increase of the rosy
apple aphids Dysaphis plantaginea Passerini (Cross et al. 1999; Solomon et al. 2000; Dib et al. 2016), thus prompting
the use of neonicotinoids to control them. 2.
Biological control and natural derived insecticides Neonicotinoids play
also a role in the control of the San José scale Diaspidiotus
perniciosus Comstock. An advantage of their use is the high
systemic activity that allows an effective control of this pest
(Buzzetti et al. 2015). In fruit orchards, other pests such as the
codling moth, Cydia pomonella (L.), and the oriental fruit
moth,Grapholita molesta (Busck), can be controlled with
neonicotinoid applications (Jones et al. 2010; Magalhaes and
Walgenbach 2011; Yang et al. 2016). Trunk injections of systemic insecticides (e.g., imidacloprid,
acephate, dinotefuran) were tested to manage avocado thrips in
California (Byrne et al. 2003). Acephate, which has been
banned in the EU, was mobilized rapidly and proved to be
effective against these thrips, but Bunacceptable^ pesticide res-
idue contents were detected in fruits. Residues of imidacloprid
and dinotefuran were significantly higher in leaves, whereas
residues in fruits were below detection limits. The authors sug-
gested that neonicotinoids may be a suitable control option
against these pests. In vineyards, neonicotinoids have been used for manage-
ment of mealybugs (e.g., Planococcus ficus Signoret)
(Wallingford et al. 2015). They were also used against ants
that interact by mutualism with mealybugs and coccids in
vineyards (Daane et al. 2008). Neonicotinoids, in particular
imidacloprid, have been suggested against Daktulosphaira
vitifoliae (Fitch) (Herbert et al. 2008), which is becoming
aggressive again in some parts of Europe. These insecticides
are effectively used to control leafhoppers (e.g., Empoasca
vitis Goethe, Erythroneura elegantula Osborn, and
Scaphoideus titanus Ball) in the vineyards of Europe and
NorthAmerica (Van Timmeren et al. 2011; Žežlina et al. 2013). In the USA, drench application of neonicotinoids has
been proposed against mealybugs and leafhoppers (Daane
et al. 2008; Van Timmeren et al. 2011). Neonicotinoids were suggested as an alternative tool in the
control of medfly (Ceratitis capitata Wiedmann) and South
American fruit fly Anastrepha fraterculus (Wiedemann) in
peach and nectarine orchards (Raga and Sato 2011; Rahman
and Broughton 2016). The results are not always successful,
and other control measures, including attract-and-kill tech-
niques, are recommended (Broughton and Rahman 2017). Alternative methods for pest control in perennial crops
1.
Mating disruption Mating disruption based on the use of synthetic sexual pher-
omones (Table 3) is an effective control tool against several
pests, particularly tortricid moths such as C. pomonella,
G. molesta, and Lobesia botrana (Den. and Schiff.)
(e.g.,Witzgall et al. 2008; Ioriatti and Lucchi 2016). These
methods can have some limitations in orchards and vineyards
with uneven topography, high pest densities, and more gener-
ally in the first years after adoption. Successful control of these
pests has been achieved using mating disruption with positive
implications for insecticide use reduction and prevention of
pesticide resistance (Trimble 1993; Angeli et al. 2007;
Bohnenblust et al. 2011; Bosch et al. 2016; Calkins and Faust
2003; Ioriatti et al. 2011). However, recent research in Spain
showed that the application of mating disruption in vineyards
was associated with an increase of minor pest incidence
(Gallardo et al. 2016). Pheromone-based mating disruption
has also been developed against grapevine mealybugs with
positive results (Walton et al. 2006; Cocco et al. 2014;
Sharon et al. 2016). Mating disruption can be induced by
substrate-borne vibrations that have been proposed in particular The spotted wing drosophila, Drosophila suzukii
Matsumura, is a serious pest of sweet cherry and other fruit
crops. In trials carried out in North America, some
neonicotinoids and OPs were effective against this pest
(Beers et al. 2011), but in other trials, neonicotinoids seem less
effective than other insecticides (Bruck et al. 2011; Shawer
et al. 2018). Laboratory experiments suggested that acetamiprid
can provide efficient control of D. suzukii when applications
are performed before egg deposition (Pavlova et al. 2017). Wise et al. (2015) suggested that the use of neonicotinoids is
not a good option in post-infestation applications. Another pest of increasing importance worldwide is the
brown marmorated stink bug (BMSB), Halyomorpha halys
(Stål) (Hemiptera: Pentatomidae). Invasive in the USA and
Europe, BMSB can attack various crops (Leskey et al. 2012). Against this pest, neonicotinoids are considered as an
efficient option for its control (Kuhar and Kamminga 2017). The Asian citrus psyllid Diaphorina citri Kuwayama
(Hemiptera: Liviidae) is an economically important pest of cit-
rus worldwide and the vector of the phloem-limited plant Another pest of increasing importance worldwide is the
brown marmorated stink bug (BMSB), Halyomorpha halys
(Stål) (Hemiptera: Pentatomidae). Invasive in the USA and
Europe, BMSB can attack various crops (Leskey et al. 2012). 2.
Exclusion netting 2. Exclusion netting The control of the woolly apple aphid Eriosoma lanigerum
Haussmann represents a successful case of classical biological
control by the hymenopteran parasitoid Aphelinus mali (Hald.). Outbreaks of the woolly aphids have often been associated with
the negative effect of non-selective pesticides on parasitoid pop-
ulations. Similar mechanisms can also be related to Cacopsilla
pyri L. outbreaks in pear orchards (Solomon et al. 1989;
Solomon et al. 2000; Vrancken et al. 2015). The role of preda-
tors in controlling peach aphids has been studied extensively
(e.g., Pappas and Koveos 2011), but their real potential to keep
pest populations under economic threshold levels needs further
investigation. Recent research in aphid management showed
that an increase in biological control can be indirectly obtained
by excluding ants or by providing alternative sugar feeding to
ants to reduce the ant-aphid mutualism (Nagy et al. 2013, 2015). Within microbial pest control agents, a number of entomo-
pathogenic fungi (e.g., Beauveria bassiana Bals. and
Paecilomyces fumosoroseus (Wize)) have been evaluated for
their activity against M. persicae and other aphid species with
promising results (Andreev et al. 2012; Lefort et al. 2014; Lee
et al. 2015). However, their effects in field conditions have
been poorly explored. The control of the woolly apple aphid Eriosoma lanigerum
Haussmann represents a successful case of classical biological
control by the hymenopteran parasitoid Aphelinus mali (Hald.). Outbreaks of the woolly aphids have often been associated with
the negative effect of non-selective pesticides on parasitoid pop-
ulations. Similar mechanisms can also be related to Cacopsilla
pyri L. outbreaks in pear orchards (Solomon et al. 1989;
Solomon et al. 2000; Vrancken et al. 2015). The role of preda-
tors in controlling peach aphids has been studied extensively
(e.g., Pappas and Koveos 2011), but their real potential to keep
pest populations under economic threshold levels needs further
investigation. Recent research in aphid management showed
that an increase in biological control can be indirectly obtained
by excluding ants or by providing alternative sugar feeding to
ants to reduce the ant-aphid mutualism (Nagy et al. 2013, 2015). Exclusion netting with insect-proof screens represents an-
other option for pest control (Table 3). Their use has been
proposed to protect orchards from moth invasion, with posi-
tive effects in aphid population densities reduction (Dib et al. 2010; Sauphanor et al. 2012). 2.
Exclusion netting More recently, the use of exclu-
sion netting has been suggested for the control of the invasive
pests H. halys and D. suzukii (Dobson et al. 2016; Rogers et al. 2016; Leach et al. 2016). These pests are generalist feeders
that can repeatedly invade orchards during a season. Exclusion netting should provide a physical obstacle for the
colonization of the crops. In addition, nets can be treated
chemically, i.e., pyrethroid-impregnated nets. This tool could
be used for the control of H. halys (Kuhar et al. 2017). Environ Sci Pollut Res (2021) 28:11798–11820 Table 3 Summary of the main
alternative methods in contrast
with extensive, conventional, and
intensive agriculture Table 3 Summary of the main
alternative methods in contrast
with extensive, conventional, and
intensive agriculture Table 3 Summary of the main
alternative methods in contrast
with extensive, conventional, and
intensive agriculture
Landscape
Farming methods
Organisms
Others
Patchy (reduced-size
fields)
Mutual funds (insurance
cover)
Macro-organisms:
Traps
Edge shrubs
Crop rotation
• Parasitoids
Attractants (traps)
Edge crops
Resistant variety:
• Predators:
Pheromones (traps)
Bund with flowers
• To insects
○Vertebrates
Repellants
Wet zones (e.g., pond)
• To diseases
○Invertebrates
Basic substances
Ecological corridors
Late sowing
Micro-organisms:
• Sugars
Trees (agroforestry)
Mixing varieties
• Fungi
• Oils
Tillage
• Bacteria
• Nettle extracts
Intercropping
• Nematodes
Mineral barrier (powders)
Netting
• Viruses
Hot water (plant nursery)
Stale seed bed
Sex confusion
Removal of plants bearing
pest
Chemical mediators
Manual pruning
Plant defense stimulators
Soil cover (e.g., grass)
Acoustic confusion
Natural-derived
insecticides
These methods are generally used in combination (without or) with low-risk pesticides for organic farming and
IPM practices. These methods contrast with the prophylactic uses of highly toxic pesticides such as neonicotinoids
and fipronil. Table adapted from Bonmatin (2016) These methods are generally used in combination (without or) with low-risk pesticides for organic farming and
IPM practices. These methods contrast with the prophylactic uses of highly toxic pesticides such as neonicotinoids
and fipronil. Table adapted from Bonmatin (2016) These methods are generally used in combination (without or) with low-risk pesticides for organic farming and
IPM practices. These methods contrast with the prophylactic uses of highly toxic pesticides such as neonicotinoids
and fipronil. Table adapted from Bonmatin (2016) against some sucking pests that are known to use vibrational
signals for communication during mating. Against these pests,
vibration-based mating disruption tools have been tested show-
ing potential for extensive applications (Polajnar et al. 2016). control not always ensures satisfactory levels of pest suppres-
sion and further research is needed to implement biological
control strategies of tortricid moths in orchards and vineyards. Natural enemy releases have been proposed for the control of
Planococcus ficus (Signoret) in vineyards of the USA, but the
full efficacy of these tactics seem to be limited by climatic
conditions (Daane et al. 2004). 2. Exclusion netting Alternative methods for pest control in perennial crops
1.
Mating disruption Against this pest, neonicotinoids are considered as an
efficient option for its control (Kuhar and Kamminga 2017). The Asian citrus psyllid Diaphorina citri Kuwayama
(Hemiptera: Liviidae) is an economically important pest of cit-
rus worldwide and the vector of the phloem-limited plant 11805 4.
Natural-derived insecticides In fruit orchards, peach aphid populations can be affected
by chemical control measures but also by cultural practices
and natural regulation. Fertilization has controversial effects
on aphid dynamics and management. In an ad hoc experi-
ment, M. persicae populations increased with a moderate
number of treatments but decreased at higher number of treat-
ments. The concentrations of primary and secondary metabo-
lites in the plant were modified by the number of treatments,
and this mechanism was suggested to be involved in these
contrasting effects (Sauge et al. 2010). Four aphid control
strategies, namely intensive, optimised, input-substitution,
and integrated control, were compared in France (Penvern
et al. 2010). The use of pesticides lowered densities of aphids
as well as those of their natural enemies while cultural
methods (e.g., ground cover and manual pruning of infested
branches) promoted high populations of both arthropod
groups. Data were critically discussed to redesign advanced
orchard protection strategies that aimed to obtain pest control
in the framework of biodiversity conservation. This approach
requires local adaptations. For example, in China, the intro-
duction of ground cover based on Trifolium repens L. in peach
orchards obtained a significant reduction in the abundances of
aphids and G. molesta (> 31%) probably due to the increase of
generalist predators (> 115%) (Wan et al. 2014). The presence
of natural enemies in pome fruit orchards can be promoted
through habitat management practices such as increasing flo-
ral diversity in the agro-ecosystem by using selected trees and
grasses (Rieux et al. 1999). The presence of hedgerows may
increase the impact of parasitism of the codling moth
(Maalouly et al. 2013; Monteiro et al. 2013). Besides biological control and mating disruption, alterna-
tives in sucking pest control in perennial crops are represented
by the application of natural-derived insecticides (Table 3). Multiple applications of kaolin, a clay mineral, in apple or-
chards have been found effective against a number of pests
including the green apple aphid Aphis pomi (DeGeer) (Markó
et al. 2008). In the same study, severe infestations of the wool-
ly apple aphid have been found in kaolin-treated plots proba-
bly because of its negative impact on aphid natural enemies. The use of kaolin has been recently suggested against the
grapevine leafhoppers in vineyards (Tacoli et al. 2017). 6.
Ecological engineering for pest suppression: habitat ma-
nipulation for pest management and cultural control Agro-ecological practices aimed at enhancing the effective-
ness of natural enemies to reduce pest pressure can offer, for
some perennial cropping systems, valid alternatives to insec-
ticides in pest management. 5.
Food-derived biorationals as insect crop protection 5. Food-derived biorationals as insect crop protection More recently, natural or food-derived insecticides (e.g.,
vegetable oils), insect repellants (e.g., nettle extract), plant
strengtheners (e.g., sucrose, fructose), and trap attractants
(e.g., di ammonium phosphate) were approved as basic pest
control substances under the European pesticide regulation,
with no maximum residue limit (MRL) (Marchand 2015,
2016, 2017). 6. Ecological engineering for pest suppression: habitat ma-
nipulation for pest management and cultural control 3.
Biological control Within microbial pest control agents, a number of entomo-
pathogenic fungi (e.g., Beauveria bassiana Bals. and
Paecilomyces fumosoroseus (Wize)) have been evaluated for
their activity against M. persicae and other aphid species with
promising results (Andreev et al. 2012; Lefort et al. 2014; Lee
et al. 2015). However, their effects in field conditions have
been poorly explored. Interest on the release of biocontrol agents to control pests
in perennial cropping systems has increased in the last decades
(Table 3). Inundative releases of egg parasitoids have been
proposed against grape berry moths L. botrana (El Wakeil
et al. 2008). However, it should be stressed that biological 11806 Environ Sci Pollut Res (2021) 28:11798–11820 Microbial products based on C. pomonella granulovirus
(CpGV-M) have been suggested as an alternative in codling
moth control (Cross et al. 1999; Beers et al. 2003), but these
products have also been involved in resistance (Schmitt et al. 2013). Treatments with the entomophathogenic nematodes
Steinernema carpocapsae (Weiser) and Steinernema feltiae
Filipjev against overwintering larvae can achieve a good con-
trol of the codling moth (Unruh and Lacey 2001; Lacey et al. 2006) but their efficacy is strongly influenced by climatic
conditions. Microbial control agents active towards tortricids
and other lepidopteran pests include Bacillus thuringiensis
Berliner (Cross et al. 1999; Lacey and Shapiro-Ilan 2008;
Vassiliou 2011), characterized by its specificity towards
Lepidoptera, Diptera, and Coleoptera and reduced risks to
human health and the environment. The efficacy of
B. thuringiensis can be limited by a number of climatic (e.g.,
temperature) and agronomic (e.g., differences in larval instar
susceptibility, spray coverage, and application rate) factors
that should be considered in practice. et al. 2014; Pozzebon et al. 2014; Beers and Schmidt 2014;
Malagnoux et al. 2015). et al. 2014; Pozzebon et al. 2014; Beers and Schmidt 2014;
Malagnoux et al. 2015). 4.
Natural-derived insecticides Another option can be the use of fatty acid salts, in particular
potassium salts, that have been used with a good efficacy
against pear psylla (Souliotis and Moschos 2008). Botanical insecticides such as those based on Azadirachta
indica or natural pyrethrins have been suggested in controlling
aphids in orchards and different other pests in vineyards
(e.g., Andreev et al. 2012; Cichon et al. 2013; Dercks et al. 2014) but their effects were not always satisfactory. More
recently, Annonaceae derivatives have been tested successful-
ly in the laboratory against M. persicae (Ribeiro et al. 2014). Among natural-derived pesticides, spinosad is a natural
mixture of toxins produced by the soil actinomycete
Saccharopolyspora spinosa (Mertz and Yao 1990). This com-
pound proved to be effective to control codling moth, grape
berry moths, leaf miners, thrips, and dipterans as a recurrent
option in organic orchards and vineyards (Reissig 2003;
Mota-Sánchez et al. 2008; Vassiliou 2011). However, its use
has been associated with negative effects on natural enemies
(e.g., predatory arthropods) of importance for fruit orchards
and vineyards (Ahmad et al. 2013; Tirello et al. 2013; Duso In vineyards, the threats by sucking pests are mainly asso-
ciated with leafhoppers (e.g., E. vitis and E. elegantula),
mealybugs (e.g., P. ficus), thrips (e.g., Drepanothrips reuteri 11807 Environ Sci Pollut Res (2021) 28:11798–11820 Uzel), and spider mites (Panonychus ulmi Koch and
Eotetranychus carpini Oudemans). They are generally con-
sidered as secondary pests that can be managed by promoting
the presence of natural enemies in vineyards (Duso et al. 2012,
Walton et al. 2012). This can be achieved by increasing habitat
complexity/diversity to provide refuges and alternative hosts
and food resources to predators and parasitoids (Costello and
Daane 2003; Duso et al. 2004; Ponti et al. 2005; Zanolli and
Pavan 2011; Pozzebon et al. 2015a; Wilson et al. 2015) and
reducing the use of non-selective pesticides (e.g., Jepsen et al. 2007, Pozzebon et al. 2015b). Biological control strategies
against sucking pests can also be enhanced by inoculative or
augmentative releases of natural enemies (Duso et al. 1985;
Daane et al. 1996; Duso and Vettorazzo 1999; Daane et al. 2008). Irrigation, fertilization, and cultivar choice can be also
managed to reduce pest incidence and economic damage
(Daane and Williams 2003; Costello 2008; Fornasiero et al. 2012, 2016; Cocco et al. 2015). cases of outbreaks of pest populations have been described
in annual crops (e.g., in Santos et al. 4.
Natural-derived insecticides 2016 for soybean
treated with imidacloprid in Brazil). More specifically,
Santos et al. (2016) studied the survival and fertility of the
Neotropical brown stink bug Euschistus heros. Newly
emerged adult females were exposed for 48 h imidacloprid
residues equivalent to 1% of the field rate dose. Females ex-
hibited reduced rates of survival but higher fecundity and fer-
tility rates compared with untreated females. The authors
showed that females of E. heros increased their reproductive
output in response to the imidacloprid sublethal exposure. These findings suggest a potential involvement of sublethal
exposure to neonicotinoids in the recent outbreaks of the
Neotropical brown stink bug E. heros observed in Brazilian
soybean-producing regions. In rice crops, resistance of the brown planthopper
(Nilaparvata lugens Stål) to imidacloprid was first detected
in Thailand in 2003, and then in Vietnam, Japan, and other
Asian countries (Matsumura et al. 2008). Resistance of this
pest to neonicotinoids is widespread in China, with resistance
ratios (RRs, the greater it is, the higher resistance) in 2012
ranging from 209- to 617-fold. These values are much higher
than in 2009. For thiamethoxam, the RR varied from 17 to 47
and for nitenpyram from 1.4 to 3.7 in 2012 (Zhang et al. 2014). Current levels of resistance are much higher than those
reported 6 years earlier by Matsumura et al. in 2008. Similarly,
populations of the whiteback planthopper Sogatella furcifera
(Horvath) are resistant to fipronil in all countries of southeast
Asia. In rice fields of Kumamoto, Japan, resistance of the
small planthopper Laodelphax striatellus to fipronil has
reached RR > 1,700, whereas new compounds with the same
MoA (e.g., fluralaner acting on GABA- and L-glutamate-
gated chloride channels) can be more effective in controlling
this pest (Asahi et al. 2015). It seems that the cytochrome
P450 CYP6ER1 is significantly overexpressed in
imidacloprid-resistant planthopper populations of southeast
Asia (i.e., N. lugens and S. furcifera), with higher tolerance
levels of 10- to 90-fold compared with a laboratory-
susceptible strain. However, other pest populations showed
different overexpression of variant P450 enzymes implicated
in imidacloprid resistance (Garrood et al. 2016). One study
found that a single mutation at a conserved position (Y151S)
in two nAChR subunits, Nla1 and Nla3, is responsible for a
substantial reduction in specific imidacloprid binding (Liu
et al. 2005). Resistance to neonicotinoids and fipronil Since their commercialization in1991, neonicotinoids have
been a useful tool for the control of various pests. The first
case of resistance to neonicotinoids was reported in 1996, and
later, a number of publications were devoted to this topic
worldwide (Gorman et al. 2010). Increased use of insecticides
exacerbates the development of resistance in most crop pests. In this regard, the use of neonicotinoids increased also rapidly
after introduction of imidacloprid in several developed coun-
tries (Simon-Delso et al. 2015). The same situation has been
observed since 2003 in the USA after the introduction of seed-
treated crops in fields. The shift toward large-scale, prophy-
lactic insecticide use was unprecedented, with 34–44% of soy-
beans and 79–100% of maize hectares treated in 2011 alone,
contradicting previous expectations of using fewer insecti-
cides than a decade or two ago (Douglas and Tooker 2015). One can expect, therefore, a rapid increase in pest resistance to
all neonicotinoids in areas treated with coated seeds, as resis-
tance mechanisms may develop rather quickly. For example,
the cotton mealybug Phenacoccus solenopsis Tinsley
(Hemiptera: Pseudococcidae) developed a 315-fold greater re-
sistance to acetamiprid after five rounds of selection in con-
trolled conditions, although at the cost of reducing the biolog-
ical fitness of the resistant populations (Afzal et al. 2015). Other authors found that the development of resistance to
acetamiprid in cotton fields takes 7 years and is slower than
resistance to other neonicotinoids, carbamates, organophos-
phorus, and pyrethroid insecticides (Ahmad and Akhtar 2016). 4.
Natural-derived insecticides Even the additional use of neonicotinoid-
specific synergists such as IPPA08 does not seem to work so
efficiently with resistant populations of planthoppers (Bao
et al. 2016). Therefore, the best option to control that pest
would be IPM strategies that do not use neonicotinoids. Interestingly, the IPM strategy has been adopted in the
Philippines. Results showed that populations of the above-
mentioned planthopper in the Philippines remain susceptible
to neonicotinoids (Matsumura et al. 2008) because of the Perennial crops In cotton crops, resistance of the tobacco thrips
(Frankliniella fusca Hinds) to imidacloprid and thiamethoxam
has developed faster than expected by earlier forecasts in
southern areas of the USA. Some 57 and 65% of the popula-
tions monitored in 2015 showed resistance to the seed coated
with neonicotinoids, with RR up to 55 and 39 for imidacloprid
and thiamethoxam, respectively (Huseth et al. 2016). Resistance to thiamethoxam by the cotton aphid Aphis gossypii
(Glover) has reached RRs between 29 and 526 in the USA
(Gore et al. 2013), while in China, this pest shows a more
moderate level of resistance to imidacloprid (RR of 42) and
lesser values than for all other neonicotinoids (Shi et al. 2011). In orchards, M. persicae has become resistant to
imidacloprid and thiacloprid in populations of southern
Europe. In Italy, 65% of the aphids studied by Panini et al. (2014) had the neonicotinoid-specific R81T mutation while a
few genotypes also revealed the involvement of P450-based
metabolic resistance processes (Panini et al. 2014). The dom-
inance level of insecticide resistance in this species suggests
that the mutant allele 81T is semi-recessive, with the wild 81R
allele being rather dominant (Mottet et al. 2016). The
neonicotinoid sulfoxaflor, a newly developed agonist of the
nicotinic receptors (Giorio et al. 2017), appears to behave in a
similar way in resistant strains of the same aphid
M. persicae (Cutler et al. 2013), although some authors say
is not affected by this mutation (Wang et al. 2016b). In popu-
lations of this aphid in Greece, over 58% of the clones collect-
ed in 2013 showed a 9- to 36-fold overexpression of the
CYP6CY3 gene that codifies for the P450 detoxification
mechanism (Voudouris et al. 2016). This is a matter of concern
as tolerance can be developed very quickly by this
mechanism. The polyphagous and cosmopolitan whitefly Bemisia
tabaci (Gennadius) is a devastating pest that can cause severe
damage to a range of vegetable, fiber, and ornamental crops by
direct feeding and by plant virus transmission. This species
was the first to show resistance to imidacloprid and other
neonicotinoids (Gorman et al. 2010). Cross-resistance among
these compounds (Prabhaker et al. 2005) is threatening the
chemical management program on genetically engineered cot-
ton, where neonicotinoids are routinely being sprayed to man-
age sucking pests that have become dominant after the reduc-
tion in bollworms in the field (Basit et al. 2012). Perennial crops In potato fields of North America, resistance of the
Colorado potato beetle Leptinotarsa decemlineata (Say) to
imidacloprid developed within 10 years after its introduction
in 1995. By 2009, resistance affected more than 95% of the
population of this pest in the Northeastern and Midwestern
USA (Szendrei et al. 2012). High levels of resistance can be
observed in areas within 100 km of the treated fields. It seems
that the upregulation of three cytochrome P450s and a gluta-
thione synthase-related protein provide a mechanistic expla-
nation of resistance evolution in multiple resistant popula-
tions, but some of the resistant mechanisms involve also ge-
netic changes and not just phenotypes (Clements et al. 2016). Resistance of this beetle species to another neonicotinoid,
thiamethoxam, was first found in 2003 in a population from
Massachusetts (Szendrei et al. 2012), further advocating that
chemical control of this pest should be replaced with more
rigorous application of IPM strategies. Bass et al. (2015) reviewed most of the literature on pest
resistance to neonicotinoids. Among pests of interest for pe-
rennial cropping systems, the green peach aphid Myzus
persicae Sulzer has been involved in the highest number of
reported cases of resistance among fruit trees. This appears to
be a case of pre-selection resulting from host-plant adaptation
(tolerance to nicotine by feeding on tobacco) and an expan-
sion in host range. Resistance seems to be associated with
metabolic detoxification by enhanced expression of cyto-
chrome P450s. In some cases, it has also been found that
modified penetration through the cuticle might contribute to
resistance together with enhanced detoxification (Puinean
et al. 2010). Target site resistance was also suggested as a
mechanism inducing resistance in M. persicae (Bass et al. 2015). This was found to be associated to the R81T mutation
in nAChR subunit genes in aphid populations from peach. Toda et al. (2017) developed a molecular diagnosis test for
detecting the R81T mutation on the D-loop region of the β1
subunit of the nAChR gene; this mutation confers resistance
to neonicotinoids in the cotton aphid Aphis gossypii
(Hemiptera: Aphididae). This mutation appears to be distrib-
uted in aphid populations on peach and closely related crops
over southern Spain, southern France, and northern and cen-
tral Italy and Greece (Bass et al. 2015; Voudouris et al. 2016). Bass et al. (2015) stressed the need to employ insecticides
with different MoAs to reduce the selection pressure induced
by neonicotinoids. Annual crops Potential for resistance to neonicotinoids in the pests of arable
crops has been described (Clements et al. 2017), and some Environ Sci Pollut Res (2021) 28:11798–11820 11808 also carries plant viruses that can decimate tomato and corn
crops, so resistance to neonicotinoids strongly suggests that
chemical control should be replaced with IPM strategies as well. many years implementing IPM with little use of insecticides in
that country (Hadi et al. 2015). Actually, the obvious increase
in resistance to systemic insecticides strongly suggests that
management strategies other than chemical treatments are ur-
gently needed to prevent damage by these pests. Perennial crops The greenhouse whitefly Trialeurodes vaporariorum
(Westwood) has also developed resistance to several
neonicotinoids applied to vegetable crops in Europe and
China (Table 4). The western flower thrip Frankliniella
occidentalis (Pergande), which has invaded many horticultural
and ornamental crops in China due to international trade, has
developed up to 24-fold resistance to imidacloprid and up to
8.7-fold to acetamiprid (Wang et al. 2016a). This invasive pest Environ Sci Pollut Res (2021) 28:11798–11820 11809 Table 4
Levels of resistance, illustrated by resistance ratios (RR), of common crop pests to neonicotinoid insecticides
Species/common name
Taxa
Chemical name
RR-fold
Crop
Country
Reference
Amrasca devastans (Distant)
Cotton leaf hopper
Hemiptera: Delphacidae
Acetamiprid
2.3 to 29.3
Cotton
Pakistan
Saeed et al. (2017)
Imidacloprid
4.8 to 95.0
Cotton
Pakistan
Saeed et al. (2017)
Thiamethoxam
19.1 to 1197.9
Cotton
Pakistan
Saeed et al. (2017)
Aphis gossypii (Glover) Cotton
aphid
Hemiptera: Aphididae
Acetamiprid
4.5
Cotton
China
Shi et al. (2011)
Clothianidin
1.2
Cotton
China
Shi et al. (2011)
Dinotefuran
1.1
Cotton
China
Shi et al. (2011)
Imidacloprid
41.7
Cotton
China
Shi et al. (2011)
Nitenpyram
5.8
Cotton
China
Shi et al. (2011)
Thiacloprid
3.7
Cotton
China
Shi et al. (2011)
Thiamethoxam
1.1
Cotton
China
Shi et al. (2011)
Thiamethoxam
29 (3d) to 526 (2d)
Cotton
USA
Gore et al. (2013)
Bemisia tabaci (Gennadius)
Silverleaf whitefly/
sweetpotato whitefly
Hemiptera: Aleyrodidae
Acetamiprid
17 to > 2727
Cotton
Australia
Bingham et al. (2008)
Acetamiprid
32 to 183
Melon, cotton,
vegetables
USA (Arizona, California)
Castle and Prabhaker (2013)
Acetamiprid
3.1
Cabbage
China
Liang et al. (2012)
Acetamiprid
5 to 8
Cotton, melon
USA (Arizona, California)
Prabhaker et al. (2005)
Acetamiprid
23
Melon
Guatemala
Prabhaker et al. (2005)
Acetamiprid
36.6
Cotton
Pakistan
Basit et al. (2012)
Dinotefuran
52 to 168
Melon, cotton,
vegetables
USA (Arizona, California)
Castle and Prabhaker (2013)
Imidacloprid
741 to > 2000
Cotton
Australia
Bingham et al. (2008)
Imidacloprid
58 to 126
Cotton
Guatemala
Byrne et al. (2003)
Imidacloprid
156 to 1830
Melon, cotton,
vegetables
USA (Arizona, California)
Castle and Prabhaker (2013)
Imidacloprid
6
Cotton
Egypt
Kady and Devine (2003)
Imidacloprid
6
Cabbage
China
Liang et al. (2012)
Imidacloprid
13 (larvae), 580 (adults)
Cabbage
Germany, Spain, UK
Nauen et al. (2008)
Imidacloprid
120 to 160
Cotton, melon
USA (Arizona, California)
Prabhaker et al. (2005)
Imidacloprid
109
Melon
Guatemala
Prabhaker et al. (2005)
Imidacloprid
8.7 to 75
Cabbage
China
Yuan et al. (2012)
Imidacloprid
7.25
Cotton
Pakistan
Basit et al. Perennial crops (2012)
Nitenpyram
5
Cabbage
China
Liang et al. (2012)
Nitenpyram
7.5 to 46.4
Cabbage
China
Yuan et al. (2012)
Nitenpyram
28.4
Cotton
Pakistan
Basit et al. (2012)
Thiacloprid
28.8
Cotton
Pakistan
Basit et al. (2012) Environ Sci Pollut Res (2021) 28:11798–11820 11810 b e
(co
ued)
Species/common name
Taxa
Chemical name
RR-fold
Crop
Country
Reference
Thiamethoxam
7 to 125
Melon, cotton,
vegetables
USA (Arizona, California)
Castle and Prabhaker (2013)
Thiamethoxam
2 to 22
Cotton, melon
USA (Arizona, California)
Prabhaker et al. (2005)
Thiamethoxam
24
Melon
Guatemala
Prabhaker et al. (2005)
Thiamethoxam
52.4
Cotton
Pakistan
Basit et al. (2012)
Cimex lectularius L Common
bed bug
Hemiptera: Cimicidae
Acetamiprid
31.7 to > 33,000
USA (Ohio, Michigan)
Romero and Anderson (2016)
Dinotefuran
46.8 to 358
USA (Ohio, Michigan)
Romero and Anderson (2016)
Imidacloprid
2.0 to 462.6
USA (Ohio, Michigan)
Romero and Anderson (2016)
Thiamethoxam
2.4 to 546
USA (Ohio, Michigan)
Romero and Anderson (2016)
Cydia pomenella Coddling moth
Lepidoptera: Tortricidae
Thiacloprid
5.5 to 16.5
Apple
Turkey
İşci and Ay (2017)
Diaeretiella rapae Parasitic wasp
Hymenoptera: Aphidiidae
Fipronil
Up to 20.8
Vegetables
China
Wu et al. (2007)
Imidacloprid
Up to 74.7
Vegetables
China
Wu et al. (2007)
Frankliniella fusca (Hinds)
Tobacco thrip
Thysanoptera: Thripidae
Imidacloprid
55
Cotton
USA
Huseth et al. (2016)
Thiamethoxam
39
Cotton
USA
Huseth et al. (2016)
Frankliniella occidentalis
(Pergande) Western flower thrip
Thysanoptera: Thripidae
Acetamiprid
Up to 8.7
Vegetables, roses
China
Wang et al. (2016a)
Imidacloprid
2.8 to 24.4
Vegetables, roses
China
Wang et al. (2016a, b)
Laodelphax striatellus Small
planthopper
Hemiptera: Delphacidae
Fipronil
> 1700
Rice
Japan
Asahi et al. (2015)
Leptinotarsa decemlineata (Say)
Colorado potato beetle
Coleoptera: Chrysomelidae
Imidacloprid
1.8 to 27
Potato
USA (Wisconsin)
Clements et al. (2016)
Imidacloprid
20 to 50
Potato
USA
Alyokhin et al. (2007)
Thiamethoxam
6 to 9
Potato
USA
Alyokhin et al. (2007)
Maconellicoccus hirsutus (Green)
Pink mealybug
Hemiptera: Pseudococcidae
Imidacloprid
10.2
Mulberry, vineyards
India
Mruthunjayaswamy et al. (2016)
Musca domestica L Housefly
Diptera: Muscidae
Thiamethoxam
6 to 76
Livestock farm
Denmark
Kristensen and Jespersen (2008)
Myzus persicae (Sulzer) Green peach
aphid/peach-potato aphid
Hemiptera: Aphididae
Imidacloprid
Not yet
Orchards, beet
root, grains
Australia
Edwards et al. (2008)
Nilaparvata lugens (Stål)
Brown planthopper
Hemiptera: Delphacidae
Fipronil
3.7 to 5.4
Rice
Japan, Taiwan, Vietnam
China
Matsumura et al. (2008)
Imidacloprid
10 to 90
Rice
South East Asia
Garrood et al. (2016)
Imidacloprid
3.5
Rice
India
Garrood et al. Perennial crops (2016)
Imidacloprid
100
Rice
India
Gorman et al. (2008)
Imidacloprid
Not resistant in 2006
Rice
China, India, Indonesia,
Malaysia, Thailand
and Vietnam
Gorman et al. (2008)
Imidacloprid
22 to 89
Rice
Japan, Taiwan, Vietnam
China
Matsumura et al. (2008)
Imidacloprid
209 to 616
Rice
China
Zhang et al. (2014)
810
Environ Sci Pollut Res (2021) 28:11798–11820 Table 4 (continued) Environ Sci Pollut Res (2021) 28:11798–11820 11811 Table 4 (continued)
Species/common name
Taxa
Chemical name
RR-fold
Crop
Country
Reference
Nitenpyram
1.4 to 3.7
Rice
China
Zhang et al. (2014)
Thiamethoxam
2 to 3.4
Rice
Japan, Taiwan, Vietnam
China
Matsumura et al. (2008)
Thiamethoxam
17.4 to 47.1
Rice
China
Zhang et al. (2014)
Phenacoccus solenopsis (Tinsley)
Cotton mealybug
Hemiptera: Pseudococcidae
Acetamiprid
9.7 to 315
Cotton
Pakistan
Afzal et al. (2015)
Acetamiprid
Up to 30
Cotton
Pakistan
Ahmad and Akhtar (2016)
Imidacloprid
7.6 to 217
Cotton
Pakistan
Afzal et al. (2015)
Imidacloprid
Up to 151
Cotton
Pakistan
Ahmad and Akhtar (2016)
Thiacloprid
Up to 338
Cotton
Pakistan
Ahmad and Akhtar (2016)
Thiamethoxam
Up to 93
Cotton
Pakistan
Ahmad and Akhtar (2016)
Sogatella furcifera (Horváth)
Whitebacked planthopper
Hemiptera: Delphacidae
Fipronil
Up to 37
Rice
Japan, Taiwan, Vietnam
China
Matsumura et al. (2008)
Imidacloprid
Up to 5
Rice
Japan, Taiwan, Vietnam
China
Matsumura et al. (2008)
Trialeurodes vaporariorum
(Westwood) Greenhouse whitefly
Hemiptera: Aleyrodidae
Acetamiprid
2.6 to 18.6
Vegetables
UK
Karatolos et al. (2010)
Imidacloprid
2.6 to 21.8
Vegetables
UK
Karatolos et al. (2010)
Imidacloprid
2.5
Eggplant
China
Liang et al. (2012)
Nitenpyram
3.7
Eggplant
China
Liang et al. (2012)
Thiamethoxam
1.3 to 20.4
Vegetables
UK
Karatolos et al. (2010)
R i t
f th b d b
it i
l
i
l d d Table 4 (continued) 11812 Environ Sci Pollut Res (2021) 28:11798–11820 The most important pest of apple trees, the codling moth
Cydia pomonella, has developed resistance to thiacloprid,
with RRs between 5.5 and 16.5 measured in orchards of
Turkey (İşci and Ay 2017). Resistance of this pest to
thiacloprid is correlated with mixed-function oxidase activity
(Reyes et al. 2007). This phenomenon seems to be spread
across the world (Bass et al. 2015; İşci and Ay 2017) and is
linked to cross-resistance to other compounds such as organ-
ophosphates: in this case, the resistance mechanism seems to
be based on detoxification enzymes. In other studies on
C. Other resistance to neonicotinoids Pest resistance to neonicotinoids has been found not only in
crops but also in the control of bed bugs (Cimex lectularius L.)
in the USA. Consequently, formulations combining two
neonicotinoids or a pyrethroid are currently becoming very
popular in that country. However, high levels of resistance to
four neonicotinoids, acetamiprid (up to RR of 33,000),
imidacloprid (RR in the 2–463 range), dinotefuran (RR in
the 47–359 range), and thiamethoxam (RR in the 2.4–546
range) have already been detected in bed bug populations. In
this case, detoxification mechanisms by induction of glutathi-
one S-transferases (GST) and cytochrome P450s are respon-
sible for the development of such resistance, thus limiting the
options for chemical control of bed bugs (Romero and
Anderson 2016). On the Asian citrus psyllid D. citri, a reduced sensitivity to
neonicotinoids in certain populations of this pest in Florida
has been found, raising concerns that resistance to
neonicotinoids can hamper management of this pest (Tiwari
et al. 2011). The promotion of effective rotations of insecti-
cides and area-wide management of D. citri seems to have
determined a reversal for insecticide resistance in this pest
(Coy et al. 2016). Western flower thrips, Frankliniella occidentalis
(Pergande), is a generalist pest that can threaten fruit orchards
and vineyards. Metabolic resistance to neonicotinoids has
been reported for this pest, probably originating by cross-
resistance with other insecticides (Zhao et al. 1995;
Minakuchi et al. 2013). In vineyards and mulberry groves of
India, resistance of the pink mealybug (Maconellicoccus
hirsutus Green) to imidacloprid have reached RR of 10.2-fold,
similar to the tolerance found with other insecticidal classes
(Mruthunjayaswamy et al. 2016). Perennial crops pomonella, a number of detoxification genes (CYP9A61,
CpGST1, and CpCE-1) were differentially induced or sup-
pressed by various insecticides (including imidacloprid) while
expression of these genes was not influenced by acetamiprid
when compared to the control (Yang et al. 2016). Indeed, among the most common pests of agricultural and
ornamental crops, resistance to systemic insecticides is now
widespread and develops quickly, as it typically involves en-
hanced detoxification by GST and P450 enzymatic systems. In the case of neonicotinoids, specific mutations of the α-
subunit of nAChRs confer long-term resistance to all
chemicals of this class (Thany 2010). Highest resistance levels
were found for imidacloprid, the first neonicotinoid launched
to the market and lowest in the newest compounds like
dinotefuran and nitenpyram (Shi et al. 2011; Zhang et al. 2014)—see Table 4. While resistance due to detoxification
mechanisms can be overcome by using synergistic mixtures
with other chemicals (Bingham et al. 2008; Basit et al. 2013;
Darriet and Chandre 2013), the mutant-resistant individuals
could be selected naturally rather quickly and eventually dom-
inate the field populations of pests. Other authors have shown that over transcription of a single
gene product, Cyp6g1, which is associated with the metabolic
resistance to neonicotinoids in Drosophila melanogaster larvae,
results in a significant increase of three imidacloprid metabo-
lites in vivo (Joussen et al. 2008; Hoi et al. 2014). The high
frequency of mutations and data obtained from these studies
confirm the existence of multiple resistance mechanisms (e.g.,
enhanced detoxification, mutations, overexpression of en-
zymes), which may require different management strategies. Concluding remarks Insecticides are expected to achieve higher yields and net in-
comes, but the relationship between yields and farmer’s
profits is not so obvious. For example, the effect of insecti-
cides on yield may be negligible (see examples above), or
quality products under organic/Bintegrated^ cultivations may
be sold at higher prices than conventional ones treated with
insecticides, thus largely compensating reduced yields. An
example at the scale of west and east Germany is given by
Batáry et al. (2017). Another large-scale example is given here
with mutual funds and IPM, which increased farmer profits
while reducing the use of pesticides without negative impact
on average yields and at the same time avoiding environmen-
tal impacts. Populations of the tea green leafhopper Empoasca vitis
have been developing resistance to a number of insecticides
in southeastern China, with high levels of cross resistance
among imidacloprid, chlorfenapyr, and indoxacarb (Wei
et al. 2017). Resistance to neonicotinoids and fipronil has also been
observed in beneficial insects. For instance, the parasitic wasp
Diaeretiella rapae (Hymenoptera: Aphidiidae) showed cross-
resistance to fipronil and imidacloprid and is now 20 and 75
times respectively more resistant to these chemicals than in the
past (Wu et al. 2004). A review of the current literature on neonicotinoids and
fipronil shows these systemic insecticides have a role in
protecting certain crops against the damaging attacks of some We hope that this review may help regulators to carefully
consider the pros and cons of the continuous, increasing, and
widespread use of these systemic insecticides. On a scientific
basis, the efficiency of neonicotinoids and fipronil for pest
control should be balanced against the drawbacks of their
damage to natural enemies and other ecosystem services that
sustain agricultural systems. We have restricted this WIA to neonicotinoids and fipronil
because they represent most of the insecticide market nowa-
days. However, regulators should consider that replacing one
molecule by another in the future is not a sustainable strategy
for agricultural production, as new molecules with the same
MoA (e.g., sulfoxaflor, flupyradifurone) are additional threats
to the environment and public health. Regulators should real-
ize that a more restrictive regulatory framework is required for
more sustainable agricultural practices such as IPM, with a
strong willingness to use (present or future) highly toxic pes-
ticides only as the last resort. Here, we highlighted that the use of neonicotinoids is lim-
ited by the rapid development of resistance in target pests. Because many of the underlying mechanisms of resistance
are common to other insecticide classes (e.g., pyrethroids,
cholinesterase, inhibitors), the use of new neonicotinoids
(e.g., sulfoxaflor, flupyradifurone; see Pisa et al. 2017) or sub-
stances with the same MoA is not the solution in the medium
and longterm. It can even worsen impacts on non-target inver-
tebrates by potential synergistic interactions with other
neonicotinoids which are now everywhere in the environment
(Mitchell et al. 2017). As Barzman et al. (2015) have said,
BThe future of crop production is now also threatened by
emergence of pest resistance and declining availability of ac-
tive substances. There is therefore a need to design cropping
systems less dependent on synthetic pesticides.^ Moreover, it
is the prophylactic uses of such systemic insecticides in seed
treatments that should be urgently stopped since they are con-
trary to IPM practices. The tools for a new cropping system
that does not rely on chemicals alone have been with us for
years, but the implementation of the IPM practices is lacking
(Hokkanen 2015) despite the initial aspirations of the EU di-
rective establishing a framework for community action to
achieve the sustainable use of pesticides (EU 2009). Acknowledgments The authors thank the Lune de Miel Foundation
(France), the David Suzuki Foundation (Canada), the SumOfUs commu-
nity (Germany), the Rovaltain Foundation (France), the Aurelia
Foundation (Germany), and IUCN for their help in the dissemination of
these updated WIA findings. The authors thank K.L. Heong, C. Pacteau, E. Lumawig-Heitzmann, J. Candilon Condiman, M. Petit, T. Brooks, and U. Drebber for the fruitful discussions and their help in this project. The TFSP
members dedicate this work to the memory of Prof. Dr. Job van Praagh. Funding information The Task Force on Systemic Pesticides (TFSP,
www.tfsp.info) has been funded by the Triodos Foundation’s Support
Fund for Independent Research on Bee Decline and Systemic
Pesticides. This Support Fund received donations from the Act Beyond
Trust (Japan), Stichting Triodos Foundation (The Netherlands), M.A.O. C. Gravin van Bylandt Stichting (The Netherlands), Zukunft Stiftung
Landwirtschaft (Germany), Hartmut Spaeter Umweltstiftung (Germany)
, and citizens. The funders had no role in the study design, data collection
and analysis, decision to publish, or preparation of the manuscript. Environ Sci Pollut Res (2021) 28:11798–11820 11813 systemic insecticides are supposed to produce (Chagnon
et al. 2015). Over-reliance on chemical control is associ-
ated with contamination of ecosystems (Bonmatin et al. 2015; Pisa et al. 2015; Mineau and Whiteside 2013;
Beketov et al. 2013; Giorio et al. 2017) and undesirable
health effects (Scott et al. 2014; Cimino et al. 2017; Wang
et al. 2018), although in the case of neonicotinoids and
fipronil, the scarcity of studies on human health to date
preclude us from making a clear assessment. More effort
is needed to investigate the effects induced by these ago-
nists of the neuronal system after chronic human exposure
(e.g., farmers and workers, exposure by drinks, food,
treated pets and breeding animals, treated wood struc-
tures, air pollution, etc., and the sum of all these expo-
sures) (Salis et al. 2017). soil pest such as wireworms and root worms and of sucking
pests, in particular aphids, leafhoppers, thrips, mealybugs, and
scale insects, as well as internal grubs that can only be reached
by chemicals translocated within the plant. However, their
efficacy does not guarantee an increase of yield of the crops
they are protecting, particularly in pollinated crops. This is not
unusual, as a recent study in France demonstrated that insec-
ticide usage hardly accounts for any yield benefit in arable
crops (Lechenet et al. 2017), mostly because plants compen-
sate for the small damage that insects inflict them while the
risk of a pest outbreak is small on a year-to-year basis. Pest management can be implemented effectively by using
the multi-faceted methods of IPM described succinctly in this
paper. In addition, economic insurance initiatives, as de-
scribed for the case of maize crops in Italy, can make up for
farmers’ losses in bad years, and they do not place any pres-
sure on the environment, whereas neonicotinoids and fipronil
do have large impacts on biodiversity, ecosystems, and eco-
system services worldwide (Pisa et al. 2017). In this sense,
Europe is committed to continuing agricultural production
while reducing significantly the amount of pesticide uses
(Lescourret 2017) by making compulsory the use of IPM
practices aimed at the preservation of environment and eco-
system services that sustain agricultural productivity
(Sgolastra et al. 2017). Other countries (e.g., Canada) have
taken regulatory decisions to reduce both non-agricultural
and agricultural uses of neonicotinoids. References Benefer CM, Knight ME, Ellis JS, Hicks H, Blackshaw RP (2012)
Understanding the relationship between adult and larval Agriotes
distributions: the effect of sampling method, species identification
and abiotic variables. Appl Soil Ecol. 53:39–48. https://doi.org/10. 1016/j.apsoil.2011.11.004 Afzal MBS, Shad SA, Abbas N, Ayyaz M, Walker WB (2015) Cross-
resistance, the stability of acetamiprid resistance and its effect on the
biological parameters of cotton mealybug, Phenacoccus solenopsis
(Homoptera: Pseudococcidae), in Pakistan. Pest Manag Sci. 71:
151–158 Bijleveld van Lexmond M, Bonmatin JM, Goulson D, Noome DA (2015)
Worldwide integrated assessment on systemic pesticides. Environ
Sci Pollut Res. 22:1–4 Ahmad M, Akhtar S (2016) Development of resistance to insecticides in
the invasive mealybug Phenacoccus solenopsis (Hemiptera:
Pseudococcidae) in Pakistan. Crop Prot 88:96–102 Bingham G, Gunning RV, Delogu G, Borzatta V, Field LM, Moores GD
(2008) Temporal synergism can enhance carbamate and
neonicotinoid insecticidal activity against resistant crop pests. Pest
Manag Sci. 64:81–85 Ahmad S, Pozzebon A, Duso C (2013) Augmentative releases of the
predatory mite Kampimodromus aberrans in organic and conven-
tional apple orchards. Crop Prot 52:47–56. https://doi.org/10.1016/j. cropro.2013.05.008 Blackshaw RP, Hicks H (2013) Distribution of adult stages of soil insect
pests across an agricultural landscape. J Pest Sci. 86:53–62. https://
doi.org/10.1007/s10340-012-0413-6 Alyokhin A, Dively G, Patterson M, Castaldo C, Rogers D, Mahoney M,
Wollam J (2007) Resistance and cross-resistance to imidacloprid
and thiamethoxam in the Colorado potato beetle Leptinotarsa
decemlineata. Pest Manag Sci. 63:32–41 Bohnenblust E, Hull LA, Krawczyk G (2011) A comparison of various
mating disruption technologies for control of two internally feeding
Lepidoptera in apples. Entomol Exp Appl. 138:202–211. https://doi. org/10.1111/j.1570-7458.2010.01089.x Andreev R, Kutinkova H, Rasheva D (2012) Non-chemical control of
Aphis spiraecola Patch. and Dysaphis plantaginea Pass. on apple. J
Biopesticides 5:239–242 Bonmatin JM (2016) Néonicotinoïdes: impacts directs ou indirects sur les
abeilles et la biodiversité & développement des alternatives. Forum
Eco-Tox de la Fondation Rovaltain, 11-13 October 2016. http://
fcsrovaltain.org/assets/4-8-s2-eco-tox2016_bonmatin.pdf. Assessed 21 Oct 2017 Angeli G, Anfora G, Baldessari M, Germinara GS, Rama F, De Cristofaro
A, Ioriatti C (2007) Mating disruption of codling moth Cydia
pomonella with high densities of Ecodian sex pheromone dis-
pensers. J Appl Entomol 131:311–318. https://doi.org/10.1111/j. 1439-0418.2007.01172.x Bonmatin JM, Giorio C, Girolami V, Goulson D, Kreutzweiser DP,
Krupke C, Liess M, Long E, Marzaro M, Mitchell EAD et al
(2015) Environmental fate and exposure; neonicotinoids and
fipronil. Environ Sci Pollut Res. References 22:35–67 Asahi M, Kobayashi M, Matsui H, Nakahira K (2015) Differential mech-
anisms of action of the novel γ-aminobutyric acid receptor antago-
nist ectoparasiticides fluralaner (A1443) and fipronil. Pest Manag
Sci. 71:91–95 Bosch D, Rodríguez MA, Avilla J (2016) Captures of MFO-resistant
Cydia pomonella adults as affected by lure, crop management sys-
tem and flight. Bull Entomol Res. 106:54–62. https://doi.org/10. 1017/S0007485315000772 Bao H, Shao X, Zhang Y, Deng Y, Xu X, Liu Z, Li Z (2016) Specific
synergist for neonicotinoid insecticides: IPPA08, a cis-neonicotinoid
compound with a unique oxabridged substructure. J Agric Food
Chem. 64:5148–5155 Brandl MA, Schumann M, Przyklenk M, Patel A, Vidal S (2017)
Wireworm damage reduction in potatoes with an attract-and-kill
strategy using Metarhizium brunneum. J Pest Sci. 90:479–493. https://doi.org/10.1007/s10340-016-0824-x Barzman M, Bàrberi P, Birch ANE, Boonekamp P, Dachbrodt-Saaydeh S,
Graf B, Hommel B, Jensen JE, Kiss J, Kudsk P et al (2015) Eight
principles of integrated pest management. Agron Sustain Develop. 35:1199–1215 Broughton S, Rahman T (2017) Evaluation of lures and traps for male and
female monitoring of Mediterranean fruit fly in pome and stone fruit. J Appl Entomol. 141:441–449 Basit M, Sayyed AH, Saeed S, Saleem MA (2012) Lack of fitness costs
associated with acetamiprid resistance in Bemisia tabaci
(Hemiptera: Aleyrodidae). J Econ Entomol. 105:1401–1406 Brück E, Elbert A, Fischer R, Krueger S, Kühnhold J, Klueken AM,
Nauen R, Niebes J, Reckmann U, Schnorbach H, Steffens R, van
Waetermeulen X (2009) Movento®, an innovative ambimobile in-
secticide for sucking insect pest control in agriculture: biological
profile and field performance. Crop Prot 28:838–844. https://doi. org/10.1016/j.cropro.2009.06.015 Basit M, Saeed S, Saleem MA, Sayyed AH (2013) Can resistance in
Bemisia tabaci (Homoptera: Aleyrodidae) be overcome with mix-
tures of neonicotinoids and insect growth regulators? Crop Prot 44:
135–141 Bass C, Denholm I, Williamson MS, Nauen R (2015) The global status of
insect resistance to neonicotinoid insecticides. Pest Biochem Physiol
121:78–87 Bruck DJ, Bolda M, Tanigoshi L, Klick J, Kleiber J, Defrancesco J,
Gerdeman B, Spitler H (2011) Laboratory and field compari-
sons of insecticides to reduce infestation of Drosophila suzukii
in berry crops. Pest Manag Sci. 67:1375–1385. https://doi.org/
10.1002/ps.2242 Batáry P, Gallé R, Riesch F, Fischer C, Dormann CF, Mußhoff O, Császár
P, Fusaro S, Gayer C, Happe AK et al (2017) The former Iron
Curtain still drives biodiversity–profit trade-offs in German agricul-
ture. Nat Ecol Evol. Compliance with ethical standards In the meantime, the overwhelming evidence of nega-
tive effects on pollinators and arthropods needs to be
weighed against the pest control benefits that these Conflict of interest
The authors declare that they have no competing
interest. 11814 Environ Sci Pollut Res (2021) 28:11798–11820 Beers EH, Suckling DM, Prokopy RJ, Avilla J (2003) Apples: botany,
production and uses. In: Ferree DC, Warrington IJ (eds) Ecology and
management of apple arthropod pests. CABI Publishing,
Wallingford, pp. 489–519. https://doi.org/10.1079/
9780851995922.0489 Open Access This article is distributed under the terms of the Creative
Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give appro-
priate credit to the original author(s) and the source, provide a link to the
Creative Commons license, and indicate if changes were made. Beers EH, Van Steenwyk RA, Shearer PW, Coates WW, Grant JA (2011)
Developing Drosophila suzukii management programs for sweet
cherry in the western United States. Pest Manag Sci. 67:1386–1395 Beketov MA, Kefford BJ, Schäfer RB, Liess M (2013) Pesticides
reduce regional biodiversity of stream invertebrates. PNAS
110:11039–11043 References https://doi.org/10.1038/s41559-017-0272-x Budge GE, Garthwaite D, Crow A, Boatman ND, Delaplane KS, Brown
MA, Thygesen HH, Pietra-Valle S (2015) Evidence for pollinator
cost and farming benefits of neonicotinoid seed coatings on oilseed
rape. Sci Rep. 5:12574. https://doi.org/10.1038/srep12574 Beers EH, Schmidt RA (2014) Impacts of orchard pesticides on
Galendromus occidentalis: Lethal and sublethal effects. Crop Prot
56:16–24. https://doi.org/10.1016/j.cropro.2013.10.010 Environ Sci Pollut Res (2021) 28:11798–11820 11815 Daane KM, Yokota GY, Zheng Y, Hagen KS (1996) Inundative release of
common green lacewings (Neuroptera: Chrysopidae) to suppress
Erythroneura variabilis and E. elegantula (Homoptera:
Cicadellidae) in vineyards. Environ Entomol. 25:1224–1234. https://doi.org/10.1093/ee/25.5.1224 Buzzetti K, Chorbadjian RA, Nauen R (2015) Resistance management
for San Jose Scale (Hemiptera: Diaspididae). J Econ Entomol. 108:
2743–2752 Byrne FJ, Castle S, Prabhaker N, Toscano NC (2003) Biochemical study
of resistance to imidacloprid in B biotype Bemisia tabaci from
Guatemala. Pest Manag Sci. 59:347–352 Daane KM, Malakar-Kuenen RD, Walton VM (2004) Temperature-
dependent development of Anagyrus pseudococci (Hymenoptera:
Encyrtidae) as a parasitoid of the vine mealybug, Planococcus ficus
(Homoptera: Pseudococcidae). Biol Control 31:123–132 Calkins CO, Faust RJ (2003) Overview of areawide programs and the
program for suppression of codling moth in the western USA direct-
ed by the United States Department of Agriculture—Agricultural
Research Service. Pest Manag Sci. 59:601–604. https://doi.org/10. 1002/ps.712 Daane KM, Bentley WJ, Millar JG, Walton VM, Cooper ML, Biscay P,
Yokota GY (2008) Integrated management of mealybugs in
California vineyards. Acta Hortic. 785:235–252. https://doi.org/10. 17660/ActaHortic.2008.785.30 Castle SJ, Prabhaker N (2013) Monitoring changes in Bemisia tabaci
(Hemiptera: Aleyrodidae) susceptibility to neonicotinoid insecti-
cides in Arizona and California. J Econ Entomol. 106:1404–1413 Darriet F, Chandre F (2013) Efficacy of six neonicotinoid insecticides
alone and in combination with deltamethrin and piperonyl butoxide
against pyrethroid-resistant Aedes aegypti and Anopheles gambiae
(Diptera: Culicidae). Pest Manag Sci. 69:905–910 Castro A, Lacerda M, Zanuncio T, Ramalho FS, Polanczyk R, Serrão J,
Zanuncio J (2012) Effect of the insect growth regulator
diflubenzuron on the predator Podisus nigrispinus (Heteroptera:
Pentatomidae). Ecotoxicology 21:96–103 Deguines N, Jono C, Baude M, Henry M, Julliard R, Fontaine C (2014)
Large-scale trade-off between agricultural intensification and crop
pollination services. Front Ecol Environ. 12:212–217 Chagnon M, Kreutzweiser D, Mitchell ED, Morrissey CA, Noome DA,
Van der Sluijs JP (2015) Risks of large-scale use of systemic insec-
ticides to ecosystem functioning and services. Environ Sci Pollut
Res. References 22:119–134 Dercks W, Hackel S, Witte H, Michaelsen M, Neuber M, Gärber U,
Kleeberg H (2014) Plant Protection with Plant Extracts
(Botanicals): background, Two Case Studies, Conclusions and
Positioning | [PflanzenschutzmitPflanzenextrakten (Botanicals):
Hintergründe, zweiFallstudien, Schlussfolgerungen und
Standortbestimmung] Gesunde Pflanzen 66: 1-16. https://link. springer.com/article/10.1007%2Fs10343-013-0313-6. Assessed 12
July 2017 Cichon LI, Garrido SAS, Lago JD, Menni MF (2013) Control of green
peach aphid Myzus persicae in organic plum orchards (conference
paper). Acta Hortic. 1001:115–120. https://doi.org/10.17660/
ActaHortic.2013.1001.11 Cimino AM, Boyles AL, Thayer KA, Perry MJ (2017) Effects of
neonicotinoid pesticide exposure on human health: a systematic re-
view. Environ Health Perspect. 125:155–162 Dib H, Sauphanor B, Capowiez Y (2010) Effect of codling moth exclu-
sion nets on the rosy apple aphid, Dysaphis plantaginea, and its
control by natural enemies. Crop Prot 29:1502–1513. https://doi. org/10.1016/j.cropro.2010.08.012 Clements J, Schoville S, Peterson N, Lan Q, Groves RL (2016)
Characterizing molecular mechanisms of imidacloprid resistance
in select populations of Leptinotarsa decemlineata in the Central
Sands Region of Wisconsin. PLoS One 11:e0147844 Dib H, Sauphanor B, Capowiez Y (2016) Effect of management strate-
gies on arthropod communities in the colonies of rosy apple aphid,
Dysaphis plantaginea Passerini (Hemiptera: Aphididae) in south-
eastern France. Agric Ecosyst Environ. 216:203–206. https://doi. org/10.1016/j.agee.2015.10.003 Clements J, Schoville S, Clements N, Chapman RGL (2017) Temporal
patterns of imidacloprid resistance throughout a growing season in
Leptinotarsa decemlineata populations. Pest Manag Sci. 3:641–650 Cocco A, Lentini A, Serra G (2014) Mating disruption of Planococcus
ficus (Hemiptera: Pseudococcidae) in vineyards using reservoir
pheromone dispensers. J Insect Sci 14(1):144. https://doi.org/10. 1093/jisesa/ieu006 Dobson RC, Rogers M, Moore JL, Bessin RT (2016) Exclusion of the
brown marmorated stink bug from organically grown peppers using
barrier screens. Hortic Technol. 26:191–198 Cocco A, Marras PM, Muscas E, Mura A, Lentini A (2015) Variation of
life-history parameters of Planococcus ficus (Hemiptera:
Pseudococcidae) in response to grapevine nitrogen fertilization. J
Appl Entomol. 139:519–528. https://doi.org/10.1111/jen.12192 Douglas M, Tooker JF (2015) Large-scale deployment of seed treatments
has driven rapid increase in use of neonicotinoid insecticides and
preemptive pest management in U.S. field crops. Environ Sci
Technol. 49:5088–5097 Douglas M, Tooker JF (2016) Meta-analysis reveals that seed-applied
neonicotinoids and pyrethroids have similar negative effects on
abundance of arthropod natural enemies. PeerJ 4:e2776. https://
doi.org/10.7717/peerj.2776 Costello MJ (2008) Regulated deficit irrigation and density of
Erythroneura spp. (Hemiptera: Cicadellidae) on grape. J Econ
Entomol. 101:1287–1294. References http://novagricoltura.edagricole.it/wp-
content/uploads/sites/10/2015/04/La-gestione-degli-elateridi-
Pagine-da-TV14-2015-2.pdf, assessed 9 September 2017 Gurr GM, Heong KL, Cheng JA, Catindig J (2012) Ecological engineer-
ing against insect pests in Asian irrigated rice. In: Gurr GM, Wratten
SD, Snyder WE, Read DMY (eds) Biodiversity and insect pests: key
issues for sustainable management. John Wiley & Sons, Ltd., UK,
pp 214–229 Fornasiero D, Duso C, Pozzebon A, Tomasi D, Gaiotti F, Pavan F (2012)
Effects of irrigation on the seasonal abundance of Empoasca vitis in
North-Italian vineyards. J Econ Entomol. 105:176–185. https://doi. org/10.1603/EC11218 Gurr GM, Lu Z, Zheng X, Xu H, Zhu P, Chen G, Yao X, Cheng J, Zhu Z,
Catindig JL et al (2016) Multi-country evidence that crop diversifi-
cation promotes ecological intensification of agriculture. Nature
Plants 2:16014. https://doi.org/10.1038/nplants.2016.14 Fornasiero D, Pavan F, Pozzebon A, Picotti P, Duso C (2016) Relative
infestation level and sensitivity of grapevine cultivars to the leafhop-
per Empoasca vitis (Hemiptera: Cicadellidae). J Econ Entomol. 109(1):416–25. https://doi.org/10.1093/jee/tov313 Hadi BAR, García CPF, Heong KL (2015) Susceptibility of Nilaparvata
lugens (Hemipteran: Delphacidae) populations in the Philippines to
insecticides. Crop Prot 76:100–102 Heong KL, Escalada MM, Chien HV, Cuong LQ (2014) Restoration of
rice landscape biodiversity by farmers in Vietnam through education
and motivation using media. Surv Perspect Integrating Environ Soc
(S.A.P.I.EN.S.) 7(2). http://sapiens.revues.org/1578. Assessed 9
Aug 2017 Furlan L (2014) IPM thresholds for Agriotes wireworm species in maize
in southern Europe. J Pest Sci. 87:609–617. https://doi.org/10.1007/
s10340-014-0583-5 Furlan L, Kreutzweiser D (2015) Alternatives to neonicotinoid insecti-
cides for pest control: case studies in agriculture and forestry. Environ Sci Pollut Res. 22:135–147. https://doi.org/10.1007/
s11356-014-3628-7 Herbert KS, Hoffmann AA, Powell KS (2008) Assaying the poten-
tial benefits of thiamethoxam and imidacloprid for phylloxera
suppression and improvements to grapevine vigour. Crop Prot
27:1229–1236 Furlan L, Contiero B, Sartori E, Fracasso F, Sartori A, Vasileiadis VP,
Sattin M (2015) Mutual funds are a key tool for IPM implementa-
tion: a case study of soil insecticides in maize shows the way. IPM
Innovation in Europe, Poznan 14–16 January, Abstract book, 159. http://www.pure-ipm.eu/sites/default/files/content/files/Book%
20of%20Abstracts%20-%20IPM%20Innovation%20in%
20Europe_sessions-annuals.pdf. Assessed 9 Sept 2017 Hermann A, Brunner N, Hann P, Wrbka T, Kromp B (2013) Correlations
between wireworm damages in potato fields and landscape structure
at different scales. J Pest Sci. 86:41–51. References 48:
1523–1530 Garrood WT, Zimmer CT, Gorman KJ, Nauen R, Bass C, Davies TGE
(2016) Field-evolved resistance to imidacloprid and ethiprole in
populations of brown planthopper Nilaparvata lugens collected
from across south and east Asia. Pest Manag Sci. 72:140–149 El Wakeil NE, Farghaly HT, Ragab ZA (2008) Efficacy of inundative
releases of Trichogramma evanescens in controlling Lobesia
botrana in vineyards in Egypt. J Pest Sci. 81:49–55. https://doi. org/10.1007/s10340-007-0184-7 Gibbons D, Morrissey C, Mineau P (2015) A review of the direct and
indirect effects of neonicotinoids and fipronil on vertebrate wildlife. Environ Sci Pollut Res. 22:103–118 EU (2009) Directive 2009/128/EC of the European Parliament and of the
Council of 21 October 2009 establishing a framework for
Community action to achieve the sustainable use of pesticides, OJ
EU L309, 71–86, 24.11.2009 Giorio C, Anton Safer A, Sánchez-Bayo F, Tapparo A, Lentola A,
Girolami V, Bijleveld van Lexmond M, Bonmatin JM (2017) An
update of the Worldwide Integrated Assessment (WIA) on systemic
insecticides. Part 1: new molecules, metabolism, fate and transport. Environ Sci Pollut Res. https://doi.org/10.1007/s11356-017-0394-3 EU (2013a) Commission implementing Regulation (EU) No 485/2013 of
24 May 2013 amending Implementing Regulation (EU) No 540/
2011, as regards the conditions of approval of the active substances
clothianidin, thiamethoxam and imidacloprid, and prohibiting the
use and sale of seeds treated with plant protection products contain-
ing those active substances. OJ EU L139, 12–26, 25.05.2013 Gore J, Cook D, Catchot A, Leonard BR, Stewart SD, Lorenz G, Kerns D
(2013) Cotton aphid (Heteroptera: Aphididae) susceptibility to com-
mercial and experimental insecticides in the southern United States. J Econ Entomol. 106:1430–1439 Gorman K, Liu Z, Denholm I, Brüggen K-U, Nauen R (2008)
Neonicotinoid resistance in rice brown planthopper, Nilaparvata
lugens. Pest Manag Sci. 64:1122–1125 EU (2013b) Commission implementing Regulation (EU) No 781/2013 of
14 August 2013 amending Implementing Regulation (EU) No 540/
2011, as regards the conditions of approval of the active substance
fipronil, and prohibiting the use and sale of seeds treated with plant
protection products containing this active substances. OJ EU L219,
22–25, 15.08.2013 Gorman K, Slater R, Blande JD, Clarke A, Wren J, McCaffery A,
Denholm I (2010) Cross-resistance relationships between
neonicotinoids and pymetrozine in Bemisia tabaci (Hemiptera:
Aleyrodidae). Pest Manag Sci. 66:1186–1190 Ferrari R, Tassini C, Furlan L, Fracasso F, Sartori E, Codato F, Bin O
(2015) La gestione degli elateridi con i fondi mutualistici. Terra e
Vita 14:42–45 (in Italian). References https://doi.org/10.1603/0022-
0493(2008)101[1287:RDIADO]2.0.CO;2 Costello MJ, Daane KM (2003) Spider and leafhopper (Erythroneura
spp.) response to vineyard ground cover. Environ Entomol. 32:
1085–1098. https://doi.org/10.1603/0046-225X-32.5.1085 Duso C, Vettorazzo E (1999) Mite population dynamics on different
grape varieties with or without phytoseiids released (Acari:
Phytoseiidae). Exp Appl Acarol 23:741–763. https://doi.org/10. 1023/A:1006297225577 Coy MR, Bin L, Stelinski LL (2016) Reversal of insecticide resistance in
Florida populations of Diaphorina citri (Hemiptera: Liviidae). Fla
Entomol 99:26–32 Duso C, Trentin R, Borgo M, Egger E (1985) Influenza della
termoregolazione estiva mediante acqua sulle popolazioni di
Planococcus ficus Sign. su vite. Rivista di Viticoltura e di
Enologia 38:567–607 Cross JV, Solomon MG, Chandler D, Jarrett P, Richardson PN,
Winstanley D, Bathon H, Huber J, Keller B, Langenbruch GA,
Zimmermann G (1999) Biocontrol of pests of apples and pears in
northern and central Europe: 1. Microbial agents and nematodes. Biocontrol Sci Technol. 9:125–149. https://doi.org/10.1080/
09583159929721 Duso C, Fontana P, Malagnini V (2004) Diversity and abundance of
phytoseiid mites (acari: phytoseiidae) in vineyards and the surround-
ing vegetation in northeastern Italy. Acarologia 44:31–47 https://
www1.montpellier.inra.fr/cbgp/acarologia/export_pdf.php?id=
42&typefile=pd. Assessed 09 September 2017 Cutler P, Slater R, Edmunds AJF, Maienfisch P, Hall RG et al (2013)
Investigating the mode of action of sulfoxaflor: a fourth-generation
neonicotinoid. Pest Manag Sci. 69:607–619 Duso C, Pozzebon A, Kreiter S, Tixier M, Candolfi M (2012)
Management of phytophagous mites in European Vineyards. In:
Bostanian NJ, Vincent C, Isaacs R (eds) Arthropod management
in vineyards: pests, approaches, and future directions. Springer,
Netherlands, pp 191–217 Daane KM, Williams LE (2003) Manipulating vineyard irrigation
amounts to reduce insect pest damage. Ecol Appl. 13:1650–1666. https://doi.org/10.1890/02-5328 11816 Environ Sci Pollut Res (2021) 28:11798–11820 Duso C, Ahmad S, Tirello P, (...), Malagnini V, Angeli G (2014) The
impact of insecticides applied in apple orchards on the predatory
mite Kampimodromus aberrans (Acari: Phytoseiidae). Exp Appl
Acarol 62:391-414 Gallardo A, López MÁ, Lara M, Maistrello L, Molejón A, Ocete R
(2016) Resurgence of minor pests following the implementa-
tion of mating disruption against Lobesia botrana (Denis &
Schiffermüller) (Lepidoptera, Tortricidae) in Sherry vineyards
(Spain). Vitis J Grapevine Res 55:37–43. https://doi.org/10. 5073/vitis.2016.55.37-43 Edwards OR, Franzmann B, Thackray D, Micic S (2008) Insecticide
resistance and implications for future aphid management in
Australian grains and pastures: a review. Aust J Exp Agric. References https://doi.org/10.1603/EC10443 Leach H, Van Timmeren S, Isaacs R (2016) Exclusion netting delays and
reduces Drosophila suzukii (Diptera: Drosophilidae) infestation in
raspberries. J Econ Entomol. 109:2151–2158 Lechenet M, Dessaint F, Py G, Makowski D, Munier-Jolain N (2017)
Reducing pesticide use while preserving crop productivity and prof-
itability on arable farms. Nat Plants 3:17008 İşci M, Ay R (2017) Determination of resistance and resistance mecha-
nisms to thiacloprid in Cydia pomonella L. (Lepidoptera:
Tortricidae) populations collected from apple orchards in Isparta
Province, Turkey. Crop Prot 91:82–88 Lee WW, Shin TY, Bae SM, Woo SD (2015) Screening and evaluation of
entomopathogenic fungi against the green peach aphid, Myzus
persicae using multiple tools. J Asia-Pac Entomol. 18:607–615 Jepsen SJ, Rosenheim JA, Bench ME (2007) The effect of sulfur on
biological control of the grape leafhopper, Erythroneura elegantula,
by the egg parasitoid Anagrus erythroneurae. BioControl 52:721–
732. https://doi.org/10.1007/s10526-006-9058-9 Lefort F, Fleury D, Fleury I, Coutant C, Kuske S, Kehrli P, Maignet P
(2014) Pathogenicity of entomopathogenic fungi to the green peach
aphid Mmyzus persicae Ssulzer (Aphididae) and the european
tarnished bug Llygus rugulipennis poppius (Miridae). Egyptian J
Biol Pest Control 24:379–386 Jones MM, Robertson JL, Weinzierl RA (2010) Susceptibility of oriental
fruit moth (Lepidoptera: Tortricidae) larvae to selected reduced-risk
insecticides. J Econ Entomol. 103:1815–1820 Lescourret F (2017) Toward a reduced use of pesticides in European
farming systems: an introduction to the PURE project. Crop Prot
97:7–9 Joussen N, Heckel DG, Haas M, Schuphan I, Schmidt B (2008)
Metabolism of imidacloprid and DDT by P450 CYP6G1 expressed
in cell cultures of Nicotiana tabacum suggests detoxification of
these insecticides in Cyp6g1-overexpressing strains of Drosophila
melanogaster, leading to resistance. Pest Manag Sci. 64:65–73 Leskey TC, Lee D, Short BD, Wright SE (2012) Impact of insecticides on
the invasive Halyomorpha halys (Hemiptera: Pentatomidae): analy-
sis of insecticide lethality. J Econ Entomol. 105:1726–1735. https://
doi.org/10.1603/EC12265 Jung J, Racca P, Schmitt J, Kleinhenz B (2014) SIMAGRIO-W: devel-
opment of a prediction model for wireworms in relation to soil
moisture, temperature and type. J Appl Entomol. 138:183–194. https://doi.org/10.1111/jen.12021 Liang P, Tian Y-A, Biondi A, Desneux N, Gao X-W (2012) Short-term
and transgenerational effects of the neonicotinoid nitenpyram on
susceptibility to insecticides in two whitefly species. Ecotoxicology 21:1889–1898 Kabaluk T (2014) Targeting adult Elateridae as a concept for wireworm
biocontrol. BioControl 59:607. References https://doi.org/10.1007/
s10340-012-0444-z Hoi KK, Daborn PJ, Battlay P, Robin C, Batterham P, O'Hair RAJ,
Donald WA (2014) Dissecting the insect metabolic machinery using
twin ion mass spectrometry: a single P450 enzyme metabolizing the
insecticide imidacloprid in vivo. Anal Chem. 86:3525–3532 Furlan L, Vasileiadis VP, Chiarini F, Huiting H, Leskovšek R, Razinger J,
Holbe JI, Sartori E, Urek G, Verschweleg A, Benevegnù I, Sattin M
(2016) Risk assessment of soil-pest damage to grain maize in
Europe within the framework of integrated pest management. Crop Prot 97:52–59. https://doi.org/10.1016/j.cropro.2016.11.029 Hokkanen HMT (2015) Integrated pest management at the crossroads:
science, politics, or business (as usual)? Arthropod-Plant
Interactions 9:543–545 Furlan L, Contiero B, Chiarini F, Colauzzi M, Sartori E, Benevegnù I,
Giandon P (2017) Risk assessment of maize damage by wireworms
(Coleoptera: Elateridae) as the first step in implementing IPM and in
reducing the environmental impact of soil insecticides. Environ Sci
Pollut Res. 24:236–251. https://doi.org/10.1007/s11356-016-7692-z Hokkanen HMT, Menzler-Hokkanen I, Keva M (2017) Long-term yield
trends of insect-pollinated crops vary regionally and are linked to
neonicotinoid use, landscape complexity, and availability of polli-
nators. Arthropod-Plant Interactions 11:449–461 Environ Sci Pollut Res (2021) 28:11798–11820 11817 invasive Halyomorpha halys (Hemiptera: Pentatomidae). J Econ
Entomol. 110:543–545 Huseth AS, Chappell TM, Langdon K, Morsello SC, Martin S et al
(2016) Frankliniella fusca resistance to neonicotinoid insecticides:
an emerging challenge for cotton pest management in the eastern
United States. Pest Manag Sci. 72:1934–1945 Lacey LA, Shapiro-Ilan DI (2008) Microbial control of insect pests in
temperate orchard systems: potential for incorporation into IPM. Annu Rev Entomol. 53:121–144. https://doi.org/10.1146/annurev. ento.53.103106.093419 United States. Pest Manag Sci. 72:1934–1945 Ichinose K, Bang DV, Tuan DH, Dien LQ (2010) Effective use of
neonicotinoids for protection of citrus seedlings from invasion
by Diaphorina citri (Hemiptera: Psyllidae). J Econ Entomol. 103:127–135 Lacey LA, Arthurs SP, Unruh TR, Headrick H, Fritts R Jr (2006)
Entomopathogenic nematodes for control of codling moth
(Lepidoptera: Tortricidae) in apple and pear orchards: effect of nem-
atode species and seasonal temperatures, adjuvants, application
equipment, and post-application irrigation. Biol Control 37:214–
223. https://doi.org/10.1016/j.biocontrol.2005.09.015 Ioriatti C, Lucchi A (2016) Semiochemical strategies for tortricid moth
control in apple orchards and vineyards in Italy. J Chem Ecol. 42:
571–583 Ioriatti C, Anfora G, Tasin M, De Cristofaro A, Witzgall P, Lucchi A
(2011) Chemical ecology and management of Lobesia botrana
(Lepidoptera: Tortricidae). J Econ Entomol. 104:1125–1137. References https://doi.org/10.1007/s10526-014-
9603-x Liu Z, Williamson MS, Lansdell SJ, Denholm I, Han Z, Millar NS,
Casida JE (2005) A nicotinic acetylcholine receptor mutation con-
ferring target-site resistance to imidacloprid in Nilaparvata lugens
(Brown Planthopper). PNAS 102:8420–8425 Kabaluk JT, Ericsson JD (2007) Metarhizium anisopliae seed treatment
increases yield of field corn when applied for wireworm control. Agron J. 99:1377–1391 Lowery DT, Smirle MJ, Foottit RG, Zurowski CL, Beers Peryea EH
(2005) Baseline susceptibilities to imidacloprid for green apple
aphid and spirea aphid (Homoptera: Aphididae) collected from ap-
ple in the Pacific Northwest. J Econ Entomol. 98:188–194. https://
doi.org/10.1603/0022-0493-98.1.188 Kabaluk JT, Vernon RS, Goettel MS (2007) Mortality and infection of
Agriotes obscurus (Coleoptera: Elateridae) with inundative field ap-
plications of Metarhizium anisopliae. Phytoprotection 88:51–56 Kabaluk JT, LaFontaine JP, Borden JH (2015) An attract and kill tactic for
click beetles based on Metarhiziumbrunneumand a new formulation
of sex pheromone. J Pest Sci. 88:707–716 Lu Z, Zhu P, Gurr GM, Zheng X, Chen G, Heong KL (2015) Rice Pest
Management by Ecological Engineering: A Pioneering Attempt in
China. In: Heong K, Cheng J, Escalada M (eds) Rice Planthoppers. Springer, Dordrecht, pp 161–178. https://doi.org/10.1007/978-94-
017-9535-7_8. Assessed 9 Aug 2017 Kady HE, Devine GJ (2003) Insecticide resistance in Egyptian popula-
tions of the cotton whitefly, Bemisia tabaci (Hemiptera:
Aleyrodidae). Pest Manag Sci. 59:865–871 Karatolos N, Denholm I, Williamson M, Nauen R, Gorman K (2010)
Incidence and characterisation of resistance to neonicotinoid insec-
ticides and pymetrozine in the greenhouse whitefly, Trialeurodes
vaporariorum Westwood (Hemiptera: Aleyrodidae). Pest Manag
Sci. 66:1304–1307 Maalouly M, Franck P, Bouvier J, Toubon J, Lavigne C (2013) Codling
moth parasitism is affected by semi-natural habitats and agricultural
practices at orchard and landscape levels. Agric Ecosyst Environ. 169:33–42. https://doi.org/10.1016/j.agee.2013.02.008 Kovács-Hostyánszki A, Espíndola A, Vanbergen AJ, Settele J, Kremen
C, Dicks LV (2017) Ecological intensification to mitigate impacts of
conventional intensive land use on pollinators and pollination. Ecol
Lett. 20:673–689 Macfadyen S, Hardie DC, Fagan L, Stefanova K, Perry KD, DeGraaf HE,
Holloway J, Spafford H, Umina PA (2014) Reducing insecticide use
in broad-acre grains production: an Australian study. PLoS One 9:
e89119 Kristensen M, Jespersen JB (2008) Susceptibility to thiamethoxam of
Musca domestica from Danish livestock farms. Pest Manag Sci. 64:126–132 Magalhaes LC, Walgenbach JF (2011) Life stage toxicity and residual
activity of insecticides to codling moth and oriental fruit moth
(Lepidoptera: Tortricidae). J Econ Entomol. References 104:1950–1959 Kuhar TP, Kamminga K (2017) Review of the chemical control research
on Halyomorpha halys in the USA. J Pest Sci. 90:1021–1031 Malagnoux L, Capowiez Y, Rault M (2015) Impact of insecticide expo-
sure on the predation activity of the European earwig Forficula
auricularia. Environ Sci Pollut Res. 22:14116–14126. https://doi. org/10.1007/s11356-015-4520-9 Kuhar TP, Short BD, Krawczyk G, Leskey TC (2017) Deltamethrin-
incorporated nets as an integrated pest management tool for the 11818 Environ Sci Pollut Res (2021) 28:11798–11820 ants to support better biological control of rosy apple aphid,
Dysaphis plantaginea Passerini in apple orchards. Crop Prot 77:
127–138. https://doi.org/10.1016/j.cropro.2015.07.015 Marchand PA (2015) Basic Substances: an approval opportunity for low
concern natural products under EU pesticide regulation. Pest Manag
Sci. 71:1197–1200. https://doi.org/10.1002/ps.3997 Marchand PA (2016) Basic substances under EC 1107/2009 phytochem-
ical regulation: experience with non-biocide and food products as
biorationals. J Plant Protect Res. 56:312–318. https://doi.org/10. 1515/jppr-2016-0041h Nauen R, Bielza P, Denholm I, Gorman K (2008) Age-specific expression
of resistance to a neonicotinoid insecticide in the whitefly Bemisia
tabaci. Pest Manag Sci. 64:1106–1110 Nogueira Soares V, da Silva Almeida A, Deuner C, Jauer A, Madruga de
Tunes L (2017) Neonicotinoid insecticide treatment improves phys-
iological performance of melon and watermelon seeds. Afr J Agric
Res 12:1678–1683 Marchand PA (2017) Basic substances under EU pesticide regulation: an
opportunity for organic production? Org Farming 3:16–19. https://
doi.org/10.12924/of2017.03010016 Markó V, Blommers LHM, Bogya S, Helsen H (2008) Kaolin particle
films suppress many apple pests, disrupt natural enemies and pro-
mote woolly apple aphid. J Appl Entomol. 132:26–35. https://doi. org/10.1111/j.1439-0418.2007.01233.x Panini M, Dradi D, Marani G, Butturini A, Mazzoni E (2014) Detecting
the presence of target-site resistance to neonicotinoids and pyre-
throids in Italian populations of Myzus persicae. Pest Manag Sci. 70:931–938 Matsumura M, Takeuchi H, Satoh M, Sanada-Morimura S, Otuka A,
Watanabe T, van Thanh D (2008) Species-specific insecticide resis-
tance to imidacloprid and fipronil in the rice planthoppers
Nilaparvata lugens and Sogatella furcifera in east and south-east
Asia. Pest Manag Sci. 64:1115–1121 Pappas ML, Koveos DS (2011) Life-history traits of the predatory lace-
wing Dichochrysa prasina (Neuroptera: Chrysopidae): temperature-
dependent effects when larvae feed on nymphs of Myzus persicae
(Hemiptera: Aphididae). Ann Entomol Soc Am. 104:43–49. https://
doi.org/10.1603/AN10036 Matyjaszczyk E, Sobczak J, Szulc M (2015) Is the possibility of replacing
seed dressings containing neonicotinoids with other means of pro-
tection viable in major Polish agricultural crops? J Plant Protect Res. References 55:329–335 Pavlova AK, Dahlmann M, Hauck M, Reineke A (2017) Laboratory bio-
assays with three different substrates to test the efficacy of insecticides
against various stages of Drosophila suzukii (Diptera: Drosophilidae). J Insect Sci. 17:8. https://doi.org/10.1093/jisesa/iew100 Mertz FP, Yao RC (1990) Saccharopolyspora spinosa sp. nov. isolated
from soil collected in a sugar mill rum still. Int J Syst Bacteriol. 40:
34–39. https://doi.org/10.1099/00207713-40-1-34 Penvern S, Bellon S, Fauriel J, Sauphanor B (2010) Peach orchard pro-
tection strategies and aphid communities: towards an integrated
agroecosystem approach. Crop Prot 29:1148–1156. https://doi.org/
10.1016/j.cropro.2010.06.010 Milosavljević I, Esser AD, Crowder DW (2016) Seasonal population
dynamics of wireworms in wheat crops in the Pacific
Northwestern United States. J Pest Sci. 90:77–86. https://doi.org/
10.1007/s10340-016-0750-y Pisa LW, Amaral-Rogers V, Belzunces LP, Bonmatin JM, Downs CA,
Goulson D, Kreutzweiser DP, Krupke C, Liess M, McField M et al
(2015) Effects of neonicotinoids and fipronil on non-target inverte-
brates. Environ Sci Pollut Res. 22:68–102 Minakuchi C, Inano Y, Shi XY, Song DL, Zhang YJ, Miura K (2013)
Neonicotinoid resistance and cDNA sequences of nicotinic acetyl-
choline receptor subunits of the western flower thrips Frankliniella
occidentalis (Thysanoptera: Thripidae). Appl Entomol Zool. 48:
507–513 Pisa L, Goulson D, Yan EC, Gibbons D, Sánchez-Bayo F, Mitchell E, van
der Sluijs J, MacQuarrie C, Giorio C, Long EY, McField M,
Bijleveld van Lexmond M, Bonmatin JM (2017) An update of the
World Integrated Assessment (WIA) on systemic insecticides. Part
2: impacts on organisms and ecosystems. Environ Sci Pollut Res. https://doi.org/10.1007/s11356-017-0341-3 Mineau P, Whiteside M (2013) Pesticide acute toxicity is a better correlate
of U.S. grassland bird declines than agricultural intensification. PLoS One 8:e57457 Polajnar J, Eriksson A, Virant-Doberlet M, Mazzoni V (2016) Mating
disruption of a grapevine pest using mechanical vibrations: from
laboratory to the field. J Pest Sci. 89:909–921. https://doi.org/10. 1007/s10340-015-0726-3 Mitchell EAD, Mulhauser B, Mulot M, Mutabazi A, Glauser G, Aebi A
(2017) A worldwide survey of neonicotinoids in honey. Science
358:109–111. https://doi.org/10.1126/science.aan3684 Monteiro LB, Lavigne C, Ricci B, Franck P, Toubon J-F, Sauphanor B
(2013) Predation of codling moth eggs is affected by pest manage-
ment practices at orchard and landscape levels. Agric Ecosyst
Environ. 166:86–93. https://doi.org/10.1016/j.agee.2011.10.012 Ponti L, Ricci C, Veronesi F, Torricelli R (2005) Natural hedges as an
element of functional biodiversity in agroecosystems: the case of a
central Italy vineyard. Bull Insectol. References https://
doi.org/10.1016/0261-2194(89)90027-6 Romero A, Anderson TD (2016) High levels of resistance in the common
bed bug, Cimex lectularius (Hemiptera: Cimicidae), to
neonicotinoid insecticides. J Med Entomol. 53:727–731 Solomon MG, Cross JV, Fitzgerald JD, Campbell CAM, Jolly RL, Olszak
RW, Niemczyk E, Vogt H (2000) Biocontrol of pests of apples and
pears in northern and central Europe—3. Predators. Biocontrol Sci
Technol. 10:91–128. https://doi.org/10.1080/09583150029260 Saeed R, Razaq M, Abbas N, Jan MT, Naveed M (2017) Toxicity
and resistance of the cotton leaf hopper, Amrasca devastans
(Distant) to neonicotinoid insecticides in Punjab, Pakistan. Crop Prot 93:143–147 Souliotis C, Moschos T (2008) Effectiveness of some pesticides against
Cacopsylla pyricola and impact on its predator Anthocoris
nemoralis in pear-orchards Bull. Insectol. 61:25–30 Salis S, Testa C, Roncada P, Armorini S, Rubattu N, Ferrari A, Miniero R,
Brambilla G (2017) Occurrence of imidacloprid, carbendazim, and
other biocides in Italian house dust: potential relevance for intakes in
children and pets. J Environ Sci Health B 52:699–709. https://doi. org/10.1080/03601234.2017.1331675 Spangenberg JH, Douguet JM, Settele J, Heong KL (2015) Escaping the
lock-in of continuous insecticide spraying in rice: developing an
integrated ecological and socio-political DPSIR analysis. Ecol
Model. 295:188–195 Santos MF, Santos RL, Tomé HVV, Barbosa WF, Martins GF, Guedes
RNC, Oliveira EE (2016) Imidacloprid-mediated effects on survival
and fertility of the Neotropical brown stink bug Euschistus heros. J
Pest Sci. 89:231–240 Stenberg JA (2017) A conceptual framework for integrated pest manage-
ment. Trends Plant Sci. 22:759–769 Szendrei Z, Grafius E, Byrne A, Ziegler A (2012) Resistance to
neonicotinoid insecticides in field populations of the Colorado
potato beetle (Coleoptera: Chrysomelidae). Pest Manag Sci. 68:
941–946 Sauge M-H, Grechi I, Poëssel J-L (2010) Nitrogen fertilization effects on
Myzus persicae aphid dynamics on peach: vegetative growth allo-
cation or chemical defence? Entomol Exp Appl. 136:123–133. https://doi.org/10.1111/j.1570-7458.2010.01008.x Tacoli F, Pavan F, Cargnus E, Tilatti E, Pozzebon A, Zandigiacomo P
(2017) Efficacy and mode of action of kaolin in the control of
Empoasca vitis and Zygina rhamni (Hemiptera: Cicadellidae) in
Vineyards. J Econ Entomol. 110:1164–1178 Sauphanor B, Severac G, Maugin S, Toubon JF, Capowiez Y (2012)
Exclusion netting may alter reproduction of the codling moth
(Cydia pomonella) and prevent associated fruit damage to apple
orchards. Entomol Exp Appl. 145:134–142. https://doi.org/10. 1111/j.1570-7458.2012.01320.x Tamindžić G, Nikolić Z, Milošević D, Ignjatov M (2016) Field & vege-
table crops research. References Crop Prot 90:170–176 Sgolastra F, Porrini C, Maini S, Bortolotti L, Medrzycki P, Mutinelli F,
Lodesani M (2017) Healthy honey bees and sustainable maize pro-
duction: why not? Bull Insectol. 70:156–160 Rebach S, French DP (1996) Effects of Dimilin on the blue crab,
Callinectes sapidus, in shallow-water habitats. Estuaries 19:
279–287 Sharon R, Zahavi T, Sokolsky T, Sofer-Arad C, Tomer M, Kedoshim R,
Harari AR (2016) Mating disruption method against the vine mealy-
bug, Planococcus ficus: effect of sequential treatment on infested
vines. Entomol Exp Appl. 161:65–69 Reissig WH (2003) Field and laboratory tests of new insecticides against
the apple maggot, Rhagoletis pomonella (Walsh) (Diptera:
Tephritidae). J Econ Entomol. 96:1463–1472. https://doi.org/10. 1603/0022-0493-96.5.1463 Shawer R, Tonina L, Tirello P, Duso C, Mori N (2018) Laboratory and
field trials to identify effective chemical control strategies for inte-
grated management of Drosophila suzukii in European cherry or-
chards. Crop Prot 103:73–80 Reyes M, Franck P, Charmillot PJ, Ioriatti C, Olivares J, Pasqualini E,
Sauphanor B (2007) Diversity of insecticide resistance mechanisms
and spectrum in European populations of the codling moth, Cydia
pomonella. Pest Manag Sci. 63:890–902 Shearer PW, Frecon JL (2002) Managing Brachycaudis persicae
(Homoptera: Aphididae) during peach orchard establishment. J
Econ Entomol. 95:368–371 Ribeiro LP, Akhtar Y, Vendramim JD, Isman MB (2014) Comparative
bioactivity of selected seed extracts from Brazilian Annona species
and an acetogenin-based commercial bioinsecticide against
Trichoplusia ni and Myzus persicae. Crop Prot 62 (Supplement
C):100–106 Shi X, Jiang L, Wang H, Qiao K, Wang D, Wang K (2011) Toxicities and
sublethal effects of seven neonicotinoid insecticides on survival,
growth and reproduction of imidacloprid-resistant cotton aphid,
Aphis gossypii. Pest Manag Sci. 67:1528–1533 Rieux R, Simon S, Defrance H (1999) Role of hedgerows and ground
cover management on arthropod populations in pear orchards. Agric
Ecosyst Environ. 73:119–127. https://doi.org/10.1016/S0167-
8809(99)00021-3 Simon-Delso N, Amaral-Rogers V, Belzunces L, Bonmatin JM, Chagnon
M, Downs C, Furlan L, Gibbons D, Giorio C, Girolami V et al
(2015) Systemic insecticides (neonicotinoids and fipronil); trends,
uses, mode of action and metabolites. Environ Sci Pollut Res 22:5–
34. https://doi.org/10.1007/s11356-014-3470-y Rogers MA, Burkness EC, Hutchison WD (2016) Evaluation of high
tunnels for management of Drosophila suzukii in fall-bearing red
raspberries: potential for reducing insecticide use. J Pest Sci. 89:
815–821 Solomon MG, Cranham JE, Easterbrook MA, Fitzgerald JD (1989)
Control of the pear psyllid, Cacopsylla pyricola, in south east
England by predators and pesticides. Crop Prot 8:197–205. References 58:19–23 Pozzebon A, Ahmad S, Tirello P, Lorenzon M, Duso C (2014) Does
pollen availability mitigate the impact of pesticides on generalist
predatory mites? BioControl 59:585–596 Mota-Sánchez D, Wise JC, Poppen RV, Gut LJ, Hollingworth RM (2008)
Resistance of codling moth, Cydia pomonella (L.) (Lepidoptera:
Tortricidae), larvae in Michigan to insecticides with different modes
of action and the impact on field residual activity. Pest Manag Sci. 64:881–890. https://doi.org/10.1002/ps.1576 Pozzebon A, Loeb GM, Duso C (2015a) Role of supplemental foods and
habitat structural complexity in persistence and coexistence of gen-
eralist predatory mites. Sci Rep. 5:14997. https://doi.org/10.1038/
srep14997 Mottet C, Fontaine S, Caddoux L, Brazier C, Mahéo F, Simon J-C,
Micoud A, Roy L (2016) Assessment of the dominance level of
the R81T target resistance to two neonicotinoid insecticides in
Myzus persicae (Hemiptera: Aphididae). J Econ Entomol. 109:
2182–2189 Pozzebon A, Tirello P, Moret R, Pederiva M, Duso C (2015b) A funda-
mental step in IPM on grapevine: evaluating the side effects of
pesticides on predatory mites. Insects 6:847–857. https://doi.org/
10.3390/insects6040847 Mruthunjayaswamy PV, Thiruvengadam J, Sushil Kumar J (2016)
Resistance in Maconellicoccus hirsutus (Green) in India to selected
insecticides and quantification of detoxifying enzymes imparting
resistance. Crop Prot 89:116–122 Prabhaker N, Castle S, Henneberry TJ, Toscano NC (2005) Assessment
of cross-resistance potential to neonicotinoid insecticides in Bemisia
tabaci (Hemiptera: Aleyrodidae). Bull Entomol Res. 95:535–543 Puinean AM, Foster SP, Oliphant L, Denholm I, Field LM, Millar NS
(2010) Amplification of a cytochrome P450 gene is associated with
resistance to neonicotinoid insecticides in the aphid Myzus persicae. PLoS Genet. 6:e1000999 Nagy C, Cross JV, Markó V (2013) Sugar feeding of the common black
ant, Lasius niger (L.), as a possible indirect method for reducing
aphid populations on apple by disturbing ant-aphid mutualism. Biol Control 65:24–36. https://doi.org/10.1016/j.biocontrol.2013. 01.005 Raga A, Sato M (2011) Toxicity of neonicotinoids to Ceratitis capitata
and Anastrepha fraterculus (Diptera: Tephritidae). J Plant Protect
Res. 51:413–419 Nagy C, Cross JV, Markó V (2015) Can artificial nectaries outcompete
aphids in ant-aphid mutualism? Applying artificial sugar sources for Environ Sci Pollut Res (2021) 28:11798–11820 11819 Seagraves MP, Lundgren JG (2012) Effects of neonicotinoid seed
treatments on soybean aphid and its natural enemies. J Pest Sci. 85:125–132 Rahman T, Broughton S (2016) Evaluation of thiacloprid and clothianidin
(neonicotinoids) as alternative to fenthion (organophosphate) for
control of Mediterranean fruit fly (Diptera: Tephritidae) in decidu-
ous fruit orchards. References Assessed 9 Oct 2017 van der Sluijs JP, Amaral-Rogers V, Belzunces LP, Bijleveld van
Lexmond M, Bonmatin JM, Chagnon M, Downs CA, Furlan L,
Gibbons DW, Giorio C et al (2015) Conclusions of the Worldwide
Integrated Assessment on the risks of neonicotinoids and fipronil to
biodiversity and ecosystem functioning. Environ Sci Pollut Res 22:
148–154 Wei Q, Mu XC, Yu HY, Niu CD, Wang LX, Zheng C, Chen Z, Gao CF
(2017) Susceptibility of Empoasca vitis (Hemiptera: Cicadellidae)
populations from the main tea-growing regions of China to thirteen
insecticides. Crop Prot 96:204–210 Van Timmeren S, Wise JC, VanderVoort C, Isaacs R (2011) Comparison
of foliar and soil formulations of neonicotinoid insecticides for con-
trol of potato leafhopper, Empoasca fabae (Homoptera:
Cicadellidae), in wine grapes. Pest Manag Sci. 67:560–567 Wilson H, Miles AF, Daane KM, Altieri MA (2015) Vineyard proximity
to riparian habitat influences Western grape leafhopper
(Erythroneura elegantula Osborn) populations. Agric Ecosyst
Environ. 211:43–50. https://doi.org/10.1016/j.agee.2015.04.021 Vassiliou VA (2011) Effectiveness of insecticides in controlling the first
and second generations of the Lobesia botrana (Lepidoptera:
Tortricidae) in table grapes. J Econ Entomol. 104:580–585. https://
doi.org/10.1603/EC10343 Wise JC, Vanderpoppen R, Vandervoort C, O’Donnell C, Isaacs R (2015)
Curative activity contributes to control of spotted-wing drosophila
(Diptera: Drosophilidae) and blueberry maggot (Diptera:
Tephritidae) in highbush blueberry. Can Entomol. 147:109–117 Voudouris CC, Kati AN, Sadikoglou E, Williamson M, Skouras PJ,
Dimotsiou O, Georgiou S, Fenton B, Skavdis G, Margaritopoulos
JT (2016) Insecticide resistance status of Myzus persicae in Greece:
long-term surveys and new diagnostics for resistance mechanisms. Pest Manag Sci. 72:671–683 Witzgall P, Stelinski L, Gut L, Thomson D (2008) Codling moth man-
agement and chemical ecology. Annu Rev Entomol. 53:503–522. https://doi.org/10.1146/annurev.ento.53.103106.093323 Vrancken K, Trekels H, Thys T, Beliën T, Bylemans D, Demaeght P, Van
Leeuwen T, De Clercq P (2015) The presence of beneficial arthro-
pods in organic versus IPM pear orchards and their ability to predate
pear suckers (Cacopsylla pyri). Acta Hortic. 1094:427–429. https://
doi.org/10.17660/ActaHortic.2015.1094.55 Wu G, Jiang S, Miyata T (2004) Effects of synergists on toxicity of six
insecticides in parasitoid Diaeretiella rapae (Hymenoptera:
Aphidiidae). J Econ Entomol. 97:2057–2066 Wu G, Miyata T, Kang CY, Xie LH (2007) Insecticide toxicity and syn-
ergism by enzyme inhibitors in 18 species of pest insect and natural
enemies in crucifer vegetable crops. References Ratarstvo i povrtarstvo 53:90–95 Saussure S, Plantegenest M, Thibord J-B, Larruodè P, Poggi S (2015)
Management of wireworm damage in maize fields using new,
landscape-scale strategies. Agron Sustain Dev. 35:793–802. https://doi.org/10.1007/s13593-014-0279-5 Thany SH (2010) Neonicotinoid insecticides. Historical evolution and
resistance mechanisms. Adv. Exp Med Biol. 683:75–83 Tirello P, Pozzebon A, Duso C (2013) The effect of insecticides on the
non-target predatory mite Kampimodromus aberrans: laboratory
studies. Chemosphere 93:1139–1144 Schmitt A, Bisutti IL, Ladurner E, Benuzzi M, Sauphanor B, Kienzle J,
Zingg D, Undorf-Spahn K, Fritsch E, Huber J, Jehle JA (2013) The
occurrence and distribution of resistance of codling moth to Cydia
pomonella granulovirus in Europe. J Appl Entomol. 137:641–649. https://doi.org/10.1111/jen.12046 Tiwari S, Mann RS, Rogers ME, Stelinski LL (2011) Insecticide resis-
tance in field populations of Asian citrus psyllid in Florida. Pest
Manag Sci. 67:1258–1268 Toda S, Hirata K, Yamamoto A, Matsuura A (2017) Molecular diagnos-
tics of the R81T mutation on the D-loop region of the β1 subunit of
the nicotinic acetylcholine receptor gene conferring resistance to
neonicotinoids in the cotton aphid, Aphis gossypii (Hemiptera:
Aphididae). Appl Entomol Zool. 52:147–151 Scott JG, Duhig M, Hamlyn J, Norman R (2014) Environmental contri-
butions to autism: explaining the rise in incidence of autistic spec-
trum disorders. J Environ Immunol Toxicol 1(2):75–79. https://
eprints.qut.edu.au/84191/ 11820 Environ Sci Pollut Res (2021) 28:11798–11820 the invasive western flower thrips Frankliniella occidentalis
(Pergande) (Thysanoptera: Thripidae) in China. Pest Manag Sci. 72:1440–1444 Trimble RM (1993) Efficacy of mating disruption for controlling the
grape berry moth, Endopiza viteana (Clemens) (Lepidoptera:
Tortricidae), a case study over three consecutive growing seasons. Can Entomol. 125:1–9. https://doi.org/10.4039/Ent1251-1 Wang NX, Watso GB, Loso MR, Sparks TC (2016b) Molecular modeling
of sulfoxaflor and neonicotinoid binding in insect nicotinic acetyl-
choline receptors: impact of the Myzus β1 R81T mutation. Pest
Manag Sci. 72:1467–1474 Unruh TR, Lacey LA (2001) Control of codling moth, Cydia pomonella
(Lepidoptera: Tortricidae), with Steinernema carpocapsae: effects
of supplemental wetting and pupation site on infection rate. Biol
Control 20:48–56. https://doi.org/10.1006/bcon.2000.0873 Wang X, Anadón A, Qinghua W, Qiao F, Ares I, Martínez-Larrañaga M-
R, Yuan Z, Martínez M-A (2018)Mechanism of neonicotinoid tox-
icity: Impact on oxidative stress and metabolism. Annu Rev
Pharmacol Toxicol 58:(1):471–507. https://doi.org/10.1146/
annurev-pharmtox-010617-052429. References Pest Manag Sci 63:500–510 Wallingford AK, Fuchs MF, Martinson T, Hesler S, Loeb GM (2015)
Slowing the spread of grapevine leafroll-associated viruses in com-
mercial vineyards with insecticide control of the vector,
Pseudococcus maritimus (hemiptera: pseudococcidae). J Insect
Sci. 15:112. https://doi.org/10.1093/jisesa/iev094 Yang XQ, Wu ZW, Zhang YL, Barros-Parada W (2016) Toxicity of six
insecticides on codling moth (Lepidoptera: Tortricidae) and effect on
expression of detoxification genes. J Econ Entomol 109:320–326 Yuan L, Wang S, Zhou J, Du Y, Zhang Y, Wang J (2012) Status of
insecticide resistance and associated mutations in Q-biotype of
whitefly, Bemisia tabaci, from eastern China. Crop Prot 31:67–71 Walton VM, Daane KM, Bentley WJ, Millar JG, Larsen TE, Malakar-
Kuenen R (2006) Pheromone-based mating disruption of
Planococcus ficus (Hemiptera: Pseudococcidae) in California
vineyards. J Econ Entomol 99:1280–1290. https://doi.org/10.1093/
jisesa/ieu006 Zanolli P, Pavan F (2011) Autumnal emergence of Anagrus wasps, egg
parasitoids of Empoasca vitis, from grapevine leaves and their mi-
gration towards brambles. Agric Forest Entomol 13:423–433. https://doi.org/10.1111/j.1461-9563.2011.00546.x Walton VM, Daane KM, Addison P (2012) Biological control of
arthropods and its application in vineyards. In: Bostanian NJ,
Vincent C, Isaacs R (eds) Arthropod Management in Vineyards:
Pests, Approaches, and Future Directions. Springer,
Netherlands, pp 91–117 Žežlina I, Škvarč A, Bohinc T, Trdan S (2013) Testing the efficacy of
single applications of five insecticides against Scaphoideus titanus
on common grapevines. Int J Pest Manag 59:1–9 pp
Wan N-F, Ji X-Y, Gu X-J, Jiang J-X, Wu J-H, Li B (2014) Ecological
engineering of ground cover vegetation promotes biocontrol ser-
vices in peach orchards. Ecol. Eng. 64:62–65. https://doi.org/10. 1016/j.ecoleng.2013.12.033 Zhang X, Liu X, Zhu F, Li J, You H, Lu P (2014) Field evolution of
insecticide resistance in the brown planthopper (Nilaparvata lugens
Stal) in China. Crop Prot 58:61–66 Zhao GY, Liu W, Brown JM, Knowles CO (1995) Insecticide resistance
in-field and laboratory strains of western flower thrips
(Thysanoptera, Thripidae). J Econ Entomol 88:1164–1170 j
g
Wang ZH, Gong YJ, Jin GH, Li BY, Chen JC, Kang ZJ, Zhu L, Gao YL,
Reitz S, Wei SJ (2016a) Field-evolved resistance to insecticides in
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Single-Entity Electrochemistry for Digital Biosensing at Ultralow Concentrations
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Analytical chemistry
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■INTRODUCTION single-molecule redox cycling,19,20 and various forms of
particle-impact electrochemistry.21−24 A salient theme in analytical science in recent decades has been
the miniaturization of detection systems. Driven by the
nanotechnology revolution, an important end point of this
process has been the development of methods capable of
detecting and interrogating individual microscopic entities
ranging from inorganic and biological nanoparticles to single
molecules. These methodological developments have opened
new windows into the microscopic world and enabled
fundamental and exploratory studies at the molecular scale in
practically all areas of the physical sciences. Typical SEE signals have a different character from that of
conventional electrochemical data. Because individual single-
entity events occur at random intervals, data typically consist of
a mixture of quiescent periods in which very little happens,
punctuated by sudden jumps or spikes in the signal
corresponding to the detection of the analyte. This
phenomenology is illustrated in Figure 1 for various
amperometry-based methods. Determining a concentration
from these signals requires counting events in time rather than
measuring an average response. Electrochemistry is no exception to this trend. Conventional
methods such as voltammetry and amperometry are in
principle straightforward to scale down, even to the nanoscale,
by decreasing the size of the electrodes employed. Interest-
ingly, the detection of individual target particles or molecules
can become possible when the size of the working electrode(s)
approaches that of the targets. This has led to the development
of so-called single-entity electrochemistry (SEE), as recently
expounded by Baker.1 Single-entity methods have however made relatively few
inroads in the sensing of ultralow analyte concentrations. This
may at first appear surprising: does not single-molecule
detection represent the ultimate sensitivity? Indeed, the ability
to detect a single analyte particle or molecule represents
remarkable mass sensitivity for that analyte. One cannot detect
fewer than one elementary entity. In practice, however, the
goal is typically to determine the concentration of an analyte,
and concentration sensitivity represents the key figure of merit. A
high mass sensitivity does not necessarily translate into a high
concentration sensitivity. Single-Entity Electrochemistry for Digital Biosensing at Ultralow
Concentrations Serge G. Lemay* and Taghi Moazzenzade Cite This: Anal. Chem. 2021, 93, 9023−9031
Read Online
ACCESS
Metrics & More
Article Recommendations
*
sı
Supporting Information
ABSTRACT: Quantifying ultralow analyte concentrations is a continuing challenge in
the analytical sciences in general and in electrochemistry in particular. Typical hurdles
for affinity sensors at low concentrations include achieving sufficiently efficient mass
transport of the analyte, dealing with slow reaction kinetics, and detecting a small
transducer signal against a background signal that itself fluctuates slowly in time. Recent
decades have seen the advent of methods capable of detecting single analytes ranging
from the nanoscale to individual molecules, representing the ultimate mass sensitivity to
these analytes. However, single-entity detection does not automatically translate into a superior concentration sensitivity. This is
largely because electrochemical transducers capable of such detection are themselves miniaturized, exacerbating mass transport and
binding kinetic limitations. In this Perspective, we discuss how these challenges can be tackled through so-called digital sensing: large
arrays of separately addressable single-entity detectors that provide real-time information on individual binding events. We discuss
the advantages of this approach and the barriers to its implementation. ABSTRACT: Quantifying ultralow analyte concentrations is a continuing challenge in
the analytical sciences in general and in electrochemistry in particular. Typical hurdles
for affinity sensors at low concentrations include achieving sufficiently efficient mass
transport of the analyte, dealing with slow reaction kinetics, and detecting a small
transducer signal against a background signal that itself fluctuates slowly in time. Recent
decades have seen the advent of methods capable of detecting single analytes ranging
from the nanoscale to individual molecules, representing the ultimate mass sensitivity to
these analytes. However, single-entity detection does not automatically translate into a superior concentration sensitivity. This is
largely because electrochemical transducers capable of such detection are themselves miniaturized, exacerbating mass transport and
binding kinetic limitations. In this Perspective, we discuss how these challenges can be tackled through so-called digital sensing: large
arrays of separately addressable single-entity detectors that provide real-time information on individual binding events. We discuss
the advantages of this approach and the barriers to its implementation. ■INTRODUCTION pubs.acs.org/ac Perspective Single-Entity Electrochemistry for Digital Biosensing at Ultralow
Concentrations
Serge G. Lemay* and Taghi Moazzenzade
Cite This: Anal. Chem. 2021, 93, 9023−9031
Read Online
ACCESS
Metrics & More
Article Recommendations
*
sı
Supporting Information
ABSTRACT: Quantifying ultralow analyte concentrations is a continuing challenge in
the analytical sciences in general and in electrochemistry in particular. Typical hurdles
for affinity sensors at low concentrations include achieving sufficiently efficient mass
transport of the analyte, dealing with slow reaction kinetics, and detecting a small
transducer signal against a background signal that itself fluctuates slowly in time. Recent
decades have seen the advent of methods capable of detecting single analytes ranging
from the nanoscale to individual molecules, representing the ultimate mass sensitivity to
these analytes. However, single-entity detection does not automatically translate into a superior concentration sensitivity. This is
largely because electrochemical transducers capable of such detection are themselves miniaturized, exacerbating mass transport and
binding kinetic limitations. In this Perspective, we discuss how these challenges can be tackled through so-called digital sensing: large
arrays of separately addressable single-entity detectors that provide real-time information on individual binding events. We discuss
the advantages of this approach and the barriers to its implementation. Single-Entity Electrochemistry for Digital Biosensing at Ultralow
Concentrations ■INTRODUCTION The very characteristics lending a p
y
The best-known example of a SEE analytical method is the
classic Coulter counter, in which modulation of the ionic
conduction through an aperture is utilized to count and size
particles.2 This concept has been scaled down to molecular
scale in both solid-state3,4 and biological5 nanopores, the latter
having become so sophisticated as to permit the sequencing of
nucleic acids.6−9 Other examples of nanoscale SEE approaches
include nanoelectrode-based studies for single-cell studies,10
electrogeneration of surface nanobubbles,11 single-ion-channel
patch clamp recordings,12,13 scanning-probe methods ranging
from electrochemical scanning tunneling microscopy14,15 to
scanning electrochemical cell microscopy (SECCM),16−18 Received:
February 3, 2021
Accepted:
June 9, 2021
Published: June 25, 2021
https://doi.org/10.1021/acs.analchem.1c00510
Anal. Chem. 2021, 93, 9023−9031 Received:
February 3, 2021
Accepted:
June 9, 2021
Published: June 25, 2021 © 2021 The Authors. Published by
American Chemical Society https://doi.org/10.1021/acs.analchem.1c00510
Anal. Chem. 2021, 93, 9023−9031 https://doi.org/10.1021/acs.analchem.1c00510
Anal. Chem. 2021, 93, 9023−9031 9023 Figure 1. Examples of discrete signals in amperometry-based SEE. (a) Mediated faradaic impact: current spikes occur due to electrocatalytic water
oxidation upon impact of IrOx nanoparticles on a NaBH4-treated Pt UME.33 Adapted with permission from ref 33. Copyright 2010 American
Chemical Society. (b) Current blockade impact: steps in the anodic current from oxidation of the Fe(CN)6
4−mediator are observed upon impact
of vesicles on a Pt ultramicroelectrode.34 Adapted with permission from ref 34. Copyright 2015 American Chemical Society. (c) Direct faradaic
impact: Ag nanoparticles are oxidized upon making contact with an electrode, leading to current spikes.35 Adapted with permission from ref 35. Copyright 2019 American Chemical Society. (d) Scanning electrochemical cell microscopy (SECCM): current spikes are observed when Au
nanoparticles are immobilized upon an alkanethiol-modified Au electrode.36 Adapted with permission from ref 36. Copyright 2015 American
Chemical Society. (e) Solid-state nanopore: transient dips occur in the ionic current through the (plasmonic) nanopore upon translocation of
single DNA molecules.37 Adapted with permission from ref 37. Copyright 2018 American Chemical Society. (f) Electroosmotically driven current
blockade: current from oxidation of the ferrocene dimethanol mediator dips during convectively driven transient passage of polystyrene beads over
a Pt ultramicroelectrode.38 Adapted with permission from ref 38. Copyright 2020 American Chemical Society. Analytical Chemistry
pubs.acs.org/ac
Perspective Analytical Chemistry pubs.acs.org/ac Perspective Figure 1. Examples of discrete signals in amperometry-based SEE. ■INTRODUCTION (a) Mediated faradaic impact: current spikes occur due to electrocatalytic water
oxidation upon impact of IrOx nanoparticles on a NaBH4-treated Pt UME.33 Adapted with permission from ref 33. Copyright 2010 American
Chemical Society. (b) Current blockade impact: steps in the anodic current from oxidation of the Fe(CN)6
4−mediator are observed upon impact
of vesicles on a Pt ultramicroelectrode.34 Adapted with permission from ref 34. Copyright 2015 American Chemical Society. (c) Direct faradaic
impact: Ag nanoparticles are oxidized upon making contact with an electrode, leading to current spikes.35 Adapted with permission from ref 35. Copyright 2019 American Chemical Society. (d) Scanning electrochemical cell microscopy (SECCM): current spikes are observed when Au
nanoparticles are immobilized upon an alkanethiol-modified Au electrode.36 Adapted with permission from ref 36. Copyright 2015 American
Chemical Society. (e) Solid-state nanopore: transient dips occur in the ionic current through the (plasmonic) nanopore upon translocation of
single DNA molecules.37 Adapted with permission from ref 37. Copyright 2018 American Chemical Society. (f) Electroosmotically driven current
blockade: current from oxidation of the ferrocene dimethanol mediator dips during convectively driven transient passage of polystyrene beads over
a Pt ultramicroelectrode.38 Adapted with permission from ref 38. Copyright 2020 American Chemical Society. ionizing radiation particle impinges upon the detector. For an
electrochemical sensor, each node consists of a separate SEE
experiment that can be independently sampled and analyzed. In its simplest form, each feature in the node signal (such as
the steps and pulses in Figure 1) can be assigned one-to-one to
the occurrence of an event of interest (the binding of an
analyte to a receptor, for example). More generally, the
amplitude, duration and shape of single SEE events can be
further analyzed to enhance specificity, as done for example in
the analysis of nanopore signals.6−9 Such fingerprinting can be
employed to selectively ignore false positives from interfering
species, suppress instrumental noise, and eliminate baseline
drift. Somewhat abstractly, the final output of the sensor as a
whole can be considered to be the aggregate events from all of
the nodes. In this sense, the output consists of a sequence of
times at which each event took place together with the nature
of that event, from which analyte concentrations are inferred. https://doi.org/10.1021/acs.analchem.1c00510
Anal. Chem. 2021, 93, 9023−9031 ■THE NEED FOR PARALLELIZATION This is best illustrated with a simple numerical example, as
emphasized by Sheehan and Whitman.39 The steady-state
diffusive flux (in analytes/second), kdiff, to a miniaturized
detector with characteristic lateral size d has the general
form40,41 γ
=
×
k
DN C d
10
diff
3
A
(1) γ
=
×
k
DN C d
10
diff
3
A (1) g
Furthermore, the minimum requirement ⟨Nocc⟩≥1 is highly
optimistic. Analyte binding is a stochastic process taking place
at independent receptors, and random fluctuations occur
around the average occupancy ⟨Nocc⟩. According to the
binomial distribution, the root-mean-square fluctuation in the
instantaneous value of the occupancy, Nocc, is Δ
=
⟨
⟩
N
Nocc
when p ≪1. In order to use a measurement of Nocc to deduce
the analyte concentration C, it is therefore necessary to
increase ⟨Nocc⟩to a point where the relative error ΔNocc/
⟨Nocc⟩is sufficiently small. For example, based on the above, a
10% and 1% accuracy in the average occupancy ⟨Nocc⟩requires
Nrec = 102/p and 104/p, respectively. Fitting these additional
receptors onto the sensor increases its minimum size by a
significant factor. Here, D is the diffusion coefficient of the analyte, NA is
Avogadro’s number, C is the molar concentration of the
analyte, d is the characteristic size of the detector, and γ is a
geometry-dependent factor of order unity (for example, γ = 2
for a shrouded disk of diameter d). The total flux is
proportional to d and thus increases with increasing electrode
size. Eq 1 is valid in the steady state, which is established after a
brief transient with a duration comparable to the diffusion
time, d2/4D.42 In practice, for miniaturized sensors, this
transient is too short to be accessed, and eq 1 holds at
essentially all times. Here, D is the diffusion coefficient of the analyte, NA is
Avogadro’s number, C is the molar concentration of the
analyte, d is the characteristic size of the detector, and γ is a
geometry-dependent factor of order unity (for example, γ = 2
for a shrouded disk of diameter d). The total flux is
proportional to d and thus increases with increasing electrode
size. Eq 1 is valid in the steady state, which is established after a
brief transient with a duration comparable to the diffusion
time, d2/4D.42 In practice, for miniaturized sensors, this
transient is too short to be accessed, and eq 1 holds at
essentially all times. ■INTRODUCTION Translating the raw signals from the individual nodes into
event counts and combining this information into a total
sensor output can be carried out as postexperimental analysis,
although performing parts of it in real time can help mitigate
the sheer volume of data. high mass sensitivity to a transducer can even explicitly
preclude a high concentration sensitivity. We argue that single-entity electrochemical detectors are
intrinsically handicapped in measuring low concentrations due
to their small physical dimensions. These limitations can
however be circumvented by employing large arrays of
independently addressable transducers. Consistent with recent
usage in both SEE and single-molecule optical measure-
ments,25−28 we employ the term “digital sensing” to refer to
this combination of single-entity, real-time, and parallelized
detection. This terminology evokes the high degree of
integration characteristic of modern electronics. It also reflects
the discrete nature of the output signals, which in an abstract
sense consist of a stream of discrete events as a function of
time rather than an averaged sensor response. This represents a
more stringent use of the term compared to digital micro-
fluidics29,30 or digital ELISA,31,32 where discrete events are
usually detected via chemical postamplification. More precisely, we define a digital sensor as an ensemble of
independent nodes, each consisting of a device capable of
single-entity detection. In practice this corresponds to
generating a (usually sudden) time-resolved signature each
time that a microscopic event of interest takes place. Photomultiplier tubes and Geiger counters are classic examples
that generate a burst of charge when a single photon or It is not necessarily a priori obvious that digital sensing can
deliver performance superior to that of conventional
approaches, which are based on large detectors integrating 9024 https://doi.org/10.1021/acs.analchem.1c00510
Anal. Chem. 2021, 93, 9023−9031 https://doi.org/10.1021/acs.analchem.1c00510
Anal. Chem. 2021, 93, 9023−9031 Analytical Chemistry pubs.acs.org/ac Perspective miniaturized electrodes.” It is true that, based on eq 1, the
analyte flux density scales as d−1 and thus increases upon
decreasing the electrode size. But, when working at the single-
analyte limit, what matters is the time needed for each analyte
to reach the detector. This is determined by the total flux, not
the flux density. Hence, the measurement time increases with
smaller detector size. over a large number of microscopic events. Here, we first
summarize fundamental challenges inherent to detecting
ultralow concentrations. ■THE NEED FOR PARALLELIZATION (2) SEE methods typically rely on electrodes with critical
dimensions of a size that is not much larger than the size of
the target itself. Simply put, it is easier to detect a small change
in an electrical signal when it is not superimposed on a much
larger background signal and the associated noise. However,
decreasing the size of the working electrode comes at a price as
it can limit the ultimate achievable performance of a sensor in
several ways. In this section, we focus on the mechanisms by
which minimization impacts theoretical detection limits. Here, p is the probability of the receptor being occupied by an
analyte at time t (starting from p = 0 at t = 0), KD is the
dissociation constant, and τ is the time constant for the
reaction. In terms of the association and dissociation rates, ka
(units M−1 s−1) and kd (units s−1), respectively, the model
parameters take the form KD = kd/ka and τ = (kaC + kd)−1. p
D
d
a
( a
d)
Eq 2 indicates that at low concentration (C ≪KD) and in
equilibrium (t →∞) the probability of the receptor being
occupied, p ≈C/KD, is much smaller than unity. That is, a
receptor is most likely to remain unoccupied even at
equilibrium. For a device incorporating a large number of
receptors, Nrec, the average number of occupied receptors is
⟨Nocc⟩= pNrec. In order to have at least one receptor occupied
on average (⟨Nocc⟩= 1), the minimum number of receptors is
then Nrec = 1/p = KD/C ≫1. If the detector is too small to
accommodate this number of receptors, then ⟨Nocc⟩< 1, and
the sensor ceases to function in a conventional sense. In
principle, it is still possible to determine ⟨Nocc⟩when ⟨Nocc⟩<
1 by measuring a time average over multiple association and
dissociation cycles, but this usually requires an impractically
long time. First, decreasing the electrode size hinders mass transport of
the analyte. Targets need to reach the sensor detection domain
in order to be detected; the smaller the sensor, the lower the
likelihood that a target will find the sensor in a given time
interval. For nanoscale sensors, the time needed for analyte
molecules to come into contact can become prohibitively large. ■INTRODUCTION We pay particular attention to the
main limitations of single-entity methods and address how
parallelization can help lift some of the main obstacles. We
then discuss the properties of the signals resulting from digital
sensing. Finally, we argue that modern electronics are poised to
provide the ideal platform for realizing digital sensors, noting
that this may involve a shift from popular amperometry-based
SEE approaches to high-frequency methods. While our focus is
on electrochemical sensors, many of the general concepts that
we consider here apply equally well to miniaturized optical and
mechanical transducers. A second complication introduced by sensor miniaturization
stems from the properties of chemical equilibria and kinetics. These can place additionaland often more severe
limitations on concentration sensitivity. As an illustration,
consider Langmuir kinetics43 between an analyte at concen-
tration C and a single receptor, as described by the equation =
+
−
τ
−
p
C
K
C (1
e
)
t
D
/
(2) https://doi.org/10.1021/acs.analchem.1c00510
Anal. Chem. 2021, 93, 9023−9031 ■THE NEED FOR PARALLELIZATION For small macromolecules (D ≈10−6 cm2/s) at a low
concentration of C = 1 pM diffusing to a nanoscale disk sensor
(d = 100 nm), steady state is achieved on the time scale of 25
μs. The probability of even a single analyte reaching the
electrode during this time is ≪1. The steady-state flux then
settles to a constant value of ∼0.01 analytes/s. That is, one
needs to wait on average ∼100 s between the moments when a
single analyte arrives at the detector. Lower concentrations or
larger analytes with lower diffusion coefficients demand
correspondingly longer times, placing increasing limitations
on the average number of detection events expected for a given
electrode size and measurement time. Finally, we have so far assumed that the receptors are in
equilibrium with solution. Reaching equilibrium requires
incubating the sample for a period equal to several times the
relaxation time τ. In the limit where C ≪KD, the expression
for τ reduces to τ ≈kd
−1. For molecules with a low dissociation
rate, τ can thus prove prohibitively long (up to days). Alternatively, one does not wait for equilibrium and makes use
of eq 2 to extract a concentration from the transient response. For short measurement times (t ≪τ) and low concentrations,
eq 2 reduces to p ≈(C/KD)(t/τ). This occupancy is a factor of
t/τ ≪1 smaller than the equilibrium result. Consequently, for The above observations may at first appear to contradict the
rule of thumb that “mass transport is more effective at 9025 https://doi.org/10.1021/acs.analchem.1c00510
Anal. Chem. 2021, 93, 9023−9031 Analytical Chemistry Analytical Chemistry pubs.acs.org/ac Perspective a given desired average occupancy ⟨Nocc⟩, the required number
of receptors further increases by a factor τ/t ≫1. a given desired average occupancy ⟨Nocc⟩, the required number
of receptors further increases by a factor τ/t ≫1. These parameters satisfy Nnode ≫(Δ/d −1)4 = 6600, hence
the diffusion-limited flux is essentially the same as that of a 360
μm electrode, yielding kdiff ≈40 analyte/s−1. This is sufficient
to accommodate the maximum binding rate kideal =
NnodeNreckaC = 1 analyte/s. Summarizing, low equilibrium occupancy and long equili-
bration times combine to place rather stringent limitations on
the minimum size of a sensor. For example, substituting typical
numbers for short DNA at a concentration of 1 pM, 10%
accuracy, and 100 s measurement time yields Nrec ≥102(KD/
C)(τ/t) ≈2 × 107. ■BENEFITS OF DIGITAL DETECTION This
remains true even when the amplitude of the discrete events is
smaller than the amplitude of the slow background
fluctuations. Consider an array of Nnode nodes arranged in the form of a
disk, each consisting of a shrouded disk electrode of diameter d
supporting Nrec receptors and separated by an average distance
Δ. Trivially, the total number of receptors in the array is
increased by a factor of Nnode compared to a single node. Mass
transport is also substantially increased. When Nnode ≪(Δ/d
−1)4, each electrode behaves independently and obeys eq 1.46
In this case, the total rate of mass transport is also boosted by a
factor of Nnode. In the opposite limit where the number of
nodes becomes large or the spacing between electrodes
becomes small such that Nnode ≫(Δ/d −1)4, mass transport
becomes limited by hemispherical diffusion from the far field
to the array as a whole, and kdiff increases by a large yet more
modest factor Δ N
d
/
node
.46 Interestingly, however, con-
vective mass transport can be much more effectively employed
in the latter case due to the larger Peclet numbers associated
with the diameter of the array compared to a single electrode.47
ll
d
h
l
l
b Insensitivity to Electrode Fouling. In electrochemical
methods, it is common for the surface of a working electrode
to become fouled after prolonged exposure to a sample. In a
conventional sensor, the corresponding change in sensitivity
can lead to error. In digital sensing, slow changes in the
response of individual nodes to discrete events do not a priori
prevent identification. As long as the individual event
signatures can be recognized, they can be counted and
analyzed independently of their exact shape. This additional
degree of robustness is especially relevant in analyses requiring
long measurement times. Insensitivity to Electrode Fouling. In electrochemical
methods, it is common for the surface of a working electrode
to become fouled after prolonged exposure to a sample. In a
conventional sensor, the corresponding change in sensitivity
can lead to error. In digital sensing, slow changes in the
response of individual nodes to discrete events do not a priori
prevent identification. As long as the individual event
signatures can be recognized, they can be counted and
analyzed independently of their exact shape. This additional
degree of robustness is especially relevant in analyses requiring
long measurement times. Accuracy. ■THE NEED FOR PARALLELIZATION This estimate is based on representative
values44 of KD = 0.2 nM and τ = 105 s. Although specific values
quoted in the literature vary substantially based on the specifics
of different experiments, the conclusion that Nrec is a very large
number holds generally. At optimal packing, these receptors
occupy ∼40 nm2/receptor or ∼1000 μm2 in total, correspond-
ing to the area of a ∼36 μm diameter disk electrode. This
footprint is much larger than the typical nanoscale dimensions
of devices capable of single-entity detection. Conversely,
detecting a mere 100 target molecules at such a large electrode
is at present essentially impossible by purely electrochemical
means. This example demonstrates how an electrode array can allow
simultaneously reconciling the demands of mass transport and
chemical kinetics. More generally, the size and number of
nodes, the geometry of the array and of the nodes, and the
spacing between nodes can be concomitantly optimized using
theoretically derived mass transport equations46 or numerical
simulations. This optimization process will necessarily be
subject to constraints imposed by the nature of the SEE
measurement, the contents of a node, and the technology
employed to implement the array. ■BENEFITS OF DIGITAL DETECTION Here, we discuss qualitatively the potential gains made by
employing SEE detection methods. While here we have focused solely on diffusive mass
transport and first-order kinetics using order-of-magnitude
estimates, these illustrate a more general pattern. On the time
scales deemed desirable for point-of-need assays, the number
of single-entity events expected for a nanoscale sensor easily
becomes far smaller than unity, let alone large enough to
provide sufficient statistics for an accurate measurement. While
performance may be somewhat improved using convection,
this is usually insufficient to significantly alter the picture
sketched above. For a more extensive and quantitative
discussion, we refer the reader to the excellent review by
Squires et al.45 Insensitivity to Baseline Drift. An important requirement
for detecting a low concentration is to be able to detect a
response above the background or baseline signal. Background
may result for example from parasitic electrochemical reactions
or small offsets in the measurement electronics. If the
background signal was perfectly stable, the ability to measure
small changes in detection signal would be limited only by the
resolution of the readout electronics. In practice, however, the
baseline signal fluctuates due to, e.g., temperature variations,
electrode aging, and the ubiquitous 1/f noise characteristic of
nonequilibrium processes. This renders the smallest signals
occurring over long time scales, such as those that occur in
affinity sensors, essentially impossible to distinguish from
baseline drift. These considerations lead to the conclusion that individual
SEE detectors are largely unsuitable for detection at ultralow
analyte concentrations, while devices large enough to satisfy
the mass transport and binding kinetics requirements are too
large to serve as SEE detectors. The same reasoning however
suggests that the main limitations can be overcome through
the massive parallelization of SEE detectors. This is most
readily achieved through pixelation: replacing the single large
electrode with a dense array of many individually addressable
small electrodes, or nodes, each capable of SEE detection. A SEE detector, on the other hand, is often relatively
insensitive to baseline drift. This is because there exists a clear
separation of time scales between abrupt SEE events and the
(usually much slower) baseline fluctuations. Intuitively, it is
clear that steps and spikes that occur on a subsecond time scale
are easily identified against a smoothly varying background
signal that evolves on the scale of minutes or longer. ■ACCURACY, PRECISION, SENSITIVITY, AND
DYNAMIC RANGE In order to paint a more quantitative picture of the noise
properties of digital sensors, we consider some performance
indicators within the context of the simple Langmuir kinetics
model introduced above. We focus on an affinity sensor at low
occupancy (p ≪1). We ignore mass transport limitations and
analyte unbinding, which corresponds to short times (t ≪τ ≈
kd
−1). Under these assumptions, the analyte binding rate for one
receptor is then given by kaC, and the total binding rate is kideal
= NnodeNreckaC. The response of the sensor consists of a series
of random events, or counts, as a function of time. This is
illustrated in Figure 2a for two values of kideal using numerically
generated data. Each trajectory has a random character that
nonetheless reflects an average rate kideal. The time required to
achieve a certain number of counts is different for each
trajectory, but it fluctuates around a mean value such that the
average response remains predictable. This is illustrated in
Figure 2b and c, which show histograms of the distribution of
times required to reach a fixed number of events, Nevent = 20. These values cluster around a mean value but with a significant
spread. could depend on either Nrec or C, and the expressions below
would need to be adapted for those cases. p
Eq 3 can be inverted to yield an expression for the analyte
concentration β
=
Δ −
i
k
jjjjj
y
{
zzzzz
C
N
k
N
N
t
k
1
rec
a
event
node
false
(4) (4) Determining the concentration in this way assumes that kfalse
and the product Nrecβka are known a priori. Determining these
parameters would entail a calibration process that would itself
be subject to statistical error. For simplicity, we assume below
that this error is sufficiently small as to be negligible in
comparison to the statistical fluctuations arising during sensor
operation. To represent imperfections in the nodes’ ability to detect
single events, we assume here for the average number of
binding events from all the nodes in a time interval Δt,
⟨Nevent⟩, the nonideal form During a measurement, the average ⟨Nevent⟩is not known;
one performs a measurement to obtain a measured value Nevent
which is subject to Poisson statistics. The probability of a false
negative result (Nevent = 0 even though ⟨Nevent⟩≠0) is thus
e−⟨Nevent⟩, which quickly becomes negligible as ⟨Nevent⟩increases. ■BENEFITS OF DIGITAL DETECTION Since the role of the node signals is to
allow the identification and counting of events, it is sufficient
that the characteristic signature of an event can be reliably
recognized. Offsets or distortion of the signal are tolerable,
insofar as they are taken into consideration so as not to
preclude event detection and identification. The absolute
accuracy of the raw output signal of the node thus becomes
largely irrelevant in determining the accuracy of the sensor as a
whole. This reduces the demands on the node instrumentation
required to achieve a high sensor accuracy. Advanced Signal Processing. There has been consid-
erable effort devoted to applying modern statistical methods to
the analysis of single-entity measurements. As a primary
example, so-called hidden Markov model descriptions48,49
permit the construction of sophisticated models of microscopic
dynamics from complex time-resolved measurements even
with limited a priori knowledge. These approaches have been
extensively applied for example to the interpretation of single-
ion-channel recordings50 and Förster resonance energy transfer
(FRET) measurements.51,52 While so far underutilized in most
SEE experiments, these methods can be harnessed in the future
for interpreting signals in digital SEE sensing, with expected
boosts in both sensitivity and selectivity. Figure 2. Simulated response of an ideal digital sensor. The sensor is
described by a Poisson process with rate kideal. (a) Typical individual
trajectories for kideal = k0 (red) and kideal = k0/3 (blue), where k0 is an
arbitrary rate constant. The solid black lines represent the average
number of events at each time, ⟨Nevent⟩, as predicted by eq 3 with kfalse
= 0. The dashed lines represent one standard deviation in Nevent as
predicted by Poisson statistics. The trajectories were generated
numerically via a simulated Poisson process.53 (b,c) Histograms
showing the distribution of the time needed to generate 20 events for
kideal = k0 (b, red) and kideal = k0/3 (c, blue) based on 104 trajectories. Apart from a factor of 3 stretching in time in c compared to b and
small statistical fluctuations, the distributions are equivalent. This
scaling property reflects eq 5, which indicates that the relative error
only depends on the number of counts. ■BENEFITS OF DIGITAL DETECTION When considering the features of a digital
sensor, it is crucial to make a distinction between the
properties of the individual nodes and those of the sensor as
a whole. Ideally, digital sensing would allow detecting every
binding event and would exhibit no false positives from
interfering species. In this idealized case, the sensor could
exhibit near-perfect accuracy (vide infra). Importantly,
however, this would not require that the individual node
signals (e.g., the current in an amperometric measurement) For illustration, consider again the numerical example above
where 2 × 107 DNA receptors are required. For d = 100 nm
electrodes, this corresponds to ∼105 electrodes. If these are
positioned 1 μm apart, the array has a diameter of 360 μm. https://doi.org/10.1021/acs.analchem.1c00510
Anal. Chem. 2021, 93, 9023−9031 9026 Figure 2. Simulated response of an ideal digital sensor. The sensor is
described by a Poisson process with rate kideal. (a) Typical individual
trajectories for kideal = k0 (red) and kideal = k0/3 (blue), where k0 is an
arbitrary rate constant. The solid black lines represent the average
number of events at each time, ⟨Nevent⟩, as predicted by eq 3 with kfalse
= 0. The dashed lines represent one standard deviation in Nevent as
predicted by Poisson statistics. The trajectories were generated
numerically via a simulated Poisson process.53 (b,c) Histograms
showing the distribution of the time needed to generate 20 events for
kideal = k0 (b, red) and kideal = k0/3 (c, blue) based on 104 trajectories. Apart from a factor of 3 stretching in time in c compared to b and
small statistical fluctuations, the distributions are equivalent. This
scaling property reflects eq 5, which indicates that the relative error
only depends on the number of counts. s.acs.org/ac
Perspective .acs.org/ac
Perspective Analytical Chemistry pubs.acs.org/ac exhibit perfect accuracy. Since the role of the node signals is to
allow the identification and counting of events, it is sufficient
that the characteristic signature of an event can be reliably
recognized. Offsets or distortion of the signal are tolerable,
insofar as they are taken into consideration so as not to
preclude event detection and identification. The absolute
accuracy of the raw output signal of the node thus becomes
largely irrelevant in determining the accuracy of the sensor as a
whole. This reduces the demands on the node instrumentation
required to achieve a high sensor accuracy. exhibit perfect accuracy. https://doi.org/10.1021/acs.analchem.1c00510
Anal. Chem. 2021, 93, 9023−9031 ■IMPLEMENTATION OF DIGITAL SENSING ■IMPLEMENTATION OF DIGITAL SENSING Accuracy and Precision. In the absence of false positive
events (kfalse = 0), eq 4 indicates that the accuracy of the
measurement depends on the degree to which the multi-
parameter product βNrecka is known. The accuracy is thus set
by the uncertainty in ka (a molecular property), Nrec (which
depends on the reliability and reproducibility of electrode
functionalization), and β (which may depend on both receptor
immobilization and the ability to recognize single events in the
node signals). A very significant disadvantage of an electrochemical digital
sensing architecture is the enormous increase in complexity
associated with implementing a (very) large array of separately
addressable SEE detectors. Approaches for doing so fall mostly
outside the laboratory-based instrumentation typically asso-
ciated with SEE. Most high-quality electrodes employed for electrochemistry
on the nanoscale rely on macroscopic techniques such as laser-
assisted pulling. These are eminently unsuited to large-scale
parallelization. Material-based methods for creating electrode
arrays down to the nanoscale have become increasingly
sophisticated, but separately reading out each electrode is
challenging (although in principle possible, e.g., by optical
methods55). Simple lithographically based approaches, in
which electrodes are patterned on the surface of a chip and
passively connected to external instrumentation via individual
wires,56 are also unfeasible due to the sheer number of
connections that would be required between the chip and the
outside world. Again for kfalse = 0, eq 5 indicates that the relative precision,
ΔC/C, depends only on ⟨Nevent⟩, independently of the value of
the physical parameters of the system. ΔC/C decreases
monotonically with increasing ⟨Nevent⟩, indicating that, not
surprisingly, the precision improves with increased measure-
ment time Δt. This also indicates that the same desired degree
of precision can be attained for measurements at different
analyte concentrations by letting Δt vary for each measure-
ment such that a desired value for Nevent (and thus ΔC/C) is
achieved.27 This property is reflected in Figure 2b and c, where
the distributions of times for achieving Nevent = 20 are seen to
be independent of kideal apart from an overall scaling factor. In
practice, taking advantage of this property would require real-
time event recognition. One way we can begin to address this problem using a
purely electrochemical strategy is to employ array architectures
for measurements involving two working electrodes. ■IMPLEMENTATION OF DIGITAL SENSING (b) Example of a generator-collector node geometry in which a
disk electrode acts as generator and a surrounding ring electrode acts
as collector. The horizontal and vertical bars at the left and bottom
represent the row and column interconnects, respectively. Figure 3. Two-dimensional array architecture. (a) Individual nodes
(red dots) can be individually addressed via their row (1, 2, 3) and
column (α, β, γ) interconnects. In a generator-collector experiment,
all of the generator electrodes on the same row are excited
simultaneously, and the collector current for each node on that row
can be read out via the corresponding column interconnect. Alternatively, in an active array, the row and column wires can
control transistor switches to dynamically control which node(s) are
active. (b) Example of a generator-collector node geometry in which a
disk electrode acts as generator and a surrounding ring electrode acts
as collector. The horizontal and vertical bars at the left and bottom
represent the row and column interconnects, respectively. (6) The sensitivity is proportional to Nnode, independently of the
values of the node parameters. The sensitivity is proportional to Nnode, independently of the
values of the node parameters. Dynamic Range. The measurement must be achievable in
a reasonable time. Combining eq 3 and eq 5 yields for the
minimum measurement time at a desired relative precision
(taking kfalse = 0 for the moment) β
Δ =
Δ
t
N
N
k C
C C
1
(
/ )
node
rec
a
2
(7) (7) This minimum time can in principle be made arbitrarily
small by increasing Nnode. On the other hand, the precision
deteriorates once C ≲kfalse/Nrecβka. This depends only on the
properties of the individual nodes and is not improved by
increasing Nnode. Even with an arbitrarily large Nnode, the limit
of detection (LOD) thus still depends on optimizing the node
properties, in particular the suppression of false positives (kfalse
→0) and missed events (β →1), through both experimental
design and signal processing. each electrode separately to the outside world, the generator
electrode from each node is short-circuited with all the other
generator electrodes from the same row, and a single external
connection exists for that row. The collector electrodes are
similarly connected in a column-wise fashion (Figure 3b). During measurements, a single row is set to a generating
potential while the remaining rows and all the columns are set
to a collecting potential. Analytical Chemistry Perspective pubs.acs.org/ac ■IMPLEMENTATION OF DIGITAL SENSING For
illustration, consider a generator-collector experiment, in
which a species is electrogenerated at one electrode and
collected at a second, nearby electrode. The measurement
nodes are arranged on a two-dimensional square grid, as
illustrated schematically in Figure 3a. Rather than connecting In cases where false positives occur (kfalse > 0), the situation
is essentially unchanged so long as the number of false
positives, NnodekfalseΔt, remains much smaller than the total
number of measured events, Nevent. When the majority of
events becomes false events, on the other hand, eq 5 indicates
that the relative error increases significantly. Reducing the rate
of false events thus becomes critical for improving precision at
the lowest achievable concentrations. Figure 3. Two-dimensional array architecture. (a) Individual nodes
(red dots) can be individually addressed via their row (1, 2, 3) and
column (α, β, γ) interconnects. In a generator-collector experiment,
all of the generator electrodes on the same row are excited
simultaneously, and the collector current for each node on that row
can be read out via the corresponding column interconnect. Alternatively, in an active array, the row and column wires can
control transistor switches to dynamically control which node(s) are
active. (b) Example of a generator-collector node geometry in which a
disk electrode acts as generator and a surrounding ring electrode acts
as collector. The horizontal and vertical bars at the left and bottom
represent the row and column interconnects, respectively. Figure 3 Two dimensional array architecture (a) Individual nodes Sensitivity. As per the IUPAC definition, the sensitivity is
the slope of the response curve.54 Eq 3 indicates that the
response is linear in C (as opposed to, e.g., logarithmic),
yielding a constant sensitivity β
=
⟨
⟩=
Δ
d N
dC
N
N
k
t
Sensitivity
event
node
rec
a
(6) Figure 3. Two-dimensional array architecture. (a) Individual nodes
(red dots) can be individually addressed via their row (1, 2, 3) and
column (α, β, γ) interconnects. In a generator-collector experiment,
all of the generator electrodes on the same row are excited
simultaneously, and the collector current for each node on that row
can be read out via the corresponding column interconnect. Alternatively, in an active array, the row and column wires can
control transistor switches to dynamically control which node(s) are
active. ■ACCURACY, PRECISION, SENSITIVITY, AND
DYNAMIC RANGE More generally, the standard deviation for Nevent is
⟨
⟩
Nevent ,
again from Poisson statistics (while the true value of ⟨Nevent⟩is
not known, it can be estimated as the measured value, Nevent, so
long as Nevent ≫1). The corresponding standard deviation is
given by (Supporting Information): β
⟨
⟩=
Δ =
+
Δ
N
k
t
N
N
k C
k
t
(
)
event
nonideal
node
rec
a
false
(3) (3) Here, we introduced two parameters, β and kfalse, to
represent nonidealities. β represents the probability that a
binding event will actually be detected: β = 1 represents a
perfect detector, 0 < β < 1 means that the sensor fails to detect
some events, and β > 1 corresponds to a situation in which a
single target can trigger multiple counts. kfalse represents a rate
of false events that cannot be distinguished from actual events;
these could be the result of noise being mistakenly identified as
an event or unwanted events caused by interfering species. Δ
=
⟨
⟩
+
Δ
−
Δ
C
C
N
N
k
t N
N
k
t N
1
(1
/
)
1
/
event
node false
event
1/2
node false
event
(5) Note that this model is highly simplified for the sake of
readability. For example, there are scenarios for which kfalse (5) 9027 Analytical Chemistry Analytical Chemistry A key ingredient of (bio)sensors that has not yet been
explicitly addressed at the experimental level for most SEE
methods is receptor compatibility. So far, many SEE
measurements have focused on the development of signal
transduction methods with minimal attention to specificity. How best to couple an affinity-based assay to most SEE
detection methods remains a largely open question. This is
further complicated by the fact that electrochemical processes
can locally influence biochemical kinetics, an encouraging
example being the enhancement of the rate of DNA
hybridization at an electrode under potential modulation.69 p
g
In recent decades, a wide variety of electrochemical
instrumentation has been implemented using complementary
metal-oxide-semiconductor (CMOS) technology, the work-
horse of the semiconductors industry. While this field is far too
broad to review here,57 some recent examples of CMOS-based
massively parallelized electrochemical devices include ion-
sensitive field-effect transistors (ISFETs) for DNA sequenc-
ing,58 microelectrode arrays (MEAs) for electrogenic cell
recording and stimulation,59,60 and high-density nanocapacitor
arrays for high-frequency detection.61 This directly exploits
some of the strengths of CMOS technology, in particular the
means to create miniaturized electrode structures (although
this can require postprocessing following standard CMOS
fabrication), the ability to arrange these in large arrays of
separately addressable nodes, high switching speeds, and the
flexibility to integrate additional functionality. Can SEE-based digital (bio)sensors advance to the point
where they can revolutionize trace-level detection? This is at
the moment an open question. Most SEE methods are still at
an early stage of development, and the technical challenges
ahead are very significant. By comparison, optical methods
provide a more straightforward entry for the development of
digital sensing concepts. This is in no small part due to digital
cameras that permit high-sensitivity measurements over large
areas and with high spatial resolution. The SEE equivalent of
mature CCD and CMOS image sensors does not exist yet. It is
difficult to underestimate the potential of CMOS technology,
however. Past the initial hurdle of development, in terms of
both time and resources, all-electrical assays incorporating
colossal numbers of active elements could be mass produced at
low cost. For this to happen, however, will require continued
close collaboration between nanoelectrochemists and circuit
engineers. Implementing electrochemical instrumentation on a chip
may however entail significant compromises. Analytical Chemistry pubs.acs.org/ac Perspective the collector current can be done for each column in
succession, for several columns at a time, or for all columns
in parallel. Such a square array with N2 nodes requires 2N
wires, a factor N reduction when compared to the 2N2 wires
needed to connect each electrode separately. This geometry
lends itself well to a lithography-based approach in which the
individual nodes and their interconnects are fabricated on an
insulating substrate. required to overcome the limitations imposed by mass
transport and binding kinetics. Large-scale parallelization
suggests a solution to this conundrum, but this comes at a
significant increase in complexity and most likely requires
implementing digital SEE sensing in the form of integrated
circuits. This necessarily involves significant design compro-
mises since microelectronic processing and circuit architectures
are not always in harmony with electrode materials and
instrumentation most commonly employed in electrochemis-
try. An important compromise in this approach is that only a
subset of the nodes is being actively monitored at any given
time. The SEE process under investigation must therefore be
sufficiently slow that it can be resolved despite each node being
only intermittently probed. Another issue is that the inactive
nodes still contribute to the noise and background current of
the column signals, degrading the overall noise properties of
the measurement. A potentially disruptive factor that we have ignored in our
analysis of mass transport and binding kinetics is targeted
active transport. There is a growing set of methods in micro-
and nanofluidics for selectively enriching the local concen-
tration of target molecules at a sensor (e.g., isotachophoresis,66
ion concentration polarization focusing,67 AC electrokinetic
methods68). These techniques are beneficial for both conven-
tional and digital sensors, but in particular they may help
alleviate the stringent requirements for implementing digital
detection by decreasing the minimum number of nodes. A more general strategy is to separate the function of
addressing individual nodes from that of performing the
electrochemical measurement. This requires active switches
being incorporated into the array to select which electrode(s)
are polarized and/or read out. This approach lends itself
naturally to an integrated circuit implementation in which the
active elements take the form of transistors that are
incorporated together with the electrodes and their inter-
connects on the same chip. Having taken that step, it becomes
possible to implement additional functionality on the chip such
as amplification and even digitization. ■SUMMARY AND OUTLOOK SEE is a rapidly evolving subfield that is extending fundamental
understanding and pushing the capabilities of electrochemical
techniques into new territory. While SEE detectors have
exquisite mass sensitivity, translating this into a high
concentration sensitivity still represents a formidable challenge. This is in no small part due to the size mismatch between
typical SEE detectors and the much larger dimensions that are Analytical Chemistry For example, 1/f
noise plays an increasingly large role upon decreasing transistor
dimensions.62 This directly impacts the noise properties of
transimpedance amplifiers employed in amperometry (the
method of choice for many SEE experiments, as illustrated in
Figure 1). While 1/f noise is not a main limiting factor in many
present-day implementations,63 it can severely limit down-
scaling amperometric arrays to the submicron scale. This may
ultimately favor transducers that are less sensitive to 1/f noise,
such as high-frequency switched capacitor methods.64,65 ■ASSOCIATED CONTENT *
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.analchem.1c00510. Derivation of eq 5 (PDF) Derivation of eq 5 (PDF) Derivation of eq 5 (PDF) ■SUMMARY AND OUTLOOK ■IMPLEMENTATION OF DIGITAL SENSING In this way, generation takes place
only in the row being addressed. The collector current from
each node in that row can then be measured by measuring the
collector current through the corresponding column, even
though each column is also connected to the collector
electrodes from the inactive rows. In order to scan the whole
array, the active row is cycled in time. For each row, readout of Eq 6 indicates that the sensitivity is independent of
concentration, but in practice a limit for the maximum
measurable concentration will be set by the ability to resolve
events that overlap in time at high event rates. This depends
largely on the details of the node and is not discussed further
here. 9028 https://doi.org/10.1021/acs.analchem.1c00510
Anal. Chem. 2021, 93, 9023−9031 Analytical Chemistry Analytical Chemistry ■REFERENCES 2004, 22, 1315−1316 (50) Chung, S. H.; Moore, J. B.; Xia, L.; Premkumar, L. S.; Gage, P. W. Philos. Trans. R. Soc., B 1990, 329, 265−285. (50) Chung, S. H.; Moore, J. B.; Xia, L.; Premkumar, L 11) Luo, L.; White, H. S. Langmuir 2013, 29, 11169−1117
) g
W. Philos. Trans. R. Soc., B 1990, 329, 265−285. (12) Neher, E.; Sakmann, B. Nature 1976, 260, 799−802. (
) (51) McKinney, S. A.; Joo, C.; Ha, T. Biophys. J. 2006, 91, 1941−
1951. (13) Hamill, O. P.; Marty, A.; Neher, E.; Sakmann, B.; Sigworth, F. J. Pfluegers Arch. 1981, 391, 85−100. (52) Bronson, J. E.; Fei, J. Y.; Hofman, J. M.; Gonzalez, R. L.;
Wiggins, C. H. Biophys. J. 2009, 97, 3196−3205. (
) (14) Liu, H. Y.; Fan, F. R. F.; Lin, C. W.; Bard, A. J. J. Am. Chem. Soc. 1986, 108, 3838−3839. (53) Kostiuchenko, Z. A.; Zhang, B.; Lemay, S. G. J. Phys. Chem. C
2016, 120, 22777−22783. (15) Itaya, K.; Tomita, E. Surf. Sci. 1988, 201, L507−L512. (
) (16) Ebejer, N.; Schnippering, M.; Colburn, A. W.; Edwards, M. A.;
Unwin, P. R. Anal. Chem. 2010, 82, 9141−9145. (54) Ekins, R.; Edwards, P. Clin. Chem. 1997, 43, 1824−1831. (
) (55) Fan, Y.; Anderson, T. J.; Zhang, B. Current Opinion in
Electrochemistry 2018, 7, 81−86. (17) Kleijn, S. E. F.; Lai, S. C. S.; Miller, T. S.; Yanson, A. I.; Koper, (17) Kleijn, S. E. F.; Lai, S. C. S.; Miller, T. S.; Ya M. T. M.; Unwin, P. R. J. Am. Chem. Soc. 2012, 134, 18558−18561. (56) Rassaei, L.; Singh, P. S.; Lemay, S. G. Anal. Chem. 2011, 83,
3974−3980. (18) Bentley, C. L.; Kang, M.; Unwin, P. R. Current Opinion in
Electrochemistry 2017, 6, 23−30. (57) Li, H. T.; Liu, X. W.; Li, L.; Mu, X. Y.; Genov, R.; Mason, A. J. Sensors 2017, 17, 74. (19) Fan, F. R. F.; Bard, A. J. Science 1995, 267, 871−874. (20) Zevenbergen, M. A. G.; Singh, P. S.; Goluch, E. D.; Wolfrum, B. (58) Rothberg, J. M.; Hinz, W.; Rearick, T. M.; Schultz, J.; Mileski,
W.; Davey, M.; Leamon, J. H.; Johnson, K.; Milgrew, M. J.; Edwards,
M.; et al. Nature 2011, 475, 348−352. (
)
g
g
L.; Lemay, S. G. Nano Lett. 2011, 11, 2881−2886. L.; Lemay, S. G. Nano Lett. 2011, 11, 2881−2886. (21) Quinn, B. M.; van’t Hof, P. G.; Lemay, S. ■ACKNOWLEDGMENTS (32) Cohen, L.; Walt, D. R. Annu. Rev. Anal. Chem. 2017, 10, 345−
363. We thank F. Widdershoven and L. Selmi for critical comments
on the manuscript, the SENTINEL network for useful
discussions (funded by the European Union’s Horizon 2020
Research and Innovation Program under Marie Skłodowska-
Curie grant agreement no. 812398), and The Netherlands
TopSector High-Tech Systems & Materials for financial
support under the TKI project “Early Cancer Diagnostics”. (33) Kwon, S. J.; Fan, F.-R. F.; Bard, A. J. J. Am. Chem. Soc. 2010,
132, 13165−13167. (34) Lebègue, E.; Anderson, C. M.; Dick, J. E.; Webb, L. J.; Bard, A. J. Langmuir 2015, 31, 11734−11739. J
g
,
,
(35) Li, X.; Batchelor-McAuley, C.; Compton, R. G. ACS Sensors
2019, 4, 464−470. (36) Chen, C.-H.; Ravenhill, E. R.; Momotenko, D.; Kim, Y.-R.; Lai,
S. C. S.; Unwin, P. R. Langmuir 2015, 31, 11932−11942. Notes (31) Rissin, D. M.; Kan, C. W.; Campbell, T. G.; Howes, S. C.;
Fournier, D. R.; Song, L.; Piech, T.; Patel, P. P.; Chang, L.; Rivnak, A. J.; et al. Nat. Biotechnol. 2010, 28, 595−599. The authors declare no competing financial interest. The authors declare no competing financial interest. Analytical Chemistry pubs.acs.org/ac Perspective Author (26) Nouri, R.; Tang, Z.; Guan, W. Anal. Chem. 2019, 91, 11178−
11184. Taghi Moazzenzade −MESA+ Institute for Nanotechnology
and Faculty of Science and Technology, University of Twente,
7500 AE Enschede, The Netherlands;
orcid.org/0000-
0002-3908-6062 (27) Jing, W.; Wang, Y.; Yang, Y.; Wang, Y.; Ma, G.; Wang, S.; Tao,
N. ACS Nano 2019, 13, 8609−8617. (28) Huang, Q.; Li, N.; Zhang, H.; Che, C.; Sun, F.; Xiong, Y.;
Canady, T. D.; Cunningham, B. T. Lab Chip 2020, 20, 2816−2840. Complete contact information is available at: y
g
p
(29) Teh, S.-Y.; Lin, R.; Hung, L.-H.; Lee, A. P. Lab Chip 2008, 8,
198−220. https://pubs.acs.org/10.1021/acs.analchem.1c00510 (30) Guo, M. T.; Rotem, A.; Heyman, J. A.; Weitz, D. A. Lab Chip
2012, 12, 2146−2155. ■REFERENCES (37) Shi, X.; Verschueren, D. V.; Dekker, C. Nano Lett. 2018, 18,
8003−8010. (1) Baker, L. A. J. Am. Chem. Soc. 2018, 140, 15549−15559. (1) Baker, L. A. J. Am. Chem. Soc. 2018, 140, 15549−15559. (38) Moazzenzade, T.; Yang, X.; Walterbos, L.; Huskens, J.; Renault,
C.; Lemay, S. G. J. Am. Chem. Soc. 2020, 142, 17908−17912. (2) Coulter, W. H. U.S. Patent US2656508, 1953. (3) Li, J.; Stein, D.; McMullan, C.; Branton, D.; Aziz, M. J.;
Golovchenko, J. A. Nature 2001, 412, 166−169. (3) Li, J.; Stein, D.; McMullan, C.; Branton, D.; Aziz, M. J.;
Golovchenko, J. A. Nature 2001, 412, 166−169. y
J
(39) Sheehan, P. E.; Whitman, L. J. Nano Lett. 2005, 5, 803−807. (40) Liu, X.; Lu, J.; Cha, C. J. Electroanal. Chem. Interfacial
Electrochem. 1990, 294, 289−292. (4) Li, J.; Gershow, M.; Stein, D.; Brandin, E.; Golovchenko, J. A. Nat. Mater. 2003, 2, 611−615. ,
,
(41) Oldham, K. B. J. Electroanal. Chem. 1992, 323, 53−76. (5) Kasianowicz, J. J.; Brandin, E.; Branton, D.; Deamer, D. W. Proc. Natl. Acad. Sci. U. S. A. 1996, 93, 13770−13773. (42) Bard, A. J.; Faulkner, L. R. Electrochemical Methods:
Fundamentals and Applications; John Wiley & Sons: New York, 2001. (
) (6) Clarke, J.; Wu, H.-C.; Jayasinghe, L.; Patel, A.; Reid, S.; Bayley,
H. Nat. Nanotechnol. 2009, 4, 265−270. (43) Langmuir, I. J. Am. Chem. Soc. 1918, 40, 1361−1403. (
) (44) Liebermann, T.; Knoll, W.; Sluka, P.; Herrmann, R. Colloids
Surf., A 2000, 169, 337−350. (7) Deamer, D.; Akeson, M.; Branton, D. Nat. Biotechnol. 2016, 34,
518−524. (45) Squires, T. M.; Messinger, R. J.; Manalis, S. R. Nat. Biotechnol. 2008, 26, 417. (8) Jain, M.; Koren, S.; Miga, K. H.; Quick, J.; Rand, A. C.; Sasani,
T. A.; Tyson, J. R.; Beggs, A. D.; Dilthey, A. T.; Fiddes, I. T.; et al. Nat. Biotechnol. 2018, 36, 338−345. ( ) (46) Morf, W. E. Anal. Chim. Acta 1996, 330, 139−149. (
) (47) Sokolov, S. V.; Bartlett, T. R.; Fair, P.; Fletcher, S.; Compton,
R. G. Anal. Chem. 2016, 88, 8908−8912. (9) Depledge, D. P.; Srinivas, K. P.; Sadaoka, T.; Bready, D.; Mori,
Y.; Placantonakis, D. G.; Mohr, I.; Wilson, A. C. Nat. Commun. 2019,
10, 754. (48) Bilmes, J. Techanical Report; University of California: Berkeley,
1998. (10) Liu, Y.; Li, M.; Zhang, F.; Zhu, A.; Shi, G. Anal. Chem. 2015,
87, 5531−5538. (49) Eddy, S. R. Nat. Biotechnol. ■AUTHOR INFORMATION Corresponding Author
Serge G. Lemay −MESA+ Institute for Nanotechnology and
Faculty of Science and Technology, University of Twente,
7500 AE Enschede, The Netherlands;
orcid.org/0000-
0002-0404-3169; Email: s.g.lemay@utwente.nl Corresponding Author
Serge G. Lemay −MESA+ Institute for Nanotechnology and
Faculty of Science and Technology, University of Twente,
7500 AE Enschede, The Netherlands;
orcid.org/0000-
0002-0404-3169; Email: s.g.lemay@utwente.nl 9029 https://doi.org/10.1021/acs.analchem.1c00510
Anal. Chem. 2021, 93, 9023−9031 Analytical Chemistry Analytical Chemistry https://doi.org/10.1021/acs.analchem.1c00510
Anal. Chem. 2021, 93, 9023−9031 ■REFERENCES G. J. Am. Chem. Soc. 2004, 126, 8360−8361. (59) Ballini, M.; Muller, J.; Livi, P.; Yihui Chen; Frey, U.; Stettler,
A.; Shadmani, A.; Viswam, V.; Lloyd Jones, I.; Jackel, D.; Radivojevic,
M.; Lewandowska, M. K.; Wei Gong; Fiscella, M.; Bakkum, D. J.;
Heer, F.; Hierlemann, A.; et al. IEEE J. Solid-State Circuits 2014, 49,
2705−2719. (22) Xiao, X.; Bard, A. J. J. Am. Chem. Soc. 2007, 129, 9610−9612. (
) (23) Zhou, Y. G.; Rees, N. V.; Compton, R. G. Angew. Chem., Int. Ed. 2011, 50, 4219−4221. 2011, 50, 4219−4221. (24) Banks, C. E.; Rees, N. V.; Compton, R. G. J. Phys. Chem. B
2002, 106, 5810−5813. (60) Tsai, D.; Sawyer, D.; Bradd, A.; Yuste, R.; Shepard, K. L. Nat. Commun. 2017, 8, 1802. (25) Visser, E. W. A.; Yan, J.; van Ijzendoorn, L. J.; Prins, M. W. J. Nat. Commun. 2018, 9, 2541. 9030 https://doi.org/10.1021/acs.analchem.1c00510
Anal. Chem. 2021, 93, 9023−9031 Analytical Chemistry Analytical Chemistry Perspective pubs.acs.org/ac Perspective (61) Laborde, C.; Pittino, F.; Verhoeven, H. A.; Lemay, S. G.; Selmi,
L.; Jongsma, M. A.; Widdershoven, F. P. Nat. Nanotechnol. 2015, 10,
791−795. (62) Tuinhout, H.; Duijnhoven, A. Z. In Evaluation of 1/F Noise
Variability in the Subthreshold Region of Mosfets, 2013 IEEE
International Conference on Microelectronic Test Structures
(ICMTS), March 25−28, 2013; pp 87−92. (63) Hartel, A. J. W.; Shekar, S.; Ong, P.; Schroeder, I.; Thiel, G.;
Shepard, K. L. Anal. Chim. Acta 2019, 1061, 13−27. (64) Widdershoven, F.; Cossettini, A.; Laborde, C.; Bandiziol, A.;
van Swinderen, P. P.; Lemay, S. G.; Selmi, L. IEEE Transactions on
Biomedical Circuits and Systems 2018, 12, 1369−1382. (65) Ieee. 2019 Ieee Biomedical Circuits and Systems Conference 2019,
1. (66) Smejkal, P.; Bottenus, D.; Breadmore, M. C.; Guijt, R. M.;
Ivory, C. F.; Foret, F.; Macka, M. Electrophoresis 2013, 34, 1493−
1509. (67) Berzina, B.; Anand, R. K. Anal. Chim. Acta 2020, 1128, 149−
173. (68) Salari, A.; Thompson, M. Sens. Actuators, B 2018, 255, 3601−
3615. (69) Tymoczko, J.; Schuhmann, W.; Gebala, M. ACS Appl. Mater. Interfaces 2014, 6, 21851−21858. 9031
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https://applbiolchem.springeropen.com/counter/pdf/10.1186/s13765-023-00842-x
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English
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Recent progress on drugs discovery study for treatment of COVID-19: repurposing existing drugs and current natural bioactive molecules
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Applied biological chemistry
| 2,023
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cc-by
| 34,810
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© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. Abstract COVID-19 has been a major global health concern for the past three years, and currently we are still experienc‑
ing coronavirus patients in the following years. The virus, known as SARS-CoV-2, shares a similar genomic identity
with previous viruses such as SARS-CoV and MERS-CoV. To combat the pandemic, modern drugs discovery tech‑
niques such as in silico experiments for docking and virtual screening have been employed to design new drugs
against COVID-19. However, the release of new drugs for human use requires two safety assessment steps consisting
of preclinical and clinical trials. To bypass these steps, scientists are exploring the potential of repurposing existing
drugs for COVID-19 treatment. This approach involves evaluating antiviral activity of drugs previously used for treat‑
ing respiratory diseases against other enveloped viruses such as HPV, HSV, and HIV. The aim of this study is to review
repurposing of existing drugs, traditional medicines, and active secondary metabolites from plant-based natural
products that target specific protein enzymes related to SARS-CoV-2. The review also analyzes the chemical structure
and activity relationship between selected active molecules, particularly flavonol groups, as ligands and proteins
or active sites of SARS-CoV-2. Keywords COVID-19, SARS-CoV-2, Coronavirus, Repurposing drugs, Natural products, Antiviral agent respiratory syndrome coronavirus 2 (SARS-CoV-2). This
disease was first discovered on December 31, 2019, in
Wuhan, China, as a cluster of pneumonia cases. Later,
on March 27, 2020, World Health Organization (WHO)
declared the outbreak a global pandemic, as it had spread
to numerous countries around the world [1, 3, 5]. Introduction COVID-19, also known as coronavirus disease 2019, is a
highly infectious illness that is caused by the novel coro-
navirus, which has been officially named severe acute [
]
An outbreak of SARS was first reported in Guang-
dong, China, in November 2002 [7]. This disease was
later identified in Hong Kong in late February 2003,
and it subsequently spread globally to North America,
Europe, and other parts of Asia [9, 11]. However, phylo-
genetic analysis showed that SARS coronavirus (SARS-
CoV) differed from previously known coronavirus [13]. In June 2012, another coronavirus-related respiratory
illness, the Middle East respiratory syndrome (MERS),
caused by MERS coronavirus (MERS-CoV), emerged in
the Middle East, particularly in Saudi Arabia, and was
spread to humans by dromedary camels [14, 16, 17]. *Correspondence:
Sri Fatmawati
fatma@chem.its.ac.id
1 Department of Chemistry, Faculty of Science and Data Analytics, Institut
Teknologi Sepuluh Nopember, Sukolilo, Surabaya 60111, East Java,
Indonesia
2 Department of Chemistry, Faculty of Mathematics and Natural Science,
Universitas Jember, Kampus Tegalboto, Jember 68121, Indonesia
3 Faculty of Applied Sciences and Technology, Universiti Tun Hussein Onn
Malaysia (UTHM), 84600 Muar, Johor, Malaysia
4 Department of Pharmaceutical Engineering, College of Biomedical
Science, Daegu Haany University, 290 Yugok‑dong, Gyeongsan‑si,
Gyeongsangbuk‑do 38610, Republic of Korea
5 Agri‑Food and Biotechnology Research Center, Institut Teknologi
Sepuluh Nopember, Sukolilo, Surabaya 60111, East Java, Indonesia *Correspondence:
Sri Fatmawati
fatma@chem.its.ac.id
1 Department of Chemistry, Faculty of Science and Data Analytics, Institut
Teknologi Sepuluh Nopember, Sukolilo, Surabaya 60111, East Java,
Indonesia
2 Department of Chemistry, Faculty of Mathematics and Natural Science,
Universitas Jember, Kampus Tegalboto, Jember 68121, Indonesia
3 Faculty of Applied Sciences and Technology, Universiti Tun Hussein Onn
Malaysia (UTHM), 84600 Muar, Johor, Malaysia
4 Department of Pharmaceutical Engineering, College of Biomedical
Science, Daegu Haany University, 290 Yugok‑dong, Gyeongsan‑si,
Gyeongsangbuk‑do 38610, Republic of Korea
5 Agri‑Food and Biotechnology Research Center, Institut Teknologi
Sepuluh Nopember, Sukolilo, Surabaya 60111, East Java, Indonesia Oktavianawati et al. Applied Biological Chemistry (2023) 66:89
https://doi.org/10.1186/s13765-023-00842-x Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Page 2 of 48 MERS-CoV is phylogenetically related to bat coronavi-
rus (SARS-CoV-2), the virus that causes COVID-19. SARS-CoV-2 within lineage B was found to be closer to
that of SARS-CoV [21]. MERS-CoV is phylogenetically related to bat coronavi-
rus (SARS-CoV-2), the virus that causes COVID-19. MERS-CoV is phylogenetically related to bat coronavi-
rus (SARS-CoV-2), the virus that causes COVID-19. However, viruses are known to enhance their infectivity
by acquiring mutations that allow viruses to evade human
immune responses, including those triggered by vaccines
and drugs. The original strain of SARS-CoV-2 underwent
mutations primarily in its spike protein (S) that resulted
in the emergence of several variants, such as α, β, γ, δ,
and Omicron variants (Table 1). The number and loca-
tion of mutations within the spike protein influences
the characteristics and potential risks of each variant in
evading infection by circumventing human antibodies
and immune responses [24]. Recent surge in COVID-
19 cases, known as the third wave, has been attributed
to the Omicron variant, specifically the B.1.1.529 strain. The high reinfection rate and greater transmissibility of
this variant are believed to be due to the large number of
mutations in the spike protein. Therefore, addressing the
nature of the virus and developing effective treatment to
overcome current and future waves of infections should
be a top priority. The genomic characteristics of SARS-CoV-2 indi-
cate that it is closely related (88% identity) to two bat-
derived SARS-like coronavirus, bat-SL-CoVZC45 and
bat-SL-CoVZXC21, were detected in Rhinolophus pusil-
lus bats from Zhoushan, eastern China, in 2018 [19,
21]. Additionally, Zhou et al. reported that a corona-
virus strain, SARSr-Ra-BatCoV-RaTG13, isolated from
Rhinolophus affinis bats in Pu’er, China, in 2013, has an
overall genome identity of 96.2% to SARS-CoV-2n. This
close phylogenetic relationship to RaTG13 suggests that
SARS-CoV-2 originated in bats [22]. According to Lam et al. [18], receptor-binding
domain (RBD) of SARS-CoV-2 spike (S) protein exhib-
its extremely high sequence similarity to Guangdong
pangolin (97.4% amino acid similarity). The amino
acids of this pangolin coronavirus, GX/P2V, are identi-
cal to the five critical residues of RBD, while RaTG13
has only one identical amino acid to SARS-CoV-2. It is
worth noting that SARS-CoV-2 rapidly spread among
human populations. The lack of insertion of the polyba-
sic cleavage sites in the spike protein of pangolin coro-
navirus contributed to this phenomenon. Structure of coronavirush The viral lipid bilayer enve-
lope, which is sensitive to desiccation, heat, and amphi-
philes such as soap and detergents, is more susceptible to
sterilization outside the human cell environment than the
non-enveloped virus. However, the glycoproteins in the
viral envelope helps the virus bind to the receptor sites
on the host membrane to avoid the human immune sys-
tem. Therefore, coronavirus binds to its primary receptor,
the cellular angiotensin-converting enzyme 2 (ACE2),
through its spike glycoproteins. Once the spike binds to
the receptor, the cell and viral membrane fuses directly,
causing the virion RNA genome inside the capsid to enter
the host cell or endocytosis [1, 3, 32, 33]. Modes of action of antiviral agents related to virus
life cycle life cycle
In accordance with previous studies, various targets
have been identified for developing antiviral drugs based
on the virus life cycle, namely (1) inhibitors of fusion or
entry, which targets the interaction between the virus and
the host cell membrane, (2) uncoating inhibitors, a tech-
nique used to acidify the viral interior to weaken electro-
static interaction, (3) nucleic acid synthesis terminators,
used to block viral enzymes, (4) integrase inhibitors,
utilized to target the attachment of host cell DNA to the
viral genome through the replication step, (5) protease
inhibitors, often combined with reverse transcriptase
inhibitors, and (6) release inhibitors, used to hinder or
block the receptor from viral protein attachment [38–41]. Drugs targets for inhibiting viral infections are started
by blocking the initial step of viral attachment to the
receptor. This was achieved through receptor blockade
by using a monoclonal antibody against the major cel-
lular receptor or by employing specific inhibitor com-
pounds [42]. For soluble receptors, blocking can be
accomplished by disrupting the interaction between the
glycosylated extracellular domains of the receptor and
the hydrophobic transmembrane region on the virus. It
usually blocks viral replication in cell culture form and
prevents the attachment of the mutant virus to the recep-
tor. This blockade effectively inhibits viral replication in In accordance with previous studies, various targets
have been identified for developing antiviral drugs based
on the virus life cycle, namely (1) inhibitors of fusion or
entry, which targets the interaction between the virus and
the host cell membrane, (2) uncoating inhibitors, a tech-
nique used to acidify the viral interior to weaken electro-
static interaction, (3) nucleic acid synthesis terminators,
used to block viral enzymes, (4) integrase inhibitors,
utilized to target the attachment of host cell DNA to the
viral genome through the replication step, (5) protease
inhibitors, often combined with reverse transcriptase
inhibitors, and (6) release inhibitors, used to hinder or
block the receptor from viral protein attachment [38–41]. Drugs targets for inhibiting viral infections are started
by blocking the initial step of viral attachment to the
receptor. This was achieved through receptor blockade
by using a monoclonal antibody against the major cel-
lular receptor or by employing specific inhibitor com-
pounds [42]. For soluble receptors, blocking can be
accomplished by disrupting the interaction between the
glycosylated extracellular domains of the receptor and
the hydrophobic transmembrane region on the virus. Structure of coronavirush The size of the virus ranges from 20 to 300 nm and it
is capable of infecting and replicating cells. It contains
genes and proteins enclosed within a lipid layer envelope
or a non-enveloped one. Specifically, SARS-CoV-2 has a
diameter ranging from 60 to 140 nm with a spike protein
size of approximately 9 to 12 nm. Its virion is spherical,
sometimes pleomorphic, with a diameter of 78 nm and
resembles a solar corona. Goldsmith et al. and Tshibangu
et al. stated that the virus contains a helical nucleocapsid
within an envelope [29, 30]. Lau et al. [23], stated that the genome backbone of
SARS-CoV-2 evolved from bat coronavirus, its RBD
region was likely acquired from pangolin coronavirus,
causing SARS-CoV-2 to become a recombinant virus. Additionally, SARS-CoV-2 RBD has distinct evolu-
tionary characteristics compared to other Sarbecovi-
rus species, particularly in terms of subunit cleavage
sites. While the genomic characteristics of SARS-CoV
and MERS-CoV are more distant from SARS-CoV-2,
with similarities of 79% and 50%, respectively, RBD of According to Wang and Liang [31], viruses associ-
ated with acute respiratory infections include influenza, Table 1 General information on some major identified SARS-CoV-2 variants/strains
Variant/strain name Country origin
First identified
Mutation sites
Refs. SARS-CoV-2 (Hu-1)
China
December 2019
Wild type
[18, 22, 23]
Alpha (α)
B.1.1.7
20I/501Y.V1
United Kingdom
September 2020 N501Y; P681H; 69/70 deletion
[2, 24]
Beta (β)
B.1.351
20H/501Y.V2
South Africa
October 2020
K417N; E484K; N501Y
[24]
Gamma (γ)
P.1
20 J/501Y.V3
Brazil
November 2020
K417T; E484K; N501Y
[24, 25]
Delta (δ)
B.1.617.2
India
December 2020
T19R; L452R; T478K; D614G; P681R; D960N; 157/158 deletion
[26, 27]
Omicron (O)
B.1.1.529
Bostwana and South Africa November 2021
N440K; G446S; G339D; E484A; A76V; Q493R; Q498R; G496S; T547K;
Y505H; N679K; H655Y; N764K; N856K; D796Y; Q954H; S375F; L981F;
N969K; S371L; L212I; S373P
[28] Table 1 General information on some major identified SARS-CoV-2 variants/strains Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Page 3 of 48 through lysis, exocytosis, or direct budding from the
plasma membrane. parainfluenza, picornaviruses, coronavirus (CoV), ade-
noviruses, and respiratory syncytial viruses. The human
coronavirus (HCoV) has a complex structure, with an
RNA genome inside the nucleocapsid protein, coated
by spike glycoproteins and an envelope on the outer side
(Fig. 1). The viral envelope is composed of structural
membrane containing spike (S), envelope protein (E), and
membrane lipoprotein (M). Modes of action of antiviral agents related to virus
life cycle It
usually blocks viral replication in cell culture form and
prevents the attachment of the mutant virus to the recep-
tor. This blockade effectively inhibits viral replication in Viruses can spread through the stages of their life cycle,
which include cellular entry, translation, replication of
the viral genome, and egress from the host cell to infect
new cells [34]. While interferon (IFN) plays a crucial role
in the host defence against viruses, [32, 35], efforts have
also been focused on disrupting specific stages of the
virus life cycle to inhibit and prevent viral infection. In
particular, disrupting the viral envelope has been iden-
tified as a promising approach to impede viral egress
[36, 37]. The lipid bilayer composition can be disrupted Fig. 1 Schematic of human coronavirus. S spike glycoprotein consisting of S1 and S2 parts, M membrane, E envelope protein, N nucleocapsids
protecting the viral genome Fig. 1 Schematic of human coronavirus. S spike glycoprotein consisting of S1 and S2 parts, M membrane, E envelope protein, N nucleocapsids
protecting the viral genome Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Page 4 of 48 cell cultures and decrease the affinity of the virus to bind
to the receptor, making it less virulent and nonviable [42]. McKinlay et al. [42], suggested a mechanism for block-
ing viral entry into host cells by inhibiting the attachment
of the virus to the host cell receptor. The first step in viral
infection involves the attachment of the virus to the cellu-
lar receptor on the surface of the host cell, with the ACE2
receptor contributing to the attachment of coronavirus to
the host cell, as shown in Fig. 2. The binding pocket of
the virion capsid protein, containing hydrophobic amino
acid side chains, interacts with the hydrophobic domains cell cultures and decrease the affinity of the virus to bind
to the receptor, making it less virulent and nonviable [42]. of the soluble receptor through van der Waals physical
interactions. This interaction dissolves the virion capsid
proteins or viral envelope in the outer space of the host
cell, releasing the virion RNA genome, which becomes
damaged by its surrounding conditions. The virion
genome cannot replicate or maintain its genomic struc-
ture unless inside the host cell. Modes of action of antiviral agents related to virus
life cycle Another mechanism for
blocking the virus from releasing its viral genome dur-
ing the uncoating process of the virion capsid or viral
envelope is by providing inhibitor compounds that act
as chelating agents within a hydrophobic pocket of the McKinlay et al. [42], suggested a mechanism for block-
ing viral entry into host cells by inhibiting the attachment
of the virus to the host cell receptor. The first step in viral
infection involves the attachment of the virus to the cellu-
lar receptor on the surface of the host cell, with the ACE2
receptor contributing to the attachment of coronavirus to
the host cell, as shown in Fig. 2. The binding pocket of
the virion capsid protein, containing hydrophobic amino
acid side chains, interacts with the hydrophobic domains Fig. 2 Coronavirus life cycle starts from entering a host cell (infection) until the production and release of a new virus. The words in the blue state
are the steps of viral transformation inside the host cell. The activation process begins when the viral glycoprotein attaches to hACE2 and TMPRSS2
receptors. The virus fuses into the cell by endocytosis and starts to enter the cell. Following this cellular entry, the virus undergoes an uncoating
process to release its viral genome. Further, structural protein synthesis is followed by RNA packaging, budding, and assembly to form a mature
virion. Exocytosis releases a new infectious virion to infect a new cell. The insert box (on the bottom right) illustrates what happens to the host cell
resulting in further cell death Fig. 2 Coronavirus life cycle starts from entering a host cell (infection) until the production and release of a new virus. The words in the blue state
are the steps of viral transformation inside the host cell. The activation process begins when the viral glycoprotein attaches to hACE2 and TMPRSS2
receptors. The virus fuses into the cell by endocytosis and starts to enter the cell. Following this cellular entry, the virus undergoes an uncoating
process to release its viral genome. Further, structural protein synthesis is followed by RNA packaging, budding, and assembly to form a mature
virion. Exocytosis releases a new infectious virion to infect a new cell. The insert box (on the bottom right) illustrates what happens to the host cell
resulting in further cell death Fig. Vaccines for COVID‑19 A vaccination campaign using emergency-approved vac-
cines is underway in many countries. According to WHO
website, there are current 13 different COVID-19 vac-
cines from four platforms that have been widely released
and administered worldwide. These vaccines include
Pfizer/BioNTech Comirnaty (BNT162b2), Moderna
mRNA-1273, AstraZeneca AZD1222 and Covishield
from the Serum Institute of India, Johnson & John-
son Ad26.COV2.S and Sinopharm COVID-19 vaccine
from China etc. Table 2 also shows two versions of the
AstraZeneca/Oxford COVID-19 vaccine produced by
AstraZeneca-SKBio (Republic of Korea) and the Serum
Institute of India as the ChAdOx1-S vaccine as mention
on WHO website on 15 February 2021 [15]. The Pfizer/
BioNTech COVID-19 (BNT162b2) vaccine is a nucleo-
side-modified mRNA vaccine that utilizes lipid nanopar-
ticles to encode the prefusion SARS-CoV-2 spike protein
[47]. The Ad26.COV2.S vaccine, an alternative to the
ChAdOx1S recombinant vaccine produced by the Serum
Institute of India, is also a recombinant vaccine that uses
a vector to encode the full-length SARS-CoV-2 from
incompetent adenovirus serotype 26 (Ad26) [48].hi p
g
y
[
]
SARS-CoV-2 virus consists of a large membrane gly-
coprotein called the structural protein (S protein), which
includes several proteins such as membrane, spike, enve-
lope, and nucleocapsid. S protein belongs to the class I
viral fusion glycoproteins and is responsible for cell entry. Among the sixteen non-structural proteins (nsp1-16),
three play a crucial role in the replication, transcription,
and host cell recognition processes. These proteins are
chymotrypsin-like protease (3CLpro), papain-like pro-
tease (PLpro), and RNA-dependent RNA polymerase
(RdRp) [33, 39, 43, 44]. Cys-proteases and papain are
protein degrading and processing enzymes, especially
during the translation process. Chymotrypsin protease
(3Cpro) contains Cys-proteases with a sulfhydryl group
that cleaves the glutamine-glycine amide bond. 3CLpro
is a highly conserved protease and plays a vital role in
coronavirus replication by overlapping polyproteins pp
1a (486 kDa) and pp 1ab (790 kDa) in SARS-CoV [31]. Both PLpro and 3CLpro are necessary for replication,
transforming polyproteins into non-structural proteins
such as RdRp and helicases. 3CLpro and PLpro contain
11 and 3 cleavage sites, respectively. Therefore, 3CLpro
is also known as the main protease (Mpro) and an ideal
target for developing antiviral drugs [45]. 3CLpro is a
highly conserved protease, and its substrate specificity
is similar to the 3Cpro of the main picornavirus. Pro-
tease inhibitors can block the proteolytic process of viral
polyproteins, leading to incorrect viral replication and
transcription. Modes of action of antiviral agents related to virus
life cycle 2 Coronavirus life cycle starts from entering a host cell (infection) until the production and release of a new virus. The words in the blue state
are the steps of viral transformation inside the host cell. The activation process begins when the viral glycoprotein attaches to hACE2 and TMPRSS2
receptors. The virus fuses into the cell by endocytosis and starts to enter the cell. Following this cellular entry, the virus undergoes an uncoating
process to release its viral genome. Further, structural protein synthesis is followed by RNA packaging, budding, and assembly to form a mature
virion. Exocytosis releases a new infectious virion to infect a new cell. The insert box (on the bottom right) illustrates what happens to the host cell
resulting in further cell death Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Page 5 of 48 virus. This approach fills the empty pocket of the virus,
preventing extensive conformational shifts that could
cause the virion to disassemble. Therefore, chelating
agents, such as a divalent cation, may block the release of
RNA from the capsid. physiological renin-angiotensin system by hydrolyzing
vasoconstricting angiotensin II into vasodilating angio-
tensin. During severe COVID-19, ACE2 expression is
downregulated, leading to inflammation or cytokine
storm and an increase in interleukins and other stimu-
lating factor proteins. Therefore, modulating ACE2
expression may be a potential strategy for controlling
COVID-19 symptoms. Most inhibitors target the glycoprotein of the virion
capsid or viral envelope, inhibiting viral attachment
to the receptor. However, positive-stranded viral RNA
is translated once the virus has attached and entered
the host cell. The viral genomic RNA is released from
the viral capsid through an uncoating mechanism,
often facilitated by a receptor such as ACE2. Inside the
host cell, viral proteins replicate and produce new ones
through mRNA synthesis (Fig. 2). The virus strain, host
cell type, pH, temperature, and multiplicity of infection
influence this process. Typically, a virus requires five to
ten hours to replicate in a single cycle [31]. Vaccines for COVID‑19 The significant advancement in vaccine develop-
ment has allowed for the production of effective vac-
cines against SARS-CoV-2 and the development of herd
immunity within the community. However, drugs and
therapeutic actions are still necessary to manage and
treat COVID-19 cases. Li et al. (2022), and Rehman et al. (2021), reported that majority of the vaccines recently
developed use novel techniques such as messenger RNA
(mRNA) to stimulate the human immune system [49, 50]. The techniques for applying messenger RNA (mRNA)
as a recognizing and reactive component of the virus to
create the immune system of the human body have been
developed as a significant advancement in public health
and vaccine development. Instead of containing a weak-
ened or inactive form of the virus, these vaccines rely on
the immune system of the body to recognize and attack
the spike protein of the virus. After vaccination, vac-
cine particles interact with immune system cells, which
deliver the mRNA message to create the spike protein in
vaccinated cells. The immune system then recognizes the
spike protein as foreign and produces antibodies for its Drugs targeting SARS-CoV-2 spike protein can prevent
membrane fusion between the spike and virus, thereby
disrupting virus entry into the host cell. The spike pro-
tein also contains SARS-CoV-2 RNA-dependent RNA
polymerase (RdRp), which recognizes the ACE2 recep-
tor [33, 46]. ACE2 is a type I transmembrane metal-
locarboxypeptidase that plays a crucial role in the Page 6 of 48 Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Table 2 List of some approved vaccines
Vaccine
Vaccine platform
Effectivity
SinoVac vaccine (Coronavac)
Inactivated virus
50% effective against P.1 in Brazil [24]; 96.8% efficacy
against COVID-19 in Indonesia [51]
Covaxin BBV152 (Bharat Biotech-Indian Council of Medi‑
cal Research)
Inactivated virus
81% interim efficacy in preventing COVID-19 (SARS-CoV-2
original variant) [52]; 78% effective against the double
mutant variant [24]; third dose neutralizes antibody
responses against β and Omicron variants (14.70
and 18.53 fold, respectively) [53]
ChAdOx1-S-/AZD1222 (AstraZeneca/University
of Oxford)
Viral vector (non-replicating)
70.4% efficacy against α variant [54]. Vaccines for COVID‑19 60–70% effi‑
cacy against ancestor and B.1.1.7 variants in UK, Brazil
and South Africa, but did not protect against B.1.351
variant [55]
Ad26.COV2.S (Janssen Pharmaceutical)
Viral vector (non-replicating)
Protects over 80% of Syrian hamster and non-human pri‑
mate SARS-CoV-2 infection models [56]; but 59% effective
against COVID-19 hospitalization [57]
SARS-CoV-2 rS (Novavax)
Protein subunit
95.6% effective against SARS-CoV-2 wild type; 85.6%
and 60% effective against α and β variants [58]
CVnCoV vaccine (Curevac AG)
Nucleic acid vaccine (RNA based) Low efficacy [59], up to 47% efficacy against SARS-CoV-2
[60]
mRNA-1273 (Moderna-NIAID)
Nucleic acid vaccine (RNA based) 94% efficacy in preventing COVID-19 illness [61]
BNT162b2 (3LNP-mRNAs) (Pfizer/BioNTech)
Nucleic acid vaccine (RNA based) 95% efficacy against COVID-19 [62, 63] 50% effective against P.1 in Brazil [24]; 96.8% efficacy
against COVID-19 in Indonesia [51] 81% interim efficacy in preventing COVID-19 (SARS-CoV-2
original variant) [52]; 78% effective against the double
mutant variant [24]; third dose neutralizes antibody
responses against β and Omicron variants (14.70
and 18.53 fold, respectively) [53] instance, Sinovac vaccines have been linked to deafness
and cerebral venous thrombosis [66, 67]. Janssen Phar-
maceutical R&D team has reported that booster shots
enhance immunity and maintain a safety profile of rela-
tively 93.7% efficacy in the US [68]. However, the vac-
cine has been associated with rare adverse effects such
as thrombocytopenia [69] and acute myocarditis [70]. The AstraZeneca vaccine has also been associated with
adverse reactions such as thrombosis and blood clots
[71–73]. Despite this, the positive benefit-risk profile
of the vaccine and its tremendous potential to prevent
infections and reduce deaths worldwide have outweighed
the adverse effects, and it continues to be used in the
public domain. Based on previous studies, Curevac,
which uses an unmodified RNA-based vaccine, has low
efficacy [59], with an efficacy rate of only 47% against
SARS-CoV-2 [60]. Other modified mRNA-based vac-
cines such as Moderna and Pfizer have demonstrated sig-
nificant efficacy and have been approved for emergency
use during COVID-19 pandemic. In addition, mRNA
vaccines are straightforward to manufacture, have a high
biosafety profile, and are a safer vector than DNA, with
no chance of infectious viruses [74, 75]. The development
of modified mRNA-based vaccines has garnered wide-
spread support. destruction. This process generates an immune response
that continues until all spike proteins have been elimi-
nated, enabling the immune system to fight the virus
upon infection. Repurposing antiviral agents as a potential way
of drugs discovery for COVID‑19 Several studies have been working to discover drugs to
combat coronavirus using various methods. Some of
these proposed drugs were discovered through in silico
studies involving bio- and immuno-informatics, while
others were discovered using conventional organic syn-
thetic chemistry based on the retrosynthetic method [76,
77]. However, all proposed drugs must undergo preclini-
cal and clinical testing, including a series of safety and
health considerations, before they can be released com-
mercially. These steps can take over five years to assess
the safety of a drugs for consumption. Based on the
emergency and pandemic nature of COVID-19, WHO
has authorized health sectors and scientists to openly
communicate their results and clinical trial assessments
for new drugs to combat the virus. Baicalin and baicalein from Scutellaria baicalensis
Georgi have been proposed as potential treatment for
COVID-19 due to their inhibitory effects on the activity
of HIV-1 reverse transcriptase, which blocks HIV-1 rep-
lication [94, 97, 98]. Since HIV is also an enveloped virus
like SARS-CoV-2, it is hypothesized that baicalein may
also inhibit COVID-19. Su et al. [103] investigated the
effects of baicalin and baicalein against SARS-CoV-2 in
silico study. The results showed that baicalein interacted
with the two catalytic residues of SARS-CoV-2, acting as
a shield to prevent further interaction with the substrates
or receptors of human cells.h The approach of repurposing drugs based on the nature
similarity of the virus is considered a promising tech-
nique to identify potential treatment for COVID-19. This method is viewed as a faster alternative to develop-
ing new drugs since existing medicines that have been
approved as safe for use are repurposed for COVID-19
treatment, thereby eliminating the need for additional
safety assessments. Consequently, this approach saves
time and expedites drugs release to the public. In light of the health concerns surrounding the use of
drugs to treat COVID-19 and the time required for their
clinical assessment, an approach to repurpose or reposi-
tion existing drugs that have previously been recognized
as effective antiviral was proposed. This approach is
based on certain criteria, including the similarity of the
virus type or group, genomic composition, and structure. Examples of some drugs that have been repurposed in
this way are shown in Fig. 3, Table 3.h The strategy of repurposing drugs is crucial in respond-
ing to the emergence of new variants of SARS-CoV-2,
which result from natural mutation and evolution of the
virus. Vaccines for COVID‑19 The COVAX initiative, under the auspices of WHO,
has facilitated the development and manufacturing of
COVID-19 vaccines, aiming to ensure equitable access
worldwide. In order to meet the demands of the pan-
demic, all recommended drugs and vaccines for SARS-
CoV-2 were assessed based on the Emergency Use Listing
(EUL) procedure, which ensures safety, efficacy, and qual-
ity standards. The EUL relies on a rigorous evaluation of
late-phase II and phase III clinical trials, which are inde-
pendently reviewed by WHO experts and teams. WHO has emphasized that a vaccine on its own will
not end the pandemic [64]. Despite the progress of the
vaccination program, numerous cases associated with
it have been reported, and doubts remain regarding the
its long-term efficacy. The emergence of new coronavi-
rus strains has become a significant challenge that needs
to be addressed promptly. Additionally, the preparation
required for administering two doses of the vaccine and
booster shots is a significant task that medical and health
services, pharmaceutical industries, and governments
must fulfil [65]. i
Alerts on several medical products have been issued to
the following release of COVID-19 vaccines to increase
the public awareness of drugs and vaccine safety. Some
rare adverse events related to vaccine use have been
reported to inform individuals in making informed
decisions about enhancing their immune systems. For As the public becomes increasingly aware of the safety
and efficacy of drugs and vaccines, new natural-based
alternatives are being explored. Despite the lack of spe-
cific drugs to cure COVID-19, recommendations for
treating the disease have emerged from informal trials,
including traditional herbal medicine. This has led to a Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Page 7 of 48 renewed interest in repurposing or repositioning drugs,
including natural products such as medicinal plants and
some commercial synthetic drugs that have previously
shown antiviral activity. as drugs for COVID-19. For instance, resveratrol has
been proven to reduce inflammation and levels of inter-
feron-gamma (IFN-γ) in human RSV A2-strain virus
infections. This was demonstrated in in vitro assays
using 9HTEo and Hep-2 cell lines, as well as in vivo
assays using BALB/c nude mice [89, 90]. Therefore, res-
veratrol is a promising candidate for treating COVID-
19 infections. Repurposing antiviral agents as a potential way
of drugs discovery for COVID‑19 The high levels of viral transmission have led to
the emergence of virus variants associated with increas-
ing viral transmissibility but not disease severity. Clini-
cally tested drugs and vaccines should also cover variant
B.1.351, which has been associated with reduced efficacy
of some previously recommended ones. Therefore, the
scientific response to the rising number of new SARS-
CoV-2 variants must adapt quickly to develop practical
antiviral activity against these emerging variants. WHO
reported that efforts to suppress transmission, protect
the vulnerable, and save lives in a comprehensive and
coordinated manner needs to be redoubled in response
to the welcoming of new variants of SARS-CoV-2 in
2021. Several new variants of SARS-CoV-2 were identi-
fied after whole-genome sequencing in samples from
Brazil (SARS-CoV-2 (P1) derived from B.1.1.28 line-
age, the United Kingdom (SARS-CoV-2 VUI 202012/01,
some listed as SARS-CoV-2 VOC 202012/01 from clus-
ter B.1.1.7 lineage), and South Africa (501Y.V2 variant
as an N501Y mutation) at the end of 2020 and into the
following year. Therefore, the acceleration of access to The phylogenetic tree can group viruses that share sim-
ilar characteristics based on their genomic composition
and structure. HCoV is a member of the Coronaviridae
group, an RNA virus that causes respiratory tract infec-
tions. This means that HCoV viruses have close relation-
ships with each other, as shown in a phylogenetic tree
analysis. SARS-CoV-2, as a member of this group, has
an 88% similarity in identity to two bat-derived SARS-
like coronavirus [20, 21]. Additionally, the viral structure
can be used as a critical factor in grouping the virus. For
example, SARS-CoV-2 has an enveloped viral design,
similar to herpes simplex virus (HSV), human immu-
nodeficiency virus (HIV), retrovirus, flavivirus, and
hepatitis B and C virus (HBV/HCV). On the other hand,
non-enveloped structures are found in human papilloma-
virus (HPV), poliovirus, norovirus, and rhinovirus. Res-
piratory tract infections can be caused by viruses such as
rhinoviruses, influenza, parainfluenza, respiratory syncy-
tial virus (RSV), enteroviruses, coronavirus, and certain
strains of adenovirus. Antiviral agents previously used to treat respiratory
tract diseases are potential candidates for repurposing Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Page 8 of 48 Fig. 3 Some compounds acting as antiviral agents have similarities in the type, genomic composition, and structure of SARS-CoV-2 [78–80, 87, 88] vaccination campaigns worldwide and the development
of drugs discovery are supported. even proposed as chemoprophylaxis in some countries,
with trials conducted in 2020 [111]. In June 2020, WHO
announced on their website that hydroxychloroquine
should be discontinued as it did not reduce mortality in
hospitalized COVID-19 patients [112–114]. Despite this,
controversy over the use of hydroxychloroquine remains,
and some countries continue to use it as a COVID-19
treatment [104, 111, 115, 116]. It may be a promising
candidate for further investigation as a treatment for
SARS-CoV-2 [117]. Ivermectin has also demonstrated
antiviral activity against SARS-CoV-2 in in vitro studies,
with an IC50 of approximately 2 μM [118, 119]. Chaccour Repurposing of existing drugs has led to individuals or
groups using commercial antiviral agents, such as chlo-
roquine or hydroxychloroquine and ivermectin, without
medical prescription. Scientific studies have shown that
these FDA-approved drugs, initially developed as antivi-
ral and antiparasitic agents, have potential in inhibiting
SARS-CoV-2 in in vitro and in silico assays [105–107]. While the efficacy and risks associated with the use
of chloroquine and hydroxychloroquine in COVID-
19 treatment have been debated [108–110], they were Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Page 9 of 48 Table 3 Antiviral Drugs or Compounds against Viruses have Similarities in the Type/Group, Genomic Composition, and Structure of SARS-CoV-2
Viruses
Compounds
Plant Sources
Assays
Activities
Refs. Human coronavirus type
Human coronavirus strains OC43
(HCoV-OC43)
Tetrandrine (TET)
Fangchinoline (FAN)
Cepharanthine (CEP)
Stephania tetrandra
In vitro: in MRC-5 cells
IC50 of TET = 0.33 ± 0.03 μM;
FAN = 1.01 ± 0.07 μM;
CEP = 0.83 ± 0.07 μM
[91]
HCoV-OC43
MERS-CoV
HCoV-NL63
MHV-A59
Lycorine (standard)
Amaryllidaceae
In vitro: in BHK-21, Vero E6, LLC-MK2,
DBT cells
In vivo: in mice against HCoV-OC43
EC50 = 0.15 – 1.63 μM
[78]
Coronavirus MHV-A59
Essential oils of the ethanol extracts
(Ah extract) containing carvacrol
(38.4%) and α-pinene (30.9%)
Anthemis hyaline (Ah)
In vitro using quantitative analysis
by enzyme-linked immunosorbent
assay (ELISA) in HeLa-CEACAM1a
cells
After 6 and 8 h post infections,
no detected virus was evaluated
with TCID50 values at 1/10 dilution
of Ah extracts
[79]
HCoV-229E
Saikosaponin B2
Bupleurum spp., Heteromorpha spp.,
Scrophularia scorodonia
In vitro using XTT assay
IC50 = 1.7 ± 0.1 μmol/L
[92, 93]
Genomic similarities
SARS-CoV strains 39,849
Baicalin
Scutellaria baicalensis (Huang Qin)
In vitro antiviral susceptibility test‑
ing on fRhK4 and Vero-E6 cell lines
EC50 in fRhK4 and Vero-E6 cell
lines = 12.5 and 100 μg/mL at 48 h
[94]
Recombinant SARS-CoV PLpro
Papyriflavonol A (prenylated
quercetin derivative)
Broussonetia papyrifera (dried roots)
In vitro using viral protease inhibi‑
tion assay on SARS-CoV based
on the FRET method
IC50 = 3.7 μM
[80]
MERS-CoV
Resveratrol
Vitis vinifera (grape), Polygonum
cuspidatum (Huzhang), Vaccinium
macrocarpon (cranberry)
In vitro using MTT assay, NRU (neu‑
ral red uptake) assay, and plaque
reduction assay in Vero E6 cells
Resveratrol reduced cell death
in a range concentration of 250–
125 μM for 48 h after infection
[81]
SARS-CoV 3CLpro
Curcumin
In vitro using FRET method
IC50 40 μM
[95]
SARS-CoV 3CLpro
Savinin
Chamaceyparis obtusa var. for-
mosana
In vitro using FRET method
IC50 25 μM
Ki = 9.1 ± 2.4 μM
[95]
SARS-CoV 3CLpro and PLpro
Xanthoangelol E
Angelica keiskei (Miq.) Koidz (etha‑
nolic leaf extract)
In vitro using cell-free based assay
IC50 of 11.4 and 1.2 μM
[82]
SARS-CoV PLpro
Coumestrol, isobavachalcone,
and psoralidin
Psoralea corylifolia L. (ethanol
extract of the seeds)
In vitro using the fluorogenic sub‑
strate Ub-AMC
IC50 of 4.2; 7.3 and 10.1 μM
[96]
Recombinant SARS-CoV 3CLpro
Dieckol
Ecklonia cava (brown algae)
In vitro using FRET method (for cell-
free transcleavage assay) and lucif‑
erase activity (for Vero cell-based
cis-cleavage assay)
IC50 of trans- and cis- cleavage
inhibitory: 2.7 and 68.1 μM, respec‑
tively
[83]
Enveloped viruses
HIV-1
Baicalin and baicalein
Scutellaria baicalensis Georgi
In vitro using ELISA on fresh normal
peripheral blood mononuclear cells
(PBMC)
IC50 = 0.5 μg/mL
[97]
HIV-1
Baicalin
Scutellaria baicalensis Georgi
In vitro using quantitative colori‑
metric assays
IC50 = 4 μM
[98] Page 10 of 48 Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Table 3 (continued)
Viruses
Compounds
Plant Sources
Assays
Activities
Refs. Murine cytomegalovirus (MCV)
Black seed oil (BSO) or habatus‑
saudah
Nigella sativa
In vivo using a viral plaque-forming
assay of BALB/c mice spleen
and liver
Undetected virus at the ratio
of the effector to target cells
was 20:1
[99]
HSV-1 and HSV-2
Ethanol extracts of flower buds of E. caryophyllus containing eugenol
Eugenia caryophyllus (Spreng.)
Bullock & S.G. Harrison
In vitro using plaque reduction
assay on green monkey kidney
(GMK)
ED50 against HSV-1 and HSV-2: 72.8
and 74.4 μg/mL
[84]
HSV-1
Isoborneol
Salvia fruticosa
In vitro using viral plaque assay
on Vero cells
0.1% isoborneol inactivated 86%
of the infectious virus within 30 min
[85]
HSV-1
Star anise oil (SAO) contains trans-
anethole (80%), eugenol, b-caryo‑
phyllene, eugenol
Illicium verum (star anise)
In vitro using plaque reduction
assay
IC50 SAO = 1 ± 0.1 μg/mL
IC50 beta-caryophyl‑
lene = 0.25 ± 0.0 μg/mL
[86]
Hepatitis B Virus (HBV)
Phyacidusin B and phllanthacidoid
A1
Phyllantus acidus (stem)
In vitro using the cytopathic end-
point assay in HepG2.2.2.15 cells
IC50 of HBsAg is 11.2 ± 0.01 μM
by Phyacidusin B and HBeAg
is 57.1 ± 0.02 μM by phllanthacidoid
[100]
HSV-1 (F strain ATCC VR733)
J. oxycedrus berries oil containing
α-pinene, β-myrcene
Juniperus oxycedrus ssp. In vitro, using visually scoring
of the virus-induced cytopatho‑
genic effect (CPE) for 72 h post-
infection on Vero cells
IC50: 200 μg/mL; SI of 5
[101]
Respiratory diseases
Influenza virus A/Germany/27,
str. Weybrigde (H7N7) and A/
Germany/34, str. Rostock
(H7N1)
(-)-thalimonine (Thl)
Thalictrum simplex L (aerial parts)
In vitro in cell cultures of chicken
embryo fibroblasts
Inhibit viral reproduction at non-
toxic concentration 0.1–6.4 μM
with a selectivity index = 640
[87]
Influenza type A (A/
Betezda/63/10/H2N2) and type B
(B/Lee/40)
Essential oil from fruits contain‑
ing pinene, limonene, a complex
of ethers of octanol and hexanol
Heracleum L species, such as H. aco-
nitifolium Woronow. H. antasiaticum
Manden., etc
In vivo using intrasanal and oral
treatments on mice
LD50 of 0.2–0.4 mL
[102]
Influenza A/PR/8/34 (PR8) virus
(H1N1 subtype)
Cinnamaldehyde (CA)
Cinnamomi cortex
In vitro using plaque reduction
assay on MCDK cells
In vivo based on therapeutic effi‑
cacy in mice
CA inhibits all of the virus growth
at 200 μM. Application of CA
in the airways led to the significant
rescue of infected mice
[88] Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Page 11 of 48 Page 11 of 48 et al. conducted randomized clinical trials on the use
of ivermectin as a COVID-19 treatment [120]. WHO
(2021c), recommends that ivermectin should only be
used in COVID-19 treatment based on clinical trials as
the evidence supporting its efficacy is inconclusive [121]. Doxycycline has also been investigated for its ability to
inhibit SARS-CoV-2 in in vitro studies, with an EC50 of
4.5 ± 2.9 mM when tested on the IHUMI-3 strain in Vero
E6 cells [122].h that stops RNA synthesis and acts as a false substrate for
RdRp, is one of the primary drugs used to treat hospital-
ized patients [125, 127, 128]. It is important to note that
the effectiveness of these drugs in treating COVID-19 is
still being evaluated. In December 2021, Pfizer released a new oral drugs for
COVID-19 called Paxlovid, which contains nirmatrelvir
(300 mg) and ritonavir (100 mg) [129–132]. Adminis-
tered orally twice daily for five days, Paxlovid has dem-
onstrated a significant reduction in COVID-19-related
deaths [133]. Nirmatrelvir (PF-07321332) has shown oral
activity against SARS-CoV-2 in vitro, and its potency was
demonstrated in phase I clinical trials with a tolerable
plasma concentration in the cell [134, 135]. The detailed
computational analysis of PF-07321332 against SARS-
CoV-2 Mpro has been clearly discussed in several stud-
ies [136, 137]. A combina-
tion of remdesivir and baricitinib, as immunotherapeutic
agents, produces better outcomes in hospitalized patients
with COVID-19 than the use of only remdesivir. The use
of two antiviral drugs, remdesivir and dexamethasone,
has resulted in reduced mortality for 30 days. The use of
combined antiviral and antibiotic therapies has also been
proven to be more effective and safer for early sympto-
matic patients [154].h Rupintrivir (AG7008) are other drugs being investi-
gated for its potential to treat COVID-19. It contains
a lactam ring that mimics Glutamine residues at the
P1 position and forms a covalent bond with the active-
site cysteine residue of the virus protease [143]. In vitro
antiviral assays using H1-HeLa and MRC-5 cells have
shown Rupintrivir to have a potent broad-spectrum anti-
viral activity against 48 HRV serotypes and four related
picornaviruses [144]. Ramajayam et al. has proven that
the fluorophenylalanine group and isoxazoyl moiety in
rupintrivir may hinder its ability to bind to Arg188 in
the S2 pocket and hydrophobic residues of SARS-CoV
3CLpro, respectively [143]. Therefore, its efficacy in
treating COVID-19 is still being studied in detail. A simi-
lar case has also been observed with amantadine, which
was previously used to treat influenza. In vitro studies
have shown an inhibitory effect of amantadine on SARS-
CoV-2 infected Vero E6 cells with an IC50 between 83 and
119 μM [145]. The dosage required for in vitro efficacy
is not feasible in vivo due to toxicity concerns. Its thera-
peutic window cannot be offered, suggesting that the oral
administration of amantadine appears obsolete. Several
studies reported that amantadine could be administered
through inhalation, as the infection of human airways
by SARS-CoV-2 covers a high concentration in the nasal
epithelium until distal pulmonary epithelium [146]. The application of repurposed drugs has yielded some
promising examples of inhibitors for SARS-CoV-2. Sev-
eral studies have identified available drugs agents that
can inhibit the protein and reproduction cycles of viruses
[155–157]. In addition, clinical trials conducted on April
2020 have shown that the combination of natural prod-
ucts, such as honey, and Nigella sativa seeds, improved
symptoms and reduced mortality without adverse effects
[158]. The Ayurvedic drugs, AYUSH 64, demonstrated
improved recovery and reduced hospitalization for mild-
moderate symptomatic patients [159]. The potential of
these natural compounds as alternative COVID-19 drugs
and therapeutic agents is described in detail in the fol-
lowing section of this study. In October 2021, Pfizermectin, new drugs
for COVID-19 treatment suspected to contain iver-
mectin, was also developed by Pfizer, but the company
has denied repackaging ivermectin inside this new pill
and selling it at a higher price than existing commercial The use of some commercial medicines has been
approved by health and medical ministries to treat
COVID-19 (Fig. 4). WHO is conducting solidarity thera-
peutic trials in over 30 countries, enrolling nearly 12,000
patients, to find effective treatment for the disease. How-
ever, after six months of attempting these trials, WHO
reported in October 2020 that remdesivir, hydroxychlo-
roquine, lopinavir or ritonavir, and IFN regimens showed
little or no effect on patients hospitalized for 28 days. Other drugs, including oseltamivir, azvudine, ribavirin,
favipiravir, and auranofin, have been recommended off-
label [106, 123–126]. Remdesivir, an adenosine analogue Fig. 4 Some commercial drugs have been recognized and approved to treat COVID-19 patients Fig. 4 Some commercial drugs have been recognized and approved to treat COVID-19 patients Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Page 12 of 48 Page 12 of 48 ivermectin. It was suspected of containing ivermectin, a
protease inhibitor proposed to kill parasites. Molnupira-
vir has been shown to successfully target the viral RdRp
on the Omicron variant [138–140]. In early 2022, WHO
recommended two new drugs for COVID-19 patients,
baricitinib and sotrovimab [141], while other monoclonal
antibodies, such as bebtelovimab and Evusheld (contain-
ing tixagevimab and cilgavimab), have also gained atten-
tion due to their high efficacy against the virus at mild to
moderate levels [142]. [150, 151]. In addition, 25-hydrocholesterol is oxidized
cholesterol products found in human peripheral blood,
cerebrospinal fluid, colostrum, and milk. Several studies
are considering 25-hydroxycholesterol and 27-hydroxy-
cholesterol, which are side-chain oxysterols, as potential
inhibitors of respiratory viruses against COVID-19 [152]. Clinical trials have been conducted using a drugs
repurposing approach, either with a single-molecule
therapy or a combination of therapies, to treat COVID-
19. However, one of the studies involving 1206 rand-
omized patients showed no improvement in the recovery
of mild to moderate COVID-19 patients using a single
treatment of ivermectin [153]. Combination therapies
involve the simultaneous repurposing of therapeutic,
antiviral, immunotherapeutic, and convalescent plasma
therapies. Remdesivir is popular antiviral drugs that has
received emergency approval from WHO. IFN are signaling proteins produced by host cells
that have shown therapeutic potential for MERS and
SARS-CoV [35, 147], making it a proposed treatment
for COVID-19. The EC50 of IFN-α and IFN-β treatment
on infected SARS-CoV-2 Vero cells is reported to be
1.35 IU/mL and 0.76 IU/mL, respectively [148]. In addi-
tion, glucocorticoids such as ciclesonide, dexamethasone,
betamethasone, hydrocortisone, fludrocortisone, and tri-
amcinolone are potential candidates for treating inflam-
mation accompanying COVID-19 [149]. Other therapies
such as convalescent plasma and anti-interleukin-6 (anti-
IL-6) inhibitors have also been explored to combat the
pandemic. In‑silico study on drugs discovery for COVID‑19 In‑silico study on drugs discovery for COVID‑19
Modern drugs discovery relies on in-silico studies involv-
ing molecular docking and dynamics. This approach uses
bioinformatics and computational modelling to design
new lead compounds and enable virtual screening of bio-
active metabolites [160]. However, by enabling prelimi-
nary screening activities, this technology accelerates the
identification and analysis of bioactive compounds, while
significantly reducing time and costs associated with lab-
oratory work. Molecular modelling is particularly useful
for repurposing existing drugs and natural products, as
it predicts the affinity and binding mode of molecules to
the active site of a receptor protein. Investigations have proven that 25-hydrocholesterol,
a type of oxidized cholesterol products found in various
human body fluids, has the potential to inhibit COVID-
19 with an IC50 of 550 nM by blocking membrane fusion Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Page 13 of 48 Page 13 of 48 Page 13 of 48 Computer modelling enables the efficient screening of
hundreds and thousands of compounds, in experiments
conducted for a brief period. Promising compounds or
drugs identified during the process are further subjected
to docking studies (Table 4, Fig. 5). In order to under-
stand the mode of action of these compounds or drugs,
molecular dynamics (MD) simulations were used to
model their interaction with the active site of the virus
[161–163]. However, through artificial neural network
analysis, medicines are classified based on their primary
role in SARS-CoV-2 infection, specifically in viral replica-
tion and immune response. This approach differentiates
between antiviral agents that prevent virus replication
and those modulating immunity to combat the virus without overreacting. Once a potential antiviral candi-
date is identified, further assays using human and non-
human cell lines are necessary. Viral entry is prevented by targeting the host recep-
tor, ACE2 and other proteins parts of coronavirus, such
as spike glycoproteins (including nsp1-16, RdRp) and
proteases (Mpro and PLpro), and has been explored as
a potential strategy [33, 157, 179–181]. In-silico investi-
gations provide valuable parameters, including RMSD,
docking scores, and binding affinities, to assess the effec-
tiveness of antiviral agents. A notable example of applying the in-silico approach
for drugs discovery is the study conducted by Elinger
et al. [182]. In‑silico study on drugs discovery for COVID‑19 They successfully generated a small set of Table 4 Antiviral drugs/compounds that have been assessed using in silico approach against SARS-CoV-2 proteins
Compounds
Antiviral activities
Molecular docking tools
Refs. 40 triterpenoids, flavonol glycosides, antho‑
cyanidins
SARS-CoV-2 Mpro (pdb id: 6LU7), RBD (pdb
id: 6M0J), RdRp (YP_009725307.1), human
trans-membrane serine protease 2 TMPRSS2
(NP_001128571.1)
AutoDock Vina
[164]
51 alkaloids, terpenoids, polyphenols, peptides
SARS-CoV-2 Mpro (pdb id: 6LU7)
AutoDock 4.2.0
[165]
19 hydrolyzable tannins
SARS-CoV-2 Mpro (pdb id: 6Y84)
Molecular Operating Environment (MOE 09)
[166]
24 natural plant-based compounds, 22 antivi‑
ral drugs, 16 anti-malarial drugs
SARS-CoV-2 Mpro (pdb id: 6LU7)
Virtual screening followed with SP and XP
docking modes using GLIDE module
MD simulations using GROMACS-2019
[167]
Four tropane alkaloids from Schizanthus por-
rigens
SARS-CoV-2 PLpro (pdb id: 6WX4)
Autodock Vina
Molecular dynamic simulations using NAMD
v.2.14
[168]
21 flavonoids
SARS-CoV-2 Mpro (pdb id: 6YNQ)
AutoDock Vina and Swiss dock
Molecular dynamic simulations using CABS
Flex 2.0
[169]
66 active flavonoids were selected from 2030
natural compounds
SARS-CoV-2 Mpro (pdb id: 6LU7)
GLIDE module
[170]
80 flavonoids
SARS-CoV-2 Mpro (pdb id: 6LU7)
Molegro Virtual Docker 7
[171]
23 flavonoids and 25 indole chalcones
SARS-CoV-2 Mpro (pdb id: 6YB7), RdRp (pdb
id: 6M71), spike protein (pdb id: 6LZG)
AutoDock Vina v.1.1.2
[172]
458 flavonoids
SARS-CoV-2 Mpro (pdb id: 6LU7), RdRp (pdb
id: 6M71), spike protein (pdb id: 6VW1)
AutoDock 4.1
[173]
12 triterpenoids isolated from Calendula
officinalis L
SARS-CoV-2 Mpro (pdb id: 6LU7)
MOE 2019 Suite
Molecular dynamic simulation using
GROMACS-2019
[174]
14 limonoids and terpenoids
SARS-CoV-2 RBD (pdb id: 6M0J)
AutoDock 4.2
Molecular dynamic simulation using Des‑
mond MD System
[175]
218 coumarins
SARS-CoV-2 Mpro (pdb id: 6LU7), viral methyl‑
transferase (nsp16/10 complex, pdb id: 6W4H),
RBD (pdb id: 6M0J), human ACE2 (pdb id:
6VW1)
AutoDock Vina
[176]
78 secoiridoids
SARS-CoV-2 Mpro (pdb id: 6LU7), and S protein
(6LZG)
AutoDock Vina
Molecular dynamic simulation using Des‑
mond MD System
[177]
6 phenyl propanoids
SARS-CoV-2 Mpro (pdb id: 6Y2F); PLpro (pdb
id: 6WX4); RdRp (pdb id: 6M71)
Genetic optimization of ligand docking (GOLD
v5.2.2)
Groningen machine for chemical simulations
(GROMACS v5.15)
[178] Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Page 14 of 48 However, in another study, the
molecular interactions and stabilities of 3,639 drugs from
the SuperDRUG2 database were analyzed using PyRx
and GROMACS v5.1.5. It was observed that colchicine
emerged as the top binding compound against SARS-
CoV-2 Mpro [204]. The
molecular
docking-based
virtual
screening
approach using AutoDock Vina was employed to iden-
tify potential inhibitors of 3CLpro of SARS-CoV-2 [200]. The top four compounds were selected from a pool of
2000 compounds in the ZINC database, based on their
low free energy binding adherence to the Lipinski rule
of five, and functional molecular interactions with the
target protein. Similarly, Barage et al. utilized AutoDock
Tool 1.5.6 to retrieve 3277 compounds from the ZINC
database and generate 10 top compounds with the low-
est binding energy against RdRp (PDB ID: 6NUR) and
Nsp15 (PDB ID: 20ZK). MD simulations performed with
GROMACS tools, was used to identify three compounds
with the highest affinity to interact with RdRp and Nsp15
namely alectinib, naldemedine, and ergotamine [201]. Sharma and Kaur investigated the potential of jense-
none, a key component of eucalyptus oil, as an inhibi-
tor for COVID-19 infection [205]. The in-silico study
revealed that jensenone formed a complex structure with
the main viral proteinase/chymotrypsin-like protein-
ase (Mpro) through hydrophobic, hydrogen bonds, and
strong ionic interactions. Paul et al. explored synthetic
molecules, peptidomimetic, and small molecules inhibi-
tors targeting viral proteinases to assess its potentials as
anti-SARS-CoV Mpro agents through computational
approaches [206]. Another study by J. K. R. da Silva et al. [207], investigated the potential of 171 essential oil com-
ponents in treating SARS-CoV-2 using molecular dock-
ing analysis. The findings showed that (E)-β-farnesene
exhibited the best normalized docking score, while (E,E)-
α-farnesene, (E)-β-farnesene, and (E,E)-farnesol were
identified as the best docking ligands. Unfortunately,
the docking energies were relatively weak, limiting their
applicability to coronavirus interactions. Potential components of Ayurvedic medicinal plants
have been assessed for their repurposing possibility as
anti-COVID-19. After screening selected compounds
from twelve medicinal plants, molecular docking and
dynamic simulations showed that curcumin, gingerol,
and quercetin were potential candidates [202]. In another
study, fluoro-substituted heterocyclic ring systems were
added to quercetin-based derivatives, which were then
screened by in silico experiments against SARS-CoV
3CLpro (PDB ID: 6LU7) using Autodock 4.2 software. The compounds L4 (5-fluoro-2H-1,2,3-triazol-4-yl), L8
(2-fluoro-4H-1,3-oxazin-4-yl), and L14 (3-fluoropiperi-
din-4-yl) showed promising results, with IC values of
0.330, 0.456, and 0.50 uM, respectively. Fig. 5 Interesting compounds with higher activities against SARS-CoV-2 proteins in terms of lower binding affinity or Docking score in rece
papers [34, 164, 165, 186–199] fi Fig. 5 Interesting compounds with higher activities against SARS-CoV-2 proteins in terms of lower binding affinity or Docking score in recent
papers [34, 164, 165, 186–199] Fig. 5 Interesting compounds with higher activities against SARS-CoV-2 proteins in terms of lower binding affinity or Docking score in recent
papers [34, 164, 165, 186–199] Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Page 15 of 48 related drugs that exhibited significant activity in terms
of IC50. A primary screening assay [183] of 5632 com-
pounds was tested for their ability to inhibit SARS-CoV-2
in human epithelial colorectal adenocarcinoma cells
(Caco-2) [184]. After the procedure, 271 compounds
were selected based on achieving more than 75% inhibi-
tion cut-off, as determined by quantifying cell viability
readouts. Subsequently, 184 compounds were further
chosen based on their clinical status. Among these, 64
compounds demonstrated an IC50 value of less than
20 μM, while 19 exhibited an IC50 value of less than
1 μM.. This study highlighted six of the 64 compounds,
namely camostat, nafamostat, lopinavir, mefloquine,
papaverine, and cetylpyridium. However, 90% of those
confirmed compounds have not been reported as SARS-
CoV-2 antiviral agents in in vitro cell assays [182, 185]. The names and structures of those compounds were not
disclosed in the present study. simulations of 14 compounds led to the identification of
six hits of phenyl propanoid compounds, including fas-
ciospongiside A, epolactoena, constanolactone B, con-
stanolatone F, debromo araplysillin I, and maniloside A as
potential anti-COVID-19 agents [178]. g
The Korea Chemical Bank Drugs Repurposing (KCB-
DR) database, consisting of 1,865 compounds, was used
to propose potential therapeutic agents for COVID-19. GOLD virtual screening identified 149 binders based on
their Goldscore and Chemscore. MD simulations were
then employed to analyze the binding modes and funda-
mental interactions, thereby revealing seven top drugs. Based on the binding free energy approaches, ceftaroline
fosamil and telaprevir emerged as potential drugs against
SARS-CoV-2 with telaprevir raising safety concerns due
to its side effects. In order to address this, a substructure
search in the PubChem database led to the identifica-
tion of 11 potential derivatives of telaprevir exhibiting
desirable pharmacokinetic properties, particularly lower
hepatotoxicity [203]. Additionally, a
study on marine natural product-based drugs-like small
molecules screened 14,492 compounds from the MNP
library, of which 7471 compounds fulfilled Lipinski rule
of five. After conducting the evaluation process through
ADMET descriptor, 2033 compounds were selected for
further analysis. Docking analysis and molecular dynamic The Searching off-lAbel drugs aNd NEtwoRk (SAve-
RUNNER) is an interesting approach for repurposing
existing drugs to treat COVID-19. This method evaluates
the interaction between drugs and target protein based
on their location and position in the same network neigh-
bourhoods. Recent study utilized 14 COVID-19-related
diseases to generate 282 repurposing drugs of 1875 FDA-
approved drugs from DrugBank v5.1.6. Ruxolitinib has
the potential to inhibite JAK and H1-antihistamines that Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Page 16 of 48 Page 16 of 48 to substantiate the health-related information or knowl-
edge associated with these natural remedies. play a vital role in controlling immune responses [208]. Asides from SAveRUNNER, other in-silico approaches
for identifying potential repurposing drugs include net-
work module separation and the RWR algorithm. Both
approaches highlight the disease module of H1N1 flu and
SARS-CoV-2 infection [209]. The development of net-
work-based mechanisms involves multimodal technology
using artificial intelligence, network diffusion, and prox-
imity algorithms. [210], stated that 76 of the 77 drugs
achieved viral effects through indirect viral protein bind-
ing targets by perturbing the host subcellular network. Molecular docking through computational approaches
was used to observe the interaction patterns of bind-
ing viral proteins to host targets. Indeed, network-based
perturbations is induced by altering the virus ability to
enter the cell or replicate within the cell. This advanced
approach of in-silico method for drugs repurposing is
beneficial in developing a faster and cheaper strategy for
drugs discovery schemes. Discovery of alternative drugs for treating viral respira-
tory diseases such as COVID-19 has led to repurposing
of natural products with new pharmacological proper-
ties. Medicinal plants from the Lamiaceae, Cupressaceae,
and Zingiberaceae families, as well as isolated natu-
ral products such as ritonavir, chloroquine phosphate,
arbidol, and ribavirin, have shown potential antiviral
activities against some viruses [212]. These natural prod-
ucts have also been found to be beneficial in preventing
and relieving the symptoms of COVID-19. Other natural
products compounds, such as vitamin D (calcitriol), vita-
min C (ascorbic acid), lactoferrin, quercetin, resveratrol,
hanfangchin A (tetrandrine), glycyrrhizin, artemisinin,
colchicine, and berberine, are current under clinical tri-
als for treatment of COVID-19 [213]. In addition, Panyod
et al. [5] stated that the use of immunomodulator foods
and herbs containing large amounts of vitamins C and
D, flavonoids, and essential oils, helps strengthen the
immune system and acts as air disinfectants or sanitiz-
ers to prevent aerosol transmission of the virus. The use
of rich and bulky spices found in tropical lands, such as
cinnamon, cloves, mint, lemon, and balm, also offers pos-
sibilities for discovering bioactive natural molecules suit-
able against viruses [214–219]. However, computer modelling is not the only approach
for determining drugs as reliable antiviral agents, even
when it shows a strong binding mode to the active sites
of the virus. Vatansever et al. [211] stated that calculated
binding energy does not necessarily correlate strongly
with the actual IC50 values. Computer modelling is the
only approach used to obtain detailed information in
relation to predicting the mode of antiviral action. The
next crucial step is to conduct in vitro and in vivo assays
in preclinical trials. These assessments help to identify a
small number of drugs or compounds for further evalu-
ation in clinical trials. As public awareness of health and
safety increases and the challenges posed by viral infec-
tions persist, there is a growing need for alternative,
nature-based medications. This alternative treatment can
complement existing approaches and offer potential solu-
tions for viral infections that are difficult to cure or pre-
sent challenges during treatment. Several food sources have been found to play a role in
the immunomodulatory system by reducing inflamma-
tion. For example, the fruit extract of Embelia schimperi
(Myrsinaceae), which contains benzoquinones, has been
proven to exhibit potent HCV-PR (hepatitis C virus pro-
tease) activity [220]. Pomegranate peel extract (PPE),
which contains polyphenols, has also been found to have
immunomodulatory effects [221–223]. In addition, Ali
et al. [224], and Wen et al. [95], reported that among 221
phytocompounds, some diterpenoids, sesquiterpenoids,
triterpenoids, and lignoids were potent inhibitors against
SARS-CoV on Vero E6 cells. Other possible sources of
natural products active compounds in treating human
diseases include endophytes and medicinal plants. atural products for treatment of viral infection a u a p odu
s o
a
o
a
o
Medicinal plants encompass all plants or herbs whose
components exhibit biological activities. These bioactive
compounds, when extracted from medicinal plants, can
be considered as lead compounds. In recent times, there
has been a growing interest in novel natural approaches
to treating viral infections, driven by increased public
awareness and concern for safety and health issues in
comparison to synthetic drugs. The utilization of natu-
ral products as remedies for various infectious diseases
often stems from the fields of ethnobotany, phytochem-
istry, and local wisdom, giving rise to ethnopharmacol-
ogy. However, it is important to note that in many cases,
there are insufficient or even lack of scientific evidences [105],
stated some molecules obtained from medicinal plants
that have been claimed to be effective against SARS-
CoV-2 in virtual assays or clinically applied, although
there is no scientific proof. Traditional herbal medicines Traditional herbal remedies are widely used as comple-
mentary or alternative medicines in many countries,
particularly in the context of eastern medicine. These
remedies consist of traditional medicinal preparations
derived from single or combined medicinal plants. Despite the lack of comprehensive studies, these thera-
pies have been employed for centuries in treatment of Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Page 17 of 48 Page 17 of 48 various ailments. Traditional Chinese Medicine (TCM),
which boasts a history of over 2000 years, and Ayur-
veda are two prominent herbal remedies enjoying trust
and popularity not only within their countries of origin,
China and India respectively, but also in other parts of
the world. medicinal plants or Chinese medical formulas. Xi et al. [229], specifically identified components of TCM herbs
as potential agents against antiviral pneumonia, while An
et al. [225] listed various TCM treatment along with their
initial symptoms, outcomes, and effects on antiviral dis-
eases. It is important to note that the evidence support-
ing TCM treatment relies on clinical evidence obtained
from their practical use. In a study conducted by Yang et al. [227], various TCM
herb formulae and extracts were identified for their
potential in treating SARS-CoV infections, along with
TCM-derived compounds exhibiting anti-HCoV activi-
ties. Notably, the Yin Qiao San formula demonstrated
positive therapeutic effects against upper respiratory
tract infections, while Ma Xin Gan Shi Tang exhibited
anti-SARS-CoV activity. Several TCM compounds were
found to possess antiviral properties, particularly against
SARS-CoV, MERS, and SARS-CoV-2. These include
plant-derived phenolic compounds from Isatis indig-
otica root extract, litchi seed extract, herbacetin, rhoifo-
lin, pectolinarin, quercetin, epigallocatechin gallate, and
gallocatechin gallate. Glycyrrhizin from Glycyrrhizae
radix, water extract of Houttuynia cordata, and emodin
derived from Rheum and Polygonum genera also exhib-
ited antiviral activity (Fig. 6). Yi et al. [228], conducted
study on 121 Chinese herbal medicines and reported that
tetra-O-galloyl-β-D-glucose and luteolin were two active
constituents effective against the wild type of SARS-
CoV. Another review reported that TCM is obtained
from a single preparation rather than the combination of TCM is believed to treat COVID-19 by inhibiting the
replication and transcription of SARS-CoV-2 through
various mechanisms such as blocking the viral functions
of RdRp, 3CLpro, spike protein, and PLpron. Addition-
ally, it can hinder the binding of the virus to host cells by
acting on ACE2 and TMPRSS2. Traditional herbal medicines TCM also has the poten-
tial to reduce cytokine production, prevent immune sys-
tem impairment, and abnormal blood clotting following
SARS-CoV-2 post-infection. Shuanghuanglian is a popular traditional Chinese pat-
ent medicine that is formulated from the extraction of
three Chinese herbal medicines, namely Lonicera japon-
ica Thunb, Scutellaria baicalensis Georgi, and Forsythia
suspense (Thunb.) Vahlv. The key constituents of this
medicine are chlorogenic acid, phillyrin, and baicalin. Despite being a traditional medicine, Shuanghuanglian
has undergone scientific investigations in China to assess
its antiviral activity. Su et al. [45] reported that the inhibi-
tory effect of Shuanghuanglian on SARS-CoV-2 3CLpro
is primarily attributed to its significant components, Fig. 6 Some parts of medicinal plants are used as traditional herbal therapies to alleviate the symptoms of respiratory diseases,
and the effectiveness of some of these remedies has been scientifically proven. However, there are other herbal remedies that have not been
listed in international monographs [225, 226]. All pictures were obtained from the Google search engine and were not recognized as the author’s
ownership or copyright Fig. 6 Some parts of medicinal plants are used as traditional herbal therapies to alleviate the symptoms of respiratory diseases,
and the effectiveness of some of these remedies has been scientifically proven. However, there are other herbal remedies that have not been
listed in international monographs [225, 226]. All pictures were obtained from the Google search engine and were not recognized as the author’s
ownership or copyright Fig. 6 Some parts of medicinal plants are used as traditional herbal therapies to alleviate the symptoms of respiratory diseases,
and the effectiveness of some of these remedies has been scientifically proven. However, there are other herbal remedies that have not been
listed in international monographs [225, 226]. All pictures were obtained from the Google search engine and were not recognized as the author’s
ownership or copyright Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Page 18 of 48 Page 18 of 48 disease. Maurya et al. [235], have virtually screened
natural products from Ayurveda to identify compounds
capable of modulating the immune system and block-
ing the entry of SARS-CoV-2. Three plants of critical
significance in Ayurvedic medicine, especially in Rasay-
ana therapy, are Whitania somnifera, Tinospora cordi-
folia, and Asparagus racemosus. Traditional herbal medicines Pudilan Xiaoyan Oral Liquid (PDL), another TCM
preparation, has been the subject of study for disease
enrichment analyses. It has shown promising potential
in treating asthma and chronic obstructive pulmonary
disease, with similar significance levels to COVID-19
(p = 2.4E−03 and p = 2.45E−03, respectively). PDL con-
tains four herbs, including Indigowoad Root (Isatis indig-
otica), Bunge Corydalis (Corydalis Bungeana), Mongolian
Dandelion (Taraxacum Mongolicum), and Scutellaria
Amoena (Scutellaria Baicalensis) [232]. Interestingly,
there is also a commercially available oral liquid in the
United States known as Respiratory Detox Shot (RDS),
which is a food supplement containing ingredients com-
monly used in TCM. These ingredients include Panax
ginseng, Lonicera japonica, Forsythia suspensa, Glycyr-
rhiza uralensis, Scrophularia ningpoensis, etc. The effect
of RDS on SARS-CoV-2 was investigated, resulting in an
IC50 value at a 1:40 dilution [233]. Ni et al. [230], reported a case in which a combination
of Shuanghuanglian and Western medicine was used to
treat three family members suffering from COVID-19,
resulting in a positive therapeutic effect. However, the use
of this method needs to be approached with caution due
to the need for early treatment and potential errors, such
as combining antibiotics and antiviral drugs. In addition
to health concerns, some TCM herbs contain nephro-
toxins and mutagens, such as aristolochic acids found in
Aristolochia and related plants [231]. The regulation of
these herbs varies among nations, China, Taiwan, and the
US are some countries that have unregulated their usage. In Romania, the native flora, including medicinal plants
such as dandelion, daisy, and fat grass [238], was utilized
as part of traditional medicine during current pandemic
[225, 239]. These plants are rich in flavonoids, saponins,
tannins, sterols, fatty acids, coumarin, and vitamins. Moreover, the Fritillaria species, known for their phar-
macological effects on the respiratory system, possess
antitussive, expectorant, and antiasthmatic properties. This genus has been included in the Ayurvedic (Fritil-
laria roylei), Korean (four species), and Chinese Phar-
macopeia (ten species known as Bei Mu in Chinese), and
are also widely used in Tibetan, Mongolian, Miao, Lisu,
Tujia, Kazakh, Uighur, Jingpo and De’ang traditional
medicine [240]. Other herbal products, Sumac, extracted
from the Rhus genus, has demonstrated interactions with
viral envelopes and host cell surfaces, exhibiting diverse
antiviral activities against influenza A and B, HSV, and
HIV. According to Korkmaz [241], it has been suggested
as a potential treatment for COVID-19 infection. Traditional herbal medicines These plants contain
active steroid compounds such as ashwagandhonolides,
whitacoagin, withaferin, and withanone, which have
shown potential against various proteins associated
with SARS-CoV-2, including spike glycoprotein (RBD),
RdRp, and Mpro [236]. Ayush-64, an Ayurvedic formu-
lation used clinically for its anti-malarial, anti-inflam-
matory, and antipyretic properties, has demonstrated
favourable binding energy to Mpro, with values of
approximately − 8.4, − 7.5, and − 7.4 kcal/mol, corre-
sponding to molecules akuammicine N-Oxide (from
Alstonia scholaris), akuammiginone, and echitaminic
acid, respectively [237]. Nimbin and curcumin, active
compounds found in Ayurvedic formulations, have
exhibited higher binding affinity than nafamostat, a
synthetic protease inhibitor [235]. Figure 7 shows some
of the active compounds found in traditional Ayurve-
dic medicines that have demonstrated efficacy against
SARS-CoV-2 in in vitro assays.l baicalin and baicalein (an aglycone of baicalin). The IC50
value for this inhibition was found to be only 0.94 μM. However, it should be noted that these two compounds
exhibited less than 50% inhibition activity against SARS-
CoV-2 PLpro at a concentration of 50 μM. The potent inhibitory activity of baicalein against
SARS-CoV-2 3CLpro can be attributed to its structural
features, including three phenolic hydroxyl groups, a car-
bonyl group, and a free phenyl ring. These features allow
baicalein to form multiple hydrogen bonds and hydro-
phobic interactions with amino acid residues, both in the
main and side chains of the viral active sites. While both
baicalein and baicalin demonstrated significant inhibi-
tion, antiviral activity of Shuanghuanglian was found to
be limited in Vero E6 cells. Su et al. [45], reported that
this could be attributed to the low permeability of the cell
membrane to the components of the preparation. p
p
p
Ni et al. [230], reported a case in which a combination
of Shuanghuanglian and Western medicine was used to
treat three family members suffering from COVID-19,
resulting in a positive therapeutic effect. However, the use
of this method needs to be approached with caution due
to the need for early treatment and potential errors, such
as combining antibiotics and antiviral drugs. In addition
to health concerns, some TCM herbs contain nephro-
toxins and mutagens, such as aristolochic acids found in
Aristolochia and related plants [231]. The regulation of
these herbs varies among nations, China, Taiwan, and the
US are some countries that have unregulated their usage. Traditional herbal medicines Pudilan Xiaoyan Oral Liquid (PDL), another TCM
preparation, has been the subject of study for disease
enrichment analyses. It has shown promising potential
in treating asthma and chronic obstructive pulmonary
disease, with similar significance levels to COVID-19
(p = 2.4E−03 and p = 2.45E−03, respectively). PDL con-
tains four herbs, including Indigowoad Root (Isatis indig-
otica), Bunge Corydalis (Corydalis Bungeana), Mongolian
Dandelion (Taraxacum Mongolicum), and Scutellaria
Amoena (Scutellaria Baicalensis) [232]. Interestingly,
there is also a commercially available oral liquid in the
United States known as Respiratory Detox Shot (RDS),
which is a food supplement containing ingredients com-
monly used in TCM. These ingredients include Panax
ginseng, Lonicera japonica, Forsythia suspensa, Glycyr-
rhiza uralensis, Scrophularia ningpoensis, etc. The effect
of RDS on SARS-CoV-2 was investigated, resulting in an
IC50 value at a 1:40 dilution [233]. Traditional medicines in the form of Jamu have been
produced in Indonesia [242–244]. Popular Jamu formu-
lations include wedhang jahe, jamu kunyit asam, jamu
teulawak, and jamu beras kencur, which typically contain
rhizomes from the ginger family (Zingiberaceae) such as
Zingiber officinale and Curcuma longa. These formula-
tions may also include additional ingredients like Cin-
namomum verum bark, Citrus aurantifolia fruit, and
starch fillers. Ayurveda, an ancient traditional medicine system
dating back to the Vedic period (1500 to 500 BCE),
has emerged as a potential remedy for mitigating the
severity of COVID-19. Care [234], reported that the
Ayurvedic approach, focuses on both preventive and
curative aspects, tailored to different stages of the Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 (2023) 66:89 Page 19 of 48 According to the Committee on Herbal Medicinal
Plant Products (EMEA/HMPC/892618/201), consum-
ing the extract of Eucalyptus globulus Labill, in the
form of dried leaves up to four times a day, is helpful
in managing respiratory diseases such as bronchitis and
rhinitis. This is because it contains active components
such as 1,8-cineol and phenolic compounds. The Brit-
ish Herbal Pharmacopeia recommends the use of garlic
products that contain sulfuric compounds, including
allicin and mercaptan, amino acids, peptides, terpe-
nes, minerals, and flavone glucosides to treat COVID-
19, as it has traditionally been used to manage colds
and whooping cough. However, it is important to note
that Traditional Herbal Medicine Products (THMP) in
Europe are not considered a treatment for COVID-19
as it is a severe, life-threatening illness [226].h Polyphenolic compounds can also act as antioxidants
due to their hydroxyl groups, which react with radicals
and oxidizing compounds. Resveratrol, a biflavonoid
compound with the IUPAC name 3,5,4-trihydroxy-trans-
stilbene, is a potent antioxidant that scavenges for reac-
tive oxygen species, such as O2
− and OH−, and lipid
hydroperoxyl free radicals. Although it has poor oral bio-
availability and water miscibility, resveratrol is rapidly
metabolized in the body. Abba et al. [282] have stated the
role of resveratrol and its action mechanisms in combat-
ing viral infections in human and animal cells. Therefore,
resveratrol is presumed to have potential therapeutic
benefits in treating COVID-19 by enhancing the immu-
nity of infected patients. Quercetin is another popu-
lar phenolic compound that has been combined with
N-acetylcysteine in the formulation of Quercinex to be
directly administered to the deep lung tissue through a
nebulizer to treat respiratory problems and multifocal
pneumonia in COVID-19 patients [283]. Rutin, another
phenolic compound, has been studied for its in-silico
binding affinity to interact with the main protease of
SARS-CoV-2 in the three-dimensional structures of PDB
IDs 6LU7 ([170] and 6YNQ [169].h The utilization of medicinal plant extracts mentioned
earlier has gained widespread popularity worldwide as
a recommendation to combat COVID-19, serving as an
alternative to drugs provided by WHO and the Minis-
try of Health in each country. Zhang et al. [254], con-
ducted in silico screening of Chinese herbal medicine
and identified 13 active compounds effective against
SARS-CoV-2. This method of analysis is beneficial in
expediting drugs discovery process based on ethnobo-
tanical reasoning. Fig. 7 Chemical structures of active components inside traditional medicines that have been tested by in vitro and in silico analysis against proteins
of SARS-CoV and SARS-CoV-2 [94, 228, 235–237, 247–253] Fig. 7 Chemical structures of active components inside traditional medicines that have been tested by in vitro and in silico analysis against proteins
of SARS-CoV and SARS-CoV-2 [94, 228, 235–237, 247–253] tures of active components inside traditional medicines that have been tested by in vitro and in silico analysis against protein
S-CoV-2 [94, 228, 235–237, 247–253] Fig. 7 Chemical structures of active components inside traditional medicines that have been tested by in vitro and in silico analysis against proteins
of SARS-CoV and SARS-CoV-2 [94, 228, 235–237, 247–253] Page 20 of 48 Page 20 of 48 Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Although the use of Jamu is not officially recom-
mended in treatment of COVID-19 patients, it has
become a popular alternative among Indonesians as an
immunostimulatory agents to prevent symptoms and
promote speedy recovery from post-infection symp-
toms [245]. Another example is the application of vir-
gin coconut oil (VCO) in the local communities as a
therapeutic adjuvant to overcome inflammation caused
by COVID-19. While no scientific evidence has been
presented, clinical trials have been conducted in four
hospitals in Yogyakarta caring for hospitalized COVID-
19 patients [246]. It is important to note that traditional
remedies should not be used as a substitute for medi-
cal treatment for COVID-19 and their effectiveness
remains unproven. Polyphenol‑based secondary metabolites Polyphenols are a prominent group of naturally occur-
ring bioactive compounds found in plants that contain
at least one substituted phenol ring or several hydroxyl
groups on aromatic ring compounds. This group com-
prises four classes, namely phenolic acids, flavonoids,
stilbenes, and lignans. The flavonoid class includes sev-
eral derivatives such as chalcones, flavones, flavanones,
flavonols, isoflavones, anthocyanins, and flavan-3-ols. Polyphenols are known for their broad antiviral activi-
ties against various viruses, including influenza A virus
(H1N1), HBV/HCV, HSV, HIV, and Epstein-Barr virus
(EBV) [255]. Table 5 shows some phenolic compounds
that have been explored as antiviral agents, particularly
against SARS-CoV-2. In silico and in vitro approaches
have been used to study subsites of the virus, including
proteins and enzymes related to SARS-CoV-2 and cell
receptors in the human body. Exploring traditional medicine and
ethnopharmacology presents a potential alternative for
drugs discovery in combating COVID-19 pandemic. However, it is crucial to exercise caution regarding
the preparation, dosage, and individual health consid-
erations associated with traditional treatment prior
to their application. Therefore, conducting a detailed
investigation into the extraction of active compounds
from traditional herbal medicines would prove advan-
tageous as it focuses on the specific or known com-
bination of active molecules responsible for their
bioactivity, eliminating unnecessary or unsafe compo-
nents that may be consumed. The polyphenols present in ethanol PPE have exhib-
ited positive in vitro activity in reducing the interac-
tion between SARS-CoV-2 spike glycoprotein and
human ACE2, along with the activity of SARS-CoV-2
Mpro. ACE2 and TMPRSS2 gene expression levels were
reduced by 30 and 70%, respectively, by applying PPE at
0.04 mg/mL on human kidney-2 cells infected by SARS-
CoV-2 Spike pseudotyped lentivirus. Furthermore, PPE
displayed the inhibition of Mpro activity by relatively 80%
when used at 0.2 mg/mL [223]. It has also been evaluated
in three commercial forms, namely pomegranate juice, a
concentrated liquid extract, and 93% PP powder extract, Page 21 of 48 Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Table 5 Potential polyphenolic compounds as antiviral agents against SARS-CoV-2
Compounds
Plant sources
Antiviral Activities
Assays
IC50/Binding Affinity*
Refs. Phillyrin (KD-1)
Forsythia suspensa
SARS-CoV-2 and HCoV-229E
In vitro using cytopathic effect
and plaque reduction assay in Vero
E6 cells
IC50 against SARS-CoV-2 and HCoV-
229E is 63.90 and 64.53 mg/mL
[256]
Phillyrin (KD-1)
Forsythia suspensa
SARS-CoV-2 and HCoV-229E
In vitro based on pro-inflammatory
cytokine expression levels in Huh-7
cells by RT-PCR assay
Phillyrin reduced the production
of proinflammatory cytokines
at mRNA levels and reduced
the protein expression of p-NF-κB
p65, NF-κB p65, and p-IκBα
[256]
Cannabidiol and Δ9-
tetrahydrocannabinol
Cannabis sativa L. (Chongsam,
leaves)
SARS-CoV-2 (βCoV/KOR/
KCDC03/2020)
In vitro using screening assay
in Vero cells
IC50 of 7.91 mM and 10.25 mM
[257]
Pelargonidin
SARS-CoV-2 Spike protein
In vitro using Spike/ACE2 Inhibitor
Screening Assay Kit and using
plaque assay in Vero E6 cells
At 50 mM reduces Spike bind‑
ing to ACE2 by about 40%. Plaque assay reduces virus entry
by about 70% at 100 mM
[258]
Juglanin
3a-protein channel of SARS-CoV
In vitro using Voltage-clamp experi‑
ments on SARS-3a protein
IC50 of 2.3 mM
[259]
Emodin
Genus Rheum and Polygonum
SNE (spike and envelope gene)-3a
protein of SARS-CoV
In vitro using Voltage-clamp experi‑
ments on SARS-3a protein
IC50 of 20 mM
[249]
Emodin
SARS-CoV spike protein
In vitro using luciferase assay
IC50 of 200 mM
[250]
ThE (composed of green tea cat‑
echin and epigallocatechin gallate
EGCG; total catechins were 85–95%
and total EGCG was 65–70%, caf‑
feine < 0.5%)
Product from Mitsui Norin Co. Ltd
SARS-CoV-2
In vivo using clinical trials. Ten
patients were treated for 15 days
sessions of inhalation plus three
capsules per day (total catechin,
840 mg; total EGCG, 595 mg)
Seven of ten patients switched
to a negative SARS-CoV-2 naso‑
pharyngeal swab test in a range
of 6–13 days
[260]
Curcumin
Hesperidin
Quercetin
hydroxychloroquine
From chemical manufacturers
SARS-CoV-2 from hCoV-19/Egypt/
NRC-3/2020 SARS-CoV-2 virus
(Accession
Number on GSAID: EPI_ISL_430820)
In vitro using plaque reduction
assay in Vero E6 cells
IC50 values: Curcumin 0.44 mM
Hesperidin 13.25 mM
Quercetin 18.2 mM
Hydroxychloroquine 1.72 mM
[194, 261]
Gallocatechin gallate (GCG)
Epigallocatechin gallate (EGCG)
Quercetin
From Sigma-Aldrich
Recombinant SARS 3CLpro trans‑
formed and expressed in Pichia
pastoris GS115 based on GenBank
accession no. AY274119
In vitro: proteolytic activity based
on fluorescence resonance energy
transfer (FRET) assay
In silico: using Autodock Tools
software with a ligand number
of 3CLpro is 2ZU5
IC50 of:
GCG = 47 mM
EGCG = quercetin = 73 mM
The binding energy of:
GCG = − 14.1 kcal/mol,
EGCG = − 11.7 kcal/mol, querce‑
tin = − 10.2 kcal/mol
SAR: EGCG and CGC have a gal‑
loyl moiety at the 3-OH position
to interact with the 3CLpro active
site pocket
[248] IC50/Binding Affinity* Page 22 of 48 Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Table 5 (continued)
Compounds
Plant sources
Antiviral Activities
Assays
IC50/Binding Affinity*
Refs. Myricetin
SARS-CoV-2 Mpro
In vitro using a proteolytic assay
based on FRET
In silico: using AMBER18
with ligand number of 3CLpro
is 6LZE
In vivo: pulmonary inflammation
in bleomycin treated mice
IC50 3.684 ± 0.076 μM
The binding free energy
is -32.98 kcal/mol
Myrcetin inhibits the infiltration
of inflammatory cells and secretion
of inflammatory factors in the lung
[262]
Ginkgolic acid (GA) and anacardic
acid (AA)
SARS-CoV-2 PLpro, SARS-CoV-2
CLpro, isolated SARS2-CoV-2 USA-
WA1/2020
In vitro using an enzymatic assay
based on fluorometric peptide
(FRET) assay. IC50/Binding Affinity* Antiviral determina‑
tion using plaque reduction assay
on Vero-E6 cells
In silico using Autodock Vina
with ligand 6m2n and 6WX4
IC50 against PLpro: GA = 16.30 ± 0.64
and AA = 17.08 ± 1.20 mM
IC50 against 3CL
pro: GA = 1.79 ± 0.58
and AA = 2.07 ± 0.35 mM
EC50 against SARS-CoV-2:
GA = 8.3 ± 0.03 mM
and AA = 9.0 ± 2.5 mM
Inhibition at 7.5 mM: GA = 42%
and AA = 13%
Binding affinity of GA to 3CLpro
and PLpro is -5.3 and -4.9 kcal/mol
[263]
Curcumin, brazilin, and theafla‑
vin-3,3’-digallate
SARS-CoV-2 RBD
In vitro using SARS-CoV-2 Surrogate
Virus Neutralization Test Kit
% binding with RBD at 0.1 mg/
mL = 100 ± 0.2; 100 ± 0.1;
and 100 ± 0.1
[34]
Broussochalcone A (BcA); Papyri‑
flavonol A (PA); 3’-(3-methylbut-
2-enyl)-3’,4’,7-trihydroxy flavane
(tHF); Broussoflavan A (BfA); Kazinol
F (KF); Kazinol J (KJ)
Broussonetia papyrifera
SARS CoV-2 Mpro
In silico using AutoDock Vina
on SARS-CoV-2 Mpro (6LU7)
Binding affinity (kcal/mol):
BcA =− 8.1
PA =− 7.9
tHF =− 8.2
BfA =− 7.8
KF =− 8.1
KJ =− 8.0
[264]
Kaempferol, quercetin, luteolin-
7-glucoside, demethoxycurcumin,
naringenin, apigenin-7-glucoside,
oleuropein, curcumin, catechin,
epicatechin-gallate
From chemical manufacturers
SARS-CoV-2 3CLpro/Mpro
and SARS-CoV 3CLpro/Mpro
In silico using
Autodock 4.2 with Lamarckian
Genetic Algorithm on Mpro (6LU7
and 2GTB)
These listed compounds were
ranked by affinities (ΔG)
[265]
Curcumin
Hesperidin
Quercetin
hydroxychloroquine
From chemical manufacturers
SARS-CoV-2: S spike protein
and main protease
In silico using MOE 2019.012
suite with S spike protein (6VW1)
and Mpro (6LU7)
Binding score to S protein
and Mpro:
Curcumin − 7.02 and − 7.28 kcal/
mol
Hesperidin − 7.92 and − 8.37 kcal/
mol
Quercetin − 6.48 and − 6.23 kcal/
mol
Hydroxychloroquine -6.60
and − 7.05 kcal/mol
[194] Antiviral Activities Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Page 23 of 48 Table 5 (continued)
Compounds
Plant sources
Antiviral Activities
Assays
IC50/Binding Affinity*
Refs. Gallocatechin gallate (GCG)
Epicatechin gallate (ECG)
Epigallocatechin gallate (EGCG)
Catechin gallate (CG)
Epicatechin (EC)
Catechin
Gallocatechin (GC)
Epigallocatechin (EGC)
SARS-CoV-2 main protease
In silico using AutoDock Vina
with Mpro (6LU7)
Three best binding energies:
GCG − 9.0 kcal/mol
ECG − 8.2 kcal/mol
EGCG − 7.6 kcal/mol
[266, 267]
Rutin
SARS-CoV-2 Mpro (6LU7 and 6YNQ)
In silico using Glide module
Docking score: above − 7.0; − 8.7;
− 9.16 kcal/mol
[169]–[171]
Calceolarioside B
SARS-CoV-2 Mpro (6LU7), Nsp15
endoribonuclease (6VWW), coro‑
navirus fusion protein (6LXT), SARS-
CoV-2 spike ectodomain (6VYB)
In silico using Molegro Virtual
Docker
MolDock score:
− 191.295
− 164.77
− 141.587
− 153.135
[268]
5-O-D-glucopyranosyl-4’-hydroxy-
7-methoxy-4-phenylcoumarin
SARS-CoV-2 Nsp15 endoribonucle‑
ase (6WXC)
In silico using windows MOE
Binding energy of − 10.1 kcal/mol
[269]
Luteolin 7-O-β-glucopyranoside
(cynaroside), acacetin 7-O-β-
rutinoside (linarin) and isoacteoside
(isoverbascoside)
Amphilophium paniculatum (L.)
Kunth (leaves)
SARS-CoV-2 Mpro (7BUY)
In silico using Molecular Operating
Environment (MOE) 2019.0102
Energy score of − 9.54, − 8.54,
− 8.46 kcal/mol
[270]
Cannabidiol and Δ9-
tetrahydrocannabinol
SARS-CoV-2 (6LU7)
In silico using Autodock and Vina
Binding energy in Autodock
is − 10.53 and − 10.42 kcal/
mol, while in Vina is − 6.43
and − 7.13 kcal/mol
[257]
Cyanidin, malvidin, pelargonidin,
peonidin, petunidin
Pimpinella anisum L. (anise)
SARS-CoV-2 3CLpro (6LU7)
In silico using AutoDock Vina
Binding energy: − 8.1; − 8.0; − 8.0;
− 7.7; − 7.5 kcal/mol
[271]
Procyanidin b2 and mangiferin
Chincona pubescenc. Antiviral Activities and from mango tree
SARS-CoV-2 3CLpro (6LU7)
In silico using AutoDock Vina
Binding affinity: -9.4 and -8.5 kcal/
mol
[272]
Heptafuhalol A
SARS-CoV-2 Mpro (6LU7)
In silico using Vina and Autodock
Average ΔG = − 14.6 kcal/mol
[273]
Oolonghomobisflavan-A
SARS-CoV-2 Mpro (6Y2F)
In silico using GROMACS
Binding free energy on MM-PBSA
calculation: -256.875 kj/mol
[274]
Epigallocatechin gallate
Green tea
SARS-CoV-2 Mpro (6LU7), NSP15
endoribonuclease (6VWW), free
enzyme Mpro (6Y2E), and 2019-
nCoV HR2 domain (6LVN),
post fusion core of S2 subunit
(6LXT), prefusion spike glycopro‑
tein (6VSB), chimeric receptor-bind‑
ing domain complexed with hACE2
(6VW1)
In silico using AutoDock
Binding energy (kcal/mol):
6LU7 = − 6.99; 6LVN = − 4.90;
6LXT = − 7.57; 6VSB = − 7.26;
6VWW = − 8.38; 6Y2E = − 9.30;
6VW1 = − 8.66
[275]
Theaflavin digallate
SARS-CoV-2 Mpro (6LU7)
In silico using GLIDE
Docking score: -10.574 kcal/mol
[167] Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Page 24 of 48 Table 5 (continued)
Compounds
Plant sources
Antiviral Activities
Assays
IC50/Binding Affinity*
Refs. Glycyrrhizic acid (GlA) and theafla‑
vin 3,3-digallate (TF3)
SARS-CoV-2 Mpro (6LU7) and ACE2
receptor (IR4L)
In silico using AutoDock Vina
Binding energy to 6LU7: GlA = − 9.3
and TF3 = − 10; and with 1R4L:
GlA = − 9.6 and TF3 = − 8.3 kcal/mol
[276]
Theaflavin
SARS-CoV-2 RBD
In silico using SwissDock
Idock score − 7.95 kcal/mol
[277]
Pedunculagin, tercatain, and casta‑
lin
SARS-CoV-2 Mpro, catalytic dyad
residues: Cys145 and His41 (6Y84)
In silico using MOE 09
S score: − 18.58; − 23.11;
and − 14.04
[166]
Hypericin, Amentoflavone, terflavin
Hypericum perforatum L. and Termi-
nalia chebula Retz. or T. catappa L
SARS-CoV-2 Mpro (6LU7)
In silico using AutoDock Vina
Binding energy: − 10.4, − 9.7
and − 9.7 kcal/mol
[164]
Eriodictyol-7-O-rutinoside, narirutin
Flavanone glycoside in lemon
and sweet orange
SARS-CoV-2 RdRp protein sequence
(YP_009725307.1)
In silico using AutoDock Vina
Binding energy: -9.9 and -9.7 kcal/
mol
[164]
Cis-miyabenol C
Foeniculum vulgare Mill. Antiviral Activities (fennel)
SARS-CoV-2 human transmem‑
brane serine protease 2 (TMPRSS2:
sequence NP_001128571.1)
In silico using AutoDock Vina
Binding energy: -9.4 kcal/mol
[164]
Ellagic acid
Prunica granatum
SARS-CoV-2 Mpro (6LU7)
In silico using AutoDock Vina
Binding affinity and IC: − 8.4 kcal/
mol and 0.7 μM
[193]
5,7,3′,4′-tetrahydroxy-2′-(3,3-
dimethylallyl) isoflavone
Myricitrin
Methyl rosmarinate
Psorothamnus arborescens
Myrica cerifera
Hyptis atrorubens Poit
SARS-CoV-2 3CLpro (PMDB ID
PM0082635)
In silico using MOE
Binding affinities:
− 29.57
− 22.13
− 20.62
[278]
Agathisflavone
SARS-CoV-2 Mpro (pdb id: 6LU7)
In silico using AutoDock 4.1
Binding energy: − 8.4 kcal/mol
[173]
Albireodelphin
SARS-CoV-2 RdRp (pdb id: 6M71)
and spike protein (pdb id: 6VW1)
In silico using AutoDock 4.1
Binding energy: − 9.8
and − 11.2 kcal/mol
[173]
Inophyllum G2
SARS-CoV-2 Mpro (pdb id: 6LU7)
In silico using AutoDock Vina
Docking score: − 8.8 kcal/mol
[176]
Daphnorin
SARS-CoV-2 viral methyltransferase
(nsp16/10 complex, pdb id: 6W4H)
and RBD (pdb id: 6M0J)
In silico using AutoDock Vina
Docking scores: − 9.8
and − 8.2 kcal/mol
[176]
Isodispar B and daphnogirin
SARS-CoV-2 hACE2 (pdb id: 6VW1)
In silico using AutoDock Vina
Docking score: − 8.0 kcal/mol
[176]
Isorhamnetin-3-O-rutinoside
(narcissin)
Salvadora persica L
SARS-CoV-2 Mpro (pdb id: 6LU7)
In silico using AutoDock
Binding energy:− 8.2530 kcal/mol
[279]
Acetoside
(polyherbal formulation
SARS-CoV-2 Mpro (pdb id: 6LU7)
In silico using iGEMDOCK
Binding energy: − 153.06 kcal/mol
[280]
Demethyloleuropein
SARS-CoV-2 Mpro (pdb id: 6LU7)
In silico using AutoDock Vina
Binding energy: -8.90 kcal/mol
IC50 prediction: 11.58 μM
[177]
Nuzhenide oleoside
SARS-CoV-2 S protein (pdb id:
6LZG)
In silico using AutoDock Vina
Binding energy: − 8.90 kcal/mol. IC50 prediction: 6.44 μM
[177]
Myricitrin
Quercetin-3-O-glucuronide
Phyllanthus amarus
SARS-CoV-2 Mpro (pdb id: 6LU7)
In silico using AutoDock Vina
Binding affinity: − 9.6 kcal/mol,
and − 9.4 kcal/mol
[281]
Isorhamnetin-3-O-rutinoside
(narcisin)
Salvadora persica
SARS-CoV-2 Mpro (pdb id: 6LU7)
In silico using AutoDock Vina
Docking score: − 8.2530 kcal/mol
[279]
* IC50 (mg/mL and mM): the concentration of particular compound or drug in inhibiting the biological process to half of the maximum. Docking score (kcal/mol): a computational result for particular program and
energy to allow in predicting binding free energy and binding affinity, or ranking the complex of ligand and receptor according to specific parameters. Binding affinity (kcal/mol): an expression of the degree of ligand
binding with the protein in complex formation. Antiviral Activities Binding energy (kcal/mol): the energy released due to the bond formation or the interaction of the ligand and protein which is calculated as a sum of all the intermolecular
interactions presented in the complex Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Page 25 of 48 to demonstrate its anti-influenza activity against PR8
(H1N1), X31 (H3N2), and H5N1 [221]. Based on this reasoning, it becomes evident that
exploring the antioxidant potential of natural phenolic
extracts, as well as other forms such as food-based
extracts and polyphenol-containing functional foods
or nutraceuticals [290–292], can be valuable in man-
aging severe COVID-19 cases with inflammatory con-
ditions like cytokine storm. Further discussion about
the correlation between the activities of polyphenolic
compounds, especially those with flavonoid structures,
against the Mpro of SARS-CoV-2 has been elaborated in
subchapter 8. Dietary intake of polyphenols at high concentrations
also regulates ACE2 expression and function, by acting as
an antioxidant. Calceolarioside B is an active compound
found in Akebia trifoliata fruit, which has been suggested
as a potential dietary treatment for COVID-19 patients
due to its various health benefits, including antimicro-
bial and anti-inflammatory effects [284]. This caffeic acid
derivative is also present in other plants such as Staunto-
nia hexaphylla (leaves), Scutellaria galericulata L. (aerial
parts), Forsythiae Fructus (fruit), and Mimulus guttatus
(seeds) [285–288]. Figure 8 shows that active phenolic
compounds, such as quercetin and vitamin C, have a syn-
ergistic effect as adjuncts in treating COVID-19 [289]. Alkaloid‑based secondary metabolites Alkaloids are a class of organic nitrogenous base com-
pounds that occur naturally in plants, microorganisms, Fig. 8 Structures of some phenolics which have been tested for SARS-CoV-2-related in vitro activities [194, 249, 250, 256–259, 262, 263] Fig. 8 Structures of some phenolics which have been tested for SARS-CoV-2-related in vitro activities [194, 249, 250, 256–259, 262, 263] Fig. 8 Structures of some phenolics which have been tested for SARS-CoV-2-related in vitro activities [194, 249, 250, 256–2 Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Page 26 of 48 from Fromia monilis and Celerina heffernani, produce a
polycyclic guanidine alkaloid skeleton in their secondary
metabolites, which act as antiviral agents [190]. and animals. Their basicity depends on the form of nitro-
gen they contain, which can be primary, secondary, or
tertiary amines. Alkaloids are categorized based on the
amino acids that make up their nitrogen content and
the structure of their alkaloid skeleton. These secondary
metabolites have been found to exhibit a diverse range
of biological and pharmacological activities, including
antimicrobial, antiparasitic, antiasthma, analgesic, anti-
hyperglycemic, anticancer, psychotropic, and stimulant
properties. Some alkaloids have been identified as poten-
tial antiviral agents against SARS-CoV-2 through both in
silico and in vitro assays, as outlined in Fig. 9, Table 6. The study conducted by Quimque et al. [308] focused
on examining 97 antiviral secondary metabolites from
fungi. They utilized computational modelling to screen
these metabolites and identified Quinadoline B as the
top-scoring compound, predicted to exhibit high bind-
ing affinity to various proteins associated with SARS-
CoV-2, including PLpro, 3CLpro, RdRp, non-structural
protein 15 (nsp15), and the spike binding domain to
GRP78. Additionally, the ADMET value analysis indi-
cated that quinadoline B is a favourable compound with
high absorptive probability in the gastrointestinal tract
and low capacity for crossing the blood–brain barrier. y
g
In 1818, quinine was discovered and isolated from
Chincona bark, which prompted the exploration of other
plant alkaloids due to their bioactivities [304]. Recent
studies found that quinine exhibited promising activ-
ity against SARS-CoV-2 with an effective concentration
of EC90 at 38.8 mM [305]. In Northern Chile, Schizan-
thus porrigens Graham, herbaceous species, contains
a tropane-derived alkaloid called Schizanthine Z that
actively binds to PLpro with docking affinity − 7.5 kcal/
mol [168]. Another promising bis-benzylisoquinoline
alkaloid, cepharanthine, showed strong activity against
SARS-CoV-2 by blocking host Ca + channels and inhibit-
ing virus fusion and entry [306]. Alkaloid‑based secondary metabolites Additionally, Cryptolepis
sanguinolenta, a plant found in West Africa, contains
antipathogenic-based alkaloids that could be a promis-
ing candidate for SARS-CoV-2 inhibitors [307]. Focus
has also been directed to marine products in the search
for secondary metabolite contents, particularly alkaloids. Some marine organisms such as sponges from Crypto-
tethya crypta, Dysidea avara, Crambe crambe, a cyano-
bacterium from Nostoc ellipsosporum, and starfishes Chowdhury [300] conducted a molecular docking
study of five secondary metabolites from Tinospora cor-
difolia (Willd.) Hook.f. & Thomson (Menispermaceae)
and found that berberine showed the best binding affin-
ity of − 7.3 kcal/mol to 3CLpro of SARS-CoV-2, lead-
ing to an inhibition constant of 4.4 μM. Berberine has
previously been shown to have antiviral activity against
influenza, with a comparable IC50 to the standard drugs
oseltamivir [309]. Garg and Roy [302] identified the
four best molecules out of twenty antiviral alkaloids for
potential scrutiny using Lipinski’s rule and docking study
based on maximum negative binding energy with Mpro
of SARS-CoV-2. Thalimonine, emetine, sophaline D, and
tomatidine exhibited binding energies of − 8.39 kcal/
mol, − 10.17 kcal/mol, − 8.79 kcal/mol, and − 9.58 kcal/
mol, respectively. Thalimonine and sophaline D were
recommended for further in vitro studies based on fil-
ters, parameters, and mechanisms of virtual bioactivity Fig. 9 Some alkaloid structures which have been tested by in vitro and in vivo assays against SARS-CoV-2 proteins [293–296, 298, 305] alkaloid structures which have been tested by in vitro and in vivo assays against SARS-CoV-2 proteins [293–296, 298, 305] Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Page 27 of 48 Table 6 Potential alkaloid compounds acting as antiviral agents against SARS-CoV-2
Compounds
Plant sources
Antiviral acitivities
Assays
IC50/Binding Affinity*
Refs. Alkaloid‑based secondary metabolites Berbamine
Berberis
SARS-CoV-2
In vitro: in Vero cells
EC50 = ~ 2.3 mM
[293]
Emetin
Homoharringtonine (HHT)
Carapichea ipecacuanha and Cephalo-
toxus fortune
SARS-CoV-2 replication step
The virus, beta-CoV/Hongkong/
VM20001061/2020, was isolated
from the nasopharynx aspirate
In vitro: in Vero E6 cells
IC50 of emetine and HHT were
0.46 mM and 2.55 mM, respectively
A combination of remdesivir
at 6.25 mM with emetine at 0.195 mM
resulted in 64.9% inhibition in viral
yield
[294]
Berbamine (BE12)
Derivate of berbamine (BE33)
SARS-CoV-2 envelope protein
containing ion permeable channels
that regulates electrolyte balance,
including potassium, sodium and cal‑
cium concentration in serum
In vitro: in Vero E6 cells and other 13
cell lines
In vivo: BE-33 was injected into mice,
which significantly reduced cytokine
secretion
IC50 of BE12 and BE33 as: an inhibitor
of envelope channels: 111.50 mM
and 5.79 mM antivirus; 14.50 mM
and 0.94 mM. Selection index of BE12
and BE33: 2.06 and 33.47
[295]
Isolated lycorine
EC50 = 15.7 ± 1.2 nM; selective
index (SI) > 900
Lycoris radiata (steam cortex)
Artemisia annua (whole plant)
Pyrrosia lingua (leaf)
Lindera aggregata (root)
SARS-CoV strain BJ001
In vitro: 3-(4,5-dimethylthiazol-2-yl)-
5-(3-carboxymethoxyphenyl)-2-(4-
sulfophenyl)-2H-tetrazolium inner
salt (MTS) assay for virus-induced
cytopathic effect (CPE)
L. radiata extract (EC50 = 2.4 mg/mL;
SI = 370)
A. annua extract (EC50 = 34.5 mg/mL;
SI = 31)
P. lingua extract (EC50 = 43.2 mg/mL;
SI = 55)
L. Alkaloid‑based secondary metabolites aggregata extract (EC50 = 88.2 mg/
mL; SI = 16)
[296]
Phycocyanobilins
Spirulina platensis and Spirulina
maxima [297]
SARS-CoV-2 Mpro and PLpro
In vitro using FRET-based cleav‑
age assay with SARS-CoV-2 Mpro
and PLpro
IC50 with Mpro and PLpro is 71 mM
and 62 mM
[298]
Chloroquine
The bark of Cinchona tree
nCoV-2019BetaCoV/Wuhan/
WIV04/2019
In vitro using qRT-PCR and immuno‑
fluorescence microscopy in Vero E6
cells
EC50 = 1.13 μM; CC50 > 100 μM,
SI > 88.50
[299]
Berberine
Beta-sitosterol
Octacosanol
Tetrahydropalmatine
Choline
Tinospora cordifolia
3CLpro of SARS-CoV-2
In silico with ligand (6LU7)
Binding affinities in order: − 7.3; − 7.1;
− 6.6; − 6.4; − 3.4 kcal/mol
Inhibition constant in order; 4.4 × mM;
6.16 × mM; 1.43 × mM; 2.01 × mM;
3.2 × mM
[300]
Schizanthine Z
Schizanthus porrigens
PLpro (6WX4) of SARS-CoV-2
In silico using Autodock Vina and PyRx
with a ligand of 6WX4
Binding affinity: − 7.5 kcal/mol
[168]
Caffeine and nicotine
Active sites of the S protein in SARS-
CoV-2
In silico using AutoDock v4.2 package
with a ligand of 6LZG and 6VW1
Binding energy:
Nicotine + favipiravir + CTD-
ACE2 = − 7.13 kcal/mass
Caffeine + ribavirin + RBD-
ACE2 = − 6.76 kcal/mol
[301]
Thalimonine
Sophaline D
Tomatidine
Emetine
Mpro of SARS-CoV-2
In silico using AutoDock
Binding energy in order: − 8.39;
− 8.79; − 9.58; − 10.17 kcal/mol
Inhibition constant in order:
0.706 mM; 0.36266 mM; 0.09544 mM;
0.03535 mM
[302] Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Page 28 of 48 * IC50 (mg/mL and mM): the concentration of particular compound or drug in inhibiting the biological process to half of the maximum. Docking score (kcal/mol): a computational result for particular program and
energy to allow in predicting binding free energy and binding affinity, or ranking the complex of ligand and receptor according to specific parameters. Binding affinity (kcal/mol): an expression of the degree of ligand
binding with the protein in complex formation. Binding energy (kcal/mol): the energy released due to the bond formation or the interaction of the ligand and protein which is calculated as a sum of all the intermolecular
interactions presented in the complex
Table 6 (continued)
Compounds
Plant sources
Antiviral acitivities
Assays
IC50/Binding Affinity*
Refs. Alkaloid‑based secondary metabolites Phycocyanobilins
SARS-CoV-2 Mpro (6LU7) and PLpro
(6WUU)
In silico using AutoDock Vina
Binding energy of Mpro and PLpro
is − 8.6 and − 9.8 kcal/mol
[298]
Bismahanine
Murraya koenigii (L.) Spreng (leaves)
SARS-CoV-2 spike protein (6M0J)
In silico using AutoDock Vina
Binding energy: − 9.1 kcal/mol
[164]
Quinine
The bark of Cinchona tree
SARS-CoV-2 Mpro (pdb id: 6m0k)
In silico using
Binding energy: − 6.2 kcal/mol
[303] * IC50 (mg/mL and mM): the concentration of particular compound or drug in inhibiting the biological process to half of the maximum. Docking score (kcal/mol): a computational result for particular program and
energy to allow in predicting binding free energy and binding affinity, or ranking the complex of ligand and receptor according to specific parameters. Binding affinity (kcal/mol): an expression of the degree of ligand
binding with the protein in complex formation. Binding energy (kcal/mol): the energy released due to the bond formation or the interaction of the ligand and protein which is calculated as a sum of all the intermolecular
interactions presented in the complex
ab e 6 (co t ued)
Compounds
Plant sources
Antiviral acitivities
Assays
IC50/Binding Affinity*
Refs. Phycocyanobilins
SARS-CoV-2 Mpro (6LU7) and PLpro
(6WUU)
In silico using AutoDock Vina
Binding energy of Mpro and PLpro
is − 8.6 and − 9.8 kcal/mol
[298]
Bismahanine
Murraya koenigii (L.) Spreng (leaves)
SARS-CoV-2 spike protein (6M0J)
In silico using AutoDock Vina
Binding energy: − 9.1 kcal/mol
[164]
Quinine
The bark of Cinchona tree
SARS-CoV-2 Mpro (pdb id: 6m0k)
In silico using
Binding energy: − 6.2 kcal/mol
[303] Terpenoid and its derivatives Terpenoids are a diverse group of natural products that
are derived from isoprene (1,3-butadiene) units. They are
formed by combining carbon skeletons from other ace-
tate and shikimate pathways such as steroidal saponins,
cardioactive glycosides, and phytosterols. Terpenoids
have many essential applications in the fields of medicine,
cosmetics, and food industries. This group of secondary
metabolites exhibits biological activities, including anti-
tumor, anti-inflammatory, antibacterial, antiviral, antima-
larial, and antidiabetic activities [310]. Some terpenoids
have also been studied for their potential bioactivity
against SARS-CoV-2 and illustrated in Fig. 10, Table 7. Terpenoids, specifically monoterpenes and sesquit-
erpenes, are highly active compounds that may inter-
act more rapidly with the primary site of infection in
COVID-19 patients when delivered via aerosol delivery
systems such as nebulizers and inhalers. This form of
drugs delivery is preferred over oral administration as
it allows for direct entry of the terpenoids through the
respiratory tract, which increases their bioavailability
[255]. The aerosol form of terpenoids contains essential
oils, which are volatile oils obtained from different parts
of plants including leaves, fruit, flowers, bark, and roots
using various extraction methods. Essential oils are clas-
sified as terpenoids because they predominantly contain
monoterpenes and sesquiterpenes, rather than deriva-
tives of phenyl, propanoid, and aromatic compounds. Tetraterpenes,
particularly
astaxanthin
from
the carotenoid class, have been extensively dis-
cussed for their potential as an adjunctive supple-
ment in COVID-19 [314]. Triterpene glycosides, such
as saikosaponins A, B, C, and D, which can be iso-
lated from Heteromorpha spp., Bupleurum spp., and
Scrophularia scorodonia, have demonstrated anti-
viral activity against HCoV-22E9 [93]. Another sapo-
nin,
3-beta-O-(alpha-L-rhamnopyranosyl-(1- > 2)
alpha-L-arabino
pyranosyl)olean-12-ene-28-O-(alpha-
L-rhamnopyranosyl-(1- > 4)-beta-D-glucopyrano-
syl-(1- > 6)-beta-D-glucopyranosyl) ester, isolated from
leaves and stems of Oreopanax guatemalensis [315],
exhibited the highest binding energy to interact with
SARS-CoV-2 S-RBD compared to other terpenes using
computer-based molecular simulation [316]. Li et al. [296] reported that extracts from Artemisia annua, Lyco-
ris radiate, Pyrrosia lingua, and Lindera agregata were
practical for anti-SARS-CoV screening analysis. The
aqueous extract of Houttuynia cordata showed inhibition
of viral 3CL protease and blockade activity of viral RNA-
dependent RNA polymerase in SARS-CoV [317]. According to Javed et al. [333], carvacrol, a phenolic
monoterpene found in thyme and oregano, has dem-
onstrated therapeutic properties against various viral
diseases such as HSV type 1, bovine diarrhea virus, res-
piratory syncytial virus, and murine norovirus in vitro. Table 6 (continued) Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Page 29 of 48 Page 29 of 48 against Mpro of COVID-19. Various alkaloids derived
from plants have been shown to have potent antiviral
activity against various viruses, including coronavirus, as
listed by Majnooni et al. [305] and Topcu et al. [304]. against Mpro of COVID-19. Various alkaloids derived
from plants have been shown to have potent antiviral
activity against various viruses, including coronavirus, as
listed by Majnooni et al. [305] and Topcu et al. [304]. Derivatives of glycyrrhizin, including the amide form,
have been shown to exhibit higher anti-SARS-CoV activ-
ity than glycyrrhizin itself. This is due to the addition of
amino acid residues on the glycoside part, while preserv-
ing the free -COOH function in C30, which appears to
be crucial for the anti-SARS-CoV effect [332]. In vitro
experiments on antiviral activity of aqueous licorice root
extract containing glycyrrhizin against SARS-CoV-2 in
Vero E6 cells have demonstrated that glycyrrhizin blocks
SARS-CoV-2 replication by inhibiting Mpro, [252].i Terpenoid and its derivatives Essential oils directly act
on enveloped viruses, such as HSV type-1 and type-2, by
binding to viral envelopes and glycoproteins. The plaque
development assay supported this statement and showed
that essential oils reduced viral load significantly during
contact with virions before the adsorption process or
during the pre-treatment step, but not when used before
HSV-1 and HSV-2 adsorption and attachment [339, 340]. Time-of-addition experiments concluded that essential
oils blocked virus adsorption [341]. Thymoquinone and
black seed fixed oil were also found to be positively active
against avian influenza virus (H9N2) and MCMV infec-
tion model 36. Pelargonium sidoides, extracted herbal
products, has been licensed and marketed for patients
with acute bronchitis, reducing rhinovirus infection and
interfering with the reproduction of multiple respiratory
viruses [163–165]. The efficacy of natural plant essential oils in reducing
virus titers (TCID50) against non-enveloped viruses at
different temperatures and times was found to be insig-
nificant. Several previous studies have investigated the
impact of essential oils on non-enveloped viruses, such as
norovirus, rotavirus, adenovirus, and HPV. For instance,
Kovac et al. [344] examined the effect of Hyssopus offic-
inalis and Thymus mastichina essential oils against
murine norovirus (MNV-1) and human adenovirus sero-
type 2 (HAdV-2). Garozzo et al. [345] investigated Mela-
leuca alternifolia essential oil (tea tree oil, TTO) against
polio type 1, ECHO 9, and Coxsackie B1. While Cermelli
et al. Evaluated eucalyptus oil against adenovirus. In all
of these [344, 346], essential oils were unable to mask
non-enveloped viruses, indicating that they may not be a
viable option for reducing foodborne viruses in the food
industry. Conversely, essential oils have shown signifi-
cant virucidal activity against enveloped viruses due to
their ability to disrupt the virus’s enveloped proteins and
interaction with host cells. Jackwood et al. [347] reported
that QR448(a), a blend of botanical oleoresins and essen-
tial oils developed by Quigley Pharma, Inc., exhibited
virucidal effects against avian infectious bronchitis virus
(IBV) in Vero E6 cells, embryonated eggs, and chickens
by reacting before virus attachment and entry. Essential oils are edible, but their potential toxicity
requires caution when ingesting orally. The non-polar
properties of essential oils make them easily permeable
through skin membranes, leading to whole-body heal-
ing. As a result, essential oils can activate specific brain
regions, influencing the hypothalamus and providing
pain relief, mood enhancement, and improved cognitive
function [334]. Terpenoid and its derivatives It was proposed that carvacrol could also have potential
mechanisms of action against SARS-CoV-2, the virus
responsible for COVID-19. Specifically, carvacrol may
interfere with ACE2 receptors in host cells, leading to
protective effects against inflammation, and potentially
hinder the virus’s interaction with viral proteases during
infection. Essential oils derived from medicinal plants and their
food matrices contain volatile compounds that can be
quickly released, making them highly therapeutically
potent compared to the original plants or herbs [334]. These essential oils can easily enter the body through
inhalation and reach the bloodstream due to their high
volatility. However, it is important to assess the duration
of essential oil diffusion to ensure that it is safe to inhale
and maintain indoor air quality [335]. Glycyrrhizin, also known as glycyrrhizic acid, is a type
of terpenoid saponin that is commonly extracted from
the roots (Glycyrrhizae radix rhizoma) of glycyrrhiza
plants, including Glycyrrhiza glabra L. and licorice root
[251, 328]. The primary compound of glycyrrhizin is a
triterpene glycoside, with its aglycone being 18b-glycyr-
rhetinic acid [329]. Due to its mode of action and char-
acteristics, glycyrrhizin has the potential to be utilized as
an anti-SARS-CoV-2 agents [251, 330]. In a case report
of a non-hospitalized COVID-19 patient who took diam-
monium glycyrrhizinate, Ding et al. [331] stated that
immune regulation against cytokine storm had improved,
and inflammation was reduced. Boukhatem (2020) conducted a literature review of
published study articles and reported antiviral activities
of essential oils and isolated compounds. The potential
of essential oils from aromatic plants as antiviral com-
pounds against coronavirus have also been explored [3,
207, 336]. Adorjan and Buchbauer [337], as well as Ojah
[338], have listed essential oils with antiviral activities Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 (2023) 66:89 Page 30 of 48 Fig. 10 Chemical structures of terpenoids with their bioactivities against SARS-CoV-2 proteins [95, 101, 311–313] Fig. 10 Chemical structures of terpenoids with their bioactivities against SARS-CoV-2 proteins [95, 101, 311–313] [342], lemon balm (Melissa officinalis) [343], peppermint
[341], dwarf lavender cotton (Santolina insularis) [339],
ginger (Zingiber officinale), thyme (Thymus vulgaris),
hyssop (Hyssopus officinalis), and sandalwood (Santalum
album) [340], have been identified. Table 7 highlights
some potential essential oils and common terpenoids
with activity against SARS-CoV-2. against human-targeting viruses. Using essential oils as a
therapeutic antiviral intervention is a safe alternative due
to their natural extract origin. Terpenoid and its derivatives Due to concerns about their bioavailabil-
ity, essential oils are recommended for topical application
to the skin. Their lipophilic nature enables them to easily
penetrate the skin and disrupt the virion envelope, inhib-
iting host cell attachment. Several essential oils with viru-
cidal activity, such as lemongrass (Cymbopogon citratus) Page 31 of 48 Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Table 7 Derivates of terpenes potentially act as antiviral agents
Compound/Extract
Plant sources
Antiviral activities
Assays
Activity*
Refs. Black seed oil (BSO) or habatus‑
saudah
Nigella sativa
Murine cytomegalo virus
In vivo using a viral plaque-forming
assay of BALB/c mice spleen and liver
Undetected virus at the ratio
of the effector to target cells
was 20:1
[99]
Manuka oil
HSV
In vitro using a plaque reduction
assay on RC-37 cells (monkey kidney
cells)
IC50 = 0.96 mg/mL
[318]
Laurus nobilis oil containing
β-ocimene, 1,8-cineole, α-pinene,
β-pinene
Laurus nobilis
SARS-CoV (isolate FFM-1 from Ger‑
many)
In vitro using visually scoring
of the virus-induced cytopathogenic
effect (CPE) for 48 h post-infection
on Vero cells
IC50:120 mg/mL; SI of 4.16
[101]
Ethyl acetate and methanol extracts
of aerial parts of D. virgatus
Daucus virgatus (Poir.) Maire
Coxsackievirus B (CV-B)
In vitro using plaque reduction assay
on Hep-2 cell line
IC50 ethylacetate and methanol
extracts = 98.16 and 60.08 mg/mL
[319]
Ethanol extracts
Mentha piperita
Desmodium canadense
Thymus vulgaris
Avian infectious bronchitis virus (IBV) In vitro using plaque reduction assay
on Vero cells
TCID = 1.83 ± 0.31–3.45 ± 0.21 log10
EC50 = 0.003–0.076 mg
[320]
Betulinic acid
SARS-CoV 3CL pro
In vitro using FRET method
IC50 10 mM
[95]
The fraction containing high of can‑
nabidiol (FCBD)
Cannabis sativa strain Arbel
Interleukin: IL-6 dan IL-8
In vitro using enzyme-link immuno‑
sorbent assay on A549 cells
IC50 of 3.45 and 3.49 mg/mL
[311]
Artemisinin content
Artemisia annua L. (dried leaves)
SARS-CoV-2 USA/WA1
In vitro using cytophatic effect assay
on Vero E6 cells infected by SARS-
CoV-2
IC50 of 0.01–0.14 mg
[313]
Cryptotanshinone
Salvia miltiorrhiza
SARS-CoV PLpro
In vitro using proteolysis
of the fluorogenic substrate
IC50 = 0.8 ± 0.2 μM
[321]
Dihydrotanshinone I
Salvia miltiorrhiza
SARS-CoV Mpro
In vitro using proteolysis
of the fluorogenic substrate
IC50 = 14.4 ± 0.7 μM
[321]
Pachymic acid
Dried sclerotia of Poria cocos (Schw.). Terpenoid and its derivatives Wolf
SARS-CoV-2 Mpro recombinant
In vitro using the fluorogenic sub‑
strate for inhibition assay
IC50: 18.607 μM
[312]
Garlic essential oil containing 17
organosulfurs
ACE2 protein and main protease
of SARS-CoV-2
In silico using MOE 2015.10 on ACE2
protein and 6LU7 (Mpro)
ACE2:
diallyl tetrasulfide and allyl disulfide
− 14.06 and − 12.84 kcal/mol
Mpro:
Allyl disulfide and allyl trisulfide
− 15.32 and − 15.02 kcal/mol
[322] Table 7 Derivates of terpenes potentially act as antiviral agents Page 32 of 48 Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Table 7 (continued)
Compound/Extract
Plant sources
Antiviral activities
Assays
Activity*
Refs. β-farnesene
α-farnesene
farnesol
α-bulnesene
SARS-CoV-2: Mpro (main protease),
Nsp15/NendoU (endoribonu‑
cleoase), ADRP (ADP-ribose-1′′-
phosphatase), rS (binding domain
of the SARS-CoV-2 spike protein),
RdRp (RNA-dependent RNA
polymerase), and hACE2 (human
angiotensin-converting enzyme)
In silico using Molegro Virtual Docker
v. Terpenoid and its derivatives 6.0.1 on SARS-CoV-2 Mpro (5R7Z,
5R80, 5R81, 5R82, 5R83, 5R84, 6LU7,
6M03, 6Y84), Nsp15/NendoU (6VWW,
6W01, 6W02), rS (6M0J, 6M17, 6VX1,
6VW1), RdRp (6M71)
Docking Score (DS)
SARS-CoV-2 Mpro = (E)-β-farnesene
− 115.4 kJ/mol
SARS-CoV Nsp15/NendoU = (E,E)-
alpha-farnesene -107.5 kJ/mol
SARS-CoV-2 ADRP = (E)-β-farnesene
-116.3 kJ/mol
SARS-CoV-2 RdRp = (E,E)-farnesol
-89.6 kJ/mol
hACE2 = alpha-bulnesene < -100 kJ/
mol
[207]
Eucalyptol (1,8-cineole) in eucalyp‑
tus oil
MPro/3CLpro of SARS-CoV-2
In silico using 1-click dock and swiss
dock tools
DS = − 4.2
ΔG = − 6.04 kcal/mol
[323]
Jensenone in eucalyptus oil
MPro/3CLpro of SARS-CoV-2
In silico using 1-click dock and swiss
dock tools
DS = − 5.5
ΔG = − 6.03 kcal/mol
[205]
Cuminal
Carvacrol
Myrtanol
Pinocarveol
Receptor binding domain (RBD)
of the S1 glycoprotein (residues
319–541)
In silico using AutoDock Vina on RBD
of SARS-CoV-2 S1 subunit (6M07)
Binding affinity (kcal/mol):
Cuminal − 4.9
Carvacrol − 4.9
Myrtanol − 5.3
Pinocarveol -5.0
[324]
Lauruside 5
Laurus nobilis
SARS-CoV-2
In silico using 1-Click Mcule on SARS-
CoV-2 Mpro (6YB4)
Binding energy − 8.2 kcal/mol
[325]
Tanshinone I
SARS-CoV PLpro
In silico using Gold software
with PLpro (6WX4)
IC50 of 8.8 mM
[92]
3-β-O-(α-L-rhamnopyranosyl-(1- > 2)
α-L-arabinopyranosyl)
olean-12-ene-28-O-(α-L-
rhamnopyranosyl-(1- > 4)-β-D-
glucopyranosyl-(1- > 6)-β-D-
glucopyranosyl)ester
SARS-CoV-2 S-RBD
In silico using Autodock Vina
with ligand 6LZG
IC50 of − 11 kcal/mol
[316]
Limonin and scopadulcic acid B
Dictamus dasycarpus, and Citrus
orange
SARS-CoV-2 RdRp (6M71), hACE2
(6M1D), and Spike glycoprotein
(2GHV)
In silico using Autodock 4.2
The docking score of limonin
against RdRp, hACE2, and spike pro‑
tein is − 9.0, − 8.9, and − 8.4. While
docking score of scopadulcic acid B
is − 8.6, − 8.2, and − 8.8
[192]
Carvacrol, anethol, cinnamyl acetate
SARS-CoV-2 RBD S1 subunit of S
glycoprotein (6M0J)
In silico using AutoDock Vina
Binding affinities of three of them
were − 5.2. kcal/mol
[326]
Coagulin N
Withania coagulans (Stocks) Dunal
SARS-CoV-2 spike protein (6M0J)
In silico using AutoDock Vina
Binding energy: − 9.1 kcal/mol
[164]
Glycyrrhizic acid
Glycyrrhiza glabra L. (liquorice roots)
SARS-CoV-2 TMPRSS2 (sequence
NP_001128571.1)
In silico using AutoDock Vina
Binding energy: − 9.5 kcal/mol
[164]
Glycyrrhizin
Glycyrrhiza glabra L
SARS-CoV-2 spike RBD (6M0J)
In silico using AutoDock 4.2
Binding affinity: − 9.47 kcal/mol
[175] Page 33 of 48 Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Table 7 (continued)
Compound/Extract
Plant sources
Antiviral activities
Assays
Activity*
Refs. Terpenoid and its derivatives Ashwagandhanolide, withacoagin,
withaferin, and withanone
Ayurveda botanical: Withania somnif-
era (roots)
SARS-CoV-2 Mpro (5R84)
In silico using AutoDock 4.2.6
Docking score: − 9.9, and three other
withanolide are − 8.8
[236]
Ashwagandhanolide, withacoagin,
27-hydroxywithanone
Ayurveda botanical: Withania somnif-
era (roots)
SARS-CoV-2 RBD spike glycoprotein
(6M17)
In silico using AutoDock 4.2.6
Docking score: − 10, and two others
are − 7.6
[236]
Ashwagandhanolide, muzanzagenin
Ayurveda botanical: Withania somnif-
era (roots)
SARS-CoV-2 RdRp (6M71)
In silico using AutoDock 4.2.6
Docking score: − 10.2 and − 9.3
[236]
Arjunic acid, theasapogenol B,
euscaphic acid
Terminalia arjuna
Camelia sasanqua
Folium eriobortryde and Geum
japonicum
SARS-CoV-2 Mpro (6LU7)
In silico using Autodock Vina
Binding affinities and inhibition
constants:
− 8.1 kcal/mol and 1.16 μM
− 8.1 kcal/mol and 1.16 μM
− 8.0 kcal/mol and 1.37 μM
[193]
Crocin, digitoxigenin, β-eudesmol,
Crocus sativus L
Nerium oleander
Lauris nobilis L
SARS-CoV-2 Mpro (6LU7)
In silico using Autodock 1.5.4
Binding energies: − 8.2, − 7.2,
− 7.1 kcal/mol
[327]
Calendulaglycoside A
Calendula officinalis L
SARS-CoV-2 Mpro (6LU7)
In silico using MOE 2019 Suite
Binding-free energy:
− 72.14 ± 38.78 kJ/mol
[174]
* IC50 (mg/mL and mM): the concentration of particular compound or drug in inhibiting the biological process to half of the maximum. Docking score (kcal/mol): a computational result for particular program and energy
to allow in predicting binding free energy and binding affinity, or ranking the complex of ligand and receptor according to specific parameters. Binding affinity (kcal/mol): an expression of the degree of ligand binding
with the protein in complex formation. Binding energy (kcal/mol): the energy released due to the bond formation or the interaction of the ligand and protein which is calculated as a sum of all the intermolecular
interactions presented in the complex Page 34 of 48 Page 34 of 48 Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Antiviral mechanism of essential oils may be use-
ful in inhibiting SARS-CoV-2 when the virus has struc-
tural similarities to other viruses. One such virus is HSV,
an enveloped virus similar to SARS-CoV-2. Eugenol,
extracted from Eugenia caryophyllus (Spreng.) Bullock &
S.G. Harrison, was found to inhibit the replication of HSV
standard strains [84] in HSV-1 and HSV-2 viruses [348]
and delay the development of herpetic keratitis in HSV-
1-infected mice. Terpenoid and its derivatives However, due to its stability concerns,
eugenol is better suited for topical treatment rather than
internal use. Eugenol in Syzygium aromaticum extract
has also been evaluated for its ability to inhibit the rep-
lication of hepatitis C virus [220]. Another essential oil
component, isoborneol from Salvia fruticosa, has dual
viricidal activity against HSV-1, and inhibits virus repli-
cation and viral glycosylation at a concentration of 0.06%. Inactivation of the virus by isoborneol may lead to the
interaction of the alcoholic moiety of isoborneol and the
lipid in the virus envelope. Clove (S. aromaticum L.) has
been reviewed as a potential therapeutic agents for anti-
COVID-19 due to its essential oil content, with eugenol
being a major component [349]. Clove extract has been
shown to inhibit HCV replication [220] and exhibit
chemopreventive activity [350]. This dried flower bud
contains approximately 11–20% of the essential oil, while
its dried leaves comprise less than 5% of the oil, with
eugenol being a major component (70–90%) [350–353]. Eugenine, an isolated compound from Syzygum aromati-
cum extract, also exhibited anti-HSV potential activity
[354]. Manuka oil was found to inactivate HSV before
entering the cell, and its virucidal activity is believed to
be due to the interference of β-triketones and other ter-
penes in adsorption and entry into host cells [337]. constituent in Cinnamomi cortex (Cinnamomum cas-
sia Blume), was able to reduce virus yields in the lungs
during an infection with lethal influenza virus-induced
pneumonia in the airways of mice. p
y
CBD is a compound belonging to the class of cannabi-
noids and is typically found in Cannabis sativa. CBD has
a unique chemical structure consisting of terpenes and
phenols, which is commonly referred to as a terpenophe-
nolic compound. Recent study by Anil et al. and Raj et al. [257, 311] suggests that CBD, along with other terpenoid
and phenolic compounds, may possess potential activ-
ity against SARS-CoV-2. CBD has been detected in the
blood plasma of healthy patients at concentrations in
the nanomolar range when using approved CBD drugs. Conversely, CBD metabolite, 7-hydroxy-cannabidiol
(7-OH-CBD), was found to be in the micromolar range. A study conducted on A549 human lung carcinoma cells
expressing exogenous human ACE-2 receptor (A549-
ACE2 cells) showed that CBD inhibited the replication
of SARS-CoV-2 with an EC50 of 1.24, while 7-OH-CBD
was 3.6 μM. Terpenoid and its derivatives Additionally, oral administration of CBD
with a high-fat meal has been shown to increase the
presence of 7-OH-CBD in the blood, which could effec-
tively inhibit SARS-CoV-2 infection. Nguyen et al. [355]
also found that CBD therapy resulted in a lower rate
of testing positive for COVID-19 in patients. Further-
more, Chatow et al. [356] reported that CBD exhibited
a synergistic effect on HCoV-229E-infected human lung
fibroblasts when combined with the NT-VRL-1 terpene
formulation. Current, CBD is available as a mouth spray
under the licensed name Nabiximols, which is intended
to reduce and relieve respiratory disease-related pain. A study by Amparo et al. [357] on molecular docking
of certain terpenoids and essential oils against SARS-
CoV-2 showed satisfactory results. AutoDock Vina was
used to investigate some compounds and the results
revealed that (E)-α-atlantone, 14-hydroxy-α-muurolene,
allo-aromadendrene
epoxide,
amorpha-4,9-dien-2-ol,
aristolochene, azulenol, germacrene A, guaia-6,9-di-
ene, hedycaryol, humulene epoxide II, α-amorphene,
α-cadinene, α-calacorene, and α-muurolene have the
highest binding energy values for PLpro, 3CLpro, S pro-
tein, and RdRp, respectively. Similarly, plant secondary
metabolites such as bismahanine, eriodictyol-7-O-ruti-
noside, glycyrrhizic acid, and hypericin showed the high-
est binding energy against S protein, RdRp, TMPRSS2,
and Mpro, respectively. In an evaluation of coronavirus inhibition in HeLa-
CEACAM1a cells, ethanol extracts of Nigella sativa,
Anthemis hyalina, and peel of Citrus sinensis, which were
presumed to contain essential oil compounds, were. Ula-
sli et al. [79] reported that A. hyaline extract molecules
have the potential to treat CoV infections. Additionally,
Salem and Hossain [99] noted that BSO from N. sativa
exhibited a remarkable antiviral effect against MCMV
infection. According to Tkachenko’s [102], essential oil extracted
from the fruit and roots of Heracleum L. species (Api-
aceae) demonstrated a toxicity LD50 of 0.2–0.4 mL
against both Influenza Types A and B. The main constitu-
ents of these essential oils were found to be octyl acetate
and octyl isobutyrate in the seeds, while the fruits con-
tained monoterpenes such as pinene and limonene, as
well as complexes of ethers of octyl and hexyl alcohols. The roots, on the other hand, were found to contain
pinene, ocimene, and sesquiterpene derivatives. Hayashi
et al. [88] reported that cinnamaldehyde, the primary Antiviral activity of plant secondary metabolites against
SARS-CoV-2 has been discussed, along with the vari-
ous drugs discovery approaches that can be employed. Polyphenols, alkaloids, and terpenoids are some of the
secondary metabolite classes that can serve as antiviral
agents. Structure–activity relationship (SAR) SAR is a theoretical concept that links chemical mole-
cules structure or structural-related properties to its bio-
logical activity or target properties. This model enables
the modification of a moleculular structure to alter its
bioactivity. Essentially, molecules with identical chemi-
cal properties that interact and bind with targets simi-
larly will have similar activities. Therefore, SAR approach
involves identifying the properties of molecules, such as
geometric and electronic properties, solubility, and cer-
tain chemical groups, to predict its physicochemical and
biological properties in targeting biological targets. SAR
model reduces costs, time, and concerns related to toxic-
ity bioassays. y
Kaempferol 1, quercetin 2, and myricetin 3 are known
structurally similar flavonols, differing only by the pres-
ence of hydroxy substituents at positions 3′ and 5′. Based
on in silico analysis of their binding energy with Mpro,
their relative binding energy values are comparable. The
number of hydroxyl substituents on the B ring does not
significantly affect their binding activity against Mpro,
as they show similar binding activity values of around
− 8.4 to − 8.5 kcal/mol, with critical energy data from
other literature ranging from − 7.307 kcal/mol [362] to
− 9.5 kcal/mol [164]. Quercetin 2 has been identified as
crucial molecules [155] in the prophylaxis and treatment
of COVID-19 patients due to its anti-inflammatory activ-
ity against cytokine storm during severe inflammation. Meanwhile, kaempferol derivatives, such as rhamnoside
4 and glucuronide 5 glycosides, exhibit higher binding
affinities to Mpro (− 8.8 and − 9.1 kcal/mol, respectively)
than the lead compound kaempferol (− 8.58 kcal/mol). The presence of two di-rhamnosides in the kaempferol
skeleton at positions 3 and 7, as kaempferitrin 7, shows
lower binding affinity to Mpro compared to kaempferol. Earlier studies by Mehaney et al. and Mengist et al. [366, 367] have discussed the detailed mechanism for
designing inhibitors for SARS-CoV-2. Ye et al.[199] have
also proposed a mechanism for inhibiting SARS-CoV-2
Mpro. The focus of this study is on the impact of flavonol
structures on the binding affinities of Mpro to SARS-
CoV-2, with Fig. 11 providing illustrations of three forms
of flavonols with varying hydroxy substituents in the B
ring skeleton of flavonol and glycosides. fi
Based on the previous discussion, the optimal position
for the hydroxy group on the B ring of the flavonol skel-
eton is on the para-substituted benzene ring, as shown
in Fig. 12. Terpenoid and its derivatives Additionally, Machado et al. [358] and Pisoschi Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Page 35 of 48 Page 35 of 48 Page 35 of 48 (− 9.5 kcal/mol) has a lower binding energy compared
to myricetin (− 8.4 kcal/mol). Myricetin has shown
promising activity against Mpro in vitro, with an IC50
of 3.684 μM [262]. Therefore, it is expected that myrice-
tin derivatives would exhibit better bioactivity against
Mpro of SARS-CoV-2 based on in vitro and in vivo
analysis. (− 9.5 kcal/mol) has a lower binding energy compared
to myricetin (− 8.4 kcal/mol). Myricetin has shown
promising activity against Mpro in vitro, with an IC50
of 3.684 μM [262]. Therefore, it is expected that myrice-
tin derivatives would exhibit better bioactivity against
Mpro of SARS-CoV-2 based on in vitro and in vivo
analysis. et al. [359] have suggested other secondary metabolites
such as polysaccharides, lipids, vitamins, and animal-
derived compounds for the modulation of early inflam-
matory responses in COVID-19 patients. Structure–activity relationship (SAR) The meta-position of the hydroxyl substituent
does not significantly impact the binding energy, espe-
cially when comparing kaempferol 1, quercetin 2, and
myricetin 3. When a glycosylated hydroxyl position is
present on carbon number 3, compounds 4, 5, 6, 9, 10,
12–15 exhibit better binding affinity against Mpro than
the original compounds or aglycon (1, 2, 3). An inter-
esting observation is that compounds 7 and 11 have
di-glycosides in their flavonol skeleton, but at different
positions. While kaempferitrin 7 has two sugar moieties
at positions 3 and 7, which reduces its binding energy
against Mpro compared to the aglycon, kaempferol,
quercetin-3,5-digalactoside 11 comprises two sugar moi-
eties at positions 3 and 5, enabling it to bind better to the
active site of Mpro than its aglycon, quercetin 2. The type
of sugar presented as glycosides in molecules is presumed
to influence molecules bioactivity against Mpro, with
glucuronic acid, rhamnose, and rutinose enhancing and
facilitating the binding affinity against Mpro, while glu-
cose does not. This SAR between flavonol derivatives and
Mpro may serve as the foundation for the development of
a novel drugs compound against Mpro of SARS-CoV-2. l
Quercetin derivatives, with mono- or di-substituents
of the glycoside, exhibited high activity against Mpro. Quercetin-3-O-glucuronide 9, quercetin-3-O-rutinoside
(rutin) 12, and quercetin-3,5-digalactoside 11 had bind-
ing affinities to Mpro of -9.4 kcal/mol, − 9.16 kcal/mol,
and − 9.6 kcal/mol, respectively, which were higher than
quercetin 2 (− 8.47 kcal/mol). This suggests that the pres-
ence of glycoside as a substituent on the quercetin skel-
eton is important for increasing binding affinity to Mpro. Most glycoside compounds have higher bioavailability in
the body than aglycon [164]. However, isoquercitrin or
isoquercetin 9, a monosubstituted quercetin glycoside,
showed less binding energy than quercetin. The pres-
ence of glucose as the glycoside did not improve binding
access to Mpro. Similar to quercetin, myricetin deriva-
tives in a glycoside form also showed high binding affinity
to Mpro compared to the lead compound, myricetin 3. Myricetin-3-O-rhamnoside (myricitrin) 13 and myrice-
tin-3-O-rutinoside 14 are two examples of this. However,
myricetin itself did not show a good binding affinity to
Mpro, and was not ranked highly in previously reported
study, with a binding energy of − 7.311 kcal/mol [164]. Myricetin derivatives have also been tested for their
binding energy against the RdRp of SARS-CoV-2,
and it has been found that myricetin-3-O-rutinoside Oktavianawati et al. Structure–activity relationship (SAR) Applied Biological Chemistry (2023) 66:89 (2023) 66:89 Page 36 of 48 nawati et al. Applied Biological Chemistry (2023) 66:89 Page 36 of 48 . 11 Correlation of flavonol-based derivatives with their in-silico bioactivities with Mpro of SARS-CoV-2. All docking scores data presente
re obtained from different references, which will be influenced by their own use on (1) docking program, (2) docking algorithm, and (3) s
-up. Refs.: *[164]; **[281]; ***[265]; ****[262]; *****[360]; ******[361]; *******[362]; ********[363]; *********[364]; **********[365] Fig. 11 Correlation of flavonol-based derivatives with their in-silico bioactivities with Mpro of SARS-CoV-2. All docking scores data presented here
were obtained from different references, which will be influenced by their own use on (1) docking program, (2) docking algorithm, and (3) system
set-up. Refs.: *[164]; **[281]; ***[265]; ****[262]; *****[360]; ******[361]; *******[362]; ********[363]; *********[364]; **********[365] Fig. 11 Correlation of flavonol-based derivatives with their in-silico bioactivities with Mpro of SARS-CoV-2. All docking scores data presented here
were obtained from different references, which will be influenced by their own use on (1) docking program, (2) docking algorithm, and (3) system
set-up. Refs.: *[164]; **[281]; ***[265]; ****[262]; *****[360]; ******[361]; *******[362]; ********[363]; *********[364]; **********[365] Fig. 11 Correlation of flavonol-based derivatives with their in-silico bioactivities with Mpro of SARS-CoV-2. All docking scores data presented here
were obtained from different references, which will be influenced by their own use on (1) docking program, (2) docking algorithm, and (3) system
set-up. Refs.: *[164]; **[281]; ***[265]; ****[262]; *****[360]; ******[361]; *******[362]; ********[363]; *********[364]; **********[365] Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Page 37 of 48 Fig. 12 SAR of flavonol against Mpro of SARS-CoV-2 based on in silico data from previous studies as shown on Fig. 11 [164, 262, 265, 278, 281, 317,
363, 368] Fig. 12 SAR of flavonol against Mpro of SARS-CoV-2 based on in silico data from previous studies as shown on Fig. 11 [164, 262, 265, 278, 281, 317, Current challenges against COVID‑19 After COVID-19 pandemic subsides, a major chal-
lenge that remains is increasing immunity to prevent
unexpected virus mutations, thereby leading to ques-
tions, such as Should people receive annual vaccinations
to prevent unexpected mutants? Further investigation on
vaccine production is ongoing, but the long-term impact
of immunization on the human body must also be inves-
tigated. Concerns have been raised about the potential
carcinogenic effects of vaccination, which may not be
detected quickly. Identifying adverse events after vacci-
nation is important, especially for children, who have a
longer expected future than older individuals. In some
countries, vaccinations are mandatory for children over
eight years old to improve their immunity. Therefore, it
is important to track the progress in the muscular, car-
diovascular, respiratory, and reproductive systems. In the
coming years, study on COVID-19 will provide valuable
scientific insights into preventing future unexpected viral
infections. g
g
New variants of SARS-CoV-2 have emerged through
mutations that increase their transmissibility, sever-
ity, and mortality. In the future, significant attention
and funding will be given to the study of drugs, with
a shift towards vaccine redesign when repurposing
drugs is not enough. However, an effective vaccine can-
not entirely prevent future mutant attacks, and not all
vaccines are suitable for worldwide application due to
factors such as environment, geography, and genetic
diversity. Each SARS-CoV-2 variant has unique char-
acteristics and infection roles, and current antibodies
may not always be effective in neutralizing these vari-
ants. Therefore, specific vaccines are needed to enhance
human immunity against each variant. The effective-
ness of vaccines, such as NVX-CoV2373, varies across
different regions and variants. In the UK, it showed
95.6% effectiveness against the original strain of SARS-
CoV-2, but its efficacy in South Africa was 60%, and it
was only 49.4% effective against the beta variant. Addi-
tionally, vaccine effectiveness diminishes over time,
with up to a 50% reduction in efficacy observed after
ten weeks of a booster dose [2]. Regarding the development of natural products into
antiviral drugs, there are some considerations about what
kind of challenges and problems researchers facing now-
adays. Firstly, the complexity of natural products leads to Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Page 38 of 48 Page 38 of 48 time consuming and labour-intensive problems due to
the long way of isolation, characterization and synthesis
process [4, 6]. Competing interests
h
h
d
l
h 18. Lam TT, Jia N, Zhang Y, Shum MH, Jiang J, Zhu H, Tong Y, Shi Y, Ni X, Liao
Y et al (2020) Identifying SARS-CoV-2-related coronaviruses in Malayan
pangolins. Nature 583:282–285 The authors declare that they have no competing interests. The authors declare that they have no competing interests. 19. Chellapandi P, Saranya S (2020) Genomics insights of SARS-CoV-2
(COVID-19) into target-based drug discovery. Med Chem Res 29:1777–
1791. https://doi.org/10.1007/s00044-020-02610-8 Received: 19 August 2023 Accepted: 17 November 2023 Received: 19 August 2023 Accepted: 17 November 2023 20. Hu D, Zhu C, Ai L, He T, Wang Y, Ye F, Yang L, Ding C, Zhu X, Lv R et al
(2018) Genomic characterization and infectivity of a novel SARS-like
coronavirus in Chinese bats. Emerg Microbes Infect 7:1–10. https://doi.
org/10.1038/s41426-018-0155-5 Availability of data and materials
Not applicable. 16. Raj VS, Osterhaus ADME, Fouchier RAM, Haagmans BL (2014) MERS:
emergence of a novel human coronavirus. Curr Opin Virol 5:58–62 Not applicable. 17. Zaki AM, van Boheemen S, Bestebroer TM, Osterhaus ADME, Fouchier
RAM (2012) Isolation of a novel coronavirus from a man with Pneumo‑
nia in Saudi Arabia. N Engl J Med 367:1814–1820. https://doi.org/10.
1056/nejmoa1211721 Author contributionsi IO wrote the first draft of the manuscript, wrote additional sections, and
created the figures. IO, MS, MFAB, YUK, and SF created the concept, edited,
and revised the manuscript together. All authors have read and agreed to the
published version of the manuscript. y y
Science 300:1394–1399. https://doi.org/10.1126/science.1085952 14. Cunha CB, Opal SM (2014) Middle East respiratory syndrome (MERS): a
new zoonotic viral pneumonia. Virulence 5:650–654. https://doi.org/10.
4161/viru.32077 15. WHO (2021) WHO lists two additional COVID-19 vaccines for emer‑
gency use and COVAX roll-out AstraZeneca/Oxford-developed vaccines
to reach countries in the coming weeks. World Heal. Organ. 3–5. https://www.who.int/news/item/15-02-2021-who-lists-two-additional-
covid-19-vaccines-for-emergency-use-and-covax-roll-out. Acknowledgements
Th
h
l
k The authors also acknowledge Indonesia Endowment Fund for Education
(Lembaga Pengelola Dana Pendidikan, LPDP) for supporting the present work. 13. Rota PA, Oberste MS, Monroe SS, Nix WA, Campagnoli R, Icenogle JP,
Peñaranda S, Bankamp B, Maher K, Chen M (2003) Characterization of a
novel coronavirus associated with severe acute respiratory syndrome. Science 300:1394–1399. https://doi.org/10.1126/science.1085952 Current challenges against COVID‑19 Secondly, standardization on the manufac-
ture of the isolate or compound from natural products is
difficult to be applied since a broad variation on bioac-
tivity potency from different sources and batches [8]. In
addition, formula stability and safety on the use of natural
product extracts require specific strategies on the pres-
ervation of its efficacy and safety assessments which also
to avoid any occurred resistances [10, 12]. The limited
sources of natural products are also being an ethical and
ecological concerns nowadays. Therefore, interdiscipli-
nary collaborations among researchers in chemistry, biol-
ogy, pharmacy, and medicine are required to commit in
addressing the various scientific, regulatory and ethical
challenges that arise. 6. Park D, Swayambhu G, Lyga T, Pfeifer BA (2021) Complex natural prod‑
uct production methods and options. Synth Syst Biotechnol 6:1–11. https://doi.org/10.1016/j.synbio.2020.12.001 7. Peiris JSM, Lai ST, Poon LLM, Guan Y, Yam LYC, Lim W, Nicholls J, Yee
WKS, Yan WW, Cheung MT et al (2003) Coronavirus as a possible cause
of severe acute respiratory syndrome. Lancet 361:1319–1325. https://
doi.org/10.1016/S0140-6736(03)13077-2 g
8. Atanasov AG, Zotchev SB, Dirsch VM, Taskforce (2021) Natural products
in drug discovery: advances and opportunities. Nat Rev Drug Discov
20:200–2016. https://doi.org/10.5772/intechopen.82860 9. Drosten C, Günther S, Preiser W, van der Werf S, Brodt H-R, Becker S,
Rabenau H, Panning M, Kolesnikova L, Fouchier RAM et al (2003) Identi‑
fication of a novel coronavirus in patients with severe acute respiratory
syndrome. N Engl J Med 348:1967–1976. https://doi.org/10.1056/nejmo
a030747 10. Thomas E, Stewart LE, Darley BA, Pham AM, Esteban I, Panda SS (2021)
Develop new antiviral drug candidates. Molecules 26:1–56. https://doi.
org/10.3390/molecules26206197 10. Thomas E, Stewart LE, Darley BA, Pham AM, Esteban I, Panda SS (2021)
Develop new antiviral drug candidates. Molecules 26:1–56. https://doi.
org/10.3390/molecules26206197 11. Lee N, Hui D, Wu A, Chan P, Cameron P, Joynt G, Ahuja A, Yung M,
Leung C, To K et al (2003) A major outbreak of severe acute respiratory
syndrome in Hong Kong. N Engl J Med 348:1986–1994. https://doi.org/
10.5222/terh.2003.09293 12. Ma J, Gu Y, Xu P (2020) A roadmap to engineering antiviral natural prod‑
ucts synthesis in microbes. Curr Opin Biotechnol 66:140–149. https://
doi.org/10.1016/j.copbio.2020.07.008 12. Ma J, Gu Y, Xu P (2020) A roadmap to engineering antiviral natural prod‑
ucts synthesis in microbes. Curr Opin Biotechnol 66:140–149. https://
doi.org/10.1016/j.copbio.2020.07.008 Funding
h
k This work is supported by Indonesia Endowment Fund for Education
(Lembaga Pengelola Dana Pendidikan, LPDP) under-Grant Number KET-235/
LPDP.4/2021 and LOG-3382/LPDP/LPDP.3/2023. Availability of data and materials
Not applicable. References 1. Ahmad A, Rehman MU, Alkharfy KM (2020) An alternative approach to
minimize the risk of coronavirus (Covid-19) and similar infections. Eur
Rev Med Pharmacol Sci 24:4030–4034. https://doi.org/10.2355/EUR‑
REV_202004_20873 21. Lu R, Zhao X, Li J, Niu P, Yang B, Wu H, Wang W, Song H, Huang B, Zhu N
et al (2020) Genomic characterisation and epidemiology of 2019 novel
coronavirus: implications for virus origins and receptor binding. Lancet
395:565–574. https://doi.org/10.1016/S0140-6736(20)30251-8 2. El-Shabasy RM, Nayel MA, Taher MM, Abdelmonem R, Shoueir KR,
Kenawy ER (2022) Three waves changes, new variant strains, and
vaccination effect against COVID-19 pandemic. Int J Biol Macromol
204:161–168. https://doi.org/10.1016/j.ijbiomac.2022.01.118 22. Zhou P, Yang XL, Wang XG, Hu B, Zhang L, Zhang W, Si HR, Zhu Y, Li B,
Huang CL et al (2020) A pneumonia outbreak associated with a new
coronavirus of probable bat origin. Nature 579:270–273. https://doi.
org/10.1038/s41586-020-2012-7 3. Mazraedoost S, Behbudi G, Mousavi SM, Hashemi SA (2020) Covid-19
treatment by plant compounds. J Adv Appl NanoBio Tech 2:23–33. https://doi.org/10.4277/AANBT/2(1)33 23. Lau SKP, Luk HKH, Wong ACP, Li KSM, Zhu L, He Z, Fung J, Chan TTY,
Fung KSC, Woo PCY (2020) Possible bat origin of severe acute respira‑
tory syndrome coronavirus 2. Emerg Infect Dis 26:1542–1547. https://
doi.org/10.3201/eid2607.200092 4. Zhang QW, Lin LG, Ye WC (2018) Techniques for extraction and isolation
of natural products: a comprehensive review. Chinese Med 13:1–26. https://doi.org/10.1186/s13020-018-0177-x 24. Vasireddy D, Vanaparthy R, Mohan G, Malayala SV, Atluri P (2021) Review
of COVID-19 Variants and COVID-19 Vaccine Efficacy: What the Clinician
Should Know? J Clin Med Res 13:317–325. https://doi.org/10.1740/
jocmr4518 5. Panyod S, Ho CT, Sheen LY (2020) Dietary therapy and herbal medicine
for COVID-19 prevention: a review and perspective. J Tradit Comple‑
ment Med 10:420–427. https://doi.org/10.1016/j.jtcme.2020.05.004 25. Nicolete VC, Rodrigues PT, Fernandes ARJ, Corder RM, Tonini J, Buss LF,
Sales FC, Faria NR, Sabino EC, Castro MC et al (2022) Epidemiology of Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Page 39 of 48 (2023) 66:89 COVID-19 after emergence of SARS-CoV-2 gamma variant, Brazilian
Amazon, 2020–2021. Emerg Infect Dis 28:709–712. https://doi.org/10.
3201/eid2803.211993 COVID-19 after emergence of SARS-CoV-2 gamma variant, Brazilian
Amazon, 2020–2021. Emerg Infect Dis 28:709–712. https://doi.org/10.
3201/eid2803.211993 COVID-19 drug discovery. J Mol Struct. https://doi.org/10.1016/j.molst
ruc.2021.130366 COVID-19 drug discovery. J Mol Struct. https://doi.org/10.1016/j.molst
ruc.2021.130366 45. References Botton J, Semenzato L, Jabagi MJ, Baricault B, Weill A, Dray-Spira R,
Zureik M (2022) Effectiveness of Ad26.COV2.S Vaccine vs BNT162b2
vaccine for COVID-19 hospitalizations. JAMA Netw Open 5:2–7. https://
doi.org/10.1001/jamanetworkopen.2022.0868 39. Gioia M, Ciaccio C, Calligari P, Simone G, Sbardella D, Tundo G,
Fasciglione G, Masi A, Pierro D, Bocedi A et al (2020) Role of proteo‑
lytic enzymes in the COVID-19 infection and promising therapeutic
approaches. Biochem Pharmacol 182:1–21. https://doi.org/10.1016/j.
bcp.2020.114225 58. Mahase E (2021) Covid-19: Novavax vaccine efficacy is 86% against UK
variant and 60% against South African variant. BMJ 372:2021. https://
doi.org/10.1136/bmj.n296 58. Mahase E (2021) Covid-19: Novavax vaccine efficacy is 86% against UK
variant and 60% against South African variant. BMJ 372:2021. https://
doi.org/10.1136/bmj.n296 40. Pommier Y, Johnson AA, Marchand C (2005) Integrase inhibitors to treat
HIV/AIDS. Nat Rev Drug Discov 4:236–248. https://doi.org/10.1038/
nrd1660 59. Cromer D, Reynaldi A, Steain M, Triccas JA, Davenport MP, Khoury DS
(2022) Relating in vitro neutralization level and protecztion in the
CVnCoV (CUREVAC) trial. Clin Infect Dis 75:E878–E879. https://doi.org/
10.1093/cid/ciac075 59. Cromer D, Reynaldi A, Steain M, Triccas JA, Davenport MP, Khoury DS
(2022) Relating in vitro neutralization level and protecztion in the
CVnCoV (CUREVAC) trial. Clin Infect Dis 75:E878–E879. https://doi.org/
10.1093/cid/ciac075 41. Pour PM, Fakhri S, Asgary S, Farzaei MH, Echeverría J (2019) The signal‑
ing pathways, and therapeutic targets of antiviral agents: focusing on
the antiviral approaches and clinical perspectives of anthocyanins in
the management of viral diseases. Front Pharmacol. https://doi.org/10.
3389/fphar.2019.01207 60. Cohen J (2021) What went wrong with CureVac’s mRNA vaccine pdf. Science. https://doi.org/10.1126/science.372.6549.1381 61. Baden LR, El Sahly HM, Essink B, Kotloff K, Frey S, Novak R, Diemert D,
Spector SA, Rouphael N, Creech CB et al (2021) Efficacy and safety
of the mRNA-1273 SARS-CoV-2 vaccine. N Engl J Med 384:403–416. https://doi.org/10.1056/nejmoa2035389i 61. Baden LR, El Sahly HM, Essink B, Kotloff K, Frey S, Novak R, Diemert D,
Spector SA, Rouphael N, Creech CB et al (2021) Efficacy and safety
of the mRNA-1273 SARS-CoV-2 vaccine. N Engl J Med 384:403–416. https://doi.org/10.1056/nejmoa2035389 42. McKinlay MA, Pevear DC, Rossmann MG (1992) Treatment of the
picorna virus common cold by inhibitors of viral uncoating and attach‑
ment. Annu Rev Microbiol 46:635–656. https://doi.org/10.1146/annur
ev.mi.46.100192.003223 43. Pathania S, Rawal RK, Singh PK (2022) RdRp (RNA-dependent RNA
polymerase): a key target providing anti-virals for the management
of various viral diseases. J Mol Struct 1250:131756. https://doi.org/10.
1016/j.molstruc.2021.131756 g
j
62. References Feuillet V, Canard B, Trautmann A (2021) Combining antivirals and
immunomodulators to fight COVID-19. Trends Immunol 42:31–44. https://doi.org/10.1016/j.it.2020.11.003 52. Darbar S, Agarwal S, Saha S (2021) COVID19 Vaccine: COVAXIN ®-India’s
first indigenous effective weapon to fight against coronavirus (a
review). Parana J Sci Educ 7:1–9 33. Wang M, Zhao R, Gao L, Gao X, Wang D, Cao J (2020) SARS-CoV-2: struc‑
ture, biology, and structure-based therapeutics development. Front Cell
Infect Microbiol 10:1–18 53. Deshpande GR, Yadav PD, Abraham P, Nyayanit DA, Sapkal GN, Shete
AM, Gupta N, Vadrevu KM, Ella R, Panda S et al (2022) Booster dose
of the inactivated COVID-19 vaccine BBV152 (Covaxin) enhances the
neutralizing antibody response against alpha, beta, delta and omicron
variants of concern. J Travel Med 29:1–3. https://doi.org/10.1093/jtm/
taac039 34. Goc A, Sumera W, Rath M, Niedzwiecki A (2021) Phenolic compounds
disrupt spike-mediated receptor-binding and entry of SARS-CoV-2
pseudo-virions. PLoS ONE 16:1–20. https://doi.org/10.1371/journal.
pone.0253489 54. Emary KRW, Golubchik T, Aley PK, Ariani CV, Angus B, Bibi S, Blane
B, Bonsall D, Cicconi P, Charlton S et al (2021) Efficacy of ChAdOx1
nCoV-19 (AZD1222) vaccine against SARS-CoV-2 variant of concern
202012/01 (B.1.1.7): an exploratory analysis of a randomised controlled
trial. Lancet 397:1351–1362. https://doi.org/10.1016/S0140-6736(21)
00628-0 p
35. Park A, Iwasaki A (2020) Type I and type iii interferons - induction, sign‑
aling, evasion, and application to combat COVID-19. Cell Host Microbe
27:870–878 36. Speerstra S, Chistov AA, Proskurin GV, Aralov AV, Ulashchik EA, Streshnev
PP, Shmanai VV, Korshun VA, Schang LM (2018) Antivirals acting on
viral envelopes via biophysical mechanisms of action. Antiviral Res
149:164–173. https://doi.org/10.1016/j.antiviral.2017.11.018 55. Mallapaty S, Callaway E (2021) What scientists do and don’t know about
the Oxford-AstraZeneca COVID vaccine. Nature 592:15–17. https://doi.
org/10.1038/d41586-021-00785-7 37. Wisskirchen K, Lucifora J, Michler T, Protzer U (2014) New pharmaco‑
logical strategies to fight enveloped viruses. Trends Pharmacol Sci
35:470–478. https://doi.org/10.1016/j.tips.2014.06.004 56. van der Lubbe JEM, Rosendahl Huber SK, Vijayan A, Dekking L, van
Huizen E, Vreugdenhil J, Choi Y, Baert MRM, Feddes-de Boer K, Izquierdo
Gil A et al (2021) Ad26.COV2.S protects Syrian hamsters against G614
spike variant SARS-CoV-2 and does not enhance respiratory disease. npj
Vaccines 6:1–12. https://doi.org/10.1038/s41541-021-00301-y p
g
j p
38. Antonelli G, Turriziani O (2012) Antiviral therapy: old and current issues. Int J Antimicrob Agents 40:95–102. https://doi.org/10.1016/j.ijant
imicag.2012.04.005 57. References Su HX, Yao S, Zhao W, Li M, Liu J, Shang W, Xie H, Ke C, Hu H, Gao M et al
(2020) Anti-SARS-CoV-2 activities in vitro of Shuanghuanglian prepara‑
tions and bioactive ingredients. Acta Pharmacol Sin 41:1167–1177 26. Shiehzadegan S, Alaghemand N, Fox M, Venketaraman V (2021) Analysis
of the delta variant B.1.617.2 COVID-19. Clin Pract 11:778–784. https://
doi.org/10.3390/clinpract11040093 46. Ma L, Liu H, Liu X, Yuan X, Xu C, Wang F, Lin J, Xu R, Zhang D (2021)
Screening S protein ACE2 blockers from natural products: Strategies
and advances in the discovery of potential inhibitors of COVID-19. Eur J
Med Chem 226:1–23. https://doi.org/10.1016/j.ejmech.2021.113857 27. Torjesen I (2021) Covid-19: delta variant is now UK’s most dominant
strain and spreading through schools. BMJ 373:n1445. https://doi.org/
10.1136/bmj.n1445 28. Akash K, Sharma A, Kumar D, Singh SK, Gupta G, Chellappan DK, Dua K,
Nagraik R (2022) Molecular aspects of Omicron, vaccine development,
and recombinant strain XE: a review. J Med Virol 94:4628–4643. https://
doi.org/10.1002/jmv.27936 47. WHO (2021) Interim recommendations for use of the Pfizer–BioNTech
COVID-19 vaccine, BNT162b2, under Emergency Use Listing. World
Heal. Organ., 1–7. 48. WHO (2021) Interim recommendations for use of the Janssen Ad26. COV2S (COVID-19) vaccine. World Heal. Organ. 1–6. g
j
29. Goldsmith CS, Tatti KM, Ksiazek TG, Rollin PE, Comer JA, Lee WW, Rota
PA, Bankamp B, Bellini WJ, Zaki SR (2004) Ultrastructural characterization
of SARS coronavirus. Emerg Infect Dis 10:320–326. https://doi.org/10.
3201/eid1002.030913 49. Li CX, Noreen S, Zhang LX, Saeed M, Wu PF, Ijaz M, Dai DF, Maqbool I,
Madni A, Akram F et al (2022) A critical analysis of SARS-CoV-2 (COVID-
19) complexities, emerging variants, and therapeutic interventions and
vaccination strategies. Biomed Pharmacother 146:112550. https://doi.
org/10.1016/j.biopha.2021.112550 30. Tshibangu DST, Matondo A, Lengbiye EM, Inkoto CL, Ngoyi EM,
Kabengele CN, Bongo GN, Gbolo BZ, Kilembe JT, Mwanangombo DT
et al (2020) Possible effect of aromatic plants and essential oils against
COVID-19: review of their antiviral activity. J Complement Altern Med
Res 11:10–22. https://doi.org/10.9734/jocamr/2020/v11i130175 50. Rehman SU, Rehman SU, Yoo HH (2021) COVID-19 challenges and its
therapeutics. Biomed Pharmacother 142:112015. https://doi.org/10.
1016/j.biopha.2021.112015 j
p
51. Kezia V, Ramatillah DL (2022) Intensive monitroing of sinovac vaccine
for safety and efficacy among Indonesian population. Int J Appl Pharm
14:44–48 p
g
j
31. Wang HM, Liang PH (2010) Picornaviral 3C protease inhibitors and the
dual 3C protease/coronaviral 3C-like protease inhibitors. Expert Opin
Ther Pat 20:59–71. https://doi.org/10.1517/13543770903460323 g
32. References Comput Struct Biotechnol J 19:2508–2517. https://doi.org/
10.1016/j.csbj.2021.04.061 92. Diniz LR, Perez-Castillo Y, Elshabrawy H, da Filho C, SM, and Sousa, D. (2021) Bioactive terpenes and their derivatives as potential sars-cov-2
proteases inhibitors from molecular modeling studies. Biomolecules
11:1–19 75. Park JW, Lagniton PNP, Liu Y, Xu RH (2021) mRNA vaccines for covid-19:
What, why and how. Int J Biol Sci 17:1446–1460. https://doi.org/10.
7150/ijbs.59233 93. Cheng PW, Ng LT, Chiang LC, Lin CC (2006) Antiviral effects of saiko‑
saponins on human coronavirus 229E in vitro. Clin Exp Pharmacol
Physiol 33:612–616. https://doi.org/10.1111/j.1440-1681.2006.04415.x 76. Ouled Aitouna A, Belghiti ME, Eşme A, Anouar E, Ouled Aitouna A,
Zeroual A, Salah M, Chekroun A, El Alaoui El Abdallaoui H, Benharref
A et al (2021) Chemical reactivities and molecular docking studies of
parthenolide with the main protease of HEP-G2 and SARS-CoV-2. J Mol
Struct 1243:1–9. https://doi.org/10.1016/j.molstruc.2021.130705 94. Chen F, Chan KH, Jiang Y, Kao RYT, Lu HT, Fan KW, Cheng VCC, Tsui
WHW, Hung IFN, Lee TSW et al (2004) In vitro susceptibility of 10 clinical
isolates of SARS coronavirus to selected antiviral compounds. J Clin
Virol 31:69–75. https://doi.org/10.1016/j.jcv.2004.03.003 95. Wen CC, Kuo YH, Jan JT, Liang PH, Wang SY, Liu HG, Lee CK, Chang
ST, Kuo CJ, Lee SS et al (2007) Specific plant terpenoids and lignoids
possess potent antiviral activities against severe acute respiratory
syndrome coronavirus. J Med Chem 50:4087–4095. https://doi.org/10.
1021/jm070295s 77. Zhang J, Xiong Y, Wang F, Zhang F, Yang X, Lin G, Tian P, Ge G, Gao
D (2022) Discovery of 9,10-dihydrophenanthrene derivatives as
SARS-CoV-2 3CLpro inhibitors for treating COVID-19. Eur J Med Chem. 228:1–16. https://doi.org/10.1016/j.ejmech.2021.114030 78. Shen L, Niu J, Wang CBH, Wang W, Zhu N, Deng Y, Wang H, Ye F, Chen
S et al (2019) High-throughput screening and identification of potent
broad-spectrum inhibitors of coronaviruses. J Virol 93(1):15 96. Kim DW, Seo KH, Curtis-Long MJ, Oh KY, Oh JW, Cho JK, Lee KH, Park KH
(2014) Phenolic phytochemical displaying SARS-CoV papain-like pro‑
tease inhibition from the seeds of Psoralea corylifolia. J Enzyme Inhib
Med Chem 29:59–63. https://doi.org/10.3109/14756366.2012.753591 79. Ulasli M, Gurses SA, Bayraktar R, Yumrutas O, Oztuzcu S, Igci M, Igci YZ,
Cakmak EA, Arslan A (2014) The effects of Nigella sativa (Ns), anthemis
hyalina (Ah) and Citrus sinensis (Cs) extracts on the replication of
coronavirus and the expression of TRP genes family. Mol Biol Rep
41:1703–1711. https://doi.org/10.1007/s11033-014-3019-7 97. References Chagla Z (2021) The BNT162b2 (BioNTech/Pfizer) vaccine had 95%
efficacy against COVID-19 ≥7 days after the 2nd dose Ann. Intern Med
https://doi.org/10.7326/ACPJ202102160-015 62. Chagla Z (2021) The BNT162b2 (BioNTech/Pfizer) vaccine had 95%
efficacy against COVID-19 ≥7 days after the 2nd dose Ann. Intern Med. https://doi.org/10.7326/ACPJ202102160-015 p
g
63. Polack FP, Thomas SJ, Kitchin N, Absalon J, Gurtman A, Lockhart S, Perez
JL, Pérez Marc G, Moreira ED, Zerbini C et al (2020) Safety and efficacy of 63. Polack FP, Thomas SJ, Kitchin N, Absalon J, Gurtman A, Lockhart S, Perez
JL, Pérez Marc G, Moreira ED, Zerbini C et al (2020) Safety and efficacy of 44. Ghosh K, Amin SA, Gayen S, Jha T (2021) Unmasking of crucial structural
fragments for coronavirus protease inhibitors and its implications in 44. Ghosh K, Amin SA, Gayen S, Jha T (2021) Unmasking of crucial structural
fragments for coronavirus protease inhibitors and its implications in Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Page 40 of 48 the BNT162b2 mRNA Covid-19 vaccine. N Engl J Med 383:2603–2615. https://doi.org/10.1056/nejmoa2034577 the BNT162b2 mRNA Covid-19 vaccine. N Engl J Med 383:2603–2615. https://doi.org/10.1056/nejmoa2034577 cysteine proteases of SARS-CoV. J Enzyme Inhib Med Chem 31:23–30. https://doi.org/10.3109/14756366.2014.1003215 p
g
83. Park JY, Kim JH, Kwon JM, Kwon HJ, Jeong HJ, Kim YM, Kim D, Lee WS,
Ryu YB (2013) Dieckol, a SARS-CoV 3CLpro inhibitor, isolated from the
edible brown algae Ecklonia cava. Bioorganic Med Chem 21:3730–
3737. https://doi.org/10.1016/j.bmc.2013.04.026 p
g
j
64. WHO (2020) Timeline: WHO’s Covid-19 response. World Heal. Organ. https://www.who.int/emergencies/diseases/novel-coronavirus-2019/
interactive-timeline#! 65. Jahan R, Paul AK, Jannat K, Rahmatullah M (2021) Plant essential oils:
possible COVID-19 therapeutics. Nat Prod Commun 16:2021. https://
doi.org/10.1177/1934578X21996149 84. Tragoolpua Y, Jatisatienr A (2007) Anti-herpes Simplex Virus activities of
Eugenia caryophyllus (Spreng.) Bullock & S. G. Harrison and essential oil. Eugenol Phyther Res 21:1153–1158. https://doi.org/10.1002/ptr g
66. Hameed S, Khan AF, Khan S, Wasay M (2022) First report of cerebral
venous thrombosis following inactivated-virus covid vaccination (Sin‑
opharm and Sinovac). J Stroke Cerebrovasc Dis 31:10–13. https://doi.
org/10.1016/j.jstrokecerebrovasdis.2021.106298 85. Armaka M, Papanikolaou E, Sivropoulou A, Arsenakis M (1999) Antiviral
properties of isoborneol, a potent inhibitor of herpes simplex virus
type 1. Antiviral Res 43:79–92. https://doi.org/10.1016/S0166-3542(99)
00036-4 67. Zhao H, Li Y, Wang Z (2022) Adverse event of sinovac coronavirus vac‑
cine: deafness. Vaccine 40:521–523. https://doi.org/10.1016/j.vaccine.
2021.11.091 86. Astani A, Reichling J, Schnitzler P (2011) Screening for antiviral activities
of isolated compounds from essential oils. References Evidence-based Compl
Altern Med 2011:1–8. https://doi.org/10.1093/ecam/nep187 68. Hardt K, Vandebosch A, Sadoff J, Gars MLe, Truyers C, Lowson D,
Dromme I Van, Vingerhoets J, Kamphuis T, Scheper G et al (2022)
Efficacy and safety of a booster regimen of Ad26COV2S vaccine against
covid-19. Medrxiv. https://doi.org/10.1101/2022.01.28.22270043 87. Serkedjieva J, Velcheva M (2003) In vitro anti-influenza virus activity of
the pavine alkaloid (-)-thalimonine isolated from Thalictrum simplex
L. Antivir Chem Chemother 14:75–80. https://doi.org/10.1177/09563
2020301400202 69. Banerjee S, Sandhu M, Tonzi E, Tambe A, Gambhir H (2021) Immune-
mediated thrombocytopenia associated with Ad26.COV2.S (Janssen;
Johnson & Johnson) vaccine. Am J Ther 28:e604–e605. https://doi.org/
10.1111/bjh 88. Hayashi K, Imanishi N, Kashiwayama Y, Kawano A, Terasawa K, Shimada
Y, Ochiai H (2007) Inhibitory effect of cinnamaldehyde, derived from
Cinnamomi cortex, on the growth of influenza A/PR/8 virus in vitro and
in vivo. Antiviral Res 74:1–8. https://doi.org/10.1016/j.antiviral.2007.01.
003 70. Sulemankhil I, Abdelrahman M, Negi SI (2022) Temporal association
between the COVID-19 Ad26.COV2.S vaccine and acute myocarditis:
a case report and literature review. Cardiovasc Revascularization Med
38:117–123. https://doi.org/10.1016/j.carrev.2021.08.012 89. Xie XH, Zang N, Li S (2012) Resveratrol Inhibits respiratory syncy‑
tial virus-induced IL-6 production, decreases viral replication, and
downregulates TRIF expression in airway epithelial cells. Inflammation
35:1392–1401. https://doi.org/10.1007/s10753-012-9452-7 71. Tobaiqy M, Elkout H, Maclure K (2021) Analysis of thrombotic adverse
reactions of covid-19 astrazeneca vaccine reported to eudravigilance
database. Vaccines 9:1–8. https://doi.org/10.3390/vaccines9040393 90. Liu T, Zang N, Zhou N, Li W, Xie X, Deng Y, Ren L, Long X, Li S, Zhou
L et al (2014) Resveratrol Inhibits the TRIF-dependent pathway by
upregulating sterile alpha and armadillo motif protein, contributing
to anti-inflammatory effects after respiratory syncytial virus infection. J
Virol 88:4229–4236. https://doi.org/10.1128/jvi.03637-13 72. Oldenburg J, Klamroth R, Langer F, Albisetti M, Von Auer C, Ay C, Korte
W, Scharf RE, Pötzsch B, Greinacher A (2021) Diagnosis and manage‑
ment of vaccine-related thrombosis following AstraZeneca COVID-19
Vaccination: guidance statement from the GTH. Hamostaseologie
41:E1. https://doi.org/10.1055/s-0041-1729135 91. Kim D, Min J, Jang M, Lee J, Shin Y, Park C, Song J, Kim H, Kim S, Jin
Y et al (2019) Natural bis-benzylisoquinoline alkaloids-tetrandrine,
fangchinoline, and cepharanthine, inhibit human coronavirus OC43
infection of MRS-5 human lung cells. Biomolecules 9:1–16 p
g
73. Wise J (2021) Covid-19: European countries suspend use of Oxford-
AstraZeneca vaccine after reports of blood clots. BMJ 372:n699. https://
doi.org/10.1136/bmj.n699 74. Nagy A, Alhatlani B (2021) An overview of current COVID-19 vaccine
platforms. References J Mol Struct 1243:130808. https://doi.org/10.1016/j.molstruc.2021.130808 103. Su H, Yao S, Zhao W, Li M, Liu J, Shang W, Xie H, Ke C, Gao M, Yu K et al
(2020) Discovery of baicalin and baicalein as novel, natural product
inhibitors of SARS-CoV-2 3CL protease in vitro. bioRxiv. https://doi.org/
10.1101/2020.04.13.038687 120. Chaccour C, Casellas A, Blanco-Di Matteo A, Pineda I, Fernandez-
Montero A, Ruiz-Castillo P, Richardson MA, Rodríguez-Mateos M,
Jordán-Iborra C, Brew J et al (2021) The effect of early treatment with
ivermectin on viral load, symptoms and humoral response in patients
with non-severe COVID-19: a pilot, double-blind, placebo-controlled,
randomized clinical trial. EClinicalMedicine 32:1–9. https://doi.org/10.
1016/j.eclinm.2020.100720 104. Lammers AJJ, Brohet RM, Theunissen REP, Koster C, Rood R, Verhagen
DWM, Brinkman K, Hassing RJ, Dofferhoff A, el Moussaoui R et al (2021)
Early hydroxychloroquine but not chloroquine use reduces ICU admis‑
sion in COVID-19 patients. Int J Infect Dis 101:283–289. https://doi.org/
10.1016/j.ijid.2020.12.008 j
121. WHO (2021) WHO advises that ivermectin only be used to treat COVID-
19 within clinical trials. World Heal Orga 1:1–14 105. Omokhua-Uyi AG, Van Staden J (2021) Natural product remedies for
COVID-19: a focus on safety. South African J Bot 139:386–398. https://
doi.org/10.1016/j.sajb.2021.03.012 122. Gendrot M, Andreani J, Jardot P, Hutter S, Delandre O, Boxberger M,
Mosnier J, Bideau M, Duflot I, Fonta I et al (2020) In vitro antiviral activity
of doxycycline against SARS-CoV-2. Molecules 25:1–10 106. de Almeida S, Soares J, dos Santos K, Alves J, Ribeiro A, Jacob I, Ferreira
Cj, dos Santos J, de Oliveira J, Junior Lb et al (2020) COVID-19 therapy:
What weapons do we bring into battle? Bioorganic Med. Chem 28:1–26 y y
g
123. Lu H (2020) Drug treatment options for the 2019-new coronavirus
(2019-nCoV). Biosci Trends 14:69–71. https://doi.org/10.1002/jmv.
25678.4 107. Murtuja S, Shilkar D, Sarkar B, Sinha BN, Jayaprakash V (2021) A short
survey of dengue protease inhibitor development in the past 6 years
(2015–2020) with an emphasis on similarities between DENV and SARS-
CoV-2 proteases. Bioorganic Med Chem 49:1–24. https://doi.org/10.
1016/j.bmc.2021.116415 124. Chakraborty R, Parvez S (2020) COVID-19: An overview of the current
pharmacological interventions, vaccines, and clinical trials biochem. Pharmacol 180(1):16. https://doi.org/10.1016/j.bcp.2020.114184 108. Hashem A, Alghamdi B, Algaissi A, Alshehri F, Bukhari A, Alfaleh M,
Memish Z (2020) Therapeutic use of chloroquine and hydroxychloro‑
quine in Covid-19 and other viral infections: a narrative review. Travel
Med Infect Dis 35:1–15 125. References Kitamura K, Honda M, Yoshizaki H, Yamamoto S, Nakane H, Fukushima
M, Ono K, Tokunaga T (1998) Baicalin, an inhibitor of HIV-1 production
in vitro. Antiviral Res 37:131–140. https://doi.org/10.1016/S0166-
3542(97)00069-7 80. Park JY, Yuk HJ, Ryu HW, Lim SH, Kim KS, Park KH, Ryu YB, Lee WS (2017)
Evaluation of polyphenols from Broussonetia papyrifera as coronavirus
protease inhibitors. J Enzyme Inhib Med Chem 32:504–512. https://doi.
org/10.1080/14756366.2016.1265519 98. Li BQ, Fu T, Dongyan Y, Mikovits JA, Ruscetti FW, Wang JM (2000) Flavo‑
noid baicalin inhibits HIV-1 infection at the level of viral entry. Biochem
Biophys Res Commun 276:534–538. https://doi.org/10.1006/bbrc.2000.
3485 81. Lin SC, Ho CT, Chuo WH, Li S, Wang TT, Lin CC (2017) Effective inhibition
of MERS-CoV infection by resveratrol. BMC Infect Dis 17:1–10. https://
doi.org/10.1186/s12879-017-2253-8 99. Salem ML, Hossain MS (2000) Protective effect of black seed oil from
Nigella sativa against murine cytomegalovirus infection. Int J Immu‑
nopharmacol 22:729–740. https://doi.org/10.1016/S0192-0561(00)
00036-9 82. Park JY, Ko JA, Kim DW, Kim YM, Kwon HJ, Jeong HJ, Kim CY, Park KH,
Lee WS, Ryu YB (2016) Chalcones isolated from Angelica keiskei inhibit 82. Park JY, Ko JA, Kim DW, Kim YM, Kwon HJ, Jeong HJ, Kim CY, Park KH,
Lee WS, Ryu YB (2016) Chalcones isolated from Angelica keiskei inhibit Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Page 41 of 48 Page 41 of 48 100. Gu C, Yin A, Yuan H, Yang K, Luo J, Zhan Y (2019) New anti-HBV norbis‑
abolane sesquiterpenes from Phyllantus acidus. Fitoterapia 137:1–7. https://doi.org/10.1016/j.fitote.2019.04.006 117. Szymkuć S, Gajewska EP, Molga K, Wołos A, Roszak R, Beker W, Moskal
M, Dittwald P, Grzybowski BA (2020) Computer-generated “synthetic
contingency” plans at times of logistics and supply problems: scenarios
for hydroxychloroquine and remdesivir. Chem Sci 11:6736–6744. https://doi.org/10.1039/d0sc01799j 101. Loizzo MR, Saab AM, Tundis R, Statti GA, Menichimi F, Lampronti D,
Gambari R, Cinatl J, Doerr HW (2008) Phytochemical analysis and
in vitro antiviral activities of the essential oils of seven Lebanon species
Chem Biodivers 5:461–470. https://doi.org/10.1002/cbdv.200890045 118. Caly L, Druce JD, Catton MG, Jans DA, Wagstaff KM (2020) The FDA-
approved drug ivermectin inhibits the replication of SARS-CoV-2
in vitro. Antiviral Res 178:3–6. https://doi.org/10.1016/j.antiviral.2020.
104787 102. Tkachenko KG (2006) Antiviral activity of the essential oils of some
Heracleum L. species. J. Herbs. Spices Med Plants 12:1–12. https://doi.
org/10.1300/J044v12n03_01 119. Zaheer T, Pal K, Abbas RZ, del Torres M (2021) COVID-19 and ivermectin:
potential threats associated with human use. References Yousefi H, Mashouri L, Okpechi S, Alahari N, Alahari S (2021) Repurpos‑
ing existing drugs for the treatment of COVID-19/SARS-CoV-2 infection:
a review describing drug mechanisms of action. Biochem Pharmacol
183:1–14. https://doi.org/10.1016/j.bcp.2020.114296 126. Kucukoglu K, Faydalı N, Bul D (2020) What are the drugs having poten‑
tial against COVID-19? Med Chem Res 29:1935–1955. https://doi.org/
10.1007/s00044-020-02625-1 109. Prodromos C, Rumschlag T (2020) Hydroxychloroquine is effective, and
consistently so when provided early, for COVID-19: a systematic review. New Microbes New Infect 38:1–11. https://doi.org/10.1016/j.nmni.2020.
100776 127. Frediansyah A, Nainu F, Dhama K, Mudatsir M, Harapan H (2021) Rem‑
desivir and its antiviral activity against COVID-19: a systematic review. Clin Epidemiol Glob Heal 9:123–127. https://doi.org/10.1016/j.cegh.
2020.07.011 110. Roy A, Das R, Roy D, Saha S, Ghosh NN, Bhattacharyya S, Roy MN (2022)
Encapsulated hydroxychloroquine and chloroquine into cyclic oligo‑
saccharides are the potential therapeutics for COVID-19: insights from
first-principles calculations. J Mol Struct 1247:131371. https://doi.org/
10.1016/j.molstruc.2021.131371 128. Tian L, Qiang T, Liang C, Ren X, Jia M, Zhang J, Li J, Wan M, YuWen X, Li
H et al (2021) RNA-dependent RNA polymerase (RdRp) inhibitors: the
current landscape and repurposing for the COVID-19 pandemic. Eur. J. Med. Chem. 213:1–19. https://doi.org/10.1016/j.ejmech.2021.113201 j
111. Singh AK, Singh A, Shaikh A, Singh R, Misra A (2020) Chloroquine and
hydroxychloroquine in the treatment of COVID-19 with or without
diabetes: A systematic search and a narrative review with a special ref‑
erence to India and other developing countries. Diabetes Metab Syndr
Clin Res Rev 14(3):241 Med. Chem. 213:1–19. https://doi.org/10.1016/j.ejmech.2021.11320 129. Hsu WS, Yen JH, Wang YS (2013) Formulas of components of citronella
oil against mosquitoes (Aedes aegypti). J. Environ. Sci. Heal. —Part B
Pestic. Food Contam Agric Wastes 48:1014–1019. https://doi.org/10.
1080/03601234.2013.816613 112. Lamback EB, de Oliveira MA, Haddad AF, Vieira AFM, Neto ALF, da Maia
T (2021) Hydroxychloroquine with azithromycin in patients hospitalized
for mild and moderate COVID-19. Brazilian J Infect Dis 25:1–6. https://
doi.org/10.1016/j.bjid.2021.101549 130. Hung Y, Lee J, Chiu C, Lee C, Tsai P, Hsu I, Ko W (2022) Oral nirmatrelvir/
ritonavir therapy for COVID-19: the dawn in the dark? Antibiotic 11:1–7. https://doi.org/10.3390/antibiotics11020220 131. Eng H, Dantonio AL, Kadar EP, Obach RS, Di L, Lin J, Patel NC, Boras B,
Walker GS, Novak JJ et al (2022) Disposition of PF-07321332 (Nirmatrel‑
vir), an orally bioavailable inhibitor of SARS-CoV-2 3CL protease, across
animals and humans. Drug Metab Dispos. https://doi.org/10.1124/dmd.
121.000801 113. References Rashid H, Ahmad N, Abdalla M, Khan K, Martines MAU, Shabana S (2021)
Molecular docking and dynamic simulations of cefixime, etoposide and
nebrodenside a against the pathogenic proteins of SARS-CoV-2. J Mol
Struct 1247:131296. https://doi.org/10.1016/j.molstruc.2021.131296 144. Binford SL, Maldonado F, Brothers MA, Weady PT, Zalman LS, Meador
JW III, Matthews DA, Patick A (2005) Conservation of amino acids in
human rhinovirus 3C protease correlates with broad-spectrum antiviral
activity of rupintrivir, a novel human rhinovirus 3C protease inhibitor. Antimicrob Agents Chemother 49:619–626 162. Erdogan T (2021) DFT, molecular docking and molecular dynamics
simulation studies on some newly introduced natural products for their
potential use against SARS-CoV-2. J Mol Struct 1242:1–14. https://doi.
org/10.1016/j.molstruc.2021.130733 145. Fink K, Nitsche A, Neumann M, Grossegesse M, Eisele KH, Danysz W
(2021) Amantadine inhibits sars-cov-2 in vitro. Viruses 13:1–10. https://
doi.org/10.3390/v13040539 163. Esam Z, Akhavan M, lotfi, M, and Bekhradnia, A, (2022) Molecular
docking and dynamics studies of nicotinamide riboside as a potential
multi-target nutraceutical against SARS-CoV-2 entry, replication, and
transcription: A new insight. J Mol Struct 1247:131394. https://doi.org/
10.1016/j.molstruc.2021.131394 146. Hou Y, Okuda K, Edwards C, Martinez D, Asakura T, Dinnon K III, Kato T,
Lee R, Yount B, Mascenik T et al (2020) SARS-CoV-2 reverse genet‑
ics reveals a variable infection gradient in the respiratory tract. Cell
182:429–446 147. Sallard E, Lescure FX, Yazdanpanah Y, Mentre F, Peiffer-Smadja N (2020)
Type 1 interferons as a potential treatment against COVID-19. Antiviral
Res 178:1–4. https://doi.org/10.1016/j.antiviral.2020.104791 164. Puttaswamy H, Gowtham HG, Ojha MD, Yadav A, Choudhir G, Ragura‑
man V, Kongkham B, Selvaraju K, Shareef S, Gehlot P et al (2020) In
silico studies evidenced the role of structurally diverse plant secondary
metabolites in reducing SARS-CoV-2 pathogenesis. Sci Rep 10:1–24. https://doi.org/10.1038/s41598-020-77602-0 148. Mantlo E, Bukreyeva N, Maruyama J, Paessler S, Huang C (2020) Antiviral
activities of type I interferons to SARS-CoV-2 infection. Antiviral Res
179:1–4. https://doi.org/10.1016/j.antiviral.2020.104811 165. Sepay N, Sekar A, Halder UC, Alarifi A, Afzal M (2021) Anti-COVID-19
terpenoid from marine sources: a docking, admet and molecular
dynamics study. J Mol Struct 1228:129433. https://doi.org/10.1016/j.
molstruc.2020.129433 149. Elmaaty AA, Alnajjar R, Hamed MIA, Khattab M, Khalifa MM, Al-
Karmalawy AA (2021) Revisiting activity of some glucocorticoids as a
potential inhibitor of SARS-CoV-2 main protease: theoretical study. RSC
Adv 11:10027–10042. https://doi.org/10.1039/d0ra10674g 150. References Ngo ST, Nguyen TH, Tung NT, Mai BK (2022) Insights into the binding
and covalent inhibition mechanism of PF-07321332 to SARS-CoV-2
Mpro. RSC Adv 12:3729–3737. https://doi.org/10.1039/d1ra08752e 156. Elzupir AO (2020) Inhibition of SARS-CoV-2 main protease 3CLpro by
means of α-ketoamide and pyridone-containing pharmaceuticals using
in silico molecular docking. J Mol Struct 1222:128878. https://doi.org/
10.1016/j.molstruc.2020.128878 138. Rosales R, Rodriguez ML, Rai DK, Cardin RD, Anderson AS, Sordillo EM,
van Bakel H, Simon V, García-Sastre A, White KM (2022) Nirmatrelvir,
molnupiravir, and remdesivir maintain potent in vitro activity against
the SARS-CoV-2 Omicron variant. BioRxiv. https://doi.org/10.1101/2022.
01.17.476685 157. Silva LR, da Silva Santos-Júnior PF, de Andrade Brandão J, Anderson L,
Bassi ÊJ, Xavier de Araújo-Júnior J, Cardoso SH, da Silva-Júnior EF (2020)
Druggable targets from coronaviruses for designing new antiviral
drugs. Bioorganic Med Chem 28:1–23. https://doi.org/10.1016/j.bmc.
2020.115745 139. Sacco MD, Hu Y, Gongora MV, Meilleur F, Kemp MT, Zhang X, Wang J,
Chen Y (2022) The P132H mutation in the main protease of Omicron
SARS-CoV-2 decreases thermal stability without compromising catalysis
or small-molecule drug inhibition. biorxiv. https://doi.org/10.1101/
2022.01.26.477774 158. Ashraf S, Ashraf S, Ashraf M, Imran M, Kalsoom L, Siddiqui U, Farooq I,
Akmal R, Akram M (2023) Honey and Nigella sativa against COVID-19 in
Pakistan (HNS-COVID-PK): a multicenter placebo-controlled rand‑
omized clinical trial. Phyther Res 37:627–644. https://doi.org/10.1002/
ptr.7640 140. Zarenezhad E, Marzi M (2022) Review on molnupiravir as a promising
oral drug for the treatment of COVID-19. Med Chem Res 31:232–243. https://doi.org/10.1007/s00044-021-02841-3 p
159. Chopra A, Tillu G, Chuadhary K, Reddy G, Srivastava A, Lakdawala M,
Gode D, Reddy H, Tamboli S, Saluja M et al (2023) Co-administration
of AYUSH 64 as an adjunct to standard of care in mild and moderate
COVID-19: a randomized, controlled, multicentric clinical trial. PLoS ONE
18:e0282688. https://doi.org/10.1371/journal.pone.0282688 141. WHO (2022) WHO recommends two new drugs to treat COVID-19. World Heal. Organ. https://www.who.int/news/item/14-01-2022-who-
recommends-two-new-drugs-to-treat-covid-19. 142. Reyes, E. This treatment can protect vulnerable people from COVID . But
many don’t know about it. Los Angeles Times. 2022. 160. Ghosh K, Amin SA, Gayen S, Jha T (2021) Chemical-informatics
approach to COVID-19 drug discovery: Exploration of important frag‑
ments and data mining based prediction of some hits from natural
origins as main protease (Mpro) inhibitors. J Mol Struct 1224:129026. https://doi.org/10.1016/j.molstruc.2020.129026 143. Ramajayam R, Tan KP, Liang PH (2011) Recent development of 3C and
3CL protease inhibitors for anti-coronavirus and anti-picornavirus drug
discovery. Biochem Soc Trans 39:1371–1375. https://doi.org/10.1042/
BST0391371 161. References Gies V, Bekaddour N, Dieudonne Y, Guffroy A, Frenger Q, Gros F, Rodero
M, Herbeuval J, Korganow A (2020) Beyond anti-viral effects of chloro‑
quine/hydroxychloroquine. Front Immunol 11:1–8 114. Omrani AS, Pathan SA, Thomas SA, Harris TRE, Coyle PV, Thomas CE,
Qureshi I, Bhutta ZA, Mawlawi NA, Kahlout RA et al (2020) Randomized
double-blinded placebo-controlled trial of hydroxychloroquine with or
without azithromycin for virologic cure of non-severe Covid-19. EClini‑
calMedicine 29–30:1–13. https://doi.org/10.1016/j.eclinm.2020.100645 132. Rai DK, Yurgelonis I, McMonagle P, Rothan HA, Hao L, Gribenko A, Titova
E, Kreiswirth B, White KM, Zhu Y et al (2022) Nirmatrelvir, an orally active
Mpro inhibitor, is a potent inhibitor of SARS-CoV-2 Variants of Concern. biorxiv 35:1–22 g
j
115. Ni Y, Liao J, Qian Z, Wu C, Zhang X, Zhang J, Xie Y, Jiang S (2022) Syn‑
thesis and evaluation of enantiomers of hydroxychloroquine against
SARS-CoV-2 in vitro. Bioorganic Med Chem 53:1–8. https://doi.org/10.
1016/j.bmc.2021.116523 133. Hammond J, Leister-Tebbe H, Gardner A, Abreu P, Bao W, Wisemandle
W, Baniecki M, Hendrick VM, Damle B, Simón-Campos A et al (2022) Oral
nirmatrelvir for high-risk, nonhospitalized adults with covid-19. N Engl J
Med. https://doi.org/10.1056/nejmoa2118542 116. Purwati M, Nasronudin A, Hendrianto E, Karsari D, Dinaryanti A, Ertanti
N, Ihsan IS, Purnama DS, Asmarawati TP et al (2021) An in vitro study
of dual drug combinations of anti-viral agents, antibiotics, and/or
hydroxychloroquine against the SARS-CoV-2 virus isolated from hospi‑
talized patients in surabaya Indonesia. PLoS ONE 16(1):27. https://doi.
org/10.1371/journal.pone.0252302 134. Owen D, Allerton CM, Anderson A, Aschenbrenner L, Avery M, Berritt
S, Boras B, Cardin R, Carlo A, Coffman K et al (2021) An oral SARS-CoV-2
Mpro inhibitor clinical candidate for the treatment of Covid-19. Science
374:1586–1593 135. Ullrich S, Ekanayake KB, Otting G, Nitsche C (2022) Main protease
mutants of SARS-CoV-2 variants remain susceptible to nirmatrelvir. Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Page 42 of 48 Bioorg Med Chem Lett 62:1–4. https://doi.org/10.1016/j.bmcl.2022.
128629 effective therapeutic combinations: lessons learned from major clinical
studies. Front Pharmacol 12:1–15. https://doi.org/10.3389/fphar.2021.
704205 136. Ramos-Guzmán CA, Ruiz-Pernía JJ, Tuñón I (2021) Computational
simulations on the binding and reactivity of a nitrile inhibitor of the
SARS-CoV-2 main protease. Chem Commun 57:9096–9099. https://doi.
org/10.1039/d1cc03953a 155. Takahashi JA, Barbosa BVR, Lima MTNS, Cardoso PG, Contigli C, Pimenta
LPS (2021) Antiviral fungal metabolites and some insights into their
contribution to the current COVID-19 pandemic. Bioorganic Med Chem
46:116366. https://doi.org/10.1016/j.bmc.2021.116366 137. References Wang S, Li W, Hui H, Tiwari SK, Zhang Q, Croker BA, Rawlings S, Smith
D, Carlin AF, Rana TM (2020) Cholesterol 25-Hydroxylase inhibits SARS
-CoV-2 and other coronaviruses by depleting membrane cholesterol. EMBO J 39:1–13. https://doi.org/10.1252/embj.2020106057 166. Khalifa I, Zhu W, Mohammed HHH, Dutta K, Li C (2020) Tannins inhibit
SARS-CoV-2 through binding with catalytic dyad residues of 3CLpro:
an in silico approach with 19 structural different hydrolysable tannins. J
Food Biochem 44:1–19. https://doi.org/10.1111/jfbc.13432 167. Peele KA, Potla Durthi C, Srihansa T, Krupanidhi S, Ayyagari VS, Babu DJ,
Indira M, Reddy AR, Venkateswarulu TC (2020) Molecular docking and
dynamic simulations for antiviral compounds against SARS-CoV-2: a
computational study. Inform Med Unlocked 19:1–6. https://doi.org/10.
1016/j.imu.2020.100345 151. Zu S, Deng Y, Zhou C, Li J, Li L, Chen Q, Li X, Zhao H, Gold S, He J et al
(2020) 25-Hydroxycholesterol is a potent SARS-CoV-2 inhibitor. Cell Res
30:1043–1045 152. Marcello A, Civra A, Bonotto R, Alves L, Rajasekharan S, Giacobone C,
Caccia C, Cavalli R, Adami M, Brambilla P et al (2020) The cholesterol
metabolite 27-hydroxycholesterol inhibits SARS-CoV-2 and is markedly
decreased in Covid-19 patients. Redox Biol 36:1–11 j
168. Alfaro M, Alfaro I, Angel C (2020) Identification of potential inhibitors of
SARS-CoV-2 papain-like protease from tropane alkaloids from Schizan‑
thus porrigens: a molecular docking study. Chem Phys Lett 761:1–10. https://doi.org/10.1016/j.cplett.2020.138068 153. Naggie S, Boulware DR, Lindsell CJ, Stewart TG, Slandzicki AJ, Lim SC,
Cohen J, Kavtaradze D, Amon AP, Gabriel A et al (2023) Effect of higher-
dose ivermectin for 6 days vs placebo on time to sustained recovery
in outpatients with COVID-19. JAMA 329:888–897. https://doi.org/10.
1001/jama.2023.1650 169. Arora S, Lohiya G, Moharir K, SYShah S (2020) Identification of potential
flavonoid inhibitors of the SARS-CoV-2 main protease 6YNQ A molecu‑
lar docking study. Digit Chinese Med 3:239–248 170. Xu Z, Yang L, Zhang X, Zhang Q, Yang Z, Liu Y, Wei S, Liu W (2020)
Discovery of potential flavonoid inhibitors against COVID-19 3CL 154. Chakraborty C, Sharma AR, Bhattacharya M, Agoramoorthy G, Lee SS
(2021) The drug repurposing for COVID-19 clinical trials provide very Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Page 43 of 48 186. Zaporozhets TS, Besednova NN (2020) Biologically active compounds
from marine organisms in the strategies for combating coronaviruses. AIMS Microbiol 6:470–494. https://doi.org/10.3934/microbiol.2020028 proteinase based on virtual screening strategy. Front Mol Biosci 7:1–8. https://doi.org/10.3389/fmolb.2020.556481 171. References Adem S, Eyupoglu V, Sarfraz I, Rasul A, Ali M (2020) Identification of
potent COVID-19 main protease (Mpro) inhibitors from natural poly‑
phenols: an in silico strategy unveils a hope against CORONA. Preprints. https://doi.org/10.2944/preprints202003.0333.v1 187. Khan MT, Ali A, Wang Q, Irfan M, Khan A, Zeb MT, Zhang YJ, Chinnasamy
S, Wei DQ (2021) Marine natural compounds as potents inhibitors
against the main protease of SARS-CoV-2—a molecular dynamic study. J Biomol Struct Dyn 39:3627–3637. https://doi.org/10.1080/07391102.
2020.1769733 172. Vijayakumar B, Ramesh D, Joji A, Prakasan J, Kannan T (2020) In silico
pharmacokinetic and molecular docking studies of natural flavonoids
and synthetic indole chalcones against essential proteins of SARS-
CoV-2. Eur J Pharmacol 886:1–11 188. Singh R, Chauhan N, Kuddus M (2021) Exploring the therapeutic
potential of marine-derived bioactive compounds against COVID-
19. Environ Sci Pollut Res 28:52798–52809. https://doi.org/10.1007/
s11356-021-16104-6 173. Rameshkumar M, Indu P, Arunagirinathan N, Venkatadri B, El-Serehy
H, Ahmad A (2021) Computational selection of flavonoid compounds
as inhibitors against SARS-CoV-2 main protease, RNA-dependent RNA
polymerase and spike proteins. a molecular docking study. Saudi J Biol
Sci 28:448–458 189. Omotuyi IO, Nash O, Ajiboye BO, Olumekun VO, Oyinloye BE, Osun‑
tokun OT, Olonisakin A, Ajayi AO, Olusanya O, Akomolafe FS et al (2021)
Aframomum melegueta secondary metabolites exhibit polypharma‑
cology against SARS-CoV-2 drug targets: in vitro validation of furin
inhibition. Phyther Res 35:908–919. https://doi.org/10.1002/ptr.6843 174. Zaki AA, Ashour A, Elhady SS, Darwish KM, Al-Karmalawy AA (2022)
Calendulaglycoside a showing potential activity against SARS-CoV-2
main protease: molecular docking, molecular dynamics, and SAR stud‑
ies. J Tradit Complement Med 12:16–34. https://doi.org/10.1016/j.jtcme.
2021.05.001 190. El-Demerdash A, Metwaly AM, Hassan A, El-Aziz TMA, Elkaeed EB, Eissa
IH, Arafa RK, Stockand JD (2021) Comprehensive virtual screening of the
antiviral potentialities of marine polycyclic guanidine alkaloids against
sars-cov-2 (Covid-19). Biomolecules 11:1–25. https://doi.org/10.3390/
biom11030460 175. Giofrè SV, Napoli E, Iraci N, Speciale A, Cimino F, Muscarà C, Molonia MS,
Ruberto G, Saija A (2021) Interaction of selected terpenoids with two
SARS-CoV-2 key therapeutic targets: an in silico study through molecu‑
lar docking and dynamics simulations. Comput Biol Med 134:1–13. https://doi.org/10.1016/j.compbiomed.2021.104538 191. Kar P, Kumar V, Vellingiri B, Sen A, Jaishee N, Anandraj A, Malhotra H,
Bhattacharyya S, Mukhopadhyay S, Kinoshita M et al (2020) Anisotine
and amarogentin as promising inhibitory candidates against SARS-
CoV-2 proteins: a computatational investigation. J Biomol Struct Dyn
40:1–11 176. References Abdelmohsen UR, Albohy A, Abdulrazik BS, Bayoumi SAL, Malak LG,
Khallaf ISA, Bringmann G, Farag SF (2021) Natural coumarins as poten‑
tial anti-SARS-CoV-2 agents supported by docking analysis. RSC Adv
11:16970–16979. https://doi.org/10.1039/d1ra01989a 192. Vardhan S, Sahoo SK (2004) (2020) Searching inhibitors for three impor‑
tant proteins of COVID-19 through molecular docking studies. arXiv
08095:1–13 177. Thangavel N, Al Bratty M, Al Hazmi HA, Najmi A, Ali Alaqi RO (2021)
Molecular docking and molecular dynamics aided virtual search of
olivenet™ directory for secoiridoids to combat SARS-CoV-2 infection
and associated hyperinflammatory responses. Front Mol Biosci 7:1–17. https://doi.org/10.3389/fmolb.2020.627767 193. Falade VA, Adelusi TI, Adedotun IO, Abdul-Hammed M, Lawal TA,
Agboluaje SA (2021) In silico investigation of saponins and tannins as
potential inhibitors of SARS-CoV-2 main protease (Mpro). Silico Pharma‑
col 9:1–15. https://doi.org/10.1007/s40203-020-00071-w 178. Kumar V, Parate S, Yoon S, Lee G, Lee KW (2021) Computational
simulations identified marine-derived natural bioactive compounds as
replication inhibitors of SARS-CoV-2. Front Microbiol 12:1–15. https://
doi.org/10.3389/fmicb.2021.647295 194. Kandeil A, Mostafa A, Kutkat O, Moatasim Y, Al-Karmalawy AA, Rashad
AA, Kayed AE, Kayed AE, El-Shesheny R, Kayali G et al (2021) Bioactive
polyphenolic compounds showing strong antiviral activities against
severe acute respiratory syndrome coronavirus 2. Pathogens 10:758. https://doi.org/10.3390/pathogens10060758 179. Gajjar ND, Dhameliya TM, Shah GB (2021) In search of RdRp and Mpro
inhibitors against SARS CoV-2: Molecular docking, molecular dynamic
simulations and ADMET analysis. J Mol Struct 1239:130488. https://doi.
org/10.1016/j.molstruc.2021.130488 195. Carroll AR, Copp BR, Davis RA, Keyzers RA, Prinsep MR (2020) Marine
natural products. Nat Prod Rep 37:175–223. https://doi.org/10.1039/
c9np00069k 180. Adelusi TI, Oyedele AQK, Monday OE, Boyenle ID, Idris MO, Ogunlana
AT, Ayoola AM, Fatoki JO, Kolawole OE, David KB et al (2022) Dietary
polyphenols mitigate SARS-CoV-2 main protease (Mpro)–molecular
dynamics, molecular mechanics, and density functional theory inves‑
tigations. J Mol Struct 1250:131879. https://doi.org/10.1016/j.molstruc.
2021.131879 196. Rathinavel T, Meganathan B, Kumarasamy S, Ammashi S, Thangas‑
wamy S, Ragunathan Y, Palanisamy S (2021) Potential COVID-19 drug
from natural phenolic compounds through in silico virtual screening
approach. Biointerf Res. Appl. Chem. 11:10161–10173. https://doi.org/
10.33263/BRIAC113.1016110173 197. Davella R, Gurrapu S, Mamidala E (2021) Phenolic compounds as
promising drug candidates against Covid-19 -an integrated molecular
docking and dynamics simulation study. Mater Today Proc 51:1–6 181. Nebigil C, Moog C, Vagner S, Jessel N, Smith D, Desaubry L (2020)
Flavaglines as natural products targeting eIF4A and prohibitins: From
traditional Chinese medicine to antiviral activity against coronaviruses. Eur J Med Chem 203:1–9. References https://doi.org/10.1016/j.ejmech.2020.112653 198. Kowalczyk M, Golonko A, Świsłocka R, Kalinowska M, Parcheta M, Swier‑
giel A, Lewandowski W (2021) Drug design strategies for the treatment
of viral disease. plant phenolic compounds and their derivatives. Front
Pharmacol 12:1–21. https://doi.org/10.3389/fphar.2021.709104 182. Ellinger B, Bojkova D, Zaliani A, Cinatl J, Claussen C, Westhaus S, Rein‑
shagen J, Kuzikov M, Wolf M, Geisslinger G et al (2020) Identification of
inhibitors of SARS-CoV-2 in-vitro cellular toxicity in human (Caco-2) cells
using a large scale drug repurposing collection. Res Sq 23:1–19. https://
doi.org/10.2203/rs.3.rs-23951/v1 199. Ye N, Caruso F, Rossi M (2021) Mechanistic insights into the inhibition of
SARS-CoV-2 main protease by clovamide and its derivatives. Silico Stud
Biophys 1:377–404. https://doi.org/10.3390/biophysica1040028 183. Bojkova D, Klann K, Koch B, Widera M, Krause D, Ciesek S, Cinatl J,
Münch C (2020) Proteomics of SARS-CoV-2 infected host cell proteom‑
ics reveal potential therapy targets. Nature 583:469–472. https://doi.
org/10.1038/s41586-020-2332-7 200. Abdusalam AAA, Murugaiyah V (2020) Identification of potential inhibi‑
tors of 3CL protease of SARS-CoV-2 From ZINC database by molecular
docking-based virtual screening. Front Mol Biosci 7:1–11. https://doi.
org/10.3389/fmolb.2020.603037 201. Barage S, Karthic A, Bavi R, Desai N, Kumar R, Kumar V, Lee KW (2022)
Identification and characterization of novel RdRp and Nsp15 inhibitors
for SARS-COV2 using computational approach. J Biomol Struct Dyn
40:2557–2574. https://doi.org/10.1080/07391102.2020.1841026 184. Hoehl S, Rabenau H, Berger A, Kortenbusch M, Cinatl J, Bojkova D,
Behrens P, Böddinghaus B, Götsch U, Naujoks F et al (2020) Evidence of
SARS-CoV-2 infection in returning travelers from Wuhan. China N Engl J
Med 382:1278–1280. https://doi.org/10.1056/nejmc2001899 202. Kumar Verma A, Kumar V, Singh S, Goswami BC, Camps I, Sekar A, Yoon
S, Lee KW (2021) Repurposing potential of Ayurvedic medicinal plants
derived active principles against SARS-CoV-2 associated target proteins
revealed by molecular docking, molecular dynamics and MM-PBSA
studies. Biomed Pharmacother 137:1–17. https://doi.org/10.1016/j.
biopha.2021.111356 185. Escalante DE, Ferguson DM (2021) Structural modeling and analysis of
the SARS-CoV-2 cell entry inhibitor camostat bound to the trypsin-like
protease TMPRSS2. Med Chem Res 30:399–409. https://doi.org/10.
1007/s00044-021-02708-7 Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Page 44 of 48 glycoproteins. Antiviral Res 88:1–9. https://doi.org/10.1016/j.antiviral.
2010.06.014 203. Kumar R, Kumar V, Lee KW (2021) A computational drug repurposing
approach in identifying the cephalosporin antibiotic and anti-hepatitis
C drug derivatives for COVID-19 treatment. Comput Biol Med 130:1–13. https://doi.org/10.1016/j.compbiomed.2020.104186 glycoproteins. Antiviral Res 88:1–9. https://doi.org/10.1016/j.antiviral.
2010.06.014 222. References Yang Y, Islam MS, Wang J, Li Y, Chen X (2020) Traditional Chinese medi‑
cine in the treatment of patients infected with 2019-new coronavirus
(SARS-CoV-2): a review and perspective. Int J Biol Sci 16:1708–1717. https://doi.org/10.7150/ijbs.45538 210. Gysi DM, Do Valle Í, Zitnik M, Ameli A, Gan X, Varol O, Ghiassian SD, Pat‑
ten JJ, Davey RA, Loscalzo J et al (2021) Network medicine framework
for identifying drug-repurposing opportunities for COVID-19. Proc Natl
Acad Sci USA 118:1–11. https://doi.org/10.1073/pnas.2025581118 228. Yi L, Li Z, Yuan K, Qu X, Chen J, Wang G, Zhang H, Luo H, Zhu L, Jiang P
et al (2004) Small molecules blocking the entry of severe acute respira‑
tory syndrome coronavirus into host cells. J Virol 78:11334–11339. https://doi.org/10.1128/jvi.78.20.11334-11339.2004 211. Vatansever EC, Yang KS, Drelich AK, Kratch KC, Cho CC, Kempaiah KR,
Hsu JC, Mellott DM, Xu S, Tseng CTK et al (2021) Bepridil is potent
against SARS-CoV-2 in vitro. Proc Natl Acad Sci USA 118:1–8. https://doi.
org/10.1073/pnas.2012201118 p
g
j
229. Xi S, Li Y, Yue L, Gong Y, Qian L, Liang T, Ye Y (2020) Role of traditional
Chinese medicine in the management of viral pneumonia. Front Phar‑
macol 11:1–26. https://doi.org/10.3389/fphar.2020.582322 212. Asdadi A, Hamdouch A, Gharby S, Hassani LM (2020) Chemical charac‑
terization of essential oil of Artemisia herba-alba asso and his possible
potential against covid-19. J Anal Sci Appl Biotechnol 2:67–72. https://
doi.org/10.4802/IMIST.PRSM/jasab-v2i2.21589 p
g
p
230. Ni L, Zhou L, Zhou M, Zhao J, Wang DW (2020) Combination of the
western medicine and the Chinese traditional patent medicine in treat‑
ing a family case of CoVID-19. Front Med 14:210–214 213. Romeo I, Mesiti F, Lupia A, Alcaro S (2021) Current updates on naturally
occurring compounds recognizing SARS-CoV-2 druggable targets. Molecules 26:1–27. https://doi.org/10.3390/molecules26030632 231. Ng AWT, Poon SL, Huang MN, Lim JQ, Boot A, Yu W, Suzuki Y, Thangaraju
S, Ng CCY, Tan P et al (2017) Aristolochic acids and their derivatives are
widely implicated in liver cancers in Taiwan and throughout Asia. Sci
Transl Med 9:1–12. https://doi.org/10.1126/scitranslmed.aan6446 214. Demeritte A, Wuest WM (2020) A look around the West Indies: The
spices of life are secondary metabolites. Bioorganic Med Chem 28:1–11. https://doi.org/10.1016/j.bmc.2020.115792 232. Deng W, Xu Y, Kong Q, Xue J, Yu P, Liu J, Lv Q, Li F, Wei Q, Bao L (2020)
Therapeutic efficacy of pudilan xiaoyan oral liquid (PDL) for COVID-19
in vitro and in vivo. Signal Transduct Target Ther 5:1–3. References Surucic R, Travar M, Petkovic M, Tubic B, Stojilkovic M, Grabez M, Savikin
K, Zdunic G, Skrbic R (2021) Pomegranate peel extract polyphenols
attenuate the SARS-CoV-2 S-glycoprotein binding ability to ACE2
Receptor. In silico and in vitro studies. Bioorg Chem 114:1–7 204. Peele KA, Kumar V, Parate S, Srirama K, Lee KW, Venkateswarulu TC
(2021) Insilico drug repurposing using FDA approved drugs against
Membrane protein of SARS-CoV-2. J Pharm Sci 110:2346–2354. https://
doi.org/10.1016/j.xphs.2021.03.004 223. Tito A, Colantuono A, Pirone L, Pedone E, Intartaglia D, Giamundo G,
Conte I, Vitaglione P, Apone F (2021) Pomegranate peel extract as an
inhibitor of SARS-CoV-2 spike binding to human ACE2 receptor (in
vitro): a promising source of novel antiviral drugs. Front Chem 9:1–11. https://doi.org/10.3389/fchem.2021.638187 205. Sharma AD, Kaur I (2020) Jensenone from eucalyptus essential oil as a
potential inhibitor of COVID 19 corona virus infection. Res Rev Biotech‑
nol Biosci 7:59–66. https://doi.org/10.5281/zenodo.3748477 224. Ali S, Alam M, Khatoon F, Fatima U, Elasbali AM, Adnan M, Islam A,
Hassan MI, Snoussi M, De Feo V (2022) Natural products can be used in
therapeutic management of COVID-19: Probable mechanistic insights. Biomed Pharmacother 147:1–11. https://doi.org/10.1016/j.biopha.2022.
112658 206. Paul A, Sarkar A, Saha S, Maji A, Janah P, Kumar Maity T (2021) Synthetic
and computational efforts towards the development of peptidomimet‑
ics and small-molecule SARS-CoV 3CLpro inhibitors. Bioorganic Med
Chem 46:1–39. https://doi.org/10.1016/j.bmc.2021.116301 207. da Silva JKR, Figueiredo PLB, Byler KG, Setzer WN (2020) Essential oils as
antiviral agents. Potential of essential oils to treat sars−cov−2 infection:
an in−silico investigation. Int J Mol Sci 21:1–35. https://doi.org/10.3390/
ijms21103426 225. An X, Zhang YH, Duan L, Jin D, Zhao S, Zhou RR, Duan Y, Lian F, Tong X
(2021) The direct evidence and mechanism of traditional Chinese medi‑
cine treatment of COVID-19. Biomed Pharmacother 137:1–17. https://
doi.org/10.1016/j.biopha.2021.111267 j
208. Fiscon G, Conte F, Farina L, Paci P (2021) SAveRUNNER: a network-based
algorithm for drug repurposing and its application to COVID-19. PLoS
Comput Biol 17:1–30. https://doi.org/10.1371/JOURNAL.PCBI.1008686 226. Silveira D, Prieto-Garcia JM, Boylan F, Estrada O, Fonseca-Bazzo YM,
Jamal CM, Magalhães PO, Pereira EO, Tomczyk M, Heinrich M (2020)
COVID-19: is there evidence for the use of herbal medicines as adjuvant
symptomatic therapy? Front Pharmacol 11:1–44. https://doi.org/10.
3389/fphar.2020.581840 209. Sibilio P, Bini S, Fiscon G, Sponziello M, Conte F, Farina L, Verrienti A
(2021) In silico drug repurposing in COVID-19: A network-based analy‑
sis. Biomed Pharmacother 142:1–10. https://doi.org/10.1016/j.biopha.
2021.111954 227. References https://doi.org/
10.1038/s41392-020-0176-0 215. Ademiluyi AO, Oyeniran OH, Oboh G (2020) tropical food spices: a
promising panacea for the novel coronavirus disease (COVID-19). efood
1:347. https://doi.org/10.2991/efood.k.201022.001 216. Sengupta S, Bhattacharyya D, Kasle G, Karmakar S, Sahu O, Ganguly A,
Addya S, Das Sarma J (2021) Potential immunomodulatory properties
of biologically active components of spices against SARS-CoV-2 and
Pan β-coronaviruses. Front Cell Infect Microbiol 11:1–12. https://doi.
org/10.3389/fcimb.2021.729622 233. Hetrick B, Yu D, Olanrewaju AA, Chilin LD, He S, Dabbagh D, Alluhaibi
G, Ma YC, Hofmann LA, Hakami RM et al (2021) A traditional medicine,
respiratory detox shot (RDS), inhibits the infection of SARS-CoV, SARS-
CoV-2, and the influenza a virus in vitro. Cell Biosci 11:1–12. https://doi.
org/10.1186/s13578-021-00609-1 234. Care H (2020) COVID - 19: opportunity for mainstreaming Ayurveda to
transform Indian COVID - 19: opportunity for mainstreaming Ayurveda
to transform indian health care. J Ayurveda 14:42–48. https://doi.org/
10.4103/joa.joa 217. Singh NA, Kumar P (2021) Spices and herbs: potential antiviral preven‑
tives and immunity boosters during COVID-19. Phyther Res 35:2745–
2757. https://doi.org/10.1002/ptr.7019 218. Bousquet J, Czarlewski W, Zuberbier T, Mullol J, Blain H, Cristol JP, De La
Torre R, Le Moing V, Lozano NP, Bedbrook A et al (2021) Spices to con‑
trol COVID-19 symptoms: yes, but not only. Int Arch Allergy Immunol
182:489–495. https://doi.org/10.1159/000513538 235. Maurya VK, Kumar S, Prasad AK, Bhatt MLB, Saxena SK (2020) Structure-
based drug designing for potential antiviral activity of selected natural
products from Ayurveda against SARS-CoV-2 spike glycoprotein and
its cellular receptor. VirusDisease 31:179–193. https://doi.org/10.1007/
s13337-020-00598-8 219. Parham S, Kharazi AZ, Bakhsheshi-Rad HR, Nur H, Ismail AF, Sharif S,
Ramakrishna S, Berto F (2020) Antioxidant, antimicrobial and antiviral
properties of herbal materials. Antioxidants 9:1–36. https://doi.org/10.
3390/antiox9121309 236. Borse S, Joshi M, Saggam A, Bhat V, Walia S, Marathe A, Sagar S, Chavan-
Gautam P, Girme A, Hingorani L et al (2021) Ayurveda botanicals in
COVID-19 management: an in silico multi-target approach. PLoS ONE
16:1–33. https://doi.org/10.1371/journal.pone.0248479 220. Hussein G, Miyashiro H, Nakamura N, Hattori M, Kakiuchi N, Shimo‑
tohno K (2000) Inhibitory effects of Sudanese medicinal plant extracts
on hepatitis C virus (HCV) protease. Phyther Res 14:510–516. https://
doi.org/10.1002/1099-1573(200011)14:7%3c510::AID-PTR646%3e3.0.
CO;2-B 237. Ram T, Munikumar M, Raju V, Devaraj P, Boiroju N, Hemalatha R, Prasad
P, Gundeti M, Sisodia B, Pawar S et al (2022) In silico evaluation of the
compounds of the ayurvedic drug, AYUSH-64, for the action against the
SARS-CoV-2 main protease. References Ho TY, Wu SL, Chen JC, Li CC, Hsiang CY (2007) Emodin blocks the
SARS coronavirus spike protein and angiotensin-converting enzyme 2
interaction. Antiviral Res 74:92–101. https://doi.org/10.1016/j.antiviral.
2006.04.014 268. Adem Ş, Eyupoglu V, Sarfraz I, Rasul A, Zahoor AF, Ali M, Abdalla M, Ibra‑
him IM, Elfiky AA (2021) Caffeic acid derivatives (CAFDs) as inhibitors of
SARS-CoV-2: CAFDs-based functional foods as a potential alternative
approach to combat COVID-19. Phytomedicine 85:1–17. https://doi.
org/10.1016/j.phymed.2020.153310 251. Bailly C, Vergoten G (2020) Glycyrrhizin: an alternative drug for the treat‑
ment of COVID-19 infection and the associated respiratory syndrome? Pharmacol Ther 214:1–16 269. Núñez MJ, Díaz-Eufracio BI, Medina-Franco JL, Olmedo DA (2021) Latin
American databases of natural products: biodiversity and drug discov‑
ery against SARS-CoV-2. RSC Adv 11:16051–16064. https://doi.org/10.
1039/d1ra01507a 252. van de Sand L, Bormann M, Alt M, Schipper L, Heilingloh CS, Stein‑
mann E, Todt D, Dittmer U, Elsner C, Witzke O et al (2021) Glycyrrhizin
effectively inhibits sars-cov-2 replication by inhibiting the viral main
protease. Viruses 13:1–10. https://doi.org/10.3390/v13040609 253. Jo S, Kim S, Shin DH, Kim MS (2020) Inhibition of SARS-CoV 3CL pro‑
tease by flavonoids. J Enzyme Inhib Med Chem 35:145–151. https://doi.
org/10.1080/14756366.2019.1690480 270. Samy MN, Attia E, Shoman M, Khalil H, Sugimoto S, Matsunami K, Fahim
J (2021) Phytochemical investigation of Amphilophium paniculatum an
underexplored bignoniaceae species as a source of SARS-CoV-2 Mpro
inhibitory metabolites. Isolation, identification, and molecular docking
study. South African J Bot 141:421–430 254. Zhang D, Wu K, Zhang X, qiong S, Peng B (2020) In silico screening of
Chinese herbal medicines with the potential to directly inhibit 2019
novel coronavirus. J Integr Med. https://doi.org/10.1016/j.joim.2020.02.
005 y
271. Hasan A, Nahar N, Jannat K, Afroze T, Jahan R, Rahmatullah M (2020)
In silico studies on phytochemicals of Pimpinella Anisum L. (Apiaceae)
as potential inhibitors of SARS-CoV-2 3C-like protease. Int J Ayurveda
5:8–14 255. Paraiso IL, Revel JS, Stevens JF (2020) Potential use of polyphenols in
the battle against COVID-19. Curr Opin Food Sci 32:149–155. https://
doi.org/10.1016/j.cofs.2020.08.004 272. Chauhan, A, and Kalra, S (2020) Identification of potent COVID-19 main
protease (MPRO) inhibitors from flavonoids. Res. Sq., 1–11.i 256. Ma Q, Li R, Pan W, Huang W, Liu B, Xie Y, Wang Z, Li C, Jiang H, Huang J
et al (2020) Phillyrin (KD-1) exerts anti-viral and anti-inflammatory activi‑
ties against novel coronavirus (SARS-CoV-2) and human coronavirus
229E (HCoV-229E) by suppressing the nuclear factor kappa B (NF-κB)
signaling pathway. References J Ayurveda Integr Med 13:100413. https://
doi.org/10.1016/j.jaim.2021.02.004 221. Sundararajan A, Ganapathy R, Huan L, Dunlap JR, Webby RJ, Kotwal
GJ, Sangster MY (2010) Influenza virus variation in susceptibility to
inactivation by pomegranate polyphenols is determined by envelope Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Page 45 of 48 Page 45 of 48 238. Segneanu A, Cepan C, Grozescu I, Cziple F, Olariu S, Ratiu S, Lazar V,
Murariu S, Velciov S, Marti T (2019) Therapeutic use of some Romanian
medicinal plants. In: Perveen S, Al-Taweel A (eds) Pharmacognosy. Intech, London 258. Biagioli M, Marchiano S, Roselli R, Giorgio C, Bellini R, Bordoni M, Gidari
A, Sabbatini S, Francisci D, Fiorillo B et al (2021) Discovery of a AHR
pelargonidin agonist that counter-regulates Ace2 expression and
attenuates ACE2-SARS-CoV-2 interaction. Biochem Pharmacol 188:1–15f p
g
g
g
p
attenuates ACE2-SARS-CoV-2 interaction. Biochem Pharmacol 188 239. Khanna K, Kohli SK, Kaur R, Bhardwaj A, Bhardwaj V, Ohri P, Sharma
A, Ahmad A, Bhardwaj R, Ahmad P (2021) Herbal immune-boosters:
Substantial warriors of pandemic Covid-19 battle. Phytomedicine
85:153361. https://doi.org/10.1016/j.phymed.2020.153361 259. Schwarz S, Sauter D, Wang K, Zhang R, Sun B, Karioti A, Bilia A, Efferth T,
Schwarz W (2014) Kaempferol derivatives as antiviral drugs against the
3a channel protein of coronavirus. Planta Med 80:177–182 260. Bettuzzi S, Gabba L, Cataldo S (2021) Efficacy of a polyphenolic, stand‑
ardized green tea extract for the treatment of COVID-19 syndrome: a
proof-of-principle study. Covid 1:2–12. https://doi.org/10.3390/covid
1010002 240. Wang Y, Hou H, Ren Q, Hu H, Yang T, Li X (2021) Natural drug sources for
respiratory diseases from Fritillaria: chemical and biological analyses. Chin Med 16:1–41. https://doi.org/10.1186/s13020-021-00450-1 241. Korkmaz H (2021) Could sumac be effective on COVID-19 treatment? J
Med Food 24:563–568. https://doi.org/10.1089/jmf.2020.0104 261. Bormann M, Alt M, Schipper L, van de Sand L, Le-Trilling VTK, Rink L,
Heinen N, Madel RJ, Otte M, Wuensch K et al (2021) Turmeric root
and its bioactive ingredient curcumin effectively neutralize sars-cov-2
in vitro. Viruses 13:1–12. https://doi.org/10.3390/v13101914 242. Wijaya SH, Afendi FM, Batubara I, Huang M, Ono N, Kanaya S, Altaf-Ul-
amin M (2021) Identification of targeted proteins by jamu formulas
for different efficacies using machine learning approach. Life 11:1–11. https://doi.org/10.3390/life11080866 262. Xiao T, Cui M, Zheng C, Wang M, Sun R, Gao D, Bao J, Ren S, Yang B, Lin
J et al (2021) Myricetin inhibits SARS-CoV-2 viral replication by target‑
ing Mpro and ameliorates pulmonary inflammation. References Front Pharmacol
12:1–9. https://doi.org/10.3389/fphar.2021.669642 243. Wijaya SH, Batubara I, Nishioka T, Altaf-Ul-Amin M, Kanaya S (2017)
Metabolomic studies of Indonesian Jamu medicines: prediction of
Jamu efficacy and identification of important metabolites. Mol Inform
36:1–16. https://doi.org/10.1002/minf.201700050 263. Chen Z, Cui Q, Cooper L, Zhang P, Lee H, Chen Z, Wang Y, Liu X, Rong
L, Du R (2021) Ginkgolic acid and anacardic acid are specific covalent
inhibitors of SARS-CoV-2 cysteine proteases. Cell Biosci 11:1–8. https://
doi.org/10.1186/s13578-021-00564-x 244. Fadli A, Kusuma W, Annisa B, Heryanto IR (2021) Screening of potential
indonesia herbal compounds based on multi-label classification for
2019 coronavirus disease _ enhanced reader.pdf. Big Data Cogn Com‑
put 5:1–19 264. Ghosh R, Chakraborty A, Biswas A, Chowdhuri S (2021) Identification of
polyphenols from Broussonetia papyrifera as SARS CoV-2 main protease
inhibitors using in silico docking and molecular dynamics simulation
approaches. J Biomol Struct Dyn 39:6747–6760. https://doi.org/10.
1080/07391102.2020.1802347 245. Hartanti D, Dhiani BA, Charisma SL, Wahyuningrum R (2020) The poten‑
tial roles of Jamu for COVID-19: a learn from the traditional Chinese
medicine. Pharm Sci Res 7:12–22. https://doi.org/10.7454/psr.v7i4.1083 246. CNN (2021) 4 RS di Yogya Kembangkan Obat Covid dari Minyak Kelapa
Murni. CNN Indones, Mampang Prapatan 265. Khaerunnisa S, Kurniawan H, Awaluddin R, Suhartati S (2020) Potential
inhibitor of COVID-19 main protease (M pro) from several medicinal
plant compounds by molecular docking study. Preprints 3:1–14. https://doi.org/10.2944/preprints202003.0226.v1 247. Lin CW, Tsai FJ, Tsai CH, Lai CC, Wan L, Ho TY, Hsieh CC, Chao PDL (2005)
Anti-SARS coronavirus 3C-like protease effects of Isatis indigotica root
and plant-derived phenolic compounds. Antiviral Res 68:36–42. https://
doi.org/10.1016/j.antiviral.2005.07.002 266. Ghosh R, Chakraborty A, Biswas A, Chowduri S (2020) Evaluation
of green tea polyphenols as novel corona virus (SARS CoV-2) main
protease (Mpro) inhibitors-an in silico docking and molecular dynamics
simulation study. J Biomol Struct Dyn. https://doi.org/10.1080/07391
102.2020.1779818 248. Nguyen TT, Woo H, Kang H, Nguyen V, Kim Y, Kim D, Ahn S, Xia Y, Kim
D (2012) Flavonoid-mediated inhibition of SARS coronavirus 3C-like
protease expressed in Pichia pastoris. Biotechnol Lett 34:831–838 267. Maiti S, Banerjee A (2021) Epigallocatechin gallate and theaflavin gallate
interaction in SARS-CoV-2 spike-protein central channel with reference
to the hydroxychloroquine interaction: Bioinformatics and molecular
docking study. Drug Dev Res 82:86–96. https://doi.org/10.1002/ddr.
21730 249. Schwarz S, Wang K, Yu W, Sun B, Schwarz W (2011) Emodin inhibits
current through SARS-associated coronavirus 3a protein. Antiviral Res
90:64–69. https://doi.org/10.1016/j.antiviral.2011.02.008 p
g
j
250. References Phytomedicine 78:1–9. https://doi.org/10.1016/j.
phymed.2020.153296 l
273. Gentile D, Patamia V, Scala A, Sciortino MT, Piperno A, Rescifina A (2020)
Putative inhibitors of SARS-COV-2 main protease from a library of
marine natural products: a virtual screening and molecular modeling
study. Mar Drugs 18:1–19. https://doi.org/10.3390/md18040225 274. Bhardwaj VK, Singh R, Sharma J, Rajendran V, Purohit R, Kumar S (2020)
Identification of bioactive molecules from tea plant as SARS-CoV-2
main protease inhibitors. J Biomol Struct Dyn 39:1–10. https://doi.org/
10.1080/07391102.2020.1766572 257. Raj V, Park J, Cho K, Choi P, Kim T, Ham J, Lee J (2021) Assessment of
antiviral potencies of cannabinoids against SARS-CoV-2 using com‑
putational and in vitro approaches. Int J Biol Macromol 168:474–485. https://doi.org/10.1016/j.ijbiomac.2020.12.020 Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Page 46 of 48 275. Khan MF, Khan MA, Khan ZA, Ahamad T, Ansari WA (2020) Identification
of dietary molecules as therapeutic agents to combat COVID-19 using
molecular docking studies. Res Sq. https://doi.org/10.21203/rs.3.rs-
19560/v1 trafficking of viral receptors. Res Sq. https://doi.org/10.21203/rs.3.rs-
30922/v1 294. Choy KT, Wong AYL, Kaewpreedee P, Sia SF, Chen D, Hui KPY, Chu DKW,
Chan MCW, Cheung PPH, Huang X et al (2020) Remdesivir, lopinavir,
emetine, and homoharringtonine inhibit SARS-CoV-2 replication
in vitro. Antiviral Res 178:1–5. https://doi.org/10.1016/j.antiviral.2020.
104786 276. Zhang J-J, Shen X, Yan YM, WANG Y, Cheng Y-X (2020) Discovery of
anti-SARS-CoV-2 agents from commercially available flavor via docking
screening. OSFPreprint. https://doi.org/10.31219/osf.io/vjch2 277. Lung J, Lin Y, Yang Y, Chou Y, Chang G, Tsai M, Hsu C, Yeh R, Shu L, Cheng
Y et al (2020) The potential SARS-CoV-2 entry inhibitor. biorxiv Prepr
Serv Biol 55:1–22 295. Xia B, Shen X, He Y, Pan X, Wang Y, Yang F, Fang S, Wu Y, Zuo X, Xie Z
et al (2021) SARS-CoV-2 envelope protein causes acute respiratory
distress syndrome (ARDS)-like pathological damage and constitutes
an antiviral target. Cell Res 31:847–860. https://doi.org/10.1038/
s41422-021-00519-4 278. ulQamar MT, Alqahtani SM, Alamri MA, Che LL (2020) Structural basis of
SARS-CoV-2 3CLpro and anti-COVID-19 drug discovery from medicinal
plants. J Pharm Anal 10:313–319. https://doi.org/10.1016/j.jpha.2020.03.
009 296. Li SY, Chen C, Zhang HQ, Guo HY, Wang H, Wang L, Zhang X, Hua SN, Yu
J, Xiao PG et al (2005) Identification of natural compounds with antiviral
activities against SARS-associated coronavirus. Antiviral Res 67:18–23. https://doi.org/10.1016/j.antiviral.2005.02.007 279. References Garg S, Roy A (2020) In silico analysis of selected alkaloids against main
protease (Mpro) of SARS-CoV-2. Chem Biol Interact 332:1–12. https://
doi.org/10.1016/j.cbi.2020.109309 285. Hwang SH, Kwon SH, Kim SB, Lim SS (2017) Inhibitory activities of
stauntonia hexaphylla leaf constituents on rat lens aldose reductase
and formation of advanced glycation end products and antioxidant. Biomed Res Int 4273257:1–9. https://doi.org/10.1155/2017/4273257 303. Roza D, Selly R, Munsirwan R, Fadhilah G (2021) Molecular docking of
quinine derivative as inhibitor in sars-Cov-2. J Phys Conf Ser 1819:1–8. https://doi.org/10.1088/1742-6596/1819/1/012053 286. Ersöz T, Taşdemir D, Çaliş I, Ireland CM (2002) Phenylethanoid glycosides
from Scutellaria galericulata. Turkish J Chem 26:465–471 304. Topcu G, Şenol H, Alim Toraman GÖ, Altan VM (2020) Natural alkaloids
as potential anti-coronavirus compounds. Bezmialem Sci 8:131–139. https://doi.org/10.12435/bas.galenos.2020.5035 287. Dong Z, Lu X, Tong X, Dong Y, Tang L, Liu M (2017) Forsythiae fructus:
a review on its phytochemistry, quality control, pharmacology and
pharmacokinetics. Molecules 22:1–49. https://doi.org/10.3390/molec
ules22091466 305. Majnooni MB, Fakhri S, Bahrami G, Naseri M, Farzaei MH, Echeverr J
(2021) Alkaloids as potential phytochemicals against SARS-CoV-2:
approaches to the associated pivotal mechanisms. Evidence-based
Complement Altern Med 2021:1–21. https://doi.org/10.1155/2021/
6632623 288. Keefover-Ring K, Holeski LM, Bowers MD, Clauss AD, Lindroth RL (2014)
Phenylpropanoid glycosides of mimulus guttatus (yellow monkey‑
flower). Phytochem Lett 10:132–139. https://doi.org/10.1016/j.phytol.
2014.08.016 306. He CL, Huang LY, Wang K, Gu CJ, Hu J, Zhang GJ, Xu W, Xie YH,
Tang N, Huang AL (2021) Identification of bis-benzylisoquinoline
alkaloids as SARS-CoV-2 entry inhibitors from a library of natural
products. Signal Transduct Target Ther 6:1–3. https://doi.org/10.1038/
s41392-021-00531-5 289. Colunga Biancatelli RML, Berrill M, Catravas JD, Marik PE (2020)
Quercetin and vitamin C: an experimental, synergistic therapy for the
prevention and treatment of SARS-CoV-2 related disease (COVID-19). Front Immunol 11:1–11. https://doi.org/10.3389/fimmu.2020.01451 307. Borquaye LS, Gasu EN, Ampomah GB, Kyei LK, Amarh MA, Mensah CN,
Nartey D, Commodore M, Adomako AK, Acheampong P et al (2020)
Alkaloids from cryptolepis sanguinolenta as potential inhibitors of
SARS-CoV-2 viral proteins: an in silico study. Biomed Res Int 2020:1–14. https://doi.org/10.1155/2020/5324560 290. Liskova A, Samec M, Koklesova L, Samuel SM, Zhai K, Al-Ishaq RK,
Abotaleb M, Nosal V, Kajo K, Ashrafizadeh M et al (2021) Flavonoids
against the SARS-CoV-2 induced inflammatory storm. Biomed Pharma‑
cother 138:111430. https://doi.org/10.1016/j.biopha.2021.111430 291. Annunziata G, Sanduzzi Zamparelli M, Santoro C, Ciampaglia R, Stor‑
naiuolo M, Tenore GC, Sanduzzi A, Novellino E (2020) May polyphenols
have a role against coronavirus infection? References Owis A, El-Hawary M, Amir D, Aly O, Abdelmohsen U, Kamel M (2020)
Molecular docking reveals the potential of Salvadora persica flavonoids
to inhibit COVID-19 virus main protease. RSC Adv 10:1–7 297. Stanic-Vucinic D, Minic S, Nikolic M, Velickovic T (2018) Spirulina phy‑
cobiliproteins as food components and complements. In: Jacob-Lopes
E, Zepka LQ, Queiroz MI (eds) Microalgal Biotechnology. IntechOpen,
London, pp 129–149 280. Vincent S, Arokiyaraj S, Saravanan M, Dhanraj M (2020) Molecular
docking studies on the anti-viral effects of compounds from kabasura
kudineer on SARS-CoV-2 3CLpro. Front Mol Biosci 7:1–12. https://doi.
org/10.3389/fmolb.2020.613401 298. Pendyala B, Patras A, Dash C (2021) Phycobilins as Potent food bioactive
broad-spectrum inhibitors against proteases of SARS-CoV-2 and other
coronaviruses: a preliminary study. Front Microbiol. https://doi.org/10.
3389/fmicb.2021.645713 g
281. Murthy T, Joshi T, Gunnan S, Kulkarni N, Venkatesh P, Kumar B, Gow‑
rishankar B (2021) In silico analysis of Phyllanthus amarus phytochemi‑
cals as potent drugs against SARS-CoV-2 main protease. Curr Res Green
Sustain Chem 4:1–14. https://doi.org/10.1016/j.crgsc.2021.100159 299. Wang M, Cao R, Zhang L, Yang X, Liu J, Xu M, Shi Z, Hu Z, Zhong W, Xiao
G (2020) Remdesivir and chloroquine effectively inhibit the recently
emerged novel coronavirus (2019-nCoV) in vitro. Cell Res 30:269–271. https://doi.org/10.1038/s41422-020-0282-0 282. Abba Y, Hassim H, Hamzah H, Noordin MM (2015) antiviral activity of
resveratrol against human and animal viruses. Adv Virol 184241:1–7 283. Schettig R, Sears T, Klein M, Tan-Lim R, Matthias R Jr, Aussems C, Hum‑
mel M, Sears R, Poteet Z, Warren D et al (2020) COVID-19 patient with
multifocal pneumonia and respiratory difficulty resolved quickly: pos‑
sible antiviral and anti-inflammatory benefits of quercinex (nebulized
quercetin-NAC) as adjuvant. Adv Infect Dis 10:45–55. https://doi.org/10.
4236/aid.2020.103006 300. Chowdhury P (2021) In silico investigation of phytoconstituents from
indian medicinal herb “Tinospora Cordifolia (Giloy)” as potential inhibi‑
tors against SARS-CoV-2 (COVID-19). J Biomol Struct Dyn 39:6792–6809 301. Mohammadi S, Heidarizadeh M, Entesari M, Esmailpour A, Esmailpour
M, Moradi R, Sakhaee N, Doustkhah E (2020) In silico investigation on
the inhibiting role of nicotine/caffeine by blocking the s protein of sars-
cov-2 versus ace2 receptor. Microorganisms 8:1–14. https://doi.org/10.
3390/microorganisms8101600 284. Wang X, Yu N, Peng H, Hu Z, Sun Y, Zhu X, Jiang L, Xiong H (2019) The
profiling of bioactives in Akebia trifoliata pericarp and metabolites
bioavailability and in vivo anti-inflammatory activities in DSS-induced
colitis mice. Food Funct. https://doi.org/10.1039/c9fo00393b 302. References Nair M, Huang Y, Fidock DAP, Wagoner SJ, Towler J, Weathers MJ, P,
(2021) Artemisia annua L. extracts inhibit the in vitro replication of
SARS-CoV-2 and two of its variants. J Ethnopharmacol 274:1–8 314. Talukdar J, Bhadra B, Dattaroy T, Nagle V, Dasgupta S (2020) Potential of
natural astaxanthin in alleviating the risk of cytokine storm in COVID-19. Biomed Pharmacother 132:110886. https://doi.org/10.1016/j.biopha.
2020.110886 332. Hoever G, Baltina L, Michaelis M, Kondratenko R, Baltina L, Tolstikov GA,
Doerr HW, Cinatl J (2005) Antiviral activity of glycyrrhizic acid deriva‑
tives against SARS-coronavirus. J Med Chem 48:1256–1259. https://doi.
org/10.1021/jm0493008 315. Melek FR, Miyase T, Abdel-Khalik SM, Hetta MH, Mahmoud II (2002)
Triterpenoid saponins from Oreopanax guatemalensis. Phytochemistry
60:185–195. https://doi.org/10.1016/S0031-9422(02)00058-4 333. Javed H, Meeran MFN, Jha NK, Ojha S (2021) Carvacrol, a plant metabo‑
lite targeting viral protease (Mpro) and ACE2 in host cells can be a
possible candidate for COVID-19. Front Plant Sci 11:1–10. https://doi.
org/10.3389/fpls.2020.601335 316. Muhseen ZT, Hameed AR, Al-Hasani HMH, Tahir ul Qamar M, Li G (2020)
Promising terpenes as SARS-CoV-2 spike receptor-binding domain
(RBD) attachment inhibitors to the human ACE2 receptor: Integrated
computational approach. J Mol Liq 320:1–10. https://doi.org/10.1016/j.
molliq.2020.114493 334. Halder D, Barik BB, Dasgupta RK, Roy SD (2018) Aroma therapy: an art of
healing. Indian Res J Pharm Sci. 5:1540–1558. https://doi.org/10.21276/
irjps.2018.5.3.2 335. Angulo-Milhem S, Verriele M, Nicolas M, Thevenet F (2021) Indoor use
of essential oils: emission rates, exposure time and impact on air quality. Atmos Environ 244:1–13 q
317. Yu MS, Lee J, Lee JM, Kim Y, Chin YW, Jee JG, Keum YS, Jeong YJ (2012)
Identification of myricetin and scutellarein as novel chemical inhibitors
of the SARS coronavirus helicase, nsP13. Bioorganic Med Chem Lett
22:4049–4054. https://doi.org/10.1016/j.bmcl.2012.04.081 336. Boukhatem MN (2020) Effective antiviral activity of essential oils and
their characteristics terpenes against coronaviruses: an update. J Phar‑
macol Clin Toxicol 8:1138 318. Reichling J, Koch C, Stahl-Biskup E, Sojka C, Schnitzler P (2005) Virucidal
activity of a β-triketone-rich essential oil of Leptospermum scoparium
(manuka oil) against HSV-1 and HSV-2 in cell culture. Planta Med
71:1123–1127. https://doi.org/10.1055/s-2005-873175 337. Adorjan B, Buchbauer G (2010) Biological properties of essential oils: An
updated review. Flavour Fragr J 25:407–426. https://doi.org/10.1002/ffj.
2024 338. Ojah EO (2020) Exploring essential oils as prospective therapy against
the ravaging Coronavirus (SARS-CoV-2). Iberoam J Med 04:322–330 p
g
319. References An overview of in vitro
evidence. Front Med 7:1–8. https://doi.org/10.3389/fmed.2020.00240 308. Quimque M, Notarte K, Fernandez R, Mendoza M, Liman R, Lim J, Pilapil
L, Ong J, Pastrana A, Khan A et al (2021) Virtual screening-driven drug
discovery of SARS-CoV2 enzyme inhibitors targeting viral attachment,
replication, post-translational modification and host immunity evasion
infection mechanisms. J Biomol Struct Dyn 39:1–18 292. Berretta A, Silveira M, Capcha J, Jong D (2020) Propolis and its potential
against SARS-CoV-2 infection mechanisms and COVID-19 disease. run‑
ning title: propolis against SARS-CoV-2 infection and COVID-19. Biomed
Pharmacother 131:1–16. https://doi.org/10.1016/j.biopha.2020.110622 y
309. Gaba S, Saini A, Singh G, Monga V (2021) An insight into the medicinal
attributes of berberine derivatives: a review. Bioorganic Med Chem
38:1–20. https://doi.org/10.1016/j.bmc.2021.116143 p
g
j
p
293. Huang L, Li H, Yuen TT-T, Ye Z, Fu Q, Sun W, Xu Q, Yang Y, Chan JF-W,
Zhang G et al (2020) Berbamine inhibits the infection of SARS-CoV-2
and flaviviruses by compromising TPRMLs-mediated endolysosomal 310. Yang W, Chen X, Li Y, Guo S, Wang Z, Yu X (2020) Advances in pharma‑
cological activities of terpenoids. Nat Prod Commun 15:1–13. https://
doi.org/10.1177/1934578X20903555 Page 47 of 48 Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 311. Anil SM, Shalev N, Vinayaka AC, Nadarajan S, Namdar D, Belausov E, Sho‑
val I, Mani KA, Mechrez G, Koltai H (2021) Cannabis compounds exhibit
anti-inflammatory activity in vitro in COVID-19-related inflammation in
lung epithelial cells and pro-inflammatory activity in macrophages. Sci
Rep 11:1–15. https://doi.org/10.1038/s41598-021-81049-2 discovery of drugs for the treatment of COVID-19. Med Chem Res
31:40–51. https://doi.org/10.1007/s00044-021-02826-2 p
g
329. Shibata S (2000) A drug over the millennia: pharmacognosy, chemistry,
and pharmacology of licorice. Yakugaku Zasshi 120:849–862. https://
doi.org/10.1248/yakushi1947.120.10_849 312. Wu Z, Chen X, Ni W, Zhou D, Chai S, Ye W, Zhang Z, Guo Y, Ren L, Zeng
Y (2021) The inhibition of Mpro, the primary protease of COVID-19, by
Poria cocos and its active compounds_ a network pharmacology and
molecular docking study. RSC Adv 11:11821–11843 330. Luo P, Liu D, Li J (2020) Pharmacological perspective: glycyrrhizin may
be an efficacious therapeutic agent for COVID-19. Int J Antimicrob
Agents 55:1–3. https://doi.org/10.1016/j.ijantimicag.2020.105995 331. Ding H, Deng W, Ding L, Ye X, Yin S, Huang W (2020) Glycyrrhetinic acid
and its derivatives as potential alternative medicine to relieve symp‑
toms in nonhospitalized COVID-19 patients. J Med Virol 92:2200–2204. https://doi.org/10.1002/jmv.26064 313. References Snene A, El Mokni R, Jmii H, Jlassi I, Jaïdane H, Falconieri D, Piras A,
Dhaouadi H, Porcedda S, Hammami S (2017) In vitro antimicrobial, anti‑
oxidant and antiviral activities of the essential oil and various extracts
of wild (Daucus virgatus (Poir.) Maire) from Tunisia. Ind Crops Prod
109:109–115. https://doi.org/10.1016/j.indcrop.2017.08.015 339. De Logu A, Loy G, Pellerano ML, Bonsignore L, Schivo ML (2000) Inac‑
tivation of HSV-1 and HSV-2 and prevention of cell-to-cell virus spread
by Santolina insularis essential oil. Antiviral Res 48:177–185. https://doi.
org/10.1016/S0166-3542(00)00127-3 g
j
320. Lelešius R, Karpovaite A, Mickiene R, Drevinskas T, Tiso N, Ragažinskiene
O, Kubiliene L, Maruška A, Šalomskas A (2019) In vitro antiviral activity of
fifteen plant extracts against avian infectious bronchitis virus. BMC Vet
Res 15:1–10. https://doi.org/10.1186/s12917-019-1925-6 340. Schnitzler P, Koch C, Reichling J (2007) Susceptibility of drug-resistant
clinical herpes simplex virus type 1 strains to essential oils of ginger,
thyme, hyssop, and sandalwood. Antimicrob Agents Chemother
51:1859–1862. https://doi.org/10.1128/AAC.00426-06 321. Park JY, Kim JH, Kim YM, Jeong HJ, Kim DW, Park KH, Kwon HJ, Park
SJ, Lee WS, Ryu YB (2012) Tanshinones as selective and slow-binding
inhibitors for SARS-CoV cysteine proteases. Bioorganic Med Chem
20:5928–5935. https://doi.org/10.1016/j.bmc.2012.07.038 341. Schuhmacher A, Reichling J, Schnitzler P (2003) Virucidal effect of
peppermint oil on the enveloped viruses herpes simplex virus type 1
and type 2 in vitro. Phytomedicine 10:504–510. https://doi.org/10.1078/
094471103322331467 322. Thuy BTP, My TTA, Hai NTT, Hieu LT, Hoa TT, Loan HTP, Triet NT, Van Anh
TT, Quy PT, Van Tat P (2020) Investigation into SARS-CoV-2 resistance of
compounds in garlic essential oil. ACS Omega. 5(8312):8320. https://
doi.org/10.1021/acsomega.0c00772 342. Minami M, Kita M, Nakaya T, Yamamoto T, Kuriyama H, Imanishi J (2003)
The inhibitory effect of essential oils on herpes simplex virus type-1
replication in vitro. Microbiol Immunol 47:681–684. https://doi.org/10.
1111/j.1348-0421.2003.tb03431.x 323. Sharma AD, Kaur I (2020) Eucalyptol (1,8 cineole) from eucalyptus
essential oil a potential inhibitor of Covid-19 corona virus infection by
molecular docking studies. Preprints. https://doi.org/10.20944/prepr
ints202003.0455.v1 343. Schnitzler P, Schuhmacher A, Astani A, Reichling J (2008) Melissa offici‑
nalis oil affects infectivity of enveloped herpesviruses. Phytomedicine
15:734–740. https://doi.org/10.1016/j.phymed.2008.04.018 344. Kovač K, Diez-Valcarce M, Raspor P, Hernández M, Rodríguez-Lázaro
D (2012) Natural plant essential oils do not inactivate non-enveloped
enteric viruses. Food Environ Virol 4:209–212. https://doi.org/10.1007/
s12560-012-9088-7 324. References Yadalam PK, Varatharajan K, Rajapandian K, Chopra P, Arumuganainar D,
Nagarathnam T, Sohn H, Madhavan T (2021) Antiviral essential oil com‑
ponents against SARS-CoV-2 in pre-procedural mouth rinses for dental
setting during Covid-19: a computational study. Front Chem 9:1–11 y
325. Roviello V, Roviello GN (2021) Lower COVID-19 mortality in Ital‑
ian forested areas suggests immunoprotection by Mediterranean
plants. Environ Chem Lett 19:699–710. https://doi.org/10.1007/
s10311-020-01063-0 345. Garozzo A, Timpanaro R, Stivala A, Bisignano G, Castro A (2011) Activity
of Melaleuca alternifolia (tea tree) oil on Influenza virus A/PR/8: study
on the mechanism of action. Antiviral Res 89:83–88. https://doi.org/10.
1016/j.antiviral.2010.11.010 346. Cermelli C, Fabio A, Fabio G, Quaglio P (2008) Effect of eucalyptus
essential oil on respiratory bacteria and viruses. Curr Microbiol
56:89–92. https://doi.org/10.1007/s00284-007-9045-0 326. Kulkarni SA, Nagarajan SK, Ramesh V, Palaniyandi V, Selvam SP, Mad‑
havan T (2020) Computational evaluation of major components from
plant essential oils as potent inhibitors of SARS-CoV-2 spike protein. J
Mol Struct 1221:128823. https://doi.org/10.1016/j.molstruc.2020.128823 347. Jackwood MW, Rosenbloom R, Petteruti M, Hilt DA, McCall AW, Williams
SM (2010) Avian coronavirus infectious bronchitis virus susceptibility
to botanical oleoresins and essential oils in vitro and in vivo. Virus Res
149:86–94. https://doi.org/10.1016/j.virusres.2010.01.006 327. Aanouz I, Belhassan A, El-Khatabi K, Lakhlifi T, El-ldrissi M, Bouachrine
M (2021) Moroccan medicinal plants as inhibitors against SARS-CoV-2
main protease: computational investigations. J Biomol Struct Dyn
39:2971–2979. https://doi.org/10.1080/07391102.2020.1758790 348. Benencia F, Courrges MC (2000) In vitro and in vivo activity of eugenol
on human herpesvirus. Phyther Res 14:495–500. https://doi.org/10.
1002/1099-1573(200011)14:7%3c495::AID-PTR650%3e3.0.CO;2-8 328. Chapman RL, Andurkar SV (2022) A review of natural products, their
effects on SARS-CoV-2 and their utility as lead compounds in the Oktavianawati et al. Applied Biological Chemistry (2023) 66:89 Page 48 of 48 Chem Chem Phys 22:25335–25343. https://doi.org/10.1039/d0cp0
3867a 349. Vicidomini C, Roviello V, Roviello GN (2021) molecular basis of the
therapeutical potential of clove (Syzygium aromaticum L.) and clues to
its anti-COVID-19 utility. Molecules 26:1–12. https://doi.org/10.3390/
molecules26071880 366. Mengist HM, Dilnessa T, Jin T (2021) Structural basis of potential inhibi‑
tors targeting SARS-CoV-2 main protease. Front Chem 9:1–19. https://
doi.org/10.3389/fchem.2021.622898 350. Banerjee S, Panda C, Das KS (2006) Clove (Syzygium aromaticum L.),
a potential chemopreventive agent for lung cancer. Carcinogenesis. 27:1645–1654 g
367. Mehany T, Khalifa I, Barakat H, Althwab SA, Alharbi YM, El-Sohaimy S
(2021) Polyphenols as promising biologically active substances for pre‑
venting SARS-CoV-2: a review with research evidence and underlying
mechanisms. Food Biosci 40:1–11. https://doi.org/10.1016/j.fbio.2021.
100891 351. Publisher’s Note
S
i
N
i 353. Pino JA, Marbot R, Agüero J, Fuentes V (2001) Essential oil from buds
and leaves of clove (Syzygium aromaticum (L.) Merr. et Perry) grown in
Cuba. J Essent Oil Res 13:278–279. https://doi.org/10.1080/10412905.
2001.9699693 Springer Nature remains neutral with regard to jurisdictional claims in pub‑
lished maps and institutional affiliations. 354. Kurokawa M, Hozumi T, Basnet P, Nakano M, Kadota S, Namba T, Kawana
T, Shiraki K (1998) Purification and characterization of eugeniin as an
anti-herpesvirus compound from Geum japonicum and Syzygium aro‑
maticum. J Pharmacol Exp Ther 284:728–735. https://doi.org/10.1146/
annurev-immunol-032713-120231 355. Nguyen LC, Yang D, Nicolaescu V, Best TJ, Ohtsuki T, Chen S, Friesen
JB, Drayman N, Mohamed A, Dann C et al (2021) Cannabidiol inhibits
SARS-CoV-2 replication and promotes the host innate immune
response. biorxiv 70:1–15 356. Chatow L, Nudel A, Nesher I, Hemo DH, Rozenberg P, Voropaev H,
Winkler I, Levy R, Kerem Z, Yaniv Z et al (2021) In vitro evaluation of the
activity of terpenes and cannabidiol against human coronavirus E229. Life 11:1–10. https://doi.org/10.3390/life11040290 357. Amparo TR, Seibert JB, Silveira BM, Costa FSF, Almeida TC, Braga SFP, da
Silva GN, dos Santos ODH, de Souza GHB (2021) Brazilian essential oils
as source for the discovery of new anti-COVID-19 drug: a review guided
by in silico study. Phytochem Rev 20:1013–1032. https://doi.org/10.
1007/s11101-021-09754-4 358. Amaral-Machado L, Oliveira WN, Rodrigues VM, Albuquerque NA, Alen‑
car ÉN, Egito EST (2021) Could natural products modulate early inflam‑
matory responses, preventing acute respiratory distress syndrome
in COVID-19-confirmed patients? Biomed Pharmacother 134:1–20. https://doi.org/10.1016/j.biopha.2020.111143 p
g
j
p
359. Pisoschi A, Pop A, Iordache F, Stanca L, Geicu O, Bilteanu L, Serban A
(2022) Antioxidant, anti-inflammatory and immunomodulatory roles of
vitamins in COVID-19 therapy. Eur J Med Chem 232:1–24. https://doi.
org/10.1016/j.ejmech.2022.114175 360. da Silva FMA, da Silva KPA, de Oliveira LPM, Costa EV, Koolen HHF,
Pinheiro MLB, de Souza AQL, de Souza ADL (2020) Flavonoid glycosides
and their putative human metabolites as potential inhibitors of the
sars-cov-2 main protease (Mpro) and RNA-dependent RNA polymerase
(RdRp). Mem Inst Oswaldo Cruz 115:1–8. https://doi.org/10.1590/0074-
02760200207 361. Antonio AD, Wiedemann LS, Junior VF (2020) Natural products’ role
against COVID-19. RSC Adv 10:23379–23393 362. Teli DM, Shah MB, Chhabria MT (2021) In silico screening of natural
compounds as potential inhibitors of SARS-CoV-2 main protease and
spike RBD: targets for COVID-19. Front Mol Biosci 7:1–25. https://doi.
org/10.3389/fmolb.2020.599079 363. References Chaieb K, Hajlaoui H, Zmantar T, Kahla-Nakbi AB, Rouabhia M, Mah‑
douani K, Bakhrouf A (2007) The chemical composition and biological
activity of clove essential oil, eugenia caryophyllata (Syzigium aromati-
cum L. myrtaceae): a short review. Phyther Res 21:501–506. https://doi.
org/10.1002/ptr 368. Verma S, Twilley D, Esmear T, Oosthuizen CB, Reid AM, Nel M, Lall N
(2020) Anti-SARS-CoV natural products with the potential to inhibit
SARS-CoV-2 (COVID-19). Front Pharmacol 11:1–20. https://doi.org/10.
3389/fphar.2020.561334 352. Raina VK, Srivastava SK, Aggarwal KK, Syamasundar KV, Kumar S (2001)
Essential oil composition of Syzygium aromaticum leaf from Little
Andaman. India Flavour Fragr J 16:334–336. https://doi.org/10.1002/ffj.
1005 Publisher’s Note
S
i
N
i Contreras-Puentes N, Alvíz-Amador A (2020) Virtual screening of natural
metabolites and antiviral drugs with potential inhibitory activity against
3CL-PRO and PL-PRO. Biomed Pharmacol J 13:933–941. https://doi.org/
10.13005/BPJ/1962 364. Joshi RS, Jagdale SS, Bansode SB, Shankar SS, Tellis MB, Pandya VK,
Chugh A, Giri AP, Kulkarni MJ (2020) Discovery of potential multi-target-
directed ligands by targeting host-specific SARS-CoV-2 structurally
conserved main protease. J Biomol Struct Dyn 1:1–16. https://doi.org/
10.1080/07391102.2020.1760137 365. Huynh T, Wang H, Luan B (2020) Structure-based lead optimization of
herbal medicine rutin for inhibiting SARS-CoV-2’s main protease. Phys 365. Huynh T, Wang H, Luan B (2020) Structure-based lead optimization of
herbal medicine rutin for inhibiting SARS-CoV-2’s main protease. Phys
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Designing Online Marketplaces: Trust and
Reputation Mechanisms
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Luca, Michael. "Designing Online Marketplaces: Trust and Reputation Mechanisms." Harvard
Business School Working Paper, No. 17-017, September 2016.
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Designing Online Marketplaces: Trust
and Reputation Mechanisms
Michael Luca
Working Paper 17-017
Designing Online Marketplaces: Trust
and Reputation Mechanisms
Michael Luca
Harvard Business School
Working Paper 17-017
Copyright © 2016 by Michael Luca
Working papers are in draft form. This working paper is distributed for purposes of comment and discussion only. It may
not be reproduced without permission of the copyright holder. Copies of working papers are available from the author.
Designing Online Marketplaces:
Trust and Reputation Mechanisms
Michael Luca*
Online marketplaces have proliferated over the past decade, creating new markets where
none existed. By reducing transaction costs, online marketplaces facilitate transactions
that otherwise would not have occurred and enable easier entry of small sellers. One
central challenge faced by designers of online marketplaces is how to build enough trust
to facilitate transactions between strangers. This paper provides an economist’s toolkit for
designing online marketplaces, focusing on trust and reputation mechanisms.
Harvard Business School, Soldiers Field Road, Boston, MA 02163. mluca@hbs.edu Kaye Kirschner,
Janet Lu, and Patrick Rooney provided excellent research assistance. I am grateful to Shane
Greenstein, Joshua Lerner, Yasin Ozcan, Scott Stern, and participants at the NBER Innovation Policy
and Economics workshop.
*
1 Introduction
Over the past decade, there has been a proliferation of online marketplaces,
ranging from eBay and Amazon to Uber and Airbnb. These online markets cannot exist
without trust. Buyers need to trust that sellers are accurately describing products or
services and fulfilling transactions as promised. Sellers need to trust that buyers will pay,
and in the case of services and rentals, will abide by the agreed-upon terms of service.
Trust between buyers and sellers is enabled by reputation systems and design choices
made by online marketplaces.
This paper surveys the design choices and mechanisms that online marketplaces
use to build trust and facilitate transactions. I focus on two central decisions faced by a
marketplace. First, online marketplaces design review systems, which allow buyers and
sellers to review each other and the product or service being transacted. Second,
marketplaces choose what information buyers and sellers should have about each other
when deciding whether to transact, and how much flexibility market participants should
have in choosing who they will transact with. I highlight challenges and tradeoffs faced
by online marketplaces in these design choices.
Historically, reviews have formed the backbone of reputation systems in the
online marketplace. Sellers (and the products and services they are offering) on online
platforms are rated and reviewed by buyers, and buyers can use this information to
choose whom to interact with. Likewise, sellers on some platforms can review buyers.
Reviews allow buyers and sellers to make sure they are transacting with someone deemed
trustworthy enough to participate in the transaction. Reviews also create incentives for
quality, as behavior of buyers and sellers is made more public. Despite the benefits of
creating online reputation systems, several design challenges have been documented in
the context of reviews. First, on platforms with reciprocal reviewing (i.e., where buyers
and sellers review each other), users can have strategic incentives to manipulate reviews
(Bolton et al. 2013, Fradkin et al. 2015). Second, reviews can suffer from selection bias
(Hu et al. 2009, Masterov et al. 2015), as the people leaving reviews may differ from
those who do not. Third, reviews may be distorted by promotional content in which
businesses attempt to leave reviews for themselves (Mayzlin, Dover, and Chevalier 2014;
Luca and Zervas 2015). Moreover, even if all reviews represent a user’s true experience,
some users may be more informative than others (Dai et al. 2015). Section 3 provides an
overview of these issues, as well as potential design solutions for these challenges.
In first-generation platforms such as eBay, reviews – along with pictures and
descriptions of the product – were the main information sources available to buyers at the
time of purchase. Buyers and sellers typically did not see each other’s name or picture –
names were exchanged after the purchase was made. Over time, platforms have become
considerably less anonymous. For example, Airbnb is an online platform for people to list
and find short-term lodging; it attempts to build trust by allowing would-be renters to
present personal profiles and post pictures of themselves. Hosts can then accept or reject
guests based on their pictures and profiles. This design choice is now the norm on many
platforms – indeed, it represents a broader trend of platforms’ providing more
information to market participants – not only about products and services, but about the
people buying and selling them. This also demonstrates the large degree of flexibility that
online marketplaces have in their market-design choices (much more than is typically
found in traditional markets).
The design choice of allowing hosts to reject guests on the basis of profile elements
such as name, picture, and number of Facebook friends is a double-edged sword. On the
one hand, it has the potential to make market participants feel more comfortable with
each other, in part by reducing social distance. However, this design choice also
facilitates discrimination – for example, African-American guests (and hosts) are
discriminated against on Airbnb (Edelman and Luca 2014; Edelman, Luca, and Svirsky
2016). This is part of a broader trend of discrimination that has been documented in
online marketplaces including eBay (Nunley et al., 2011), Craigslist (Doleac and Stein,
2013), and Prosper (Pope and Sydnor, 2011). In labor market contexts, there is extensive
evidence of discrimination against minority job applicants (Bertrand and Mullainathan
2004). Online labor markets such as Upwork are designed in a way that makes it easy for
discrimination to manifest in online contexts as well. For example, Ghani et al. (2014)
find that employers of Indian ethnicity are more likely to hire workers from India.
Overall, these findings highlight the promise and peril of online marketplaces. The
existence of new online markets creates value for society, enabled by trust and reputation
mechanisms. However, the same design choices allow for unintended consequences such
as discrimination. In this paper, I document the evolution of new online markets, focusing
on the design choices pertaining to trust and reputation. I highlight the key features of
reputation systems and the problems that can arise. I then survey the evidence on
discrimination in online markets and consider potential solutions that can be implemented
by platforms.
2 The Rise of Online Marketplaces
The founding of Amazon and eBay in 1994 and 1995, respectively, ushered in the
first generation of online marketplaces. These platforms were remarkable in their ability
to facilitate transactions between strangers. Someone in upstate New York could order a
used book from a stranger in southern California and trust that it would arrive in a few
days. These platforms facilitated transactions that would not otherwise occur, supported
in part by reputation systems. At the same time, buyers and sellers generally did not
provide pictures or names until after a purchase was made. Both platforms had review
systems to facilitate transactions.
In the 20 years since, a variety of more specialized platforms such as Airbnb,
Uber, and Upwork have emerged, creating new markets and pushing a growing
proportion of the economy onto the Internet. To give context for how quickly the industry
is growing: Airbnb was founded in 2008 and is now valued at $26 billion. It currently has
more than 2 million listings, which is more than the largest hotel chain. Similarly
remarkable is the statistic that there are now more Uber drivers –affiliated vehicles than
traditional taxis in New York City. As of 2015, Uber is valued at roughly $50 billion.
Relative to first-generation platforms, these marketplaces have less anonymity – pictures
and names are fast becoming the norm – but still have the reputation systems that were
staples of earlier platforms. Table 1 provides a sample of marketplaces that are active as
of 2016. Table 2 provides data on a subsample of those.
3 Reputation Systems
In this section, I highlight the main challenges – and potential solutions – to
building a robust reputation in an online marketplace, focusing on the role of user
reviews and drawing on Luca (2015). Broadly speaking, there are two main mechanisms
that a platform can use to improve the quality of its review system, taking into account
the types of biases that commonly arise. First, it can improve the incentives to leave high
quality reviews. Second, taking the reviews as given, it can aggregate the reviews in a
way to increase their informational content (Dai et al. 2015). I then look beyond reviews
for other tools to facilitate trust.
3.1 Reciprocal Reviewing
The process of reciprocal reviewing (i.e., of buyers and sellers reviewing each
other) is valuable because it builds trust on both sides of the market. However, this can
also create incentives for upward-biased reporting if reviewers fear retaliation. For
example, when Airbnb’s policy allowed renters’ reviews to be posted before hosts’ (as
was previously the case), guests might have been hesitant to leave bad reviews out of
concern that hosts would retaliate.
To circumvent this problem, platforms such as Airbnb have moved to a system
sometimes referred to as “simultaneous reveal”: they do not display reviews until both
sides have left a review (or until the time to review has expired). Simultaneous revelation
of reviews reduces the strategic problems associated with reciprocal reviewing (Bolton et
al. 2013, Fradkin et al. 2015). However, even in a simultaneous-reveal system, strategic
incentives persist. For example, buyers may be reluctant to provide negative feedback if
they suspect that it would discourage other sellers from transacting with them.
One potential solution to the problems created by reciprocal reviewing would be
to allow users to leave anonymous ratings (in situations where this is feasible), which
could allow users to be more honest without fearing direct or indirect retaliation. To
increase anonymity, platforms might consider showing only aggregated feedback. While
this reduces the total amount of information being provided to users, it may increase the
quality of that information. A second solution would be to have private ratings that are
provided from a user to the platform, but not shared publicly. This would again reduce
fear of retaliation on the part of users, while allowing the platform to use the information.
The platform could use this private feedback in a variety of ways, such as updating its
sorting algorithms, or using it to address concerns directly with the user being reviewed.
3.2 Self-Selection
Because reviews in online marketplaces are voluntary, they can suffer from
selection bias, in that reviews are left by users who chose both to purchase the product or
service and to leave a review online. In particular, users may be more likely to leave a
review after an especially positive or negative experience. Indeed, Hu et al. (2009) find
that reviews on Amazon tend to exhibit an asymmetric bimodal (J-shaped) distribution,
with more positive than negative reviews. They argue that experiences for many products
are more likely to resemble a normal distribution, and hence the J-shape suggests that
people are more likely to leave reviews after extreme experiences. Masterov et al. (2015)
find consistent evidence from eBay, where buyers are more likely to leave a review after
a good experience.
There are a variety of tools that online marketplaces can use to improve this
selection problem, ranging from sending repeated email notifications encouraging people
to report their experiences to paying people who leave reviews. Alternatively, to the
extent that platforms know who is leaving a review, they can incorporate this information
into the reputation score – for example, penalizing sellers who receive low rates of
feedback.
3.3 Promotional Content
Another potential bias in online reviews occurs when people or businesses
surreptitiously leave reviews about themselves or competitors. Promotional content is
driven, at least in part, due to changing economic incentives for a business (Mayzlin,
Dover, and Chevalier 2014, Luca and Zervas 2015). It is more prevalent among
independent businesses, when there is nearby competition, and when there is a negative
shock to a business’s reputation. One mechanism for reducing promotional reviews is to
verify whether a transaction has occurred. While this may help to reduce fake or
promotional reviews, it may also prevent legitimate reviews by increasing the barriers to
contributing content.
Beyond verification of transactions, there are several other potential approaches to
reducing promotional content: spam can be identified through algorithms that mine
review text and characteristics (e.g., Ott et al. 2011, Akoglu et al. 2013). Platforms can
also give more weight to reviewers who are less likely to be contributing promotional or
fake content – for example, reviewers with longer transaction histories could receive
more weight.
3.4 Social Distance
Social distance has the potential to affect a variety of other behaviors, such as
generosity (Hoffman et al. et al. 1996) and reciprocity (Charness et al. et al. 2007) – even
in one-shot games. With pictures and profiles becoming an increasingly common design
choice, online marketplaces are beginning to shrink the social distance between buyers
and sellers. One might expect this to lead to higher ratings. This could be good if it makes
users more comfortable with each other, but may distort reviews to the extent that higher
ratings reflect a reluctance to leave negative feedback after a bad experience. The level of
social distance can be a choice variable for platforms.
3.5 Beyond Reviews
Online marketplaces can supplement reviews through other trust-building
mechanisms. The marketplace itself can do more to screen or authenticate information
about buyers and sellers. For example, Airbnb could conduct interviews of renters and
hosts, or they could run background checks. For example, Care.com, a marketplace for
childcare providers, has incorporated a greater degree of platform-driven screening.
Platforms can also provide insurance – and clear liability rules – for situations where
something does go wrong.
4 Discrimination in Online Marketplaces
Another area in which online platforms can fall short is in their potential to allow
discrimination. In the early days of electronic commerce, economists hypothesized that
online platforms might decrease the amount of discrimination in commercial transactions.
For example, consider a customer looking to purchase a car. This is a market where
prices are negotiated separately for each buyer, and the final price paid is opaque – and
this is also a market where racial discrimination is prevalent (Ayres and Siegelman 1995).
However, when a purchase is initiated through an online platform, Morton et al. (2003)
find no difference in outcomes on the basis of race. Similarly, eBay has less scope for
discrimination because indicators of race and gender are generally not very salient on the
platform. On other platforms, such as Amazon and Expedia, sellers essentially pre-
commit to accepting all buyers regardless of race or ethnicity. Marketplaces have the
potential to facilitate transactions while reducing discrimination.
Over time, though, the design of online platforms has changed, moving toward
systems with less anonymity and fuller user profiles, coupled with more flexibility on the
part of sellers and buyers to do business based on these attributes. For example, Expedia
effectively prevents a hotel from rejecting a guest based on perceived race, ethnicity, or
almost any other factor. But if the same hotel lists a room on Airbnb, it could reject a
guest based on these or other factors. This highlights the fact that, while the Internet has
the potential to reduce discrimination, this benefit depends on the design choices made by
platforms.
In part because online platforms have evolved in this way, the conditions that
made online markets potential havens free from discrimination are not prevalent on all
platforms. To see the contrast between the prominent norms of online and offline
markets, suppose that a senior executive at a hotel chain were to propose the following
change to its reservation policy:
Let’s start encouraging guests to upload their pictures, and let’s allow branch
managers to reject guests if they don’t like the way they look. Potential guests
would continue to make reservations through the website as before, but they would
be nudged to upload pictures and links to Facebook pages or LinkedIn accounts.
Branch managers would then have 24 hours to decide whether the guest looked
sufficiently trustworthy. If the guest looked trustworthy, he or she would be
accepted for a reservation. If not, the guest would be rejected. Managers would like
the policy because they could accept the guests that they are most comfortable with.
We’d also save money because the most costly guests would go elsewhere.
Of course, this idea sounds unrealistic. While it is conceivable that a branch
manager could distinguish some of the trustworthy guests from the bad on the basis of
looks or name, the adoption of screening on this basis is fraught with risk. In particular,
what sorts of pictures and names should a manager screen for? The Civil Rights Act of
1964 prohibits discrimination on the basis of race, color, religion, or national origin.
While the manager could certainly look for subtler cues, incorporating appearance in the
reservations process would likely be too risky to justify any benefit the hotel might
receive. Yet this design choice is the emerging norm in online marketplaces for shortterm housing – and in many other marketplaces.
Discrimination has now been documented in a variety of online marketplaces. Table
3 summarizes the evidence on discrimination across different online marketplaces. This
section documents the evidence, as well as design features that might facilitate
discrimination.
4.1 Housing rental markets
Most searches for accommodations – both short term and long term – now begin
online. For long-term apartment leases, platforms including Craigslist, Zillow, and many
of their competitors provide rental information. In these markets, the platform typically
provides the initial connection between potential landlords and lessees (and often charges
a fee for advertising), but does not facilitate actual payment or charge fees based on
transacted leases.
In short-term rentals on marketplaces such as Airbnb and HomeAway, the market
looks very different. Transactions are often agreed upon from afar, and the platform does
facilitate payment. On Airbnb, prospective hosts and guests provide information not just
about their listings and preferences, but also about themselves – often posting photos,
providing their name, etc. All of these factors are provided before a host decides whether
to accept or reject a guest.
My collaborators and I find that these design choices matter – African-American
guests (and hosts) are discriminated against on Airbnb in a way that would be infeasible
under different design choices (Edelman and Luca 2014, Edelman, Luca, and Svirsky
2016). Looking at listings in New York City, Edelman and Luca (2014) find that AfricanAmerican hosts earn about 12% less than White hosts for similar listings. Edelman, Luca,
and Svirsky (2016) implement a field experiment across five cities, varying only the race
of the person requesting to stay with a host. African-American guests are roughly 15%
less likely to be accepted relative to White guests, holding all else constant.
4.2 Labor markets
Looking at the online labor market Upwork (which was called oDesk at the time),
Ghani et al. (2014) find that relative to other employers, employers of Indian descent are
more likely to hire Indian nationals.[1] Given the design of Upwork, which provides
pictures of employees during the recruiting process, one might expect other forms of
discrimination to be prevalent as well. Figure 2 presents results from a search for
potential employees on Upwork. A variety of more specialized labor markets have arisen
in the digital age as well, ranging from Topcoder (specializing in programmers) to Uber
[1]
There is extensive evidence of discrimination in offline labor markets, which is beyond the scope of this
chapter. See, for example, Bertrand and Mullainathan (2004) and Lang and Lehman (2011).
and Lyft (specializing in drivers). As of 2015, roughly 0.5% of all U.S. workers provided
contract services through online marketplaces (Katz and Krueger 2016).
The rapid growth of online labor markets has the potential to increase or decrease
discrimination. For example, consider the taxi industry, an industry in which men are
historically overrepresented. As of 2014, there were more females on Uber relative to the
traditional taxi industry (Hall and Krueger 2015). New data from a survey done by the
Benenson Strategy Group shows that these numbers are far from static – in fact, there has
been a 30% increase in female Uber drivers in the past year alone.[2]
Online labor markets have a unique opportunity to prevent discrimination using
various tools, some of which we discuss in the next section.
4.3 Credit markets
Credit and funding markets are rapidly changing as peer-to-peer lending and
crowdfunding platforms are on the rise. On peer-to-peer lending platforms such as
Prosper.com, would-be borrowers post profiles and an amount they are looking to
borrow. Would-be lenders select among borrowers that they would like to fund. Looking
at Prosper, Pope and Snydor (2011) find that loan listings for Black borrowers are 2535% less likely to get funded than loan listings for White borrowers with similar credit
scores. However, Black borrowers are also more likely to default on a loan through
Prosper, leading the authors to conclude that this is statistical discrimination. In their
context, reducing discrimination would also reduce efficiency, unless the platform were
to supplement listings with further information to predict default rates.
[2]
Benenson Strategy Group: “Uber: The Driver Roadmap 2.0.”
4.4 Other markets
Other markets face discrimination challenges as well. For example, Doleac and
Stein (2013) implement a field experiment on Craigslist in which they sell used iPods. In
the posted pictures, they vary the hand that is holding the iPod. They find that the demand
for the iPod is lower when the hand holding it is African-American. Looking at sales of
baseball cards on eBay using a similar design, Ayres et al. find that cards held by
African-American sellers earn roughly 20% less. Nunley et al. (2011) also find
discrimination on eBay, but note that the extent of discrimination also depends on the
amount of competition. In their design, the name of the seller is varied (as opposed to the
picture). Relative to platforms such as Airbnb and Upwork, both eBay and Craigslist
have less of a norm for sellers to post personal pictures of themselves. Hence, while
discrimination can exist on eBay and Craigslist, these platforms are doing less to
facilitate it than are platforms that encourage (and in some cases require) users to publicly
post information that signals their race, gender, or other personal information.
4.5 Debiasing marketplaces
There are many market design solutions that online platforms could implement in
order to reduce discrimination. Moreover, there is variation in the choices that platforms
make in this area that can influence the extent of discrimination. In this section, I lay out
potential ways to reduce discrimination in an online marketplace. This is not intended to
be a comprehensive list, but rather to highlight the fact that there are a variety of market
design solutions that could be implemented by a well-intentioned marketplace.
4.5.1 Optimal information provision
Until recently, most musicians within major symphony orchestras were male.
Over time, gender equality has increased. Goldin and Rouse (2001) find that roughly
25% of the change in composition was driven by one simple change – blind auditions.
Historically, when a musician would audition for the orchestra, he or she would be both
heard and watched by the evaluating committee. Over time, there has been a shift in
norms toward blind auditions; now, there is typically a screen between the musician and
the evaluating committee. This makes factors such as looks, race, and gender less salient,
allowing the committee to focus on the quality of the music.
This highlights the fact that while information is necessary to make markets
function, more information is not always better. Providing information about factors such
as race and gender can facilitate discrimination; removing this information can moderate
discrimination effects.
In offline settings, it is often difficult to blind transactions. For example, it would
be challenging for an interviewer to put a screen up in front of a candidate that she is
interviewing. One of the unique features about online marketplaces is that they provide
an opportunity to decide when and where virtual screens should be placed. In the context
of Airbnb, it would be a trivial exercise to remove identifying information about guests
and hosts. For example, they could limit pictures, names, and racial identifiers of hosts
until after a transaction is completed. Similarly, they could remove elements of guest
profiles that provided race information until after the host had accepted a reservation
request.
In practice, there is considerable variation in the amount and timing of
information that is provided to buyers and sellers in online marketplaces. For example,
Airbnb and HomeAway (a main competitor of Airbnb) take different approaches to
information provision. Complete guest profiles (many of which have pictures – in our
sample, we saw roughly 40% had pictures) are more the norm on Airbnb. Figures 1 and 3
display typical guest profiles on Airbnb, highlighting the differential prevalence of
pictures and social profiles across the two platforms. Differences also become apparent
when looking at the search process. When searching on Airbnb, results show the pictures
of hosts alongside pictures of listings on the main results page. In contrast, HomeAway
shows only the pictures of listings; users need to click on a listing before seeing host
information. Figures 4 and 5 provide sample results for each platform.
An online platform could choose not to provide any identifying characteristics of
buyers and sellers until after the transaction is completed. For example, both eBay and
Amazon function successfully with little information to identify personal characteristics
such as race and gender and rely mostly on user ratings.
4.5.2 Increasing salience of diversity goals
An alternative (and potentially complementary) approach would be to make the
objective of increasing diversity more top-of-mind for users. For example, Airbnb could
require hosts to check an acknowledgment box that says, “Airbnb prohibits
discriminatory behavior” each time that a host chooses whether to accept or reject a
guest. Similar to the way that priming ethics can induce ethical behavior (Shu et al.
2014), this might prime users to think more carefully about the possibility that bias is
creeping into their decisions.
4.5.3 Automating the transaction process
Another approach to reducing discrimination on online platforms is to further
automate the transaction process. For example, Uber riders do not see the picture or name
of the driver until after the ride is booked. While in principle, riders could cancel a ride
and look for a new driver, this design choice can greatly reduce the scope for
discrimination. Similarly, Airbnb has a feature called “instant book,” in which hosts can
opt to automatically accept qualified guests. By removing the ability to reject guests on
the basis of features such as name and picture, this virtually eliminates the potential to
discriminate. Yet instant booking is currently only used by a small fraction of Airbnb
hosts. If online platforms were to move toward further automation of booking, this could
help to reduce discrimination in a variety of online contexts.
5 Conclusion
The evolution of trust on the Internet and development of principles for
developing reputation systems have allowed online marketplaces to thrive. Early work
suggested that the arms-length nature of online transactions would reduce the amount of
discrimination we see. And in some cases, platforms likely are bringing us closer to the
bias-free ideal. For example, Ayres et al. (2005) find evidence of racial bias in the context
of tipping taxi drivers. Because it disallows tipping and instead provides market prices,
Uber’s design choice eliminates inequality among driver wages.
But the amount of discrimination in an online marketplace is a choice variable,
determined by the design features that the marketplace selects. Ultimately, the decisions
made by platforms will depend on ethical considerations, legal considerations,
operational
considerations,
competition
considerations,
and
public
relations
considerations. This paper has provided insight into the design principles underlying
robust reputation systems, and a toolkit for helping to reducing discrimination in the
digital age.
References
Ayres, Ian, and Peter Siegelman, 1995. “Race and Gender Discrimination in Bargaining
for a New Car,” American Economic Review, Vol. 85, No. 3, 304–321.
Ayres, Ian, Frederick Vars, and Nasser Zakariya, 2005. “To Insure Prejudice: Racial
Disparities in Taxicab Tipping,” Yale Law Journal, Vol. 114, No. 7, 1613-1674.
Becker, Gary, 1957. The Economics of Discrimination. The University of Chicago Press.
Bertrand, Marianne, and Sendhil Mullainathan, 2004. “Are Emily and Greg More
Employable Than Lakisha and Jamal? A field experiment on labor market
discrimination,” American Economic Review, Vol. 94, No. 4, 991–1013.
Bolton, Gary, Ben Greiner, and Axel Ockenfels, 2013. “Engineering Trust: Reciprocity in
the Production of Reputation Information,” Management Science, Vol. 59, No. 2, 265285.
Charness, Gary, Eman Haruvy, and Doron Sonsino, 2007. “Social Distance and
Reciprocity: An Internet Experiment,” Journal of Economic Behavior and Organization,
Vol. 63, No. 1, 88-103.
Che, Yeon-Koo, and Johannes Hörner, 2014. “Optimal Design for Social Learning,”
Working Paper.
Cullen, Zoe and Chiara Farronato, 2014. “Outsourcing Tasks Online: Matching Supply
and Demand on Peer-to-Peer Internet Platforms,” Working paper.
Dai, Weijia, Ginger Jin, Jungmin Lee, & Michael Luca, 2014. “Optimal Aggregation of
Consumer Ratings: An Application to Yelp.com,” NBER Working Paper.
Dobrescu, Loretti, Michael Luca, and Alberto Motta, 2013. “What makes a critic tick?
Connected authors and the determinants of book reviews,” Journal of Economic Behavior
and Organization Vol. 96, 85-103.
Doleac, Jennifer and Luke Stein, 2013. “The Visible Hand: Race and Online Market
Outcomes,” The Economic Journal, Vol. 123, No. 572, F469-F492.
Edelman, Benjamin, Michael Luca, and Daniel Svirsky, 2016. “Racial Discrimination in
the Sharing Economy: Evidence from a Field Experiment,” Harvard Business School
Working Paper, No. 16-069.
Edelman, Benjamin and Michael Luca, 2014. “Digital Discrimination: The Case of
Airbnb.com,” Harvard Business School Working Paper, No. 14-054.
Edelman, Benjamin, and Michael Luca. 2011. “Airbnb (A).” Harvard Business School Case No.
912-019.
Einav, Liran, Chiara Farronato, and Jonathan Levin. “Peer-to-peer Markets,” Forthcoming in the
Annual Review of Economics.
Fradkin, Andrey, Elena Grewal, David Holtz, and Matthew Pearson. 2015. “Bias and Reciprocity
in Online Reviews: Evidence from Field Experiments on Airbnb.com.” In Proceedings of the
Sixteenth ACM Conference on Economics and Computation (EC ’15), 641. New York, NY:
Association for Computing Machinery. doi: http://dx.doi.org/10.1145/2764468.2764528
Ghani, Ejaz, William Kerr, and Christopher Stanton. 2014. “Diasporas and Outsourcing:
Evidence from oDesk and India.” Management Science 60(7): 1677–97.
Hall, Jonathan, and Alan Kruger. 2015. “An Analysis of the Labor Market for Uber’s DriverPartners in the United States.” Working Paper.
Hoffman, Elizabeth, Kevin McCabe, and Vernon Smith. 1996. “Social Distance and OtherRegarding Behavior in Dictator Games.” American Economic Review 86(3): 653–60.
Horton, John. “The Effects of Algorithmic Labor Market Recommendations: Evidence from a
Field Experiment.” Forthcoming in the Journal of Labor Economics.
Katz, Lawrence, and Alan Krueger. 2016. “The Rise and Nature of Alternative Work
Arrangements in the United States, 1995–2015.” Working Paper.
Luca, Michael. 2015. “User-generated Content and Social Media.” Handbook of Media
Economics.
Luca, Michael, and Georgios Zervas. 2015. “Fake It till You Make It: Reputation, Competition,
and Yelp Review Fraud.” Harvard Business School Working Paper No. 14-006.
Masterov, Dimitriy, Uwe Meyer, and Steven Tadelis. 2015. “Canary in the e-Commerce Coal
Mine: Detecting and Predicting Poor Experiences Using Buyer-to-Seller Messages.”
Proceedings of the Sixteenth ACM Conference on Economics and Computation.
Pope, Devin, and Justin Sydnor. 2011. “What’s in a Picture? Evidence of Discrimination from
Prosper.com.” Journal of Human Resources 46(1): 53–92.
Resnick, Paul, Richard Zeckhauser, John Swanson, and Kate Lockwood. 2006. “The Value of
Reputation on eBay: A Controlled Experiment.” Experimental Economics 9(2): 79–101.
Scott Morton, Fiona, Florian Zettelmeyer, and Jorge Silva-Risso, 2003. “Consumer
Information and Discrimination: Does the Internet Affect the Pricing of New Cars to
Women and Minorities?” Quantitative Marketing and Economics, Vol. 1, No. 1, 65-92.
Todisco, Michael, 2015. “Share and Share Alike? Considering Racial Discrimination in
the Nascent Room-Sharing Economy,” Stanford Law Review Online, No. 67, 121–129.
Figure 1: Sample guests on Airbnb
Figure 2: Applicants on Upwork
Figure 3: Sample guests on HomeAway
Figure 4: Sample search result on Airbnb
Figure 5: Sample search result on HomeAway
Tables
Table 1: Sample Markets with Online Marketplaces
Market
Sample Platforms
Short-term Housing
Airbnb, HomeAway, Couchsurfing
Labor
TaskRabbit, Upwork, MechanicalTurk
Pet sitting
Dogvacay, rover, fetch!
Home Services
HouseCall, Handy
Senior and Child Care
Care.com
Food Delivery
Grubhub, Postmates, Eat24
Funding
Dating
Prosper, Lending Club, Kickstarter,
Indiegogo
Tinder, OkCupid, CoffeeMeetsBagel,
Match
Handmade Goods
Etsy, CustomMade
Fashion
Renttherunway, Rocksbox, LeTote
Car Rentals
Getaround, car2go
Car Rides
Uber, Lyft, PickupPal, BlaBlaCar
Table 2: Sample Online Marketplaces
Marketplace
Year
Founded
Valuation
$ Billions
(as of 2015)
Revenue
$ Millions
(as of 2015)
Amazon
1994
250
107,010
eBay
1995
69
6,103
Craigslist
1995
3
381
Priceline
1997
62
9,220
Etsy
2005
2
273
Airbnb
2008
26
900
Uber
2009
50
2,000
User Base
Millions
304
(active customer accounts)
162
(active buyers)
60
(monthly users in U.S.)
13
(unique monthly users)
24
(active buyers)
60
(cumulative guests)
8
(cumulative riders)
Table 3: Discrimination in Online Marketplaces
Airbnb
eBay
Craigslist
Upwork
Prosper
African-American guests 15% less likely to be accepted.
Bids 20% lower for identifiably African-American sellers.
Buyers less likely to contact identifiably African-American sellers.
Employers of Indian ethnicity more likely to hire workers from
India.
Loan listings for Black borrowers are 25-35% less likely to get
funded than loan listings for White borrowers with similar credit
scores.
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Behaviour Inquiry of Floor Vibration on Composite Structures
|
International journal of engineering and advanced technology
| 2,020
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cc-by
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S. Karunanidhi, G. Gajalakshmi, Y M S D Sastry Vibrations due to this activity have created problems in office
buildings, foot-bridges, shopping malls and sport stadia7. Recently the dynamic behavior of these composite floor
systems is being investigated by various authors analytically
using Finite Element Method is a modern computational
analytical tool which is for structural dynamic analysis. In
order to increase slender and lightness of the structural
systems, designers continuously progress to safety margins. More and more composite floors, steel floors are constructed
as light weight structures with low damping and low
frequencies, but perceptive to vibration consequently
changing the serviceability and ultimate limit state associated
with their design. Abstract: In contemporary times, building construction
requires light weight with slender structures rather than using
conventional materials like concrete. Now a day's Structural
Engineers concentrate much more on such slender structures with
longer span. The impact of vibration due to human rhythmic
activities like aerobics, jumping and dancing on these slender
structures is a notable phenomenon. As per the various
researchers contemplate, the floor vibrations annoyance not only
affect the structure and also its impact over the occupants of the
buildings in health affecting aspects. The aim of this paper is to
analyse the vibration behavior of composite steel floor
structuresunder gymnastic activities like jumping as human
rhythmic activities by FEM analysis. The Finite Element Method
analysis is done by using ANSYS software. From the Transient
analysis of Finite Element method, the peak acceleration values
are found out. These peak acceleration values are compared with
the recommended values of IS 800-2007 and ISO 2631 – part II. The annoyance of such acceleration values under human
rhythmic activities may induce vibration in terms of resonance;
the natural frequency of the structural floor may coincide with
any of the frequency of such activities. When resonance occurs,
even fatigue failure of structures may happen. Hence it is essential
for the structural Engineer to undergo the vibration analysis of
composite floor structures during design stage itself.In order to
check over such problems, in this paper as a novelty; a
mathematical model is developed using SPSS software.This
mathematical model is for peak acceleration values which helps
the structural designer to analyse vibration problems under
human rhythmic activities. Starting from Reiherand meister[12], Bruce Ellingwood3
and more number of researchers attempted to find solution for
vibration excitation. Allen [1], developed a design procedure
by solving problems for different structural properties for
different usage. S. International Journal of Engineering and Advanced Technology (IJEAT)
ISSN: 2249 – 8958 (Online), Volume-9 Issue-4, April, 2020 International Journal of Engineering and Advanced Technology (IJEAT)
ISSN: 2249 – 8958 (Online), Volume-9 Issue-4, April, 2020 International Journal of Engineering and Advanced Technology (IJEAT)
ISSN: 2249 – 8958 (Online), Volume-9 Issue-4, April, 2020 International Journal of Engineering and Advanced Technology (IJEAT)
ISSN: 2249 – 8958 (Online), Volume-9 Issue-4, April, 2020 S. Karunanidhi, G. Gajalakshmi, Y M S D Sastry Sanden de Silva et al [9] states that steel
deck composite floor with increased acceleration cause
discomfort to the occupants. Nor Hayati and Abd Ghafer[11] made a research work on
dynamic behaviour of LVL concrete composite floor system
and concluded the natural frequency must exceed 8 Hz to
prevent vibration problems. The collapse of Hyatt Regency Hotel walkway in Kansas
City US happened weekend tea dance in 1981(McGrath and
Foote 1981) [11] which caused many lives was a typical
example for structural failure. As no information and
appropriate theories were then available the people were kept
in darkness regarding cause of the destruction. Keywords
:
Vibration;
Rhythmic
activity;
Composite
structures; Acceleration; ANSYS; SPSS software Behaviour Inquiry of Floor Vibration on
Composite Structures S. Karunanidhi, G. Gajalakshmi, Y M S D Sastry Behaviour Inquiry of Floor Vibration on Composite Structures Span of the Composite floor systems are: 16m x
12m,8mx 6m, 4mx 3m,8mx 12m, 6mx 10m, 2mx 1m people which induce activities such as jumping; aerobics,
walking etc determine the dynamic response. As the type of activity carried out by a single person might
generate perceptible levels of vibration in a number of floors,
no straight calculation is in existence for fundamental
frequency. As such all the results obtained in various
applications are not specific, ambiguous since the data are
sparse, incoherent and devoid of continuity. Youngs Modulus for the steel beams : 2.4x 105 Mpa. 3 Density of Steel: 7850 kg/m3. 3 Density of Steel: 7850 kg/m3. 3 Density of Concrete: 2500 kg/m3 Yield stress: 300 Mpa. Yield stress: 300 Mpa. The above composite floor system panels are analysed
using FEM are a) Modal analysis b)Harmonic analysis and c)Transient
analysis. A. Objective The aim of this paper is to analyse the dynamic behavior for
composite steel construction like steel floors with concrete
topping for different occupancy usage under serviceable
condition for human rhythmic activity (Jumping). The various
structure models are to be analysed using Finite Element
Method -ANSYS2 software. The analytical values obtained
were compared with code recommendations. Fig 2.1. Structural model three-dimensional plan view II. DYNAMIC ANALYSIS OF STRUCTURAL
MEMBERS For
dynamic
floor
analysis
for
the
proposed
computational model, adopted the usual mesh refinement
techniques present in finite element method simulations
implemented in the ANSYS program (ANSYS, 11). In this
model, the floor beams are represented by three-dimensional
beam elements (BEAM44), tension, compression, bending
and torsion capabilities. The floor slab is represented by shell
finite elements (SHELL63 and SHELL 99). B. Novelty In order to mitigate the setbacks relating to the vibration
problem, a novel mathematical model based on software
SPSS has been arrived with reference for composite steel
structures. Fig 2.1. Structural model three-dimensional plan view I. INTRODUCTION Kromanis R and Kripakaran P[8] investigated the
application of novel computational techniques for structural
performance monitoring of National Physical Laboratory
(NPL) footbridge bridges truss and concrete footbridge that
enable quantification of temperature-induced response during
the strain measurement interpretation process. The Results
show that the methodology is capable of accurately predicting
thermal response and can therefore help with interpreting
measurements from continuous National Physical Laboratory
(NPL) footbridge bridges truss and concrete footbridge
monitoring. Of late, the modern architecture and construction trends are
motivating the structural engineer to search for challenging
solutions. The recent evolution in building construction could
be attributed to fast erection assembly with optimum weight
duly supporting large spans with lesser columns facilitating
greater space flexibility. Owing to the impacts like human rhythmic activities, the
structures floor systems are vulnerable to excess vibrations. Revised Manuscript Received on April 25, 2020. * Correspondence Author
Mr. S. Karunanidhi*, Civil Engineering, Wollega University, Ethiopia,
Email: karunabalasingam@gmail.com
Dr.G.Gajalakshmi, Civil Engineering,, Wollega University, Ethiopia. Mr. YMSD Sastry, CE, BVC Engineering College, JNTUK, Odalarevu,
India. Email: dattasastry@gmail.com It could be concluded from the above literature study that
the understanding of human interaction to the structure is a
complex phenomenon. Some equations and formula are
arrived at by all researchers from their input data based on the
corresponding human activities. Moreover many factors like
the posture of the occupants, dynamic properties of the
structure and the mass of the © The Authors. Published by Blue Eyes Intelligence Engineering and
Sciences Publication (BEIESP). This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) 1365
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& Sciences Publication
© Copyright: All rights reserved. d
C 1365
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& Sciences Publication
© Copyright: All rights reserved. Retrieval Number: D8351049420/2020©BEIESP
DOI: 10.35940/ijeat.D8351.049420
Journal Website: www.ijeat.org
Sciences Publication (BEIESP). This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) 1365
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& Sciences Publication
© Copyright: All rights reserved. Retrieval Number: D8351049420/2020©BEIESP
DOI: 10.35940/ijeat.D8351.049420
Journal Website: www.ijeat.org Retrieval Number: D8351049420/2020©BEIESP
DOI: 10.35940/ijeat.D8351.049420
Journal Website: www.ijeat.org A. Loads generated by human activities The dynamic loads have considered by Faisca (2003)4
who achieved the same through a long series of various
experiments executed with human activities as dynamic loads
are described as jumps with and without stimulation, aerobics,
soccer, rock concert audiences and dancing. based on results
achieved through a long series of experimental tests made
with individuals carrying out rhythmic and non-rhythmic
activities. Human activities such as aerobics, Gymnastics
dancing and free jumps can be simulated by the load
modelling. Fig 2.2. 3-D Elastic Tapered Unsymmetric The mathematical representation is very much analogous
to the signal force generated through experiments and it is
described by the equation(2.1). Some parameters like the
activity period T, contact period with the structure Tc, period
without contact with the model Ts, impact coefficient Kp, and
phase coefficient CD are required for this equation. Fig 2.2. 3-D Elastic Tapered Unsymmetric am Beam F(t) = 0, for Tc< t T (2.1)
Where
F (t)
-
Dynamic loading, in (N);
t
-
Time, in (s);
T
-
Activity period (s);
Tc
-
Activity contact period (s);
P
-
Weight of the individual (N);
Kp
-
Impact coefficient;
CD
-
Phase coefficient. (2.1) Results for three persons loading The following graphs shows the dynamic response of FE
model composite floor system with three persons loading p
y
p
g
Fig 2.6. Frequency Vs Displacement for Three persons at
Measuring Point A
Transient Analysis Results for five persons loading E. Harmonic Analysis Likewise the dynamic analysis for other panels like 10mx
8m, 8mx 6m, 6mx 10m, 8mx 12m, 4mx 3m, 2m x 1m are
analysed and their corresponding structural properties and
results are given as inputs for generating a regression
mathematical model using SPSS software. D. Modal analysis and the mode shapes Table 2.1. Mode shapes and natural frequencies
Mode
shapes
1
2
3
4
5
6
Natural
frequencie
s (Hz)
5.48
3
9.48
0
12.67
5
1
5.836
1
6.260
22.01
0 Table 2.1. Mode shapes and natural frequencies
Mode
shapes
1
2
3
4
5
6
Natural
frequencie
s (Hz)
5.48
3
9.48
0
12.67
5
1
5.836
1
6.260
22.01
0 Fig 2. 7. Time Vs Acceleration Response for 5 persons at Fig 2. 7. Time Vs Acceleration Response for 5 persons at
Measuring Point C
Table 2.2. Comparison of peak acceleration results with
Threshold limits
Number
of
Persons
Peak
Acceleration
(m/sq.s)
Max
peak
acceleration
Threshold
limits alim
Poi
nt A
Point
B
Point
C
1
0.27 0.247
0.24
0.27
0.5% g as
per
IS800-2007
6
3
0.737 0.644
0.557
0.737
5
0.94 0.824
0.767
0.94
7
1.41
1.17
1.16
1.41
5% g as per
ISO-2631
(PART-II) 5
11
2.01
1.66
1.59
2.01
19
3.54
2.95
2.69
3.54
ik
i
h d
i
l
i f
h
l lik 10 Fig 2.5. Vibration mode associated to the 2nd
natural frequency: F02=9.480 Fig 2.5. Vibration mode associated to the 2nd
natural frequency: F02=9.480 Fig 2.5. Vibration mode associated to the 2nd
natural frequency: F02=9.480 Fig 2.3. Elastic Shell Fig 2.3. Elastic Shell Model results of ANSYS for 16mx 12m are- modal shape,
natural frequency, harmonic analysis and transient analysis
are given below. 1366
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B. Structural Configuration Details
Structural Floor Details:
q
y
are given below. Retrieval Number: D8351049420/2020©BEIESP
DOI: 10.35940/ijeat.D8351.049420
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B. Structural Configuration Details
Structural Floor Details: 1366
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© Copyright: All rights reserved. International Journal of Engineering and Advanced Technology (IJEAT)
ISSN: 2249 – 8958 (Online), Volume-9 Issue-4, April, 2020 International Jou
ISS
Fig 2.4. Load distribution Scheme associated to eighteen
individuals practicing
D. Modal analysis and the mode shapes
Table 2.1. Mode shapes and natural frequencies
Mode
shapes
1
2
3
4
5
6
Natural
frequencie
s (Hz)
5.48
3
9.48
0
12.67
5
1
5.836
1
6.260
22.01
0
Fig 2.5. Vibration mode associated to the 2nd
natural frequency: F02=9.480
E. Harmonic Analysis
Results for three persons loading International Journal of Engineering and Advanced Technology (IJEAT)
ISSN: 2249 – 8958 (Online), Volume-9 Issue-4, April, 2020 The following Figure presents the Acceleration versus Time
graphs for the analyzed Composite Concrete Floor at
measuring point A , B, C, when only five persons load is
acting on the structural Model(Aerobic). g
Fig 2. 7. Time Vs Acceleration Response for 5 persons at Fig 2 7 Time Vs Acceleration Response for 5 persons at Fig 2.4. Load distribution Scheme associated to eighteen
individuals practicing D. Modal analysis and the mode shapes D. Modal analysis and the mode shapes Multiple regression model development Regression analysis is mathematical measure of average
relationship between two or more variables in terms of
original units of data. Regression is used to create an equation
or transfer function from the measurements of the system's
inputs or outputs acquired during a passive or active
experiment. Multiple regression analysis was conducted using
displacement (d) and Acceleration (a) as a dependent variable
and area (A), Moment of Inertia (IZ1, IY1). Web thickness
(TKyB1) as the independent variables. Mathematical model using SPSS software ANOVAb
Model
Sum of
Squares
df
Mean
Square
F
Sig. Behaviour Inquiry of Floor Vibration on Composite Structures Behaviour Inquiry of Floor Vibration on Composite Structures IY
1
Pearson
Correlatio
n
.9
90**
. 969*
*
1 .971
**
.253* .940**
.564** .619
**
Sig. (2-tailed)
.0
00
. 000
.000
.044
.000
.000 .000
N
6
4
6
4
64
64
64
64
64
64
IX
1
Pearson
Correlatio
n
.9
49**
1
.000
**
.971
**
1
.262* .894**
.592** .552
**
Sig. (2-tailed)
.0
00
. 000
.000
.036
.000
.000 .000
N
6
4
6
4
64
64
64
64
64
64
TK
ZB
1
Pearson
Correlatio
n
.2
46*
. 262*
.253
*
.262
*
1 .287*
.206 .247
*
Sig. (2-tailed)
.0
50
. 036
.044 .036
.022
.103 .049
N
6
4
6
4
64
64
64
64
64
64
TK
YB
1
Pearson
Correlatio
n
.9
44**
. 891*
*
.940
**
.894
**
.287*
1
.590** .720
**
Sig. (2-tailed)
.0
00
. 000
.000 .000
.022
.000 .000
N
6
4
6
4
64
64
64
64
64
64
dis
pla
ce
me
nt
Pearson
Correlatio
n
.5
41**
. 593*
*
.564
**
.592
**
.206 .590**
1 .735
**
Sig. (2-tailed)
.0
00
. 000
.000 .000
.103
.000
.000
N
6
4
6
4
64
64
64
64
64
64
acc Pearson
Correlatio
n
.6
11**
. 548*
*
.619
**
.552
**
.247* .720**
.735**
1
Sig. (2-tailed)
.0
00
. 000
.000 .000
.049
.000
.000
N
6
4
6
4
64
64
64
64
64
64
**. Correlation is significant at the 0.01 level (2-tailed). *. Correlation is significant at the 0.05 level (2-tailed). Acceleration
Regression Full model Regression Analysis For acceleration For acceleration SPSS 16 was used to analyze the response of this study. Person correlation was use to analyze correlation among the 7
variables. All the variables were significantly correlated with
one another at 0.001 d=f(A,IZ¬¬¬¬¬¬1, IY1,KZB1, TKYB1) (3.1) III. RESULTS AND DISCUSSION ON ANALYTICAL
RESULTS From the results with reference to Table 2., the threshold
limit for acceleration as per IS 800 2007 for human rhythmic
activity is 0.5%g. For one person loading condition for
jumping activity, the peak acceleration value is 0.27m/sec2. Nearly 81% value is increased when compared with
IS800-20075. Therefore composite steel floor is vulnerable to
vibration due to the impact of jumping activity. The floor is
not supported to jumping activity from 3 persons loading
condition when compared to the threshold limit of 5%g as per
ISO 2631(part II)6. Fig 2.6. Frequency Vs Displacement for Three persons at
Measuring Point A
Transient Analysis Results for five persons loading 1367
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Measuring Point A
Transient Analysis Results for five persons loading 1367
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Journal Website: www.ijeat.org Mathematical model using SPSS software SPSS software is used for data analysis using statistical
techniques. There are many statistical packages are available
for data analysis Minitab, SAS, STATA. But SPSS is the most
widely used software. This software can be used in all
domains like finance, healthcare, bio engineering etc. SPSS
(Statistical Package for the Social Sciences) is a software
package used for statistical analysis. Long produced by SPSS
Inc., it was acquired by IBM in 2009. The current versions
(2015) are officially named IBM SPSS Statistics. Companion
products in the same family are used for survey authoring and
deployment (IBM SPSS Data Collection), data mining (IBM
SPSS Modeler), text analytics, and collaboration and
deployment (batch and automated scoring services). SPSS
contains two views. They are two views in the SPSS such as
data view and variable view. Data view is used to store data
and variable view is used to define variables. 368
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© Copyright: All rights reserved. (2 tailed)
00
000
N
6
4
6
4
64
64
64
64
64
64
TK
ZB
1
Pearson
Correlatio
n
.2
46*
. 262*
.253
*
.262
*
1 .287*
.206 .247
*
Sig. (2-tailed)
.0
50
. 036
.044 .036
.022
.103 .049
N
6
4
6
4
64
64
64
64
64
64
TK
YB
1
Pearson
Correlatio
n
.9
44**
. 891*
*
.940
**
.894
**
.287*
1
.590** .720
**
Sig. (2-tailed)
.0
00
. 000
.000 .000
.022
.000 .000
N
6
4
6
4
64
64
64
64
64
64
dis
pla
ce
me
nt
Pearson
Correlatio
n
.5
41**
. 593*
*
.564
**
.592
**
.206 .590**
1 .735
**
Sig. (2-tailed)
.0
00
. 000
.000 .000
.103
.000
.000
N
6
4
6
4
64
64
64
64
64
64
acc Pearson
Correlatio
n
.6
11**
. 548*
*
.619
**
.552
**
.247* .720**
.735**
1
Sig. (2-tailed)
.0
00
. 000
.000 .000
.049
.000
.000
N
6
4
6
4
64
64
64
64
64
64
**. Correlation is significant at the 0.01 level (2-tailed). *. Correlation is significant at the 0.05 level (2-tailed). Acceleration - Regression Full model
Acceleration full model
Table 3.2. Model Summary
Model
R
R Square
Adjusted R
Square
Std. Error of the
Estimate
1
.878a
.771
.747
.65743038408
Table 3.3. d=f(A,IZ¬¬¬¬¬¬1, IY1,KZB1, TKYB1) (3.1) KZB1, TKYB1 are not significant in the explaining the
variation in acceleration we developed the reduced regression
that excluded the variables. The reduced model has the following form: d= f (A, IZ1, IY1) (3.2)
Predictors: (Constant), TKYB1, IZ1, A Table 3.1. Correlations
A
I
Z1 IY1 IX1
TKZB1
TKY
B1
displacem
ent
acc
A
Pearson
Correlatio
n
1
. 946*
*
.990
**
.949
**
.246* .944**
.541** .611
**
Sig. (2-tailed)
. 000
.000 .000
.050
.000
.000 .000
N
6
4
6
4
64
64
64
64
64
64
IZ1 Pearson
Correlatio
n
.9
46**
1
.969
**
1.00
0**
.262* .891**
.593** .548
**
Sig. (2-tailed)
.0
00
.000 .000
.036
.000
.000 .000
N
6
4
6
4
64
64
64
64
64
64 Acceleration - Regression Full model Table 3.2. Model Summary
Model
R
R Square
Adjusted R
Square
Std. Error of the
Estimate
1
.878a
.771
.747
.65743038408 Table 3.2. Model Summary
Model
R
R Square
Adjusted R
Square
Std. Error of the
Estimate
1
.878a
.771
.747
.65743038408 Retrieval Number: D8351049420/2020©BEIESP
DOI: 10.35940/ijeat.D8351.049420
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DOI: 10.35940/ijeat.D8351.049420
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DOI: 10.35940/ijeat.D8351.049420
Journal Website: www.ijeat.org International Journal of Engineering and Advanced Technology (IJEAT)
ISSN: 2249 – 8958 (Online), Volume-9 Issue-4, April, 2020
1
Regression
82.920
6
13.820
31.975
.000a
Residual
24.636
57
.432
Total
107.556
63
a. Predictors: (Constant), displacement, TKZB1, A, TKYB1, IZ1, IY1
IZ1
-25051.631
12646.276
-.730 -1.981 .052
IY1
1155100.942 650877.390
1.500
1.775 .081
TKYB1
2321.652
494.234
1.209
4.697 .000
a. Dependent Variable: acc International Journal of Engineering and Advanced Technology (IJEAT)
ISSN: 2249 – 8958 (Online), Volume-9 Issue-4, April, 2020 a. Predictors: (Constant), displacement, TKZB1, A, TKYB1, IZ1, IY1 Regression Full model
Regression Reduced model
(3.4)
Model Validation: Regression Full model
Regression Reduced model
(3.4)
Model Validation: Regression Full model
Regression Reduced model
(3.4)
Model Validation: b. Dependent Variable: acc
Table 3.4 Coefficientsa
Model
Unstandardized
Coefficients
Standardized
Coefficients
t
Sig. B
Std. Error
Beta
1
(Constant)
-1.756
.804
-2.185 .033
A
-602.190
341.613
-.871
-1.763 .083
IZ1
-41399.111
9928.032
-1.206
-4.170 .000
IY1
1229817.785 494641.797
1.598
2.486 .016
TKZB1
.605
1.079
.037
.561
.577
TKYB1
1490.666
398.890
.776
3.737 .000
displacement
.851
.127
.555
6.703 .000
a. d=f(A,IZ¬¬¬¬¬¬1, IY1,KZB1, TKYB1) (3.1) Dependent Variable: acc Table 3.4 Coefficientsa Regression Reduced model
(3.4)
Model Validation: Model Validation: In the above Equation , the significant error is at and
TKZB1 as 0.577.Hence the Equation is reduced as shown in
Equation , in which significant errors are at 0.01 level. The
reduced regression model has explained the variation
accounts for 58.9% which is the best model for validation. The resulting multiple regression model is From above
Equation and, there is a positive relationship between
Acceleration with IY1 and TKZB1. But there is a negative
relationship between the area and acceleration. ACC reduced IV. CONCLUSION Table 3.5. Model Summary
Model
R
R Square
Adjusted R
Square
Std. Error of the
Estimate
1
.767a
.589
.561
.86586205000
P di t
(C
t
t) TKYB1 IZ1 A IY1 The floor vibration of composite steel structures are
analysed using Finite Element method under ANSYS
software for human rhythmic activities. The peak acceleration
values are obtained from transient analysis and they are
compared with IS 800-2007 and ISO 2631. The resonance formation under deviated acceleration
values lead occupancy health problems and also fatigue
failure to structures. To mitigate such vibration resonance
problems, a mathematical model for acceleration parameter is
developed using SPSS software. This mathematical model
incorporates the various properties of composite steel
structures and helps the structural Engineer to check and
control the vibration problems due to human rhythmic
activities at the design process itself. a. Predictors: (Constant), TKYB1, IZ1, A, IY1 Table 3.6. ANOVAb
Model
Sum of
Squares
df
Mean
Square
F
Sig. 1
Regression
63.323
4
15.831 21.116
.000a
Residual
44.233
59
.750
Total
107.556
63
a Predictors: (Constant) TKYB1 IZ1 A IY1 Some of the suggestions to control the vibration problems
over engineering structures for human rhythmic activities are
summarized below: • To install a steel girder as a supporting structure
underneath the existing floor b. Dependent Variable: acc b. Dependent Variable: acc
Table 3.7. Coefficientsa
Model
Unstandardized
Coefficients
Standardized
Coefficients
t
Sig. B
Std. Error
Beta
1
(
Constant)
-2.911
1.034
-2.814 .007
A
-915.125
445.620
-1.324 -2.054 .044 • To increase damping, it is recommended to use partition
walls and planters with low cost method to increase damping. • By
providing
separate
ceilings
and
partitions
immediately below the jumping floor by supporting the
ceiling on its own framing and by not extending partition to
the floor above. • By
providing
separate
ceilings
and
partitions
immediately below the jumping floor by supporting the
ceiling on its own framing and by not extending partition to
the floor above. 1369
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© Copyright: All rights reserved. 44 Retrieval Number: D8351049420/2020©BEIESP
DOI: 10.35940/ijeat.D8351.049420
Journal Website: www.ijeat.org REFERENCES conferences. Now he is working as Assistant Professor in the department of
civil Engineering, Bonam Venkata Chalamayya Engineering College,
Autonomous, Odalarevu, Allavaram Mandal, Affiliated to JNTUK,
Kakinada, East Godavari District, Andhra Pradesh, with more than 18 years
of teaching, research and industrial experience. He is a life time member of
ISTE, CSI and Institute of Engineers (India). 1. Allen, D.E. (1990), “Building Vibrations from Human Activities,
Reprinted from American Concrete Institute”, Concrete International;
Design and Construction, Vol.12, No.6. pp.66-73, (IRC Paper
No:1679). 2. ANSYS, Swanson Analysis Systems (2003), Inc.P.O. Box 65, Johnson
Road, Houston, P.A.15342-0065, Version11.0. Basic analysis
procedures. 3. Bruce Ellingwood, M.and Andrew Tallin, M. (1983), “Structural
serviceability:Floor vibrations”, Journal of Structural Engineering,
ASCE, Vol.110, No.2 @ASCE, ISSN 0733-9445/84/0002-0401, Paper
no:18597. 4. Faisca,
R.G. (2003),
“Caracterizacao
de
cargas
dynamics
geradesporatividades humans (Characterization of Dynamic Loads due
to Human activities)”, “Ph.D Thesis (in Portuguese), COPPE/UFRJ, Rio
de Janerio, RJ, Brazil, pp.1-240. 5. International standards of Organisation/ISO 2631-2: Evaluation of
Human exposure to whole-body vibration. Part-2: Continuous and
shock-induced vibration in buildings (1-80 Hz), “1989. 6. IS 800:2007, Indian Standard general construction in Steel code of
practice- Third Revision. 7. Ivan M. Diaz and Paul Reynolds (2010), “Acceleration feedback control
of human - induced floor vibrations”, Journal of Engineering Structures,
Vol.32, pp.163-173. 8. Kromanis.R. and Kripakaran.R. (2014), “Predicting thermal response of
bridges using regression models derived from measurement histories”,
Journal of Computers and Structures, Vol.136, pp. 64-67. 8. Kromanis.R. and Kripakaran.R. (2014), “Predicting thermal response of
bridges using regression models derived from measurement histories”, Journal of Computers and Structures, Vol.136, pp. 64-67. 9. Langer, N.A.dos S., Silva,J.G.S. daVellasco, P.C.G. da S, Lima,L.R.O
and de, Neves, L.F. da C. (2009), “Vibration analysis of composite
floors induced by human rhythmic activities”, Proceedings of the 12th
International Conference on Civil, Structural and Environmental
Engineering Computing, CC, Funchal, IIha da Maderia, Portugal,
CD-ROM, pp.1-14. 10. McGrath and Foote, D (1981), “What happened at the Hyatt? Newsweek
National Affairs, United states edition, pp.26. 11. Nor Hayati and AbdGhafar (2015), “Dynamic Behaviour ofLVL –
Concrete Composite Flooring Systems”. 12. Reiher, H. and Meister, F.J. (1931), “The effect of Vibration on People”,
published
in
German
English
Translation
in
Report
No. F-TS-616-R.E.H.Q. Air Material Command, Wright Field, Ohio, 1949. AUTHORS PROFILE Mr. S. Karunanidhi, obtained his bachelors ‘degree
in Civil Engineering from PSNA College of Engineering
& Technology, Dindigul affiliated to Madurai Kamaraj
university in 1998.He was awarded his masters degree in
structural engineering from AC Tech, karaikudi affiliated
to Anna university in 2009.He has published 5
international journals and also presented 4 papers in national and
international conferences. Presently he is working as an Assistant professor
in the department of civil Engineering, College of Engineering &
Technology, Wollega University, Ethiopia since 2013 with more than 19
years of teaching and industrial experience. He is a life time member of ISTE
and Institute of Engineers (India). Dr.G.Gajalakshmi, completed her bachelor’s
degree in Civil Engineering from Periyar Maniammai
college of Tech for women affiliated to Bharadidasan
University in 1998. She obtained her degree M.E from
Sathyabama University in 2008. She has been awarded
her PhD degree in Civil Engineering/Structural Engg division from
sathyabama University, Chennai, Tamilnadu, India in 2017.She has
published 8 papers in international and national journals. Also she has
published and presented 13 papers in national and international conferences. Now she is working as a lecturer in the department of civil Engineering,
college of Engg & Tech, Wollega University, Ethiopia with more than 17.5
years of teaching, research and industrial experience. Mr. Y M S D Sastry, completed his bachelor’s degree
in Civil Engineering (Associate Member in Institute of
Engineers) from Institute of Engineers India, Kolkata in
2003. He obtained his Masters degree M. Tech from
Acharya Nagarjna University in 2010. He published
good number of papers in international and national journals. Also he had
published and presented good number of papers in national and international 70
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|
https://openalex.org/W4243761174
|
https://www.researchsquare.com/article/rs-200749/latest.pdf
|
English
| null |
Synovial Fluid Neutrophils in Oligoarticular Juvenile Idiopathic Arthritis Have an Altered Phenotype and Impaired Effector Functions
|
Research Square (Research Square)
| 2,021
|
cc-by
| 8,419
|
Sabine Arve-Butler Robin Kahn
(
Robin.kahn@med.lu.se
)
Lund University
https://orcid.org/0000-0002-3167-1179 Synovial Fluid Neutrophils in Oligoarticular Juvenile Idiopathic Arthritis
Have an Altered Phenotype and Impaired Effector Functions Synovial Fluid Neutrophils in Oligoarticular Juvenile Idiopathic Arthritis
Have an Altered Phenotype and Impaired Effector Functions Sabine Arve-Butler
Lund University: Lunds Universitet
Tobias Schmidt
Lund University: Lunds Universitet
Anki Mossberg
Lund University: Lunds Universitet
Elisabet Berthold
Lund University: Lunds Universitet
Birgitta Gullstrand
Lund University: Lunds Universitet
Anders A Bengtsson
Lund University: Lunds Universitet
Fredrik Kahn
Lund University: Lunds Universitet
Robin Kahn
(
Robin.kahn@med.lu.s
Lund University
https://orcid.org/00 Synovial Fluid Neutrophils in Oligoarticular Juvenile Idiopathic Arthritis
Have an Altered Phenotype and Impaired Effector Functions
Sabine Arve-Butler
Lund University: Lunds Universitet Synovial Fluid Neutrophils in Oligoarticular Juvenile Idiopathic Arthritis
Have an Altered Phenotype and Impaired Effector Functions
Sabine Arve-Butler
Lund University: Lunds Universitet Results Neutrophils in synovial fluid had an activated phenotype, characterized by increased CD66b and CD11b levels, and most neutrophils had a CD16hi
CD62Llow aged phenotype. A large proportion of the synovial fluid neutrophils expressed CD206, a mannose receptor not commonly expressed by
neutrophils but by monocytes, macrophages and dendritic cells. CD206 expressing neutrophils were also found in synovial tissue biopsies. The
synovial fluid neutrophil phenotype was not dependent on transmigration alone. Functionally, synovial fluid neutrophils had reduced phagocytic
capacity and a trend towards impaired oxidative burst compared to blood neutrophils. In addition, the effector functions of the synovial fluid
neutrophils correlated negatively with the proportion of CD206+ neutrophils. Conclusions Neutrophils in the inflamed joint in oligoarticular JIA were altered, both regarding phenotype and function. Neutrophils in the synovial fluid were
activated, had an aged phenotype, had gained monocyte-like features and had impaired phagocytic capacity. The impairment in phagocytosis
and oxidative burst was associated to the phenotype shift. We speculate that these neutrophil alterations might play a role in the sustained joint
inflammation seen in JIA. Research article Keywords: juvenile idiopathic arthritis, neutrophil, synovial fluid, phenotype, phagocytosis, oxidative burst, reactive oxygen species
Posted Date: February 19th, 2021
DOI: https://doi org/10 21203/rs 3 rs-200749/v1 Keywords: juvenile idiopathic arthritis, neutrophil, synovial fluid, phenotype, phagocytosis, oxidative bu DOI: https://doi.org/10.21203/rs.3.rs-200749/v1 DOI: https://doi.org/10.21203/rs.3.rs-200749/v1 License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Version of Record: A version of this preprint was published at Arthritis Research & Therapy on April 9th, 2021. See the published version at
https://doi.org/10.1186/s13075-021-02483-1. Page 1/15 Page 1/15 Methods Neutrophils obtained from paired blood and synovial fluid from patients with active oligoarticular JIA were investigated phenotypically (n=17) and
functionally (phagocytosis and oxidative burst, n=13) by flow cytometry. In a subset of patients (n=6), blood samples were also obtained during
inactive disease at a follow-up visit. Presence of CD206-expressing neutrophils was investigated in synovial biopsies from four patients by
immunofluorescence. Abstract Neutrophils are the most prevalent immune cells in synovial fluid in inflamed joints of children with oligoarticular juvenile idiopathic arthritis (JIA). Despite this, little is known about neutrophil function at the site of inflammation in JIA and how local neutrophils contribute to disease
pathogenesis. This study aimed to characterize phenotype and function of synovial fluid neutrophils in oligoarticular JIA. Background Juvenile idiopathic arthritis (JIA), is an inflammatory rheumatic joint disease affecting children. Despite disease onset being at a young age,
symptoms may be lifelong and include irreversible joint damage or growth disturbances (1,2). The JIA diagnosis is an umbrella term including
several subtypes with the common denominator of unexplained persistent arthritis occurring before the age of sixteen (3). The most common
subtype in the western world is oligoarticular JIA (4), commonly characterized by asymmetric disease onset with inflammation in one to four large
joints (3,5). Previously, the immunopathogenesis of oligoarticular JIA has been thought to be driven primarily by adaptive immune responses, as the disease
is associated with HLA class II genetic variants and presence of autoantibodies (5). However, the importance of the innate immune system in
oligoarticular JIA is becoming recognized, as abnormalities in the adaptive immune system cannot fully explain the pathology. This is reflected in
the treatment of oligoarticular JIA, where commonly used drugs are either non-specific or target important components of the innate immune
system such as tumor necrosis factor alpha (TNFa) and interleukin 6 (IL-6) (6,7). Neutrophils represent a major part of the innate immune system, and the pathology of several autoimmune rheumatic diseases are at least partly
driven by dysfunctional neutrophils (8,9). Neutrophils are the most common immune cell present in synovial fluid from inflamed joints in
oligoarticular JIA, and studies of several JIA subtypes suggest that circulating neutrophils are activated (6,10–12). It is reasonable to hypothesize
that neutrophils are important also in oligoarticular JIA. However, neutrophils at the site of inflammation have rarely been studied in JIA, except
for a recent study by Metzemaekers et al. which demonstrated that synovial fluid neutrophils in oligo- and polyarticular JIA have a distinct
phenotype compared to circulating neutrophils (13). The major neutrophil effector functions include phagocytosis and oxidative burst. Neutrophil phagocytosis is important not only as a defense
against infection but also for clearance of potential autoantigens. Reactive oxygen species (ROS) were long considered harmful byproducts in
sterile inflammation, but are now recognized for their immunosuppressive properties and impaired ROS production is associated with rheumatic
disease both in patients and animal models of disease (8,14–20). Thus far, no studies have investigated synovial fluid neutrophil effector
functions in oligoarticular JIA and their relation to the pathogenesis. Study population Children fulfilling international league of associations for rheumatology (ILAR) criteria for persistent oligoarticular JIA, untreated or solely on non-
steroid anti-inflammatory drugs (NSAIDs) for at least six months, undergoing therapeutic arthrocentesis between 2016 and 2019 were included in
the study, n=17. One patient (no 5) had received intraarticular steroids in another joint, two months prior to sampling. Synovial fluid was obtained
from knee in all patients. Blood and synovial fluid during active arthritis were collected from 17 patients. Synovial tissue biopsies were obtained
from four patients, one of which is not included in phenotype or function analyses due to ongoing treatment with methotrexate (patient no 18), in
conjunction with synovial fluid aspiration. Follow-up blood samples from periods of inactive disease were collected from six of the patients. During periods of inactive disease some of the patients were treated with conventional or biologic disease-modifying antirheumatic drugs
(DMARD). Patient characteristics are described in table 1. Twelve of the patients were also included in a previous study (25), indicated in table 1. Healthy controls were included after informed consent. All controls were adults and contributed with blood and oral samples. Preparation of synovial cells and cell-free synovial fluid Synovial fluid was centrifuged at 400 g, 10 min to pellet the cells. Synovial cells were counted and resuspended in phosphate buffered saline
(PBS) at a final concentration of 1x106/ml for use in flow cytometric staining and downstream purification procedures. The synovial fluid
supernatant was transferred to a new tube and centrifuged a second time at 800 g, 10 min to pellet potential remaining cells or debris. The cell-
free synovial fluid was collected and stored at -80 °C. Neutrophil phenotyping and definitions Equal volumes of blood, synovial cell suspension or oral cell suspension were stained with antibody panels described in Supplemental figure 1. Whole blood samples were prepared after staining using TQ-prep with Immunoprep reagent system (Beckman Coulter) for red blood cell lysis and
fixation of white cells. Synovial, oral and purified cell samples were washed with PBS after staining. Samples were analyzed on a FACS Canto II
flow cytometer (BD Biosciences) or Cytoflex (Beckman Coulter) according to Supplemental figure 1. Kaluza software (Beckman Coulter) was used
for data processing. Gating strategies are described in Supplemental figure 1. Background Page 2/15 Page 2/15 The phenotype of synovial fluid neutrophils in JIA is insufficiently characterized, and two contradicting studies describe them as being highly
activated (13) or resting (21). The study by Metzemaekers et al. further describes the synovial fluid neutrophils as hypersegmented and that a
significant proportion of the neutrophils express markers not usually found on neutrophils, such as HLA-DR (13). Neutrophils with acquired traits
of other myeloid cells, including antigen presenting capacity and expression of the mannose receptor CD206, have been described in other
rheumatic diseases such as rheumatoid arthritis (RA) (22,23) and adult-onset Still’s disease (24). In this study, we set out to investigate both phenotype and effector functions of neutrophils in paired blood and synovial fluid from children with
oligoarticular JIA. To characterize several important aspects of neutrophil biology, we investigated neutrophil phenotype by surface marker
expression related to activation, tissue migration, maturity and monocyte-like phenotype. Neutrophil function was analyzed in regard to both
phagocytosis and ROS production. We hypothesized that synovial fluid neutrophils would differ from circulating neutrophils both regarding
phenotype and function, and that the neutrophil alterations can be important in driving and maintaining the local joint inflammation in
oligoarticular JIA. Stimulation of healthy blood neutrophils with JIA synovial fluid Statistics Paired samples were analyzed using Wilcoxon’s matched-pairs signed-rank test or paired t-test when indicated. Comparisons between groups of
independent data were analyzed using Mann-Whitney U-test. Correlations were analyzed using Pearson r correlation coefficient. Statistical
analyses were performed in Prism 9. Preparation of neutrophils from healthy oral cavity Healthy volunteers performed a mouth rinse with 5 ml 0.9 % NaCl for 30 seconds in the morning, prior to breakfast and toothbrushing. The
collected rinse fluid was filtered through 10 mm syringe filcons (BD Biosciences) and centrifuged 300 g, 5 min. The pellet was resuspended in PBS
with 0.1 % BSA and 2 mM EDTA. Epithelial cells were removed by incubation of biotinylated anti-E-cadherin (Bioss) at a final concentration of 4
mg/ml for 30 minutes followed by streptavidin-Dynabeads M280 (Invitrogen) and removal by magnet. The remaining cells were washed with
PBS/BSA/EDTA. Leukocytes were isolated using DSB-XTM biotin labelled anti-CD45 at 2 mg/ml (Dynabeads FlowComp Flexi kit, Invitrogen) and
collected with Dynabeads FlowCompTM beads on a magnet. Cells were released from the beads using FlowCompTM release buffer, pelleted and
resuspended in PBS. Results Description of the study population Neutrophil effector functions Phagocytosis and oxidative burst were analyzed using PhagoburstTM and PhagotestTM assay kit (BD Biosciences) according to manufacturer’s
instructions. Both methods are flow cytometric assays performed on 100 ml heparinized whole blood or synovial fluid, measuring ROS production
upon stimulation with phorbol-myristate-acetate (PMA) or opsonized E. coli and phagocytosis of fluorescently labelled opsonized E. coli
respectively. When indicated, 90 ml serum or synovial fluid was added to 10 ml whole blood before analysis. Study population Neutrophil maturity was determined using surface markers CD62L and CD16, where mature neutrophils are CD16hi, CD62Lhi, immature
neutrophils are CD16mid, CD62Lhi and aged neutrophils are CD62Llow (26–28). Activated neutrophils were defined as having high levels of CD11b
and CD66b. High levels of CD14 and CD206 were considered features of macrophage/monocyte like phenotype. Immunofluorescence staining of synovial tissue biopsies Paraffin embedded biopsies in 3 mm sections mounted on microscope glass slides were baked for 1 h at 60 °C followed by deparaffinization in
Neo-Clear (Merck) and a series of washes in ethanol of decreasing concentration. Antigen retrieval was performed in citrate buffer pH 6.0. Slides
were blocked with 5 % bovine serum albumin (BSA) and 5 % normal goat serum (Abcam) for 30 min at room temperature prior to staining with
primary antibodies rabbit anti-human MPO 1:200 (Dako A0398), and mouse anti-CD206 1:200 (Antibodies-Online clone:22–130) in PBS with 1%
BSA at 4 °C over night. After PBS washes, the slides were stained with secondary antibodies goat anti-rabbit Alexa Fluor 488 (Invitrogen) and
donkey anti-mouse Alexa Fluor 568 (Abcam). Slides were mounted with Prolong Gold AntiFade with DAPI (Invitrogen). Images were taken using a
Zeiss fluorescence microscope and processed in Fiji software. Stimulation of healthy blood neutrophils with JIA synovial fluid Page 3/15 Neutrophils were isolated from heparinized whole blood from healthy controls using Lymphoprep (Axis-Shield) density gradient centrifugation
according to manufacturer’s instructions followed by sedimentation of red blood cells in saline with 1,5 % dextran T500 (Pharmacosmos). Neutrophils in supernatant were collected, pelleted and contaminating red blood cells lysed with sterile H2O. The purified neutrophils, 1x106/ml,
were incubated in MCDB 131 medium (Gibco) with 20 % synovial fluid or 20 % serum from blood donor at 37 °C for 1 h. Following incubation,
neutrophils were washed with PBS and phenotypically analyzed by flow cytometry. Neutrophils were isolated from heparinized whole blood from healthy controls using Lymphoprep (Axis-Shield) density gradient centrifugation
according to manufacturer’s instructions followed by sedimentation of red blood cells in saline with 1,5 % dextran T500 (Pharmacosmos). Neutrophils in supernatant were collected, pelleted and contaminating red blood cells lysed with sterile H2O. The purified neutrophils, 1x106/ml,
were incubated in MCDB 131 medium (Gibco) with 20 % synovial fluid or 20 % serum from blood donor at 37 °C for 1 h. Following incubation,
neutrophils were washed with PBS and phenotypically analyzed by flow cytometry. Study population Preparation of neutrophils from healthy oral cavity Circulating neutrophils are similar during inactive disease and flares Patients included in the study did not exhibit systemic symptoms although previous literature suggest that circulating neutrophils in JIA are
activated during flares (10,12,29). We therefore compared blood neutrophil phenotypes during flares and inactive disease. We did not observe
systemic activation of neutrophils during flares; neutrophil activation markers CD66b and CD11b were mostly unchanged, CD62L levels slightly
increased and cell surface S100A8/A9 was present on less than 3 % of the neutrophils (Figure 3A-D). CD14 levels were markedly increased during
flares (Figure 3E), while levels of CD16 and CD10 were not significantly altered (Figure 3F-G). CD206 expression, which was low or non-existent on
blood neutrophils during flares, was equally low during inactive disease (Figure 3H). The synovial neutrophil phenotype is not dependent on transmigration or synovial fluid alone To investigate if the synovial fluid neutrophil phenotype found in the JIA patients could be explained by exposure to synovial fluid, healthy blood
neutrophils were treated with synovial fluid in vitro. Exposure to synovial fluid did not induce any major phenotype shift on healthy blood
neutrophils (Figure 4A). Most surface markers remained unchanged except CD11b, which, in opposite to the findings in patients, was significantly
lower after exposure to synovial fluid. There was a very modest increase of CD206 (Figure 4A). These results suggests that the phenotype shift
between circulating and synovial fluid neutrophils cannot be explained by exposure to synovial fluid alone. To investigate if tissue transmigration towards other sites would result in a phenotype shift similar to synovial fluid neutrophils, we investigated
transmigrated neutrophils from the oral cavity of healthy controls. The phenotype shifts between healthy blood- and oral cavity neutrophils were
compared with JIA blood- and synovial fluid neutrophils. Neutrophils in both oral cavity and joint were activated compared to their blood
counterparts (Figure 4B-C). CD11b surface expression was elevated in the transmigrated neutrophils at both sites but more pronounced in
synovial fluid compared to oral neutrophils (Figure 4C). On the other hand, neutrophil surface S100A8/A9 was markedly increased on oral cavity
neutrophils but not on synovial fluid neutrophils (Figure 4D). Levels of CD62L were lower in oral neutrophils than synovial fluid neutrophils,
compared to their circulating counterparts (Figure 4E). Oral neutrophils had dramatically decreased levels of CD16 while synovial fluid neutrophils
had a small increase in this marker (Figure 4F). Both oral and synovial fluid neutrophils had increased levels of CD14 and CD206 (Figure 4G-H). Circulating neutrophils are similar during inactive disease and flares CD10 levels were more increased in synovial compared to oral neutrophils, despite not reaching statistical significance (Figure 4I). Taken together,
transmigration towards both sites induced a shift in neutrophil phenotypes, but with distinctly different surface marker patterns. Synovial fluid neutrophils have impaired phagocytosis and oxidative burst Neutrophil effector functions, phagocytosis and oxidative burst, were evaluated in blood and synovial fluid. Synovial fluid neutrophils had a
significantly decreased ability to phagocytose opsonized E. coli compared to circulating neutrophils (Figure 5A). The impaired phagocytosis was
not due to the presence of synovial fluid in the assay. Dilution of healthy control blood in cell-free JIA synovial fluid led to an increase in neutrophil
phagocytic ability, in contrast to addition of JIA serum which led to a slight impairment in phagocytosis (Figure 5B). Upon stimulation with PMA,
there was a trend towards impaired ROS production in synovial fluid neutrophils compared to circulating neutrophils, although not reaching
statistical significance (Figure 5C). ROS production upon stimulation with opsonized E. coli was similar in synovial and circulating neutrophils
(Figure 5D). Neutrophil capacity of phagocytosis and oxidative burst was not influenced by patient age (Supplemental Figure 2). Proportion of CD206+ neutrophils correlates with impaired phagocytosis and oxidative burst Monocytes typically have lower capacity for both phagocytosis and oxidative burst compared to neutrophils, and we therefore hypothesized that
CD206 expression on synovial fluid neutrophils might be associated with altered effector functions. Indeed, we observed a negative correlation of
the proportion of CD206+ neutrophils with both phagocytosis of opsonized E. coli (Figure 5E) and ROS production (Figure 5F-G) in synovial fluid
neutrophils, supporting the hypothesis that synovial fluid neutrophils have attained a more monocyte-like phenotype. Description of the study population A total of 17 patients with active oligoarticular JIA, undergoing therapeutical synovial fluid aspiration, were included in the study. The patient
cohort and samples are described in Table 1. Synovial fluid neutrophils have an aged and activated phenotype Blood neutrophils were primarily of a mature phenotype (CD16hi, CD62Lhi), with very few immature (CD16mid, CD62Lhi) or aged (CD62Llow)
neutrophils. However, in synovial fluid, most neutrophils had an aged phenotype (range 41-99 %, median 76 %) and no immature neutrophils were
observed (Figure 1A-D). Synovial fluid neutrophils also had higher levels of the age-related marker CD10 compared to neutrophils in the circulation
(Figure 1E). Neutrophils in synovial fluid had an activated phenotype, demonstrated by increased levels of activation markers CD11b and CD66b (Figure 1F-
G), and low levels of CD62L, which is shed upon neutrophil activation and/or migration. Despite the activated phenotype, S100A8/A9, a protein
released from activated neutrophils, was only present on the surface of less than 3 % of both synovial fluid and blood neutrophils (Figure 1H). Synovial fluid neutrophils express monocyte/macrophage-related surface markers Synovial fluid neutrophils had increased CD14 levels compared to the circulating neutrophils (Figure 1I). Mannose receptor CD206, not commonly
known as a neutrophil receptor, but expressed by monocytes, macrophages and dendritic cells, was found on a significant proportion of synovial
fluid neutrophils (range 6-92 %, median 56 %), while being low or non-existent on circulating neutrophils (Figure 1J). The increased expression of
both CD14 and CD206 might suggest that the neutrophils gain a monocyte/macrophage related phenotype. CD206-expressing neutrophils are found in tissue biopsies To confirm the finding of CD206+ neutrophils in synovial fluid we investigated whether neutrophils migrating through synovial tissue express
CD206. Synovial tissue biopsies obtained from four patients were stained for CD206 and myeloperoxidase (MPO). Neutrophils co-expressing both
MPO and CD206 were observed in tissue biopsies from patient 4, 8 and 18 (Figure 2, arrows), demonstrating that CD206-expressing neutrophils
can be present in synovial tissue. In all biopsies there were also neutrophils without CD206 expression (Figure 2, arrow heads). In two of biopsies Page 4/15 Page 4/15 we observed synovial blood vessels, and the circulating neutrophils within the vessels had no or low CD206 expression (Figure 2B-C). The biopsy
from patient 17 did not contain any area with neutrophils (data not shown). we observed synovial blood vessels, and the circulating neutrophils within the vessels had no or low CD206 expression (Figure 2B-C). Description of the study population The biopsy
from patient 17 did not contain any area with neutrophils (data not shown). Circulating neutrophils are similar during inactive disease and flares Discussion In this study, we show that synovial fluid neutrophils in active oligoarticular JIA have an activated, aged and transmigrated phenotype. The cells
also have monocyte-like surface marker expression both in the synovial fluid and on migrating neutrophils within the synovial membrane. This
monocyte-like phenotype is associated with reduced phagocytic capacity and ROS production. Thus, we suggest that neutrophils with altered
functions are driving or sustaining the local inflammation in the joint of children with oligoarticular JIA. In a healthy joint, the synovial fluid normally contains no or few immune cells. During arthritis, immune cells are recruited from the bloodstream
by chemotactic stimuli. Neutrophils found in synovial fluid have extravasated from the synovial blood vessels and migrated through the synovial
tissue that contains both synovial fibroblasts and infiltrating immune cells. In our study, the majority of these transmigrated neutrophils in
synovial fluid had an aged phenotype, characterized by low surface levels of L-selectin (CD62L) and increased CD10 compared to neutrophils in Page 5/15 the blood. The high amount of neutrophils with an aged phenotype in the joint could be due to anti-apoptotic effects of synovial fluid and
inflammatory stimulation (30,31). The presence of neutrophils with an aged phenotype in synovial fluid are supported by a recent study on
synovial fluid neutrophils in oligo- and polyarticular JIA, which described the majority of synovial fluid neutrophils to be of a CD62Llow and
hypersegmented phenotype (13). However, CD62L is also shed upon neutrophil activation and migration (32). Thus, the CD62Llow neutrophils
seen in synovial fluid could be a consequence of both ageing and/or activation and tissue migration. Neutrophils may become activated by a
wide variety of stimuli, such as inflammatory cytokines and chemotactic stimulation (33), and activated neutrophils present at a site of
inflammation is expected. Indeed, synovial fluid neutrophils were highly activated, measured as increased levels of CD11b and CD66b. Nevertheless, neutrophils in the non-inflammatory oral cavity also had an increase in CD11b and CD66b, as these adhesion molecules are
increased upon tissue migration. The activated phenotype of synovial fluid neutrophils is probably a consequence of combined inflammatory
stimulation and tissue migration. A large proportion of the synovial fluid neutrophils expressed the mannose receptor CD206, a receptor usually expressed by monocytes,
macrophages and dendritic cells, which is upregulated on synovial fluid monocytes in JIA (25). To our knowledge, only one study has described
CD206 on human neutrophils. Discussion In this study of adult onset Still’s disease the authors found that CD206 appeared on circulating neutrophils during
flare but not during inactive disease (24). CD206+ neutrophils are also described in a few publications of mouse models of stroke and myocardial
infarction (34–36). We confirmed the presence of CD206+ neutrophils in synovial biopsies where we found that some, but not all, of the
neutrophils scattered within the synovial tissue had expression of CD206. The CD206 mannose receptor recognizes mannosylated and sulfated
sugars, and has a wide variety of functions including phagocytosis and promotion of antigen presentation (37). In macrophages, increased
expression of CD206 is associated with the “M2” phenotype and anti-inflammatory functions (38), but the role of CD206 on neutrophils is
unknown. It seems as if the synovial fluid neutrophils are gaining a phenotype with features of both the neutrophil and monocyte lineages, based
on the high amount of CD206+ neutrophils and increased levels of the TLR4 co-receptor CD14. Neutrophils expressing surface markers related to
other myeloid phenotypes have previously been found in synovial fluid in JIA and RA. In the recent study of JIA synovial fluid neutrophils by
Metzemaekers et al., a significant portion of the synovial fluid neutrophils had gained expression of HLA-DR (13), and synovial fluid neutrophils
from RA patients have also been found to have antigen-presenting abilities (22,23). As CD206 is described to be involved in cross-presentation of
antigens (37), it is possible that expression of this receptor mediates antigen-presenting capacity of the neutrophils. Neutrophil plasticity to
transdifferentiate into cells of other myeloid lineages is well established in vitro (39–41), and we believe that the microenvironment in the
inflamed joint is affecting neutrophils to acquire other myeloid traits. The phenotype shift seen in synovial fluid neutrophils is not dependent on either synovial fluid or transmigration separately. In vitro exposure of
JIA synovial fluid to healthy blood neutrophils did not cause a phenotypical shift. We further studied oral cavity neutrophils as a control for tissue
migration towards a non-inflammatory site and found that neutrophils at both sites shared some common features (activation, loss of CD62L and
gain of CD14 and CD206). However, the extent of the alterations was different in the two locations and other changes occurred in only one of the
sites (increase of CD10 in synovial fluid, increase of S100A8/A9 in oral cavity and decrease of CD16 in oral cavity). Discussion The findings of distinct
phenotypes varying between synovial fluid and oral cavity is supported by previous publications studying neutrophils in synovial fluid compared
to skin blister fluid or pleural effusions (13,21). Our results indicate that the synovial neutrophil phenotype and function alterations are induced by
a combination of multiple factors and stimuli, and further studies are needed to characterize this. Importantly, we found a significant decrease in phagocytic capacity of the synovial fluid neutrophils compared to circulating neutrophils. In
addition, the phenotype shift of synovial neutrophils towards a monocyte-like pattern with increased surface expression of CD206 was associated
with both reduced capacity of phagocytosis and oxidative burst, suggesting that alterations in phenotype and function are connected. Impaired
neutrophil function may drive inflammation and autoimmunity, as neutrophil clearance of debris and dead cell remnants is essential to maintain
homeostasis and minimize the autoantigenic burden (8). Extracellular ROS produced by neutrophils are also important for immunoregulation
(17,42) and impaired neutrophil ROS production in the joint could prolong the resolution of inflammation. As we demonstrate that addition of
synovial fluid to healthy control neutrophils does not impair phagocytic capacity, we suggest that the impairment in effector functions is an
inherent trait of the synovial fluid neutrophils and not a consequence of the local microenvironment. The neutrophil gating strategy for patient neutrophils, and the fact that more aspects of synovial fluid neutrophil effector functions were not
investigated present some limitations to the study. The panels were designed for negative selection of neutrophils, but as synovial fluid may
contain cells and particles with neutrophil size and granularity without leukocyte surface markers, a positive selection marker was also needed. After negative selection, neutrophils were therefore selected by CD16 positivity, which may inadvertently lose immature CD16low neutrophils. Neutrophil function is not limited to oxidative burst and phagocytosis, and to further characterize the functional differences between circulating
and synovial fluid neutrophils in JIA including chemotaxis, release of neutrophil extracellular traps (NETs) and acquired ability to present antigens
would be of interest and importance. These possible functional alterations will be investigated in further studies. Funding This study was supported by grants from the Crown Princess Lovisa’s Society for Child Care, the Swedish Rheumatism Association, Greta and
Johan Kock’s Foundation, the Hedberg Foundation, the Anna-Greta Crafoord Foundation, the Crafoord Foundation, the Swedish Medical Society,
the Jerring Foundation, Alfred Österlunds Foundation, Magnus Bergvall Foundation, Thelma Zoega’s foundation, Lion research foundation, King
Gustaf V’s 80-year foundation, The Knut and Alice Wallenberg foundation, the Medical Faculty at Lund University and Region Skåne (all to RK). Authors' contributions SAB carried out the experiments and data analysis, interpreted data, and wrote the manuscript. SAB carried out the experiments and data analysis, interpreted data, and wrote the manuscript. TS, AM, BG carried out the experiments and reviewed and revised the manuscript. TS, AM, BG carried out the experiments and reviewed and revised the manuscript. EB collected clinical data and samples, as well as reviewed and revised the manuscript. FK and AAB interpreted data, reviewed and revised the manuscript. RK conceptualized and designed the study, collected clinical data and samples, interpreted data, and wrote the manuscript. RK conceptualized and designed the study, collected clinical data and samples, interpreted data, and wrote the manuscript. All authors have read and approved the final manuscript. We wish to acknowledge Åsa Pettersson for support in the phagotest and phagoburst experiments, and Daniel Butler for lending us secondary
antibodies and microscope access for immunofluorescence. We wish to acknowledge Åsa Pettersson for support in the phagotest and phagoburst experiments, and Daniel Butler for lending us secondary
antibodies and microscope access for immunofluorescence. Abbreviations JIA – juvenile idiopathic arthritis, ROS – reactive oxygen species, PMA – phorbol 12-myristate 13-acetate, NSAID – non-steroid anti-inflammatory
drug, MFI – median fluorescence intensity, RA – rheumatoid arthritis Page 6/15 This study demonstrates that neutrophils in synovial fluid of children with active oligoarticular JIA are altered both phenotypically and
functionally compared to paired neutrophils from the circulation. We observed that synovial fluid neutrophils are activated, aged and has gained a
monocyte-like phenotype, which is associated with an impairment in neutrophil phagocytosis and oxidative burst. We suggest that these
neutrophil alterations could be of importance in sustaining joint inflammation and thus interesting possible targets for specific therapies. Ethics approval and consent to participate Ethics approval and consent to participate Collection of JIA patient samples and data and healthy controls were approved by the Regional Ethical Review Board for southern Sweden ((LU
2015/734, LU2016/128 and LU2017/473). Informed consent was obtained from the healthy control, patient and/or the patient’s guardian. Consent for publication Not applicable. Anonymized datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Anonymized datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare no financial or competing interests. The authors declare no financial or competing interests. References Kelkka T, Kienhöfer D, Hoffmann M, Linja M, Wing K, Sareila O, et al. Reactive oxygen species deficiency induces autoimmunity with type 1
interferon signature. Antioxid Redox Signal. 2014 Dec 1;21(16):2231–45. 19. Kelkka T, Kienhöfer D, Hoffmann M, Linja M, Wing K, Sareila O, et al. Reactive oxygen species deficiency induces autoimmunity with type 1
interferon signature. Antioxid Redox Signal. 2014 Dec 1;21(16):2231–45. 20. Zhao J, Ma J, Deng Y, Kelly JA, Kim K, Bang S-Y, et al. A missense variant in NCF1 is associated with susceptibility to multiple autoimmune
diseases. Nat Genet. 2017 Mar;49(3):433–7. 21. Björkman L, Christenson K, Davidsson L, Mårtensson J, Amirbeagi F, Welin A, et al. Neutrophil recruitment to inflamed joints can occur
without cellular priming. J Leukoc Biol. 2018 Dec 20; 22. Iking-Konert C, Ostendorf B, Sander O, Jost M, Wagner C, Joosten L, et al. Transdifferentiation of polymorphonuclear neutrophils to dendritic-
like cells at the site of inflammation in rheumatoid arthritis: evidence for activation by T cells. Ann Rheum Dis. 2005 Oct;64(10):1436–42. 23. Cross A, Bucknall RC, Cassatella MA, Edwards SW, Moots RJ. Synovial fluid neutrophils transcribe and express class II major
histocompatibility complex molecules in rheumatoid arthritis. Arthritis Rheum. 2003 Oct;48(10):2796–806. 23. Cross A, Bucknall RC, Cassatella MA, Edwards SW, Moots RJ. Synovial fluid neutrophils transcribe and express class II major
histocompatibility complex molecules in rheumatoid arthritis. Arthritis Rheum. 2003 Oct;48(10):2796–806. 24. Kim H-A, Choi B, Suh C-H, Han MH, Jung J-Y, Sayeed HM, et al. Highly Expression of CD11b and CD32 on Peripheral Blood Mononuclear Cells
from Patients with Adult-Onset Still’s Disease. Int J Mol Sci. 2017 Jan 19;18(1). 24. Kim H-A, Choi B, Suh C-H, Han MH, Jung J-Y, Sayeed HM, et al. Highly Expression of CD11b and CD32 on Peripheral Blood Mononuclear Cells
from Patients with Adult-Onset Still’s Disease. Int J Mol Sci. 2017 Jan 19;18(1). 25. Schmidt T, Berthold E, Arve-Butler S, Gullstrand B, Mossberg A, Kahn F, et al. Children with oligoarticular juvenile idiopathic arthritis have
skewed synovial monocyte polarization pattern with functional impairment-a distinct inflammatory pattern for oligoarticular juvenile arthritis. Arthritis Res Ther. 2020 Aug 12;22(1):186. 25. Schmidt T, Berthold E, Arve-Butler S, Gullstrand B, Mossberg A, Kahn F, et al. Children with oligoarticular juvenile idiopathic arthritis have
skewed synovial monocyte polarization pattern with functional impairment-a distinct inflammatory pattern for oligoarticular juvenile arthritis. Arthritis Res Ther. 2020 Aug 12;22(1):186. 26. Grieshaber-Bouyer R, Nigrovic PA. References 1. Glerup M, Rypdal V, Arnstad ED, Ekelund M, Peltoniemi S, Aalto K, et al. Long-Term Outcomes in Juvenile Idiopathic Arthritis: Eighteen Years of
Follow-Up in the Population-Based Nordic Juvenile Idiopathic Arthritis Cohort. Arthritis Care Res (Hoboken). 2020;72(4):507–16. 2. Matsumoto T, Matsui T, Hirano F, Tohma S, Mori M. Disease activity, treatment and long-term prognosis of adult juvenile idiopathic arthritis
patients compared with rheumatoid arthritis patients. Mod Rheumatol. 2020 Jan;30(1):78–84. 2. Matsumoto T, Matsui T, Hirano F, Tohma S, Mori M. Disease activity, treatment and long-term prognosis of adult juvenile idiopathic arthritis
patients compared with rheumatoid arthritis patients. Mod Rheumatol. 2020 Jan;30(1):78–84. 3. Prakken B, Albani S, Martini A. Juvenile idiopathic arthritis. Lancet. 2011 Jun 18;377(9783):2138–49. Page 7/15 Page 7/15 4. Berthold E, Månsson B, Kahn R. Outcome in juvenile idiopathic arthritis: a population-based study from Sweden. Arthritis Res Ther. 2019 Oct
28;21(1):218. 5. Macaubas C, Nguyen K, Milojevic D, Park JL, Mellins ED. Oligoarticular and polyarticular JIA: epidemiology and pathogenesis. Nat Rev
Rheumatol. 2009 Nov;5(11):616–26. 6. Jarvis JN, Jiang K, Petty HR, Centola M. Neutrophils: the forgotten cell in JIA disease pathogenesis. Pediatr Rheumatol Online J. 2007 Jun
13;5:13. 7. Giancane G, Alongi A, Ravelli A. Update on the pathogenesis and treatment of juvenile idiopathic arthritis. Curr Opin Rheumatol. 2017
Sep;29(5):523–9. 8. Wirestam L, Arve S, Linge P, Bengtsson AA. Neutrophils-Important Communicators in Systemic Lupus Erythematosus and Antiphospholipid
Syndrome. Front Immunol. 2019 Nov 22;10:2734. 9. Wright HL, Moots RJ, Edwards SW. The multifactorial role of neutrophils in rheumatoid arthritis. Nat Rev Rheumatol. 2014 Oct;10(10):593–
601. 10. Jarvis JN, Petty HR, Tang Y, Frank MB, Tessier PA, Dozmorov I, et al. Evidence for chronic, peripheral activation of neutrophils in polyarticular
juvenile rheumatoid arthritis. Arthritis Res Ther. 2006;8(5):R154. 11. Brown RA, Henderlight M, Do T, Yasin S, Grom AA, DeLay M, et al. Neutrophils From Children With Systemic Juvenile Idiopathic Arthritis
Exhibit Persistent Proinflammatory Activation Despite Long-Standing Clinically Inactive Disease. Front Immunol. 2018 Dec 18;9:2995. 12. Ramanathan K, Glaser A, Lythgoe H, Ong J, Beresford MW, Midgley A, et al. Neutrophil activation signature in juvenile idiopathic arthritis
indicates the presence of low-density granulocytes. Rheumatology. 2018 Mar 1;57(3):488–98. 12. Ramanathan K, Glaser A, Lythgoe H, Ong J, Beresford MW, Midgley A, et al. Neutrophil activation signature in juvenile idiopathic arthritis
indicates the presence of low-density granulocytes. Rheumatology. 2018 Mar 1;57(3):488–98. 13. References Metzemaekers M, Malengier-Devlies B, Yu K, Vandendriessche S, Yserbyt J, Matthys P, et al. Synovial fluid neutrophils from patients with
juvenile idiopathic arthritis display a hyperactivated phenotype. Arthritis Rheumatol. 2020 Dec 2; 13. Metzemaekers M, Malengier-Devlies B, Yu K, Vandendriessche S, Yserbyt J, Matthys P, et al. Synovial fluid neutrophils from patients with
juvenile idiopathic arthritis display a hyperactivated phenotype. Arthritis Rheumatol. 2020 Dec 2; 3. Metzemaekers M, Malengier-Devlies B, Yu K, Vandendriessche S, Yserbyt J, Matthys P, et al. Synovial f
juvenile idiopathic arthritis display a hyperactivated phenotype. Arthritis Rheumatol. 2020 Dec 2; 14. Bengtsson AA, Pettersson Å, Wichert S, Gullstrand B, Hansson M, Hellmark T, et al. Low production of reactive oxygen species in granulocytes
is associated with organ damage in systemic lupus erythematosus. Arthritis Res Ther. 2014 Jun 5;16(3):R120. 14. Bengtsson AA, Pettersson Å, Wichert S, Gullstrand B, Hansson M, Hellmark T, et al. Low production of reactive oxygen species in granulocytes
is associated with organ damage in systemic lupus erythematosus. Arthritis Res Ther. 2014 Jun 5;16(3):R120. 15. Olsson LM, Johansson ÅC, Gullstrand B, Jönsen A, Saevarsdottir S, Rönnblom L, et al. A single nucleotide polymorphism in the NCF1 gene
leading to reduced oxidative burst is associated with systemic lupus erythematosus. Ann Rheum Dis. 2017 Jun 12; 15. Olsson LM, Johansson ÅC, Gullstrand B, Jönsen A, Saevarsdottir S, Rönnblom L, et al. A single nucleotide polymorphism in the NCF1 gene
leading to reduced oxidative burst is associated with systemic lupus erythematosus. Ann Rheum Dis. 2017 Jun 12; 16. Linge P, Arve S, Olsson LM, Leonard D, Sjöwall C, Frodlund M, et al. NCF1-339 polymorphism is associated with altered formation of
neutrophil extracellular traps, high serum interferon activity and antiphospholipid syndrome in systemic lupus erythematosus. Ann Rheum
Dis. 2020 Feb;79(2):254–61. 17. Hoffmann MH, Griffiths HR. The dual role of ROS in autoimmune and inflammatory diseases: Evidence from preclinical models. Free Radic
Biol Med. 2018 Mar 15;125:62–71. 17. Hoffmann MH, Griffiths HR. The dual role of ROS in autoimmune and inflammatory diseases: Evidence from preclinical models. Free Radic
Biol Med. 2018 Mar 15;125:62–71. 18. Urbonaviciute V, Luo H, Sjöwall C, Bengtsson A, Holmdahl R. Low production of reactive oxygen species drives systemic lupus
erythematosus. Trends Mol Med. 2019 Jul 11;25(10):826–35. 18. Urbonaviciute V, Luo H, Sjöwall C, Bengtsson A, Holmdahl R. Low production of reactive oxygen species drives systemic lupus
erythematosus. Trends Mol Med. 2019 Jul 11;25(10):826–35. 19. References Mantovani A, Sica A, Sozzani S, Allavena P, Vecchi A, Locati M. The chemokine system in diverse forms of macrophage activation and
polarization. Trends Immunol. 2004 Dec;25(12):677–86. 38. Mantovani A, Sica A, Sozzani S, Allavena P, Vecchi A, Locati M. The chemokine system in diverse forms of macrophage activation and
polarization. Trends Immunol. 2004 Dec;25(12):677–86. 39. Araki H, Katayama N, Yamashita Y, Mano H, Fujieda A, Usui E, et al. Reprogramming of human postmitotic neutrophils into macrophages by
growth factors. Blood. 2004 Apr 15;103(8):2973–80. 39. Araki H, Katayama N, Yamashita Y, Mano H, Fujieda A, Usui E, et al. Reprogramming of human postmitotic neutrophils into macrophages by
growth factors. Blood. 2004 Apr 15;103(8):2973–80. 40. Köffel R, Meshcheryakova A, Warszawska J, Hennig A, Wagner K, Jörgl A, et al. Monocytic cell differentiation from band-stage neutrophils
under inflammatory conditions via MKK6 activation. Blood. 2014 Oct 23;124(17):2713–24. 40. Köffel R, Meshcheryakova A, Warszawska J, Hennig A, Wagner K, Jörgl A, et al. Monocytic cell differentiation from band-stage neutrophils
under inflammatory conditions via MKK6 activation. Blood. 2014 Oct 23;124(17):2713–24. 41. Takashima A, Yao Y. Neutrophil plasticity: acquisition of phenotype and functionality of antigen-presenting cell. J Leukoc Biol. 2015
Oct;98(4):489–96. 41. Takashima A, Yao Y. Neutrophil plasticity: acquisition of phenotype and functionality of antigen-presenting cell. J Leukoc Biol. 2015
Oct;98(4):489–96. 42. Hultqvist M, Olsson LM, Gelderman KA, Holmdahl R. The protective role of ROS in autoimmune disease. Trends Immunol. 2009
May;30(5):201–8. 42. Hultqvist M, Olsson LM, Gelderman KA, Holmdahl R. The protective role of ROS in autoimmune disease. Trends Immunol. 2009
May;30(5):201–8. References Neutrophil Heterogeneity as Therapeutic Opportunity in Immune-Mediated Disease. Front Immunol. 2019
Mar 4;10:346. 26. Grieshaber-Bouyer R, Nigrovic PA. Neutrophil Heterogeneity as Therapeutic Opportunity in Immune-Mediated Disease. Front Immunol. 2019
Mar 4;10:346. 27. van Grinsven E, Textor J, Hustin LSP, Wolf K, Koenderman L, Vrisekoop N. Immature Neutrophils Released in Acute Inflammation Exhibit
Efficient Migration despite Incomplete Segmentation of the Nucleus. J Immunol. 2019 Jan 1;202(1):207–17. 27. van Grinsven E, Textor J, Hustin LSP, Wolf K, Koenderman L, Vrisekoop N. Immature Neutrophils Released in Acute Inflammation Exhibit
Efficient Migration despite Incomplete Segmentation of the Nucleus. J Immunol. 2019 Jan 1;202(1):207–17. Page 8/15 28. Pillay J, Kamp VM, van Hoffen E, Visser T, Tak T, Lammers J-W, et al. A subset of neutrophils in human systemic inflammation inhibits T cell
responses through Mac-1. J Clin Invest. 2012 Jan;122(1):327–36. 29. Foell D, Wittkowski H, Hammerschmidt I, Wulffraat N, Schmeling H, Frosch M, et al. Monitoring neutrophil activation in juvenile rheumatoid
arthritis by S100A12 serum concentrations. Arthritis Rheum. 2004 Apr;50(4):1286–95. 30. Ottonello L, Cutolo M, Frumento G, Arduino N, Bertolotto M, Mancini M, et al. Synovial fluid from patients with rheumatoid arthritis inhibits
neutrophil apoptosis: role of adenosine and proinflammatory cytokines. Rheumatology. 2002 Nov;41(11):1249–60. 31. Cross A, Barnes T, Bucknall RC, Edwards SW, Moots RJ. Neutrophil apoptosis in rheumatoid arthritis is regulated by local oxygen tensions
within joints. J Leukoc Biol. 2006 Sep;80(3):521–8. view of L-selectin and its impact on neutrophil behaviour. Cell Tissue Res. 2018 Jan 20;371(3):437–53. 2. Ivetic A. A head-to-tail view of L-selectin and its impact on neutrophil behaviour. Cell Tissue Res. 2018 33. Vogt KL, Summers C, Chilvers ER, Condliffe AM. Priming and de-priming of neutrophil responses in vitro and in vivo. Eur J Clin Invest. 2018
Nov;48 Suppl 2:e12967. 34. Cuartero MI, Ballesteros I, Moraga A, Nombela F, Vivancos J, Hamilton JA, et al. N2 neutrophils, novel players in brain inflammation after
stroke: modulation by the PPARγ agonist rosiglitazone. Stroke. 2013 Dec;44(12):3498–508. 35. Cai W, Liu S, Hu M, Huang F, Zhu Q, Qiu W, et al. Functional dynamics of neutrophils after ischemic stroke. Transl Stroke Res. 2020
Feb;11(1):108–21. 36. Ma Y, Yabluchanskiy A, Iyer RP, Cannon PL, Flynn ER, Jung M, et al. Temporal neutrophil polarization following myocardial infarction. Cardiovasc Res. 2016 May 1;110(1):51–61. 37. Martinez-Pomares L. The mannose receptor. J Leukoc Biol. 2012 Dec;92(6):1177–86. 37. Martinez-Pomares L. The mannose receptor. J Leukoc Biol. 2012 Dec;92(6):1177–86. 38. Tables Table I. Description of patient cohort. Page 9/15 Pat#
Sex
Disease
duration
(months)
Age
(years)
Treatment
Uveitis
ANA
Phenotype
analysis
Functional
assay
Biopsy
Follow-
up
sample
Time to
follow-
up
(months)
Treatment
at follow
up
1*
M
132
15
No
No
Pos
Yes
No
No
No
2
F
26
7
NSAID
No
Pos
Yes
No
No
No
3*
F
88
11
No
No
Pos
Yes
Yes
No
Yes
40
No
4*
F
0
6
NSAID
Yes
Pos
Yes
Yes
Yes
No
5*
F
3
11
NSAID
Yes
Pos
Yes
Yes
No
Yes
34
NSAID
6*
F
49
7.5
NSAID
No
Pos
Yes
No
No
Yes
31
TNFi
7*
F
48
16.5
No
Yes
Pos
Yes
Yes
No
No
8*
F
2
3.5
No
No
Pos
Yes
Yes
Yes
No
9*
F
1
15
NSAID
No
Pos
Yes
Yes
No
Yes
20
Mtx,
NSAID
10*
M
1
12
NSAID
No
Neg
Yes
Yes
No
No
11
M
4
10
NSAID
No
Pos
Yes
Yes
No
Yes
19
Mtx
12*
F
0
2
No
No
Pos
Yes
Yes
No
Yes
16
Mtx
13
F
144
17
NSAID
No
Neg
Yes
Yes
No
No
14*
F
1
13
NSAID
No
Neg
Yes
Yes
No
No
15
M
2
4
NSAID
No
Pos
Yes
No
No
No
16
M
0
6
NSAID
No
Pos
Yes
Yes
No
No
17*
F
1
11
No
No
Neg
Yes
Yes
Yes
No
18
F
50
5
Mtx
Yes
Pos
No
No
Yes
No
Clinical and sample data Disease duration is calculated as months since date of diagnosis Abbreviations: TNFi
TNF inhibitor Mtx Clinical and sample data. Disease duration is calculated as months since date of diagnosis. Abbreviations: TNFi – TNF inhibitor, Mtx –
methotrexate. * patients are also included in Schmidt et al. (25) * patients are also included in Schmidt et al. (25) ease duration is calculated as months since date of diagnosis. Abbreviations: TNFi – TNF inhibitor, Mtx Figures Page 10/15 Figure 1
Synovial fluid neutrophils have an aged and activated phenotype and express monocyte related markers. Neutrophi
blood and synovial fluid from patients with oligoarticular JIA were analyzed by flow cytometry. A) Neutrophilphenot
synovial fluid based on CD62L and CD16 expression. B) Representative plot of neutrophil maturity phenotype gatin
CD16hi CD62Lhi, immature neutrophils (Im) are CD16mid CD62Lhi and neutrophils with decreased CD62L are CD62
fluorescence intensity (MFI) of neutrophil expression of C) CD16, D) CD62L, E) CD10, F) CD11b, G) CD66b and I) CD
neutrophils, presented as % of total neutrophils, with cell surface expression of H) S100A8/A9, and J) CD206. * p<0
signed-rank test Figure 1 Figure 1 Synovial fluid neutrophils have an aged and activated phenotype and express monocyte related markers. Neutrophils from paired samples of
blood and synovial fluid from patients with oligoarticular JIA were analyzed by flow cytometry. A) Neutrophilphenotypical maturities in blood and
synovial fluid based on CD62L and CD16 expression. B) Representative plot of neutrophil maturity phenotype gating. Mature neutrophils (M) are
CD16hi CD62Lhi, immature neutrophils (Im) are CD16mid CD62Lhi and neutrophils with decreased CD62L are CD62Llow (L). C-G & I) Median
fluorescence intensity (MFI) of neutrophil expression of C) CD16, D) CD62L, E) CD10, F) CD11b, G) CD66b and I) CD14. H & J) Proportion of
neutrophils, presented as % of total neutrophils, with cell surface expression of H) S100A8/A9, and J) CD206. * p<0.05, *** p<0.001, Wilcoxon
signed-rank test Page 11/15 Figure 2
CD206 expressing neutrophils are found in synovial tissue. Synovial tissue biopsies from three patients, stained for MPO (green), CD206 (
DAPI (blue). Representative images of A) patient 4, B) patient 8, and C) patient 18. Neutrophils expressing CD206, indicated with arrows, w
found in all biopsies. Neutrophils without CD206 expression, indicated with arrow heads, were found in all three biopsies. Synovial vessels
indicated with dotted ellipses. Neutrophils were found both scattered in the synovial tissue and inside synovial blood vessels. Fourth imag
each panel represents a magnification of the area indicated in merged image. Scalebar is 20 μm. All images are taken at 40X magnificatio Figure 2
CD206 expressing neutrophils are found in synovial tissue. Synovial tissue biopsies from three patients, stained for MPO (green), CD206 (red) a
DAPI (blue). Representative images of A) patient 4, B) patient 8, and C) patient 18. Neutrophils expressing CD206, indicated with arrows, were
found in all biopsies. Neutrophils without CD206 expression, indicated with arrow heads, were found in all three biopsies. Synovial vessels are
indicated with dotted ellipses. Neutrophils were found both scattered in the synovial tissue and inside synovial blood vessels. Fourth image in
each panel represents a magnification of the area indicated in merged image. Scalebar is 20 μm. All images are taken at 40X magnification. Figure 2 CD206 expressing neutrophils are found in synovial tissue. Synovial tissue biopsies from three patients, stained for MPO (green), CD206 (red) and
DAPI (blue). Representative images of A) patient 4, B) patient 8, and C) patient 18. Neutrophils expressing CD206, indicated with arrows, were
found in all biopsies. Figure 1 Neutrophils without CD206 expression, indicated with arrow heads, were found in all three biopsies. Synovial vessels are
indicated with dotted ellipses. Neutrophils were found both scattered in the synovial tissue and inside synovial blood vessels. Fourth image in
each panel represents a magnification of the area indicated in merged image. Scalebar is 20 μm. All images are taken at 40X magnification. Page 12/15 Figure 3
Neutrophil phenotypes in JIA blood are similar during flare and inactive disease. Neutrophil surface marker levels in paired blood samples tak
during arthritis flare or inactive disease without arthritis. A-C & E-G) Median fluorescence intensity (MFI) of neutrophil expression of A) CD66b
CD11b, C) CD62L, E) CD14, F) CD16 and G) CD10. D & H) Proportion of neutrophils, presented as % of total neutrophils, with cell surface
expression of D) S100A8/A9 and H) CD206. * p<0.05, Wilcoxon signed-rank test Figure 3 Neutrophil phenotypes in JIA blood are similar during flare and inactive disease. Neutrophil surface marker levels in paired blood samples taken
during arthritis flare or inactive disease without arthritis. A-C & E-G) Median fluorescence intensity (MFI) of neutrophil expression of A) CD66b, B)
CD11b, C) CD62L, E) CD14, F) CD16 and G) CD10. D & H) Proportion of neutrophils, presented as % of total neutrophils, with cell surface
expression of D) S100A8/A9 and H) CD206. * p<0.05, Wilcoxon signed-rank test Page 13/15 Figure 4
The neutrophil phenotype shift is not explained by exposure to synovial fluid or transmigration. A) Healthy c
challenged with 20 % JIA synovial fluid (n=12) or 20 % serum from the healthy blood donor for 1 h followed
expression. Control serum stimulated neutrophils are represented by the dotted line. Data are presented as f
intensity (MFI) in synovial fluid stimulated compared to control neutrophils. B-I) Neutrophils in JIA synovial
neutrophils from the oral cavity (OC) of healthy controls, to investigate the alterations in surface markers be
samples of blood and SF or blood and OC. Blood values are represented by the dotted line. Data are present
CD66b, C) CD11b, D) S100A8/A9, E) CD62L, F) CD16, G) CD14, H) CD206 and I) CD10 in tissue neutrophils
Line at median. ** p<0.01, *** p<0.001, Mann-Whitney U-test. Figure 4 Figure 4 Supplementalfigures.pdf Figure 5 Neutrophil effector functions are impaired in JIA blood and synovial fluid. A) Phagocytosis of fluorescently labelled opsonized E. coli in
neutrophils from paired samples of JIA blood and synovial fluid (SF). B) Phagocytosis of fluorescently labelled opsonized E. coli in healthy
control neutrophils in a mixture of 10 % whole blood and 90 % serum or cell-free synovial fluid from four JIA patients. Results are presented as
phagocytosis, measured as MFI, in serum/SF-treated samples compared to nontreated whole blood. C-D) Neutrophil ROS production, quantified
using DHR-123 fluorescence, after stimulation with C) PMA or D) opsonized E. coli. E-G) Pearson r correlation analysis, with 95 % confidence
interval, of proportion of CD206+ neutrophils in synovial fluid with E) phagocytosis of opsonized E. coli, F) ROS production by PMA-stimulation
and G) ROS production by opsonized E. coli stimulation. *** p<0.001, Wilcoxon signed-rank test. MFI - median fluorescence intensity Figure 4 The neutrophil phenotype shift is not explained by exposure to synovial fluid or transmigration. A) Healthy control blood neutrophils were
challenged with 20 % JIA synovial fluid (n=12) or 20 % serum from the healthy blood donor for 1 h followed by analysis of surface marker
expression. Control serum stimulated neutrophils are represented by the dotted line. Data are presented as fold change of median fluorescence
intensity (MFI) in synovial fluid stimulated compared to control neutrophils. B-I) Neutrophils in JIA synovial fluid (SF) were compared to
neutrophils from the oral cavity (OC) of healthy controls, to investigate the alterations in surface markers between blood and tissue in paired
samples of blood and SF or blood and OC. Blood values are represented by the dotted line. Data are presented as fold change of MFI values of B)
CD66b, C) CD11b, D) S100A8/A9, E) CD62L, F) CD16, G) CD14, H) CD206 and I) CD10 in tissue neutrophils compared to paired blood neutrophils. Line at median. ** p<0.01, *** p<0.001, Mann-Whitney U-test. Page 14/15 Figure 5
Neutrophil effector functions are impaired in JIA blood and synovial fluid. A) Phagocytosis of fluorescently labelled opsonized E. coli in
neutrophils from paired samples of JIA blood and synovial fluid (SF). B) Phagocytosis of fluorescently labelled opsonized E. coli in healthy
control neutrophils in a mixture of 10 % whole blood and 90 % serum or cell-free synovial fluid from four JIA patients. Results are presented
phagocytosis, measured as MFI, in serum/SF-treated samples compared to nontreated whole blood. C-D) Neutrophil ROS production, quanti
using DHR-123 fluorescence, after stimulation with C) PMA or D) opsonized E. coli. E-G) Pearson r correlation analysis, with 95 % confidence
interval, of proportion of CD206+ neutrophils in synovial fluid with E) phagocytosis of opsonized E. coli, F) ROS production by PMA-stimulat
and G) ROS production by opsonized E. coli stimulation. *** p<0.001, Wilcoxon signed-rank test. MFI - median fluorescence intensity Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Page 15/15
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https://openalex.org/W2479386100
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https://ueaeprints.uea.ac.uk/id/eprint/81368/1/Published_Version.pdf
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Echoic Sensory Substitution Information in a Single Obstacle Circumvention Task
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PloS one
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cc-by
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RESEARCH ARTICLE Data Availability Statement: All relevant data are
within the paper and its Supporting Information files. Funding: This research was supported by the Vision
and Eye Research Unit, Postgraduate Medical
Institute at Anglia Ruskin University (awarded to SP),
and the Medical Research Council (awarded to
BCJM, Grant number G0701870), http://www.anglia. ac.uk/postgraduate-medical-institute/groups/vision-
and-eyeresearch-unit; http://www.mrc.ac.uk. The
funders had no role in study design, data collection
and analysis, decision to publish, or preparation of
the manuscript. Echoic Sensory Substitution Information in a
Single Obstacle Circumvention Task Andrew J. Kolarik1,2,3*, Amy C. Scarfe2,4, Brian C. J. Moore3, Shahina Pardhan2 1 Centre for the Study of the Senses, Institute of Philosophy, University of London, London, United Kingdom,
2 Vision and Eye Research Unit (VERU), Postgraduate Medical Institute, Anglia Ruskin University,
Cambridge, United Kingdom, 3 Department of Psychology, University of Cambridge, Cambridge, United
Kingdom, 4 Department of Clinical Engineering, Medical Imaging and Medical Physics Directorate, Sheffield
Teaching Hospitals NHS Foundation Trust, Sheffield, United Kingdom a1111 * ak771@cam.ac.uk Abstract Accurate motor control is required when walking around obstacles in order to avoid colli-
sions. When vision is unavailable, sensory substitution can be used to improve locomotion
through the environment. Tactile sensory substitution devices (SSDs) are electronic travel
aids, some of which indicate the distance of an obstacle using the rate of vibration of a trans-
ducer on the skin. We investigated how accurately such an SSD guided navigation in an
obstacle circumvention task. Using an SSD, 12 blindfolded participants navigated around a
single flat 0.6 x 2 m obstacle. A 3-dimensional Vicon motion capture system was used to
quantify various kinematic indices of human movement. Navigation performance under full
vision was used as a baseline for comparison. The obstacle position was varied from trial to
trial relative to the participant, being placed at two distances 25 cm to the left, right or directly
ahead. Under SSD guidance, participants navigated without collision in 93% of trials. No
collisions occurred under visual guidance. Buffer space (clearance between the obstacle
and shoulder) was larger by a factor of 2.1 with SSD guidance than with visual guidance,
movement times were longer by a factor of 9.4, and numbers of velocity corrections were
larger by a factor of 5 (all p<0.05). Participants passed the obstacle on the side affording the
most space in the majority of trials for both SSD and visual guidance conditions. The results
are consistent with the idea that SSD information can be used to generate a protective
envelope during locomotion in order to avoid collisions when navigating around obstacles,
and to pass on the side of the obstacle affording the most space in the majority of trials. OPEN ACCESS OPEN ACCESS
Citation: Kolarik AJ, Scarfe AC, Moore BCJ,
Pardhan S (2016) Echoic Sensory Substitution
Information in a Single Obstacle Circumvention Task. PLoS ONE 11(8): e0160872. doi:10.1371/journal. pone.0160872
Editor: Michael J Proulx, University of Bath, UNITED
KINGDOM
Received: January 12, 2016
Accepted: July 26, 2016
Published: August 5, 2016
Copyright: © 2016 Kolarik et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited. Citation: Kolarik AJ, Scarfe AC, Moore BCJ,
Pardhan S (2016) Echoic Sensory Substitution
Information in a Single Obstacle Circumvention Task. PLoS ONE 11(8): e0160872. doi:10.1371/journal. pone.0160872 Editor: Michael J Proulx, University of Bath, UNITED
KINGDOM Editor: Michael J Proulx, University of Bath, UNITED
KINGDOM Received: January 12, 2016
Accepted: July 26, 2016
Published: August 5, 2016 Received: January 12, 2016
Accepted: July 26, 2016
Published: August 5, 2016 Copyright: © 2016 Kolarik et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited. Data Availability Statement: All relevant data are
within the paper and its Supporting Information files. Echoic Sensory Substitution can also help visually impaired individuals to circumvent obstacles. The current study
addressed three questions: 1) Can SSD information be used to create a ‘protective-envelope’, or
buffer space, to help protect people during visionless navigation? 2) What are the exact kine-
matics of obstacle circumvention using an echoic SSD, and how do they compare to those for
echolocation-guided locomotion, as measured previously [6]? 3) Do blindfolded participants
using an SSD pass on the side of an obstacle affording the most space, as they typically do
when visual information is available? Competing Interests: The authors have declared
that no competing interests exist. Previous work has investigated obstacle avoidance under visual guidance [7], and using sen-
sory substitution information when vision is absent [8–11]. Some SSDs convert visual informa-
tion into an auditory or a haptic signal using a predetermined transformation algorithm [12]. Such SSDs, referred to here as visual pattern SSDs, include the vOICe (the central letters stand
for “oh I see,” [13–15]), and the “Prosthesis Substituting Vision with Audition” (PSVA, [16,
17]). Other SSDs, referred to here as echoic SSDs, work on an echolocation principle, and
include Kay’s Advanced Spatial Perception Aid (KASPA, [18]) and the Miniguide [19, 20]. Using an ultrasound source and a receiver, an echoic SSD detects signal echoes, which are used
to calculate the distance to an obstacle using the time delay between the emission and echo. This information is then converted into a haptic or auditory signal. A number of studies have demonstrated that echoes from self-generated sounds can be used
to perceive the spatial location of an obstacle, and to approach it as closely as possible without
touching it, for both blind [21–24] and blindfolded sighted participants [22, 25, 26]. Spatiotem-
poral flow fields from echoic SSDs can also provide information regarding the layout of the
environment to inform safe locomotion. Maidenbaum et al. [8] showed that blindfolded-
sighted participants were able to use an echoic SSD (Eyecane) for distance estimation, naviga-
tion and obstacle detection. Chebat et al. [10] showed that blind participants and blindfolded-
sighted controls could use the Eyecane to navigate through real and virtual mazes. In virtual
environments, the increased sensory range of 5 m provided by a virtual Eyecane allowed partic-
ipants to take shorter navigation paths and make fewer collisions than when a virtual white
cane was used [9]. Introduction A requirement of travelling through urban environments is the ability to safely navigate around
obstacles [1]. Visual loss substantially reduces the spatial information available for safe locomo-
tion, although aids such as canes and guide dogs can partially compensate for this. Echoes
from self-generated sound (for reviews, see [2–4]), or from electronic travel aids called sensory
substitution devices (SSDs), which convey spatial information via an unimpaired modality [5], 1 / 16 PLOS ONE | DOI:10.1371/journal.pone.0160872
August 5, 2016 PLOS ONE | DOI:10.1371/journal.pone.0160872
August 5, 2016 Although SSDs can be highly useful as a navigation aid, collisions do sometimes occur. Levy-Tzedek et al. [27] reported that the rate of collisions was higher using the Eyecane SSD
than using vision to navigate in virtual mazes. Veraart et al. [28] reported that binocularly
blinded cats were able to use echoic SSD information to assess depth in a jumping task and to
avoid obstacles when moving through a maze. Veraart and Wanet-Defalque [29] showed that
use of an echoic SSD by early-blind humans increased performance for judging the distance
and direction of obstacles located along various routes. Hughes [30] reported that echoic SSD
information allowed blindfolded normally sighted participants to judge whether narrow aper-
tures were passable in the majority of trials. Kolarik et al. [20] showed that echoic SSD informa-
tion allowed blindfolded participants to make the shoulder rotations needed to move through
narrow apertures. As expected, compared to visual guidance, shoulder rotations made under
SSD guidance were greater, movement times were longer, and collisions sometimes occurred. Hicks et al. [11] developed a depth-based visual display as an assistive device for people with
residual vision. Sighted participants were able to use the device to navigate an obstacle course,
and partially sighted individuals were able to respond to illuminated objects presented to their
residual visual fields using the device. Chebat et al. [31] showed that blind and normally sighted blindfolded participants were
able to navigate an obstacle course using a visual pattern SSD called the Tongue Display Unit
(TDU). Blind and sighted participants successfully avoided large obstacles on approximately
78% and 71% of trials, respectively. Kolarik et al. [6] showed that echoes from sounds that were
self-generated by sighted blindfolded participants could effectively guide locomotion around 2 / 16 PLOS ONE | DOI:10.1371/journal.pone.0160872
August 5, 2016 Echoic Sensory Substitution an obstacle in the majority of trials. Performance was evaluated by successful avoidance as well
as using 3-dimensional motion capture to quantify various indices including buffer space (the
clearance between the shoulders and the obstacle, following Franchak et al. [32], who used the
term buffer space to indicate the margin between the body and sides of an aperture during
locomotion). Compared to visual guidance, buffer space, movement times, and the number of
velocity corrections were greater when using echolocation. Under visual navigation, people appear to generate a protective envelope during locomotion
that allows sufficient time and distance to perceive hazards in the local environment, and plan
gait adaptations to avoid collisions [33]. If SSD information can be used to generate a protec-
tive envelope during visionless navigation, then under SSD guidance participants should be
able to navigate around an obstacle on the majority of trials without collision and they should
do this in an efficient manner once the obstacle has been detected. We assessed whether this
was the case. Locomotion under visual guidance was measured to provide a baseline for com-
parison. The experiment was designed to allow comparison with a previous study [6] using
echoes from self-generated mouth clicks, as well as a no-click/no-vision condition in which
participants wore blindfolds and did not produce mouth clicks, as the task, procedure, and
kinematics measured were similar to those used by Kolarik et al. [6]. In their review of the effectiveness of electronic mobility devices, Roentgen et al. [34] noted
that although existing studies showed that the use of SSDs by those with visual impairment was
generally beneficial, no standardized methods or existing measurement instruments were used
in the studies that were reviewed (apart from one study that reported preferred walking speed). Kinematic measures of SSD-guided obstacle circumvention have not been obtained in previous
studies, and an examination of the kinematics would be relevant to blindness and rehabilitation
training, providing objective assessments of how practical and efficient SSD information is for
everyday navigation. Although previous work has shown that SSDs can help prevent collision
with obstacles [31], other important practical information is lacking, including an assessment
of the time needed to scan and safely navigate around an obstacle using an SSD and the size of
the buffer space. Reduced walking speed, indicated by increased movement time, is also an
important safety mechanism, since if a collision does occur it is less likely to result in injury. Previous research [35] demonstrated that participants with substantially reduced visual fields
slowed down and increased the clearance between the obstacle and body when navigating
around multiple obstacles. The participants in that study appeared to optimize safety (collision
avoidance) at the cost of spending more energy (greater clearance). The measure “velocity corrections” was chosen for the present study to characterize the flu-
idity of movement and navigation around an obstacle. PLOS ONE | DOI:10.1371/journal.pone.0160872
August 5, 2016 Participants 12 participants took part (8 males and 4 females, mean age 31 yrs, range 21–42 yrs). All had
normal or near-normal hearing, defined as better-ear average (BEA) hearing thresholds across
the frequencies 500, 1000, 2000 and 4000 Hz 25 dB HL, as measured using an Interacoustics
AS608 audiometer. All participants reported normal or corrected-to-normal vision. None of
the participants reported having any prior experience with SSDs. The experiments followed the
tenets of the Declaration of Helsinki. Written informed consent was obtained from all partici-
pants following an explanation of the nature and possible consequences of the study. The
experiments were approved by the Anglia Ruskin University Ethics committee. If participants navigating under SSD
guidance frequently stop and start, this would have implications for the practicality of use in
non-laboratory settings, and may also affect energy consumption. In addition, we investigated
whether participants passed obstacles on the side affording the most space under SSD guid-
ance, as has been previously shown for vision [7]. We also measured initial path deviations (the
distance from the obstacle where participants first deviated from moving in a straight line),
indicative of path planning. Kinematic information may highlight possible limitations in the
practicality of SSDs outside a research setting [36]. In summary, the current study measured
for the first time the kinematics of SSD-guided single-obstacle circumvention. The kinematics
were directly compared to those for echolocation-guided obstacle circumvention, as measured
in a previous study [6]. We hypothesized that echoic SSD information would enable blindfolded normally sighted
participants to safely navigate around a single obstacle in the majority of trials, passing on the
side affording the most space. Navigation under SSD guidance was hypothesized to be less PLOS ONE | DOI:10.1371/journal.pone.0160872
August 5, 2016 3 / 16 Echoic Sensory Substitution accurate than in a visual baseline condition, indicated by a greater number of collisions and
velocity corrections, larger buffer space, and longer movement times. accurate than in a visual baseline condition, indicated by a greater number of collisions and
velocity corrections, larger buffer space, and longer movement times. Apparatus and data acquisition doi:10.1371/journal.pone.0160872.g001 doi:10.1371/journal.pone.0160872.g001 4 / 16 PLOS ONE | DOI:10.1371/journal.pone.0160872
August 5, 2016 Echoic Sensory Substitution against their side when using the SSD to prevent arm movements in an anterior/posterior
direction and to ensure that the hand holding the device was perpendicular to the body, in
order to standardize the SSD feedback received across the trials without any influence from the
movements of the arm [20]. The experimenter recorded the trials when a collision occurred between any part of the par-
ticipant’s body or the SSD and the obstacle, and whether the participant passed the obstacle on
the left or right side. An 8-camera motion capture system (Vicon Bonita; Oxford Metrics Ltd)
collected 3-D kinematic data at 50 Hz. Retro-reflective spherical markers were attached bilater-
ally to the participant at the following anatomical locations: the antero-lateral and postero-lat-
eral aspects of the head, the most distal, superior aspect of the 1st toe, the most distal, superior
aspect of the 5th toe, the posterior aspect of the calcanei, and the acromio-clavicular joint. A
marker was also placed on the posterior aspect of the dominant hand and another on the ster-
num. In order to define the obstacle within the Vicon coordinate system, three markers were
attached to the front aspect of the obstacle. Marker trajectory data were filtered using the cross
validatory quintic spline smoothing routine, with ‘smoothing’ options set at a predicted mean-
squared error value of 10 and processed using the Plug-in gait software (Oxford Metrics). Although reflections from the aluminium foil covering the obstacle were sometimes recorded
by the Vicon system, care was taken to label each marker individually for each trial to exclude
h
fl
i
f
h
l Although reflections from the aluminium foil covering the obstacle were sometimes recorded
by the Vicon system, care was taken to label each marker individually for each trial to exclude
these erroneous reflections from the analyses. Kinematic variables for the obstacle circumvention task were assessed using custom written
Visual Basic scripts (Table 1). A velocity correction was deemed to occur when the participant
accelerated or decelerated in the anterior/posterior direction. To avoid including very small
fluctuations in velocity, this was required to occur for the duration of 50 frames (1 sec). Stop-
ping and starting were counted as velocity corrections, as were slowing down and speeding up. Apparatus and data acquisition Testing took place in a quiet room measuring 5.7 × 3.5 m with a ceiling height of 2.8 m, with
an ambient sound level of approximately 36 dBA. The floor was carpeted, the walls were
painted, and the ceiling was tiled. The obstacle measured 0.6 (width) × 2 m (height). It was con-
structed of wood and was covered by smooth aluminium foil, following Arnott et al. [37], to
achieve high reflectivity. It was movable, flat and rectangular with a thickness of 0.6 cm, with a
small plastic triangular frame at the bottom on the side away from the participant, mounted on
castors. See Fig 1 for a schematic of the experimental layout. The obstacle was positioned in the
approximate center of the room to minimize SSD reflections from surfaces other than the
obstacle. The experimenter and participants maintained silence during testing. The SSD used in the study was the Miniguide, manufactured by GDP Research [19, 20]. The
device emitted an ultrasound signal. The distance between the device and the obstacle was esti-
mated from the time taken for the emitted signal to reflect back from the obstacle to a receptor
on the device. Spatial information was provided via a tactile vibration signal; the rate of vibra-
tion was proportional to the distance between the device and the obstacle. The range of the
device (the distance over which an obstacle could be detected) was set to 1 m [20]. The device
was held in the participant’s dominant hand. Participants were instructed to tuck their elbow Fig 1. Two views of the layout for obstacle circumvention. The 0.6-m wide obstacle was either straight
ahead on the midline relative to the participant, or ±25 cm to the left or right (right part of figure), at an
approach distance of either 1.5 or 2 m (left part of figure). doi:10 1371/journal pone 0160872 g001 Fig 1. Two views of the layout for obstacle circumvention. The 0.6-m wide obstacle was either straight
ahead on the midline relative to the participant, or ±25 cm to the left or right (right part of figure), at an
approach distance of either 1.5 or 2 m (left part of figure). Apparatus and data acquisition A change in trajectory was not deemed to be a velocity correction unless it involved a forward
acceleration/deceleration. If the participant took a side step without slowing down in the ante-
rior/posterior direction then this was not counted. However if they stopped, took a side step
and then continued forward again, this would count as two velocity corrections (1 for deceler-
ating prior to the side step, and 1 for accelerating after the side step). Procedures In the first 10 minutes, the
obstacle was placed straight ahead in front of the participant, and for the last 5 minutes the
location of the obstacle was varied randomly among three positions: straight ahead and 25 cm
to the participants’ left or right. In the testing phase, participants were blindfolded and instructed to maintain a straight line
of travel until the obstacle was detected using the device and then to circumnavigate around
the obstacle without collision. Participants were told that the obstacle would sometimes be
absent, and in this case they should maintain a straight line of travel until the experimenter
instructed them to stop. Trials were terminated when the participant had successfully moved
past the obstacle, when a collision occurred, or, for trials where the obstacle was absent, when
they moved more than 2 m forward from the starting position. Participants commenced each
trial from the same starting position, and the obstacle was initially 1.5 m from the participant
for half of the trials and 2 m away for the other half, selected randomly. These distances were
shorter than typically used for visual obstacle circumvention tasks (e.g. 5 m [1, 7]), as pilot data
showed that participants often stopped a short distance in front of the obstacle and then
explored it using the SSD, so a longer distance was not required. This has also been shown for
locomotion through apertures using an SSD [20], or using echolocation to avoid an obstacle
[6]. The obstacle was randomly varied in lateral location relative to the participant (midline, or
25 cm to the right or left), with 3 repetitions for each obstacle location, and 6 ‘no obstacle’
catch trials. In total, each participant completed 24 trials. The full vision condition consisted of a testing phase only, similar to previous studies of
visually guided navigation around an obstacle [6, 7]. This was the same as for the SSD testing
phase, except that participants were blindfolded between trials only and they removed the
blindfold at the start of each trial when signaled by the experimenter. For both conditions, before each trial, the participants’ feet were aligned against a removable
plastic box so that they faced directly forward, to ensure that they would walk straight ahead
(this was checked by the experimenter). A shoulder tap from the experimenter signaled the
beginning of the trial. Procedures There were two locomotion guidance conditions: SSD and full vision. Following previous stud-
ies [6, 20], the vision condition was performed second to avoid ‘training’ the participant with
regard to the range of distances and obstacle positions tested. Table 1. Kinematic variables assessed for the single obstacle circumvention task. Table 1. Kinematic variables assessed for the single obstacle circumvention task. Variable
Buffer space
The medio-lateral distance between the shoulder marker and obstacle, at the point of
passing the obstacle, specified as the point where the shoulder marker passed the
marker attached to the front aspect of the obstacle. Movement time
The time taken to complete the movement, measured from when the sternum marker
was 1 m from the obstacle in the anterior-posterior direction, until the point of passing
the obstacle marker (or the spatial position of the virtual obstacle marker for ‘no
obstacle’ catch trials). Velocity
corrections
The number of changes in velocity i.e. changes from acceleration to deceleration or vice
versa, measured from when the sternum was 1 m away from the obstacle until the point
of passing the obstacle marker (or the spatial position of the virtual obstacle marker for
‘no obstacle’ catch trials). See main text for additional information. Initial path
deviation
The distance between the obstacle marker and the sternum marker when the final
lateral change of direction occurred prior to the trajectory of the sternum marker
departing from that for the average of the obstacle-absent trials by more than ±1
standard deviation (SD, measured within participants). doi:10.1371/journal.pone.0160872.t001 5 / 16 Echoic Sensory Substitution The SSD condition consisted of a training phase followed by a testing phase. Previous stud-
ies that investigated the use of echoic SSDs for passing through apertures trained participants
for approximately 5 minutes [30, 38]. We provided more extensive training than used in these
studies; participants practiced navigating around the obstacle using the SSD from an approach
distance of 1.75 m for a minimum of 15 minutes. For the first 5 minutes the participants were
allowed to keep their eyes open, for the next five minutes they were encouraged to close their
eyes, and for the last five minutes they were blindfolded [6, 20]. Procedures Once completed, the experimenter led participants back to the starting
point, and stood in the same place to the side for the duration of each trial. Except when navi-
gating under visual guidance, participants wore close-fitting blindfolds, and their ears were
occluded using headphones with sound-attenuating muffs. Participants were not allowed to
use their hands to touch the obstacle and no feedback was provided during the testing phase. The sound-attenuating headphones prevented participants from hearing the obstacle being
moved (this was checked during pilot testing; participants were asked to report whether the
obstacle was present or had been moved at the start of each trial while blindfolded. None
reported being able to do this). The experiment took approximately 1.5 hours to complete for
each participant. Results The proportions of obstacle-present trials for which no collisions occurred under SSD guidance
were 0.90, 0.96, and 0.92 for obstacles located to the left, midline and right, respectively. No col-
lisions occurred under visual guidance. See S1 File for participant data. The proportion of suc-
cessful obstacle circumventions under SSD and visual guidance was substantially greater than
for the no-click/no-vision condition of Kolarik et al. [6], for which the proportion of obstacle-
present trials when no collisions occurred was 0.22, 0.22 and 0.06, for obstacles located to the
left, midline and right, respectively. The results of Kolarik et al. [6] suggest that under condi-
tions of no-click/no-vision guidance participants had severe difficulty even detecting the pres-
ence of the obstacle, and were not able to pass the obstacle on the side affording the most space
or to generate a protective envelope to avoid collision in the majority of trials. Fig 2 shows trajectories for a representative participant under visual guidance (dashed line)
and SSD guidance (solid line). The obstacle was 25 cm to the left (black rectangle). Under SSD
guidance, participants showed distinct deviations from the straight-ahead direction only when
the obstacle was within the operating range of the SSD (1 m from the obstacle), whereas under
visual guidance, participants deviated from straight ahead immediately upon initiating their
movement. Under SSD guidance, the mean distances from the obstacle at which participants
deviated by more than ± 1 SD from obstacle absent direction (with standard errors in parenthe-
ses) were 752 (46), 776 (51), and 782 (59) mm for the left, midline, and right obstacles, respec-
tively. Under both visual and SSD guidance participants moved roughly straight ahead in no-
obstacle catch trials, indicating that they only deviated markedly from straight ahead when an
obstacle was present. Fig 3 shows that for obstacle-present trials on which collisions did not occur, buffer space
was greater under SSD guidance than under visual guidance for all obstacle positions. The
mean buffer spaces under SSD guidance were 480, 422, and 468 mm for the left, midline, and
right obstacles, respectively, while those under visual guidance were 233, 196, and 233 mm,
respectively. There was a main effect of guidance condition [F(1, 11) = 42.4, p = 0.001] but no
effect of obstacle location. Statistical analyses Unless otherwise stated, repeated-measures analyses of variance (ANOVAs) were used to ana-
lyze how the buffer space, the side of obstacle chosen (right or left), the overall movement time,
and the number of velocity corrections were affected by guidance condition (SSD and vision), 6 / 16 PLOS ONE | DOI:10.1371/journal.pone.0160872
August 5, 2016 Echoic Sensory Substitution obstacle lateral location (left, midline, or right relative to the participant), and repetition (trial
1–3). The significance level was chosen as p<0.05. As preliminary analyses indicated that
scores for all measures were not significantly different for the two approach distances
(p>0.05), the results for these were pooled. Proportional data for side of avoidance were sub-
jected to arcsine transformation prior to analysis, as recommended by Howell [39]. Post hoc
analyses were performed using Bonferroni correction. PLOS ONE | DOI:10.1371/journal.pone.0160872
August 5, 2016 Results To assess the variability of the buffer space, the SD of the buffer
space across trials was calculated for each participant and each condition, and then the SDs
were averaged across participants for each condition. Fig 4 shows the variability estimated in
this way under SSD and visual guidance. Variability under SSD guidance (100, 69, and 119 mm
for the left, midline, and right obstacles, respectively) was greater than under vision (39, 33,
and 35 mm, respectively), suggesting that participants were less accurate in judging the posi-
tion of the nearest edge of the obstacle under SSD guidance than under vision. Under both SSD and visual guidance, participants generally passed the obstacle on the side
that afforded the most space. In other words, participants mostly passed on the right side when
the obstacle was positioned to the left, and passed on the left side when the obstacle was posi-
tioned to the right (Fig 5). The data for the side that was chosen showed a main effect of obsta-
cle position (F(2, 22) = 75.01, p = 0.001), but not guidance condition (F(1, 11) = 0.58, p = 0.46),
and a significant interaction between obstacle position and guidance condition (F(2, 22) = 7.95,
p = 0.003). In both conditions, the right side was chosen significantly more often when the 7 / 16 PLOS ONE | DOI:10.1371/journal.pone.0160872
August 5, 2016 Echoic Sensory Substitution Fig 2. Representative participant trajectories under visual guidance (dashed line) and SSD guidance
(solid line) when circumventing an obstacle (black rectangle). Data are shown for the left shoulder
marker, for a participant approaching the obstacle from a distance of 1.5 m. The obstacle’s lateral location
was 25 cm to the left of the participant. doi:10 1371/journal pone 0160872 g002 Fig 2. Representative participant trajectories under visual guidance (dashed line) and SSD guidance
(solid line) when circumventing an obstacle (black rectangle). Data are shown for the left shoulder
marker, for a participant approaching the obstacle from a distance of 1.5 m. The obstacle’s lateral location
was 25 cm to the left of the participant. Fig 2. Representative participant trajectories under visual guidance (dashed line) and SSD guidance
(solid line) when circumventing an obstacle (black rectangle). Data are shown for the left shoulder
marker, for a participant approaching the obstacle from a distance of 1.5 m. The obstacle’s lateral location
was 25 cm to the left of the participant. Results Fig 3. Mean buffer space between the shoulder and obstacle at the point of passing the obstacle, for
each obstacle location. Open bars show buffer space under SSD guidance and grey bars show buffer
space using vision. Here and in subsequent figures error bars denote ±1 standard error. Fig 3. Mean buffer space between the shoulder and obstacle at the point of passing the obstacle, for
each obstacle location. Open bars show buffer space under SSD guidance and grey bars show buffer
space using vision. Here and in subsequent figures error bars denote ±1 standard error. doi:10.1371/journal.pone.0160872.g003 PLOS ONE | DOI:10.1371/journal.pone.0160872
August 5, 2016 8 / 16 Echoic Sensory Substitution Fig 4. Mean buffer space variability under SSD guidance (open bars) and visual guidance (grey bars)
for each obstacle location. doi:10.1371/journal.pone.0160872.g004 Fig 4. Mean buffer space variability under SSD guidance (open bars) and visual guidance (grey bars)
for each obstacle location. Fig 4. Mean buffer space variability under SSD guidance (open bars) and visual guidance (grey bars)
for each obstacle location. doi:10.1371/journal.pone.0160872.g004 doi:10.1371/journal.pone.0160872.g004 obstacle was on the left than when it was on the midline or the right, and when the obstacle
was on the midline than when it was on the right (all p < 0.01). The mean movement times to circumvent the obstacle, and the mean number of velocity
corrections (Fig 6, upper and lower panels respectively) were larger with the SSD than with
vision. When navigating using the SSD, the mean movement times were 11, 11 and 10 s for
obstacles located to the left, midline and right, respectively, compared to 1.1, 1.2, and 1.1 s,
respectively, for vision. There was a main effect of guidance condition [F(1, 11) = 28.96,
p = 0.001], but no effect of obstacle location. When navigating using the SSD, the mean num-
bers of velocity corrections were 23, 21, and 20 for obstacles that were located to the left, mid-
line and right, respectively, while using vision they were 4, 5, and 4, respectively. There was a
significant main effect of guidance condition [F(1, 11) = 31.05, p = 0.001], but not of obstacle The mean movement times to circumvent the obstacle, and the mean number of velocity
corrections (Fig 6, upper and lower panels respectively) were larger with the SSD than with
vision. Results When navigating using the SSD, the mean movement times were 11, 11 and 10 s for
obstacles located to the left, midline and right, respectively, compared to 1.1, 1.2, and 1.1 s,
respectively, for vision. There was a main effect of guidance condition [F(1, 11) = 28.96,
p = 0.001], but no effect of obstacle location. When navigating using the SSD, the mean num-
bers of elocit corrections
ere 23 21 and 20 for obstacles that
ere located to the left mid p
y
g
p = 0.001], but no effect of obstacle location. When navigating using the SSD, the mean num-
bers of velocity corrections were 23, 21, and 20 for obstacles that were located to the left, mid-
line and right, respectively, while using vision they were 4, 5, and 4, respectively. There was a
significant main effect of guidance condition [F(1, 11) = 31.05, p = 0.001], but not of obstacle
location. p = 0.001], but no effect of obstacle location. When navigating using the SSD, the mean num-
bers of velocity corrections were 23, 21, and 20 for obstacles that were located to the left, mid-
line and right, respectively, while using vision they were 4, 5, and 4, respectively. There was a
significant main effect of guidance condition [F(1, 11) = 31.05, p = 0.001], but not of obstacle
location. To investigate whether there was any reduction in movement speed simply due to being
blindfolded, regardless of obstacle circumvention, movement times were assessed for no-obsta-
cle catch trials. The mean movement times were 1, 5, and 8 s when navigating using vision,
SSD guidance, and in the no-click/no-vision condition of Kolarik et al. [6], respectively. The
mean numbers of velocity corrections were 4, 9, and 20 using vision, the SSD and in the no-
click/no-vision condition, respectively. These findings suggest that participants moved more
slowly when using the SSD than when using vision mainly because of the absence of vision,
rather than because of SSD use per se. Fig 5. Percentage of times the right side was chosen for each obstacle location under SSD guidance
(open bars) and visual guidance (grey bars) for each obstacle location. doi:10 1371/journal pone 0160872 g005 Fig 5. Percentage of times the right side was chosen for each obstacle location under SSD guidance
(open bars) and visual guidance (grey bars) for each obstacle location. Fig 5. PLOS ONE | DOI:10.1371/journal.pone.0160872
August 5, 2016 Discussion The results show that: 1) echoic SSD information guided locomotion by blindfolded partici-
pants in a single-obstacle circumvention task with 93% accuracy; 2) Buffer space using the SSD
was larger by a factor of 2.1 than for visual guidance; 3) Movement times were longer by a fac-
tor of 9.4, and number of velocity corrections were larger by a factor of 5 for SSD than for visual
guidance; 4) Using SSD information, participants generally passed the obstacle on the side
affording the most space. These results can be compared to those for a previous study in which
blindfolded, normally sighted, non-expert echolocators performed a similar task using echolo-
cation to guide their movements around an obstacle [6]. In that study, participants circum-
vented the obstacle without collision in 67% of the trials. Compared to visual guidance, the
buffer space was larger by a factor of 1.8, movement times were longer by a factor of 27, and
number of velocity corrections was larger by a factor of 14. Also, in that study participants did
not generally pass on the side of the obstacle affording the most space. The results suggest that
for blindfolded participants, locomotion using an echoic SSD is more accurate and faster than
when using echolocation based on self-generated sounds. Further investigation is needed to
test the magnitude of deviation from the midline required for participants to choose the path
affording most space when using SSD guidance. The current findings are consistent with the idea that echoic SSD information enables a pro-
tective envelope to be generated during locomotion, similar to that generated using visual
information. The buffer space was larger under SSD guidance than under visual guidance. One
possible reason for this is that the wavelength of ultrasound imposes a limit on the spatial detail
available from echoic SSDs [40, 41]. The distance and location information available with the
SSD were less precise than provided by vision, and participants may have allowed for this (per-
haps without conscious awareness) by increasing the size of the protective envelope under SSD
guidance. Consistent with this, the measure of variability of the buffer space was greater under
SSD guidance than under vision. Another possibility is that the size of the protective envelope
was not altered by the perceptual system, but buffer space increased due to misjudging the
obstacle location or overestimating the obstacle width. Results Percentage of times the right side was chosen for each obstacle location under SSD guidance
(open bars) and visual guidance (grey bars) for each obstacle location. doi:10.1371/journal.pone.0160872.g005 doi:10.1371/journal.pone.0160872.g005 PLOS ONE | DOI:10.1371/journal.pone.0160872
August 5, 2016 9 / 16 Echoic Sensory Substitution Fig 6. Mean movement times (upper panel) and numbers of velocity corrections (lower panel) under
SSD guidance (open bars), and visual guidance (grey bars) for each obstacle location. doi:10.1371/journal.pone.0160872.g006 Fig 6. Mean movement times (upper panel) and numbers of velocity corrections (lower panel) under
SSD guidance (open bars), and visual guidance (grey bars) for each obstacle location. Fig 6. Mean movement times (upper panel) and numbers of velocity corrections (lower panel) under
SSD guidance (open bars), and visual guidance (grey bars) for each obstacle location. doi:10.1371/journal.pone.0160872.g006 doi:10.1371/journal.pone.0160872.g006 doi:10.1371/journal.pone.0160872.g006 PLOS ONE | DOI:10.1371/journal.pone.0160872
August 5, 2016 Discussion A third possibility is that the increased
buffer space under SSD guidance was partly due to increased postural sway, since the partici-
pants were blindfolded. Franchak et al. [32] described how a larger buffer space may be PLOS ONE | DOI:10.1371/journal.pone.0160872
August 5, 2016 10 / 16 Echoic Sensory Substitution produced either when the participant is exercising greater caution to avoid high speed collision
or when lateral sway increases, requiring larger spatial buffers for passage. One potential limitation of our study arises from the possible obstacle locations being fixed. Participants may have learned these locations during the last part of the training or early in the
main experiment and might then have required only minimal cues from the SSD to move in an
appropriate way. However, we think that this is unlikely, for the following reasons. Firstly,
seven possible predetermined locations were used (left, right, or directly ahead, at closer and
further distances, and, for catch trials, the obstacle was absent). During training only a single
distance was used (different to the distances used in the testing phase) and each lateral position
was presented only once or twice. During the testing phase, each location was used only three
times (plus six catch trials) and participants were not informed about the number of possible
locations, minimizing the opportunity for learning. Consistent with this, there was no signifi-
cant effect of repetition, indicating that performance did not improve over trials. Thus, we
believe it is highly unlikely that participants learned the possible locations of the obstacle. The present results are consistent with previous work suggesting that the representation of
space is amodal [42], although they are also consistent with other interpretations; see [43] for a
review. Blindfolded participants were able to generate a representation of space using SSD
vibration information and adopt a strategy that is similar to the one used during visual naviga-
tion, as has been shown in previous work [10]. The finding that movement times and velocity
corrections were greater under SSD guidance than under vision is consistent with results from
a recent study [27] which showed that the number of pauses and time taken to complete a trial
when navigating through virtual mazes were greater when using an SSD than when using
vision. Next, we discuss possible interpretations of how participants use sensory information
from a substituted modality to navigate around an obstacle. PLOS ONE | DOI:10.1371/journal.pone.0160872
August 5, 2016 Discussion In terms of the representation-based control approach,
increasing the range of the SSD may allow participants to plan their movements further in
advance, improving circumvention performance. A previous study using the EyeCane SSD
with a 5 m range showed that participants were indeed able to plan their movements in a maze
accurately and represent the configuration of the maze [10]. An alternative, information-based control approach [49, 50] is based on the assumption
that the perceptual system uses some law of control to guide navigation on a moment-by-
moment basis, using information from one or more relevant variables, such as optic flow in the
case of vision [51]. Advance path planning or internal models of space are not needed to avoid
an obstacle, and the variable used for control of movement may not even provide information
regarding spatial layout. In our experiment, participants may have avoided the obstacle by
using SSD information to inform local steering dynamics, rather than by planning the route in
advance using an internal representation of space. The variable used under SSD guidance may
have been less informative than the variable used under visual guidance, leading to increased
buffer space, velocity corrections and movement times for the former. On the other hand, the
variable used under SSD guidance may have been more informative than the variable used by
the non-expert echolocating participants tested in a previous study [6], leading to faster and
more accurate performance for the former. The representation-based and information-based control approaches are not mutually
exclusive, and both approaches can provide time-to-contact information. Information regard-
ing time-to-contact to an obstacle can be obtained using visual or auditory information [52]. By monitoring the rate of change of a relevant variable such as echoic intensity when approach-
ing an obstacle, accuracy in locating its position may increase [25, 53]. In the current study,
participants could have monitored the rate of change in SSD vibration to judge their position
relative to the obstacle and to avoid contacting the obstacle. Discussion According to representation-based control approaches [44, 45] to path planning (see [46]
for a review of visually guided obstacle avoidance), participants may use the SSD to gather
information about the spatial positions of the boundaries of the obstacle as they move, thus
generating an internal perceptual representation similar to that obtained over a series of sac-
cadic eye movements [47] or when combining information from a series of echolocation clicks
made while moving the head [48]. Such internal representations are then used to make loco-
motor adjustments, similar to using visual information during locomotor actions, where repre-
sentation of the body may also play a critical role [46]. The findings of the current study and of
our previous study [20] suggest that, across different tasks (single obstacle circumvention and
aperture navigation) and substituted modalities (haptic and auditory), internal representations
based on the SSD and echoes from self-generated sound are less accurate than those based on
vision. In the current study, less accurate internal representations with the SSD may have led to
increased buffer space, velocity corrections and movement times. However, internal represen-
tations derived from the SSD may have been more accurate than those derived by non-expert
echolocators using self-generated sounds [6]. According to the representation-based control approach, an action path is planned based
on the internal representation, prior to obstacle circumvention. The greater the effective range
over which the obstacle’s position can be detected, the further in advance can the path be
planned, potentially allowing minimization of energy expenditure and reducing the chance of
collision. In our study, vision provided an effective range covering the full length of the room
(5.7 m). Thus, participants could plan their path from the start of each trial. The effective range
for non-expert echolocators performing a similar obstacle circumvention task was approxi-
mately 60 cm [6]. In the current study the range of the SSD was set to 1 m, following a previous
study of locomotion through apertures [20]. Under SSD guidance, participants deviated from a
straight-ahead path approximately 77 cm from the obstacle. Overall, these results indicate that 11 / 16 Echoic Sensory Substitution participants were able to plan their movements from the onset of the trial under visual guid-
ance, whereas under SSD guidance they planned their movement once they were within the
effective range of the SSD (1 m). PLOS ONE | DOI:10.1371/journal.pone.0160872
August 5, 2016 Discussion It is possible that echoic SSDs provide an aspect of vision (depth) more efficiently than
echolocation alone, and for this reason the participants in the current study were able to pass
on the side affording the most space in the majority of trials, whereas non-expert echolocators
in a previous study [6], who did not pass on the side affording the most space in the majority of
trials, did not have access to this information. The blindfolded sighted participants moved
more slowly under SSD guidance than under visual guidance, but this was probably caused by
the presence/absence of vision rather than to SSD use per se. Congenitally blind participants
might not take longer to navigate around an obstacle with an SSD than without one, as they are
used to navigating without vision. Chebat et al. [31] showed that blind participants were better than blindfolded sighted con-
trols at detecting and avoiding obstacles using the TDU. The improved performance of blind
than of blindfolded sighted participants when using SSDs was shown to reflect cortical reorga-
nization by Kupers et al. [54], who used the TDU to guide navigation in a virtual environment
in an fMRI scanner and demonstrated that the occipital (visual) cortex was activated in con-
genitally blind participants but not sighted controls. The activation of the occipital cortex in
blind but not sighted controls during a distance evaluation task using an echoic SSD was also
reported by De Volder et al. [55]. Such cortical reorganization may result in different move-
ment kinematics under SSD guidance between blind and sighted participants. However, this
requires testing. Peripersonal space describes space near the individual e.g. within reaching and grasping dis-
tance, and extrapersonal space is space farther from the body [4, 10, 56–58]. Sensory events in
peripersonal space often require rapid motor responses such as avoidance in response to a 12 / 16 PLOS ONE | DOI:10.1371/journal.pone.0160872
August 5, 2016 Echoic Sensory Substitution perceived threat [56], and multisensory information may be processed differently depending
on whether it is presented in peripersonal or extrapersonal space [57]. Peripersonal space can
be extended or projected by altering the reaching distance [57, 58], and Chebat et al. [10] previ-
ously suggested that use of the EyeCane extended peripersonal space, contributing to success
navigating through real or virtual mazes. Supporting Information S1 File. Participant data for number of collisions, side of obstacle avoidance, buffer space,
overall movement times, and the number of velocity corrections for each guidance condi-
tion (SSD and vision), for each obstacle lateral location. (XLSX) Discussion Given the strong adaptive value of peripersonal space
in detecting stimuli in close proximity to the body prior to contact [58] and the similar role of
the protective envelope in obstacle avoidance during locomotion, the spatial boundary of peri-
personal space may be similar to the protective envelope, and/or the buffer space measured in
the current study, however this requires further investigation. Finally, we turn to some practical aspects of the use of SSDs. In the present study, partici-
pants took approximately 11 s to move 1 m, equivalent to a walking speed of only about 0.33
km/hr. However, further training with an SSD may lead to improved performance, decreasing
collisions and resulting in smoother and faster movements. Benefits of training have been
shown previously. For example Kim and Zatorre [59] showed that long-term training with the
vOICe SSD improved shape and pattern identification of visual images using visual-to-auditory
substitution with sighted participants. Chebat et al. [31] pointed out that the visual pattern SSD that they used to assess navigation
through a maze was tested under optimal experimental conditions, and that the findings may
not generalize to less ideal real-world conditions, such as when low-contrast obstacles are
encountered in varied light conditions. The echoic SSD tested here was also tested under opti-
mal experimental conditions, and caution is needed in generalizing the findings to real-world
conditions, where, for example, noisy environments could reduce its effectiveness. Echoic SSDs
are as effective in dark and low-level light conditions as in well-lit conditions (as are self-gener-
ated echoes used for human echolocation). However, less acoustically reflective objects may
lead to lower locomotion performance based on sound echoes, while visual pattern SSDs
would not be affected by acoustic reflectivity. Further investigation comparing the two classes
of SSD across various environmental conditions may serve to provide greater insight regarding
their applicability for practical visual rehabilitation of visually impaired individuals. Acknowledgments We thank the Editor Michael Proulx, Daniel-Robert Chebat, and two anonymous reviewers,
whose extensive comments were most helpful. We also thank Silvia Cirstea for valuable early
discussions regarding an earlier experiment, and John Peters and George Oluwafemi for their
assistance in collecting data. Author Contributions Conceptualization: AJK ACS BCJM SP. References 1. Gérin-Lajoie M, Richards CL, Fung J, McFadyen BJ. Characteristics of personal space during obstacle
circumvention in physical and virtual environments. Gait Posture. 2008; 27:239–47. PMID: 17512201 2. Stoffregen TA, Pittenger JB. Human echolocation as a basic form of perception and action. Ecol Psy-
chol. 1995; 7:181–216. 3. Kolarik AJ, Cirstea S, Pardhan S, Moore BCJ. A summary of research investigating echolocation abili-
ties of blind and sighted humans. Hear Res. 2014; 310:60–8. doi: 10.1016/j.heares.2014.01.010 PMID:
24524865 4. Kolarik AJ, Moore BCJ, Zahorik P, Cirstea S, Pardhan S. Auditory distance perception in humans: A
review of cues, development, neuronal bases and effects of sensory loss. Atten Percept Psychophys. 2016; 78:373–95. doi: 10.3758/s13414-015-1015-1 PMID: 26590050 5. Bach-y-Rita P, Collins CC, Saunders FA, White B, Scadden L. Vision substitution by tactile image pro-
jection. Nature. 1969; 221:963–4. PMID: 5818337 6. Kolarik AJ, Scarfe AC, Moore BCJ, Pardhan S. An assessment of auditory-guided locomotion in an
obstacle circumvention task. Exp Brain Res. 2016; 234:1725–35. doi: 10.1007/s00221-016-4567-y
PMID: 26879767 7. Hackney AL, Van Ruymbeke N, Bryden PJ, Cinelli ME. Direction of single obstacle circumvention in mid-
dle-aged children. Gait Posture. 2014; 40:113–7. doi: 10.1016/j.gaitpost.2014.03.005 PMID: 24679592 8. Maidenbaum S, Hanassy S, Abboud S, Buchs G, Chebat DR, Levy-Tzedek S, et al. The “EyeCane”, a
new electronic travel aid for the blind: Technology, behavior & swift learning. Restor Neurol Neurosci. 2014; 32:813–24. doi: 10.3233/RNN-130351 PMID: 25201814 9. Maidenbaum S, Levy-Tzedek S, Chebat DR, Namer-Furstenberg R, Amedi A. The effect of extended
sensory range via the EyeCane sensory substitution device on the characteristics of visionless virtual
navigation. Multisens Res. 2014; 27:379–97. PMID: 25693302 10. Chebat DR, Maidenbaum S, Amedi A. Navigation using sensory substitution in real and virtual mazes. PloS One. 2015; 10:e0126307. doi: 10.1371/journal.pone.0126307 PMID: 26039580 11. Hicks SL, Wilson I, Muhammed L, Worsfold J, Downes SM, Kennard C. A depth-based head-mounted
visual display to aid navigation in partially sighted individuals. PloS one. 2013; 8:e67695. doi: 10.1371/
journal.pone.0067695 PMID: 23844067 12. Maidenbaum S, Levy-Tzedek S, Chebat DR, Amedi A. Increasing accessibility to the blind of virtual
environments, using a virtual mobility aid based on the "EyeCane": Feasibility study. PloS One. 2013;
8:e72555. doi: 10.1371/journal.pone.0072555 PMID: 23977316 13. Meijer PBL. An experimental system for auditory image representations. IEEE Transactions on Bio-
medical Engineering. 1992; 39:112–21. PMID: 1612614 14. Striem-Amit E, Guendelman M, Amedi A. 'Visual' acuity of the congenitally blind using visual-to-auditory
sensory substitution. PLoS One. Conceptualization: AJK ACS BCJM SP. Data curation: AJK ACS. Data curation: AJK ACS. Formal analysis: AJK ACS. Funding acquisition: SP BCJM. 13 / 16 PLOS ONE | DOI:10.1371/journal.pone.0160872
August 5, 2016 Echoic Sensory Substitution Investigation: AJK ACS. Methodology: AJK ACS BCJM SP. Project administration: AJK ACS BCJM SP. Resources: SP BCJM. Software: ACS. Supervision: AJK ACS BCJM SP. Validation: AJK ACS. Visualization: AJK ACS BCJM SP. Writing - original draft: AJK. Writing - review & editing: AJK ACS BCJM SP Methodology: AJK ACS BCJM SP. Project administration: AJK ACS BCJM SP. Resources: SP BCJM. Software: ACS. Supervision: AJK ACS BCJM SP. Visualization: AJK ACS BCJM SP. PLOS ONE | DOI:10.1371/journal.pone.0160872
August 5, 2016 References 2012; 7:e33136. doi: 10.1371/journal.pone.0033136 PMID: 22438894 15. Striem-Amit E, Cohen L, Dehaene S, Amedi A. Reading with sounds: Sensory substitution selectively
activates the visual word form area in the blind. Neuron. 2012; 76:640–52. doi: 10.1016/j.neuron.2012. 08.026 PMID: 23141074 PLOS ONE | DOI:10.1371/journal.pone.0160872
August 5, 2016 14 / 16 Echoic Sensory Substitution 16. Renier L, Collignon O, Poirier C, Tranduy D, Vanlierde A, Bol A, et al. Cross-modal activation of visual
cortex during depth perception using auditory substitution of vision. NeuroImage. 2005; 26:573–80. PMID: 15907314 17. Renier L, Laloyaux C, Collignon O, Tranduy D, Vanlierde A, Bruyer R, et al. The Ponzo illusion with
auditory substitution of vision in sighted and early-blind subjects. Perception. 2005; 34:857–67. PMID:
16124271 18. Kay L. A sonar aid to enhance spatial perception of the blind: Engineering design and evaluation. Radio
and Electronic Engineer. 1974; 44:605–27. 19. Vincent C, Routhier F, Martel V, Mottard M-È, Dumont F, Côté L, et al. Field testing of two electronic
mobility aid devices for persons who are deaf-blind. Disabil Rehabil Assist Technol. 2014; 9:414–20. doi: 10.3109/17483107.2013.825929 PMID: 24266810 20. Kolarik AJ, Timmis MA, Cirstea S, Pardhan S. Sensory substitution information informs locomotor
adjustments when walking through apertures. Exp Brain Res. 2014; 232:975–84. doi: 10.1007/s00221-
013-3809-5 PMID: 24370580 21. Worchel P, Mauney J, Andrew JG. The perception of obstacles by the blind. J Exp Psychol. 1950;
40:746–51. PMID: 14803650 22. Supa M, Cotzin M, Dallenbach KM. "Facial vision": The perception of obstacles by the blind. Am J Psy-
chol. 1944; 57:133–83. 23. Worchel P, Mauney J. The effect of practice on the perception of obstacles by the blind. J Exp Psychol. 1951; 41:170–6. PMID: 14841326 24. Ammons CH, Worchel P, Dallenbach KM. "Facial vision": The perception of obstacles out of doors by
blindfolded and blindfolded-deafened subjects. Am J Psychol. 1953; 66:519–53. PMID: 13124562 25. Rosenblum LD, Gordon MS, Jarquin L. Echolocating distance by moving and stationary listeners. Ecol
Psychol. 2000; 12:181–206. 26. Carlson-Smith C, Weiner WR. The auditory skills necessary for echolocation: A new explanation. J Vis
Impair Blind. 1996; 90:21–35. 27. Levy-Tzedek S, Maidenbaum S, Amedi A, Lackner J. Aging and Sensory Substitution in a Virtual Navi-
gation Task. PloS One. 2016; 11(3):e0151593. doi: 10.1371/journal.pone.0151593 PMID: 27007812 28. Veraart C, Crémieux J, Wanet-Defalque MC. Use of an ultrasonic echolocation prosthesis by early visu-
ally deprived cats. Behav Neurosci. 1992; 106:203–16. PMID: 1554432 29. Veraart C, Wanet-Defalque MC. PLOS ONE | DOI:10.1371/journal.pone.0160872
August 5, 2016 References Representation of locomotor space by the blind. Percept Psychophys. 1987; 42:132–9. PMID: 3627933 30. Hughes B. Active artificial echolocation and the nonvisual perception of aperture passability. Hum Mov
Sci. 2001; 20:371–400. PMID: 11750669 31. Chebat DR, Schneider FC, Kupers R, Ptito M. Navigation with a sensory substitution device in congeni-
tally blind individuals. NeuroReport. 2011; 22:342–7. doi: 10.1097/WNR.0b013e3283462def PMID:
21451425 32. Franchak JM, Celano EC, Adolph KE. Perception of passage through openings depends on the size of
the body in motion. Exp Brain Res. 2012; 223:301–10. doi: 10.1007/s00221-012-3261-y PMID:
22990292 33. Gérin-Lajoie M, Richards CL, McFadyen BJ. The negotiation of stationary and moving obstructions dur-
ing walking: anticipatory locomotor adaptations and preservation of personal space. Motor Control. 2005; 9:242–69. PMID: 16239715 34. Roentgen UR, Gelderblom GJ, Soede M, de Witte LP. The impact of electronic mobility devices for per-
sons who are visually impaired: A systematic review of effects and effectiveness. J Vis Impair Blind. 2009; 103:743–53. 35. Jansen SE, Toet A, Werkhoven PJ. Human locomotion through a multiple obstacle environment: Strat-
egy changes as a result of visual field limitation. Exp Brain Res. 2011; 212:449–56. doi: 10.1007/
s00221-011-2757-1 PMID: 21687987 36. Maidenbaum S, Abboud S, Amedi A. Sensory substitution: Closing the gap between basic research
and widespread practical visual rehabilitation. Neurosci Biobehav Rev. 2014; 41:3–15. doi: 10.1016/j. neubiorev.2013.11.007 PMID: 24275274 37. Arnott SR, Thaler L, Milne JL, Kish D, Goodale MA. Shape-specific activation of occipital cortex in an
early blind echolocation expert. Neuropsychologia. 2013; 51:938–49. doi: 10.1016/j.neuropsychologia. 2013.01.024 PMID: 23391560 38. Davies TC, Pinder SD, Burns CM. What's that sound? Distance determination and aperture passage
from ultrasound echoes. Disabil Rehabil Assist Technol. 2011; 6:500–10. doi: 10.3109/17483107. 2010.542569 PMID: 21184624 PLOS ONE | DOI:10.1371/journal.pone.0160872
August 5, 2016 15 / 16 Echoic Sensory Substitution 39. Howell DC. Statistical Methods for Psychology. Belmont, CA: Duxbury Press; 1997. 40. Lee DN. Body-environment coupling. In: Neisser U, editor. The perceived self: Ecological and interper-
sonal sources of self-knowledge. Cambridge: Cambridge University Press; 1993. p. 43–67. 41. Easton RD. Inherent problems of attempts to apply sonar and vibrotactile sensory aid technology to the
perceptual needs of the blind. Optom Vis Sci. 1992; 69:3–14. PMID: 1741108 42. Levy-Tzedek S, Novick I, Arbel R, Abboud S, Maidenbaum S, Vaadia E, et al. Cross-sensory transfer of
sensory-motor information: Visuomotor learning affects performance on an audiomotor task, using sen-
sory-substitution. Sci Rep. 2012; 2:949. doi: 10.1038/srep00949 PMID: 23230514 43. References Schinazi VR, Thrash T, Chebat DR. Spatial navigation by congenitally blind individuals. Wiley Interdis-
cip Rev Cogn Sci. 2016; 7:37–58. doi: 10.1002/wcs.1375 PMID: 26683114 44. Frenz H, Lappe M. Absolute travel distance from optic flow. Vision Res. 2005; 45:1679–92. PMID:
15792843 45. Turano KA, Yu D, Hao L, Hicks JC. Optic-flow and egocentric-direction strategies in walking: Central vs
peripheral visual field. Vision Res. 2005; 45:3117–32. PMID: 16084556 46. Higuchi T, Imanaka K, Patla AE. Action-oriented representation of peripersonal and extrapersonal
space: Insights from manual and locomotor actions. Jpn Psychol Res. 2006; 48:126–40. 47. Prime SL, Vesia M, Crawford JD. Cortical mechanisms for trans-saccadic memory and integration of
multiple object features. Phil Trans R Soc B. 2011; 366:540–53. doi: 10.1098/rstb.2010.0184 PMID:
21242142 48. Milne JL, Goodale MA, Thaler L. The role of head movements in the discrimination of 2-D shape by
blind echolocation experts. Atten Percept Psychophys. 2014; 76:1828–37. doi: 10.3758/s13414-014-
0695-2 PMID: 24874262 49. Fajen BR, Warren WH. Behavioral dynamics of steering, obstacle avoidance, and route selection. J
Exp Psychol Hum Percept Perform. 2003; 29:343–62. PMID: 12760620 50. Warren WH. Visually controlled locomotion: 40 years later. Ecol Psychol. 1998; 10:177–219. 51. Fajen BR. Affordance-based control of visually guided action. Ecol Psychol. 2007; 19:383–410. 52. Schiff W, Oldak R. Accuracy of judging time to arrival: Effects of modality, trajectory, and gender. J Exp
Psychol Hum Percept Perform. 1990; 16:303–16. PMID: 2142201 53. Lee DN, van der Weel FR, Hitchcock T, Matejowsky E, Pettigrew JD. Common principle of guidance by
echolocation and vision. J Comp Physiol A. 1992; 171:563–71. PMID: 1494137 54. Kupers R, Chebat DR, Madsen KH, Paulson OB, Ptito M. Neural correlates of virtual route recognition
in congenital blindness. Proc Natl Acad Sci USA. 2010; 107:12716–21. doi: 10.1073/pnas.1006199107
PMID: 20616025 55. De Volder AG, Catalan-Ahumada M, Robert A, Bol A, Labar D, Coppens A, et al. Changes in occipital
cortex activity in early blind humans using a sensory substitution device. Brain Res. 1999; 826:128–34. PMID: 10216204 56. Serino A, Canzoneri E, Avenanti A. Fronto-parietal areas necessary for a multisensory representation
of peripersonal space in humans: An rTMS study. J Cognitive Neurosci. 2011; 23:2956–67. 57. Van der Stoep N, Nijboer T, Van der Stigchel S, Spence C. Multisensory interactions in the depth plane
in front and rear space: A review. Neuropsychologia. 2015; 70:335–49. doi: 10.1016/j. neuropsychologia.2014.12.007 PMID: 25498407 58. Serino A, Bassolino M, Farnè A, Làdavas E. PLOS ONE | DOI:10.1371/journal.pone.0160872
August 5, 2016 References Extended multisensory space in blind cane users. Psych
Sci. 2007; 18:642–8. 59. Kim JK, Zatorre RJ. Generalized learning of visual-to-auditory substitution in sighted individuals. Brain
Res. 2008; 1242:263–75. doi: 10.1016/j.brainres.2008.06.038 PMID: 18602373 16 / 16
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Breaking interfacial charge transfer barrier by sulfite for efficient pollutants degradation: a case of BiVO4
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npj clean water
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INTRODUCTION particular, the holes of non-radical photocatalysts belong to short-
range active species, which can only oxidize the molecules on the
surface. The results of first-principles and ab initio molecular
dynamics studies show that water molecules mainly exist in two
interface interaction modes on the most stable (010) plane of
BiVO4: water molecules coordinating with Bi3+ sites through lone
pair electrons and interacting with oxygen terminal of VO4
tetrahedron through hydrogen bonds15–17. Strong BiVO4-water
interface interaction may significantly reduce the reaction rate of
photogenerated holes with organic molecules. However, the
influence of solvation effects on the degradation efficiency of
organics in non-radical-type heterogeneous photocatalytic sys-
tems is seriously neglected. Therefore, it is necessary to further
explore the effect of solid–liquid interface on the organic
degradation kinetics in the non-radical-type photocatalytic system
and decrease the interfacial electron transfer barrier from organic
molecule to photocatalyst. Heterogeneous photocatalysis is considered as the potential
strategy for water decontamination due to its high efficiency,
environmental benignity, cost effectiveness, and potential utiliza-
tion of solar energy1,2. The photocatalytic reaction mechanisms
can be mainly divided into radical and non-radical pathways. Radical-type photocatalysts can generate hydroxyl radical (•OH)
under light irradiation and form a gradient distribution of radical
at the solid-liquid interface3. Meanwhile, the reactivity of •OH is
independent of its production method, leading to its high
reactivity and the universality of organic degradation1. In contrast,
the holes generated by non-radical-type photocatalysts are only
localized on the surface to undergo redox reactions. Compared
with radical systems, the reactivity of non-radical-type photo-
catalysts towards organic molecules is more complicated. In
particular, BiVO4, as a typical non-radical-type photocatalyst,
exhibits inhibited surface kinetics for the degradation of organic
molecules including phenol, ciprofloxacin, and sulfonamides4–7. However, although the photogenerated hole of BiVO4 has higher
redox potential (ca. +2.5 V vs. NHE)8, the active species [•OH
(+1.8 ~ 2.7 V vs. NHE)9, high-valent metal-oxo10–12 and singlet
oxygen (1O2, +1.88 V vs. NHE)13 with lower redox potentials
generated in advanced oxidation processes exhibit higher rate
constant for organics3,14. It suggests a contradiction between the
reactivity of non-radical-type photocatalytic system for organics
and the ideal photocatalytic model controlled by thermody-
namics. Generally, similar to the overpotential theory, the redox
potential of photogenerated holes should be positively correlated
with the oxidation reaction trend of organic molecule. www.nature.com/npjcleanwater ARTICLE
OPEN
Breaking interfacial charge transfer barrier by sulfite for
efficient pollutants degradation: a case of BiVO4 Xin Gao
1, Peifang Wang1✉, Huinan Che1, Wei Liu1 and Yanhui Ao
1✉ Heterogeneous photocatalytic systems generally lack thermodynamic dependence on the degradation of organic pollutants in
aqueous solution. Therefore, it is important to reveal the reasons for the inhibited surface kinetics but still be neglected. Herein, we
reveal the mechanism that BiVO4 can’t degrade organics although it is thermodynamically feasible. The surface solvation and
formation of double layer (compact layer and diffuse layer) makes low-polarity organics far away from the surface of BiVO4. We
found that the introduction of sulfite can solve this problem. Theory calculation illustrates that sulfite can enter into the compact
layer because of its higher adsorption energy on BiVO4 and lower adiabatic ionization potential (AIP). Then, photogenerated holes
initiate the chain transformation of sulfite and produce strong oxidizing species which can diffuse out to degrade organics. This
paper provides an insight into the understand the effects of solid-liquid interface on heterogeneously photocatalytic degradation of
organic pollutants. npj Clean Water (2023) 6:42 ; https://doi.org/10.1038/s41545-023-00254-w 1Key Laboratory of Integrated Regulation and Resource Development on Shallow Lakes, Ministry of Education, College of Environment, Hohai University, No.1, Xikang road,
Nanjing 210098, China. ✉email: pfwang2005@hhu.edu.cn; andyao@hhu.edu.cn Key Laboratory of Integrated Regulation and Resource Development on Shallow Lakes, Ministry of Education, College of Environmen
anjing 210098, China. ✉email: pfwang2005@hhu.edu.cn; andyao@hhu.edu.cn Resource Development on Shallow Lakes, Ministry of Education, College of Environment, Hohai University, No.1, Xikang road,
hhu.edu.cn; andyao@hhu.edu.cn Mechanism on sulfite activation in the system y
3
To further investigate the active species generation, quenching
experiments and ESR were performed. Since SO4•– are more
sensitive to α-H, the rate constant (k) of tert-butyl alcohol (TBA)
and ethanol (EtOH) with SO4•– [kTBA;SO-
4 ¼ ð4:0 9:1Þ ´ 105M1s1,
kEtOH;SO--
4
¼ ð1:6 7:7Þ ´ 107M1s1] are quite different, while
•OH have less
effect
[kTBA;OH ¼ 6:0 ´ 108M1s1,
kEtOH;OH ¼
1:9 ´ 109M1s1]11,25. As observed in Fig. 2b, the addition of TBA
(1 mM and 10 mM) just slightly retarded the oxidation of SMX. Significant inhibitory action can be observed only when TBA
concentration is increased to 100 mM. In contrast, there is a strong
inhibition with the addition of 1 mM EtOH, and the inhibitory
effect gradually enhance with the increase of EtOH concentration
(Fig. 2c). Therefore, SO4•– may be the major active species in the
Na2SO3/BiVO4 system (kSMX;SO-
4 ¼ 0:93 ´ 109M1s1)26, while •OH
play an auxiliary role (kSMX;OH ¼ 6:78 ´ 109M1s1)12. In addition,
ESR was carried out to probe the production of active species
(Fig. 2d). Strangely, there is a distinct signal of DMPO-•OH in the
BiVO4 system under visible light, which opposite to the degrada-
tion of SMX in the BiVO4 system (kSMX;OH ¼ 6:78 ´ 109M1s1). This may originate from the hole-assisted H2O nucleophilic attack
for DMPO (Supplementary Fig. 7a), but does not actually generate
•OH25. When sulfite is added to BiVO4 suspension, the signal of
DMPO-•OH was further enhanced, indicating that the introduction
of sulfite significantly promoted the generation of strong oxidative
radicals. However, the DMPO-SO4•– adduct signal is not clearly
detected, which may be attributed to the poor stability of DMPO-
SO4•– and easy decomposition to DMPO-•OH (Supplementary
Fig. 7b)25. In addition, SO5•– are also produced during the chain
transformation of sulfite. However, according to previous reports,
SO5•– has weak reactivity, indicating that SO5•– almost did not take
part in the degradation process of SMX23. Therefore, we can
conclude that SO4•– is the predominant active species for SMX According to the activation mechanism, sulfite is converted to a
SO3•– via one-electron oxidation (Eq. 1)20. Since the ground-state
O2 is a double-radical with triplet state, it will rapidly add to the
SO3•– to form SO5•– (Eq. 2)18,23,24. Subsequently, SO5•– is reduced
by SO3•– into peroxymonosulfate (PMS) and SO4•– (Eqs. 3, 4)23. Fig. 1
Kinetics of interfacial reaction of BiVO4 activated by sulfite. Oxidation kinetics of SMX in the Na2SO3/BiVO4 system (8) According to the structural characterization (Supplementary
Figs. 1–5), BiVO4 is a monoclinic decahedral structure with
exposed (010)/(110) facets, which has an excellent visible light
response. However, no change in SMX concentration is recorded
in the presence of BiVO4 under visible light (Fig. 1), which
indicates the limited kinetics of SMX degradation on the surface
of BiVO4. Furthermore, the photolysis of SMX in the absence of
photocatalysts can be neglected, due to its lack of visible light
absorption22. Since the chain transformation of sulfite (Na2SO3)
is difficult to trigger under visible light, SMX is not degraded in
the presence of sulfite alone. In contrast, once 0.5 mM sulfite is
added to the photocatalytic system of BiVO4, SMX exhibits a
rapid degradation with a removal efficiency of 85.6% within
15 min. Therefore, it is necessary to further explore the sulfite-
mediated mechanism and predominant active species in the
Na2SO3/BiVO4 system. Initially, the photocatalytic degradation of SMX by BiVO4 in the
presence of sulfite under different atmospheres is carried out. From Fig. 2a, SMX has the highest degradation performance under
the condition of air purging, while the dissolved oxygen (DO)
concentration is rapidly consumed to the lowest at the initial
reaction stage (Supplementary Fig. 6). After that, due to stirring
and air purging, the DO concentration gradually increases. According to the sulfite activation mechanism, O2 only can be
consumed by SO3•–, implying that sulfite is rapidly oxidized at the
initial reaction stage. Furthermore, the degradation rates of SMX
slightly decrease without air purging, indicating that higher DO
concentration is conducive to the production of active species. In
addition, under anaerobic conditions (Ar purging), the degrada-
tion of SMX can be neglected, demonstrating that O2 acts as the
initiator of the active species generation and SMX is difficult to be
oxidized by SO3•–. INTRODUCTION SO
3
(1)
SO
3
þ O2 ! SO
5
(2)
SO
5
þ SO2
3
! SO
4
þ SO2
3
(3)
SO
5
þ SO2
3
! SO2
5
þ SO
3
(4)
SO
4
þ H2O ! SO2
4
þ OH þ Hþ
(5)
SO
4
þ OH ! SO2
4
þ OH
(6)
SO
4
þ SO2
3
! SO2
4
þ SO
3
(7)
OH þ SO2
3
! SO
3
þ OH
(8) SO2
3
þ hþðBiVO4Þ ! SO
3
(1)
SO
3
þ O2 ! SO
5
(2)
SO
5
þ SO2
3
! SO
4
þ SO2
3
(3)
SO
5
þ SO2
3
! SO2
5
þ SO
3
(4)
SO
4
þ H2O ! SO2
4
þ OH þ Hþ
(5)
SO
4
þ OH ! SO2
4
þ OH
(6)
SO
4
þ SO2
3
! SO2
4
þ SO
3
(7)
OH þ SO2
3
! SO
3
þ OH
(8) (1) INTRODUCTION Therefore,
under the satisfied thermodynamic conditions, the reason why
organic molecules are difficult to be degraded by non-radical-type
heterogeneous photocatalytic system may be the limited interface
reaction kinetics. According to kinetic theory, solvation is a key
influencing factor in aqueous reactions. Among them, the
stabilization
of
the
reactants
by
the
solvation
effect
will
significantly increase the activation energy of the reaction. In In various strategies to improve interfacial electron transfer,
sulfite can effectively trap the photogenerated holes of the
photocatalyst to generate active radicals (e.g., SO3•–, SO5•–, SO4•‒,
•OH), which can promote the gradient distribution of active
species at the solid-liquid interface and the degradation of organic
molecules18,19. For example, Deng et al. found that BiOBr
combined
with
20 mM
sulfite
could
completely
degrade
20 mg L−1 methyl orange within 30 min under visible light20. Wei et al. combined g-C3N4 with sulfite under visible light to
significantly improve the removal performance of organic dyes
and phenol21. However, in previous reports, the solid-liquid
interface structure, interaction mode between sulfite and photo-
catalysts, and the photocatalytic activation law of sulfite have not
been thoroughly elucidated. g y
Sulfamethoxazole (SMX) are the oldest group of antibiotics used
in human and veterinary medicines for bacterial infections
treatment. Considering its polarity, amphotericity, photo- and
thermal stability, SMX has a high migration ability in the
environment, which has become an emerging issue and potential Published in partnership with King Fahd University of Petroleum & Minerals X. Gao et al. 2 Among them, SO4•– with the high redox potential can further
oxidize H2O/OH- to generate •OH (Eqs. 5, 6)23. Therefore, SO3•–,
SO5•–, SO4•– and •OH may be formed in the Na2SO3/BiVO4 system. threat to marine life and human health. Searching for suitable
methods with high-efficiency degradation of SMX is one of the
most challenging missions. Herein, we achieved sulfite-mediated
indirect interfacial electron transfer between BiVO4 and organic
molecules. The differences in interfacial interaction and electron
transfer of sulfite and SMX on the surface of BiVO4 were explored
by electrochemistry and theoretical calculations, while their
existence mode at the solid-liquid interface was revealed. Furthermore, the chain transformation of sulfite at the solid-
liquid interface was confirmed by quenching experiments and
electron spin resonance (ESR). Finally, combined with semicon-
ductor physics, interfacial chemistry, and double-layer theory, the
role of sulfite in the interfacial electron transfer of the non-radical
BiVO4 photocatalytic system is elaborated. SO2
3
þ hþðBiVO4Þ ! Published in partnership with King Fahd University of Petroleum & Minerals Published in partnership with King Fahd University of Petroleum & Minerals Mechanism on sulfite activation in the system Degradation of SMX by BiVO4, Na2SO3, Na2SO3/BiVO4, and blank
systems under visible light. Experimental conditions: [SMX] = 4 μM,
[Na2SO3] = 0.50 mM, [BiVO4] = 1.0 g L−1, initial pH = 7.0, λ ≥420 nm,
and T = 25 °C. The center of the data point is the mean of the two
sets of data, and error bars represent the standard deviation of the
two sets of data. Fig. 1
Kinetics of interfacial reaction of BiVO4 activated by sulfite. Degradation of SMX by BiVO4, Na2SO3, Na2SO3/BiVO4, and blank
systems under visible light. Experimental conditions: [SMX] = 4 μM,
[Na2SO3] = 0.50 mM, [BiVO4] = 1.0 g L−1, initial pH = 7.0, λ ≥420 nm,
and T = 25 °C. The center of the data point is the mean of the two
sets of data, and error bars represent the standard deviation of the
two sets of data. npj Clean Water (2023) 42 X. Gao et al. 3 Fig. 2
Dominated active species for SMX degradation in the Na2SO3/BiVO4 system. a Degradation of SMX in different atmosphere
conditions. Effects of b TBA and c EtOH on SMX degradation in the Na2SO3/BiVO4 system; d ESR signals for the detection of reactive species
generated in the Na2SO3/BiVO4 system. Experimental conditions: a [SMX] = 4 μM, [Na2SO3] = 0.50 mM, [BiVO4] = 1.0 g L−1, [TBA] = 1–100 mM,
[EtOH] = 1–100 mM, [DMPO] = 100 mM, initial pH = 7.0, λ ≥420 nm, and T = 25 °C. The center of the data point is the mean of the two sets of
data, and error bars represent the standard deviation of the two sets of data. Fig. 2
Dominated active species for SMX degradation in the Na2SO3/BiVO4 system. a Degradation of SMX in different atmosphere
conditions. Effects of b TBA and c EtOH on SMX degradation in the Na2SO3/BiVO4 system; d ESR signals for the detection of reactive species
generated in the Na2SO3/BiVO4 system. Experimental conditions: a [SMX] = 4 μM, [Na2SO3] = 0.50 mM, [BiVO4] = 1.0 g L−1, [TBA] = 1–100 mM,
[EtOH] = 1–100 mM, [DMPO] = 100 mM, initial pH = 7.0, λ ≥420 nm, and T = 25 °C. The center of the data point is the mean of the two sets of
data, and error bars represent the standard deviation of the two sets of data. Fig. 3
Controlled experiment. Published in partnership with King Fahd University of Petroleum & Minerals Mechanism on sulfite activation in the system In summary, under the
consistent
of
sulfite
concentration,
the
increase
of
BiVO4
concentration and the blue-shift of the cutoff wavelength
promotes the higher proportion of SO4•– production. Meanwhile,
there is an optimal dosage of sulfite under the consistent of the
BiVO4 concentration and light irradiation conditions. Wherein,
insufficient sulfite reduced the yield of active species, and
excessive sulfite produced a stronger self-quenching effect. According to DO (Supplementary Fig. 6) and pH (Supplementary
Fig. 8) change of solution, it can be seen that a large amount of
sulfite is consumed at the initial reaction stage. This indicates that
the higher proportion of sulfite oxidation at the initial stage will be
conducive to the generation of strong oxidative radicals (SO4•–
and •OH). In addition, the Na2SO3/BiVO4 system presents obvious
pH dependence (Fig. 3d), and displays highest degradation
performance for SMX under neutral conditions. Wherein, under
acidic conditions, the protonation of sulfite enhances its ionization
potential and inhibits its activation (Supplementary Table 1). It
could be seen from Supplementary Figs. 9, 10 that under alkaline
conditions, BiVO427, SO32- and SMX are all negatively charged,
which increases the electrostatic repulsion among BiVO4, SMX,
and SO4•–, resulting in inhibition of SMX degradation. According to the above experimental and theoretical analysis
results, the interaction and reaction of sulfite and SMX on the
surface of BiVO4 can be summarized. The main active species for
the degradation of organic molecules in the photocatalytic system
include superoxide radical (•O2−), hydroxyl radical (•OH), and hole
(h+). The contribution of •O2−to the degradation of organic
molecules is excluded due to its weak oxidation, while •OH is not
generated in the BiVO4 photocatalytic system. Therefore, photo-
generated holes are the only active species in the BiVO4
photocatalytic system. In particular, h+ belonging to short-range
active species can only oxidize molecules in the compact layer3. According to the ideal photocatalysis model, the non-radical
pathway of the photocatalysis system includes diffusion of organic
molecules to the photocatalyst surface, reaction with photogen-
erated h+, and desorption into bulk solution. Simultaneously, the
thermodynamics of surface reaction is determined by the
potential difference between the photogenerated h+ and organic
molecules. The higher the overpotential, the stronger the reaction
trend. According to previous reports, the top of the valence band
of BiVO4 is ~2.5 V vs. NHE8. Compared with BiVO4, the active
species [•OH (+1.8 ~ 2.7 V vs. NHE)9, high-valent metal-oxo10–12
and 1O2 (+1.88 V vs. Mechanism on sulfite activation in the system Effect of a BiVO4 concentration, b light irradiation range, c Na2SO2 concentration, and d pH on the
degradation of SMX. Experimental conditions: [SMX] = 4 μM, [Na2SO3] = 0.50 mM, [BiVO4] = 1.0 g L−1, initial pH = 7.0, λ ≥420 nm, and
T = 25 °C. The center of the data point is the mean of the two sets of data, and error bars represent the standard deviation of the two sets of
data. Fig. 3
Controlled experiment. Effect of a BiVO4 concentration, b light irradiation range, c Na2SO2 concentration, and d pH on the
degradation of SMX. Experimental conditions: [SMX] = 4 μM, [Na2SO3] = 0.50 mM, [BiVO4] = 1.0 g L−1, initial pH = 7.0, λ ≥420 nm, and
T = 25 °C. The center of the data point is the mean of the two sets of data, and error bars represent the standard deviation of the two sets of
data. degradation in the Na2SO3/BiVO4 system, while the contribution
from •OH, SO5•–, SO3•– is driven to a lesser extent. The mechanism of sulfite activation by BiVO4 under visible light
was further explored by control experiments. It could be seen
from Fig. 3a that with the increase of BiVO4 concentration, the
degradation performance of SMX is gradually improved, implying
that the higher reactive surface area of BiVO4 is conducive to the degradation in the Na2SO3/BiVO4 system, while the contribution
from •OH, SO5•–, SO3•– is driven to a lesser extent. increase of SO4•– generation proportion. Meanwhile, there is an
observe obvious wavelength dependence of light irradiation in
the Na2SO3/BiVO4 system (Fig. 3b). This due to the blue-shift of the
cutoff wavelength increasing the carrier concentration of BiVO4. Moreover, the degradation performance of SMX display a volcanic
relationship with the change of sulfite concentration (Fig. 3c). This
may originate from that excessive sulfite will quench the active The mechanism of sulfite activation by BiVO4 under visible light
was further explored by control experiments. It could be seen
from Fig. 3a that with the increase of BiVO4 concentration, the
degradation performance of SMX is gradually improved, implying
that the higher reactive surface area of BiVO4 is conducive to the npj Clean Water (2023) 42 Published in partnership with King Fahd University of Petroleum & Minerals X. Gao et al. 4 calculations. Figure 5a and Supplementary Fig. Investigation of the interface reaction The interfacial reaction of sulfite on the surface of BiVO4 was
further investigated by chopping LSV. As revealed in Fig. 4, the
current density of BiVO4 gradually enhances with the increase of
positive bias voltage under visible light, which displays the
excellent photoelectrochemical properties of BiVO4. However,
when SMX was added to the solution, the current density of BiVO4
is obviously suppressed, indicating that SMX is difficult to undergo
oxidation by direct electron transfer on the surface of BiVO4. Interestingly, the current curve of BiVO4 is drastically red-shifted
and its current density is essentially increased with the addition of
sulfite, which indicates that the holes of BiVO4 can be effectively
trapped by sulfite. When SMX was further added to the electrolyte,
the current density is further enhanced, indicating that SMX, as a
receptor of SO4•–, significantly promoted the interface electron
transfer of BiVO4. Mechanism on sulfite activation in the system NHE)13] with lower redox potential exhibit
higher reactivity to organic molecules. And graphitic carbon
nitride (g-C3N4), which is also a photocatalyst, has only the
position of valence band of 1.5–2.0 V vs. NHE29,30, showing
excellent SMX degradation performance31. Therefore, the limited
SMX degradation reaction of BiVO4 might be controlled by
kinetics. Especially, the identification of the difference between
g-C3N4 and BiVO4 can better reflect the kinetic control effect in the
heterogeneous photocatalytic system. From the structural point of
view, the surface of BiVO4 is composed of oxygen terminal or
hydroxyl group, because both Bi3+ and V5+ are extremely
oxyphilic. However, g-C3N4 formed from thermal polymerization
is relatively weak in hydrophilicity, and its structure is a heptazine-
ring conjugated organic polymer linked by amino nitrogen32. Therefore, g-C3N4 lacking surface hydrophilic groups are less
bound to surface water molecules. Meanwhile, according to our
previous studies, pi-pi interactions can be formed between g-C3N4
and organic molecules with aromatic rings33,34. Among them, the
pi-pi interaction can cause the organic molecules with aromatic
rings to dislodge the water molecules in the compact layer to
adsorb on the g-C3N4 surface, while the partial overlap of the
wave function in space between organic molecule and g-C3N4 is
conducive to their charge transfer. Therefore, g-C3N4 has general
organic molecular degradation performance31. In contrast, the
surface of BiVO4 composed of terminal oxygen or hydroxyl groups
is more hydrophilic, leading to stronger interactions between
BiVO4 and water molecules in the compact layer. Compared with
water molecules, organic molecules have weaker polarity and
larger volume. This results in a weaker interaction between Published in partnership with King Fahd University of Petroleum & Minerals Mechanism on sulfite activation in the system 11 illustrates the
valence band (VB) of BiVO4 is mainly composed of hybrid
orbitals of Bi6s and O2p, while its conduction band (CB) is mainly
composed of V3d, indicating that the Bi-O structure acts as the
region where holes principally accumulate28. Meanwhile, the
valence band top of BiVO4 composed of Bi6s orbitals reduces
the effective mass of holes and increases the mobility, which
effectively promotes the surface oxidation reaction. Fortunately,
the (110) plane of BiVO4 is mainly composed of Bi-O layers,
forming the hole gathering plane (Fig. 5c). As shown in Fig. 5c
and Supplementary Table 2, sulfite has higher adsorption energy
and charge transfer amount on the surface of BiVO4 compared
with SMX (Supplementary Fig. 12). In addition, sulfite has a lower
adiabatic ionization potential (AIP) than that SMX (Supplemen-
tary Table 1), indicating that sulfite is more prone to one-
electron oxidation. In summary, there is a strong coupling effect
between adsorption and single-electron oxidation of sulfite on
the surface of BiVO4. species (e.g., SO4•– and •OH) (Eqs. 7, 8). In summary, under the
consistent
of
sulfite
concentration,
the
increase
of
BiVO4
concentration and the blue-shift of the cutoff wavelength
promotes the higher proportion of SO4•– production. Meanwhile,
there is an optimal dosage of sulfite under the consistent of the
BiVO4 concentration and light irradiation conditions. Wherein,
insufficient sulfite reduced the yield of active species, and
excessive sulfite produced a stronger self-quenching effect. According to DO (Supplementary Fig. 6) and pH (Supplementary
Fig. 8) change of solution, it can be seen that a large amount of
sulfite is consumed at the initial reaction stage. This indicates that
the higher proportion of sulfite oxidation at the initial stage will be
conducive to the generation of strong oxidative radicals (SO4•–
and •OH). In addition, the Na2SO3/BiVO4 system presents obvious
pH dependence (Fig. 3d), and displays highest degradation
performance for SMX under neutral conditions. Wherein, under
acidic conditions, the protonation of sulfite enhances its ionization
potential and inhibits its activation (Supplementary Table 1). It
could be seen from Supplementary Figs. 9, 10 that under alkaline
conditions, BiVO427, SO32- and SMX are all negatively charged,
which increases the electrostatic repulsion among BiVO4, SMX,
and SO4•–, resulting in inhibition of SMX degradation. species (e.g., SO4•– and •OH) (Eqs. 7, 8). Published in partnership with King Fahd University of Petroleum & Minerals Mechanism of interface reaction in the BiVO4 photocatalytic
system The photoexcitation of BiVO4 and the interfacial interaction
between sulfite and BiVO4 were investigated by theoretical Fig. 4
Photoelectrochemical
experiment. Photocurrent
linear
sweep voltammetry (LSV) scan of BiVO4 photoanodes with Na2SO3
and SMX (0.1 M Na2SO4, [Na2SO3] = 0.5 mM, [SMX] = 4 μM, pH = 7.0)
under chopped illumination (λ = 510 ± 60 nm). Fig. 4
Photoelectrochemical
experiment. Photocurrent
linear
sweep voltammetry (LSV) scan of BiVO4 photoanodes with Na2SO3
and SMX (0.1 M Na2SO4, [Na2SO3] = 0.5 mM, [SMX] = 4 μM, pH = 7.0)
under chopped illumination (λ = 510 ± 60 nm). npj Clean Water (2023) 42 X. Gao et al. Fig. 5
Electronic structure and solid–liquid interface reaction mechanism of BiVO4. a DOS calculation for BiVO4; b the proposed
photocatalytic decomposition process of SO32−on the BiVO4; c adsorption of monodentate SO32−on the BiVO4 with (110) facet; d quasistatic
energy profile and charge transfer pathways of BiVO4 under continuous illumination in contact with the aqueous solution. Jredox is the target
charge transfer from the valence band to the redox reagent, SCL is space charge layer, Vph is the open-circuit photovoltage. EF,n and EF,p are the
quasi-Fermi levels of electrons and holes under illumination, respectively; e model of the double-layer structure of BiVO4 in contact with the
aqueous solution under equilibrium conditions; f transformation pathways of major species in Na2SO3/BiVO4. 5 Fig. 5
Electronic structure and solid–liquid interface reaction mechanism of BiVO4. a DOS calculation for BiVO4; b the proposed
photocatalytic decomposition process of SO32−on the BiVO4; c adsorption of monodentate SO32−on the BiVO4 with (110) facet; d quasistatic
energy profile and charge transfer pathways of BiVO4 under continuous illumination in contact with the aqueous solution. Jredox is the target
charge transfer from the valence band to the redox reagent, SCL is space charge layer, Vph is the open-circuit photovoltage. EF,n and EF,p are the
quasi-Fermi levels of electrons and holes under illumination, respectively; e model of the double-layer structure of BiVO4 in contact with the
aqueous solution under equilibrium conditions; f transformation pathways of major species in Na2SO3/BiVO4. organic molecules and BiVO4, and is not conducive to the
diffusion of organic molecules from the bulk solution to the solid-
liquid
interface. Mechanism of interface reaction in the BiVO4 photocatalytic
system Therefore,
combined
with
the
non-radical
mechanism and surface structural features of BiVO4, it is difficult
for organic molecules with low polarity and high ionization
potential to enter the compact layer of BiVO4 and perform non-
radical electron transfer. The steric segregation between organic
molecules and holes triggers the deactivation of the photocata-
lytic system. This phenomenon is named surface solvation-
induced inactivation (SSII). Compared with organic molecules,
the wave function of sulfite in the form of anion is more diffuse in
space, which is conducive to the formation of electrostatic
interaction-dominated hydrogen bonds with the hydroxyl on the
surface of BiVO4 and into the compact layer. Meanwhile, sulfites
with low ionization energy are favorably oxidized by the holes of
BiVO4 (Supplementary Tables 1 and 2), thereby promoting the
generation of active species. Therefore, the introduction of sulfite
realizes the indirect degradation of SMX by BiVO4, which expands
the steric reaction ability of BiVO4 to organic molecules. electron
transfer
between
substrate
molecules
and
BiVO4. According to the above results, SMX can only be in the diffuse
layer, while sulfite can enter the compact layer of BiVO4. Once
sulfite is oxidized by the hole of BiVO4 with one electron, the
generated SO3•−will then undergo chain transformation to
generate active species and enter into the diffuse layer to
participate in the oxidation of SMX (Fig. 5e, f). Published in partnership with King Fahd University of Petroleum & Minerals Quantitative structure-activity relationships In addition, the degradation of various pollutants by Na2SO3/BiVO4
system are further investigated. According to the above results,
SO4•– is the predominant active species in the Na2SO3/BiVO4
system, while •OH play an auxiliary role. Among them, •OH are
non-selective active species, while SO4•– have certain selectivity. It
could be seen from Supplementary Fig. 13 that no significant
degradation of phenol, aniline, and benzoic acid is observed in the
BiVO4 photocatalytic system. However, the concentration of
nitrobenzene
exhibits
a
significant
decrease,
which
might
originate from the reduction of the nitro group by solvated
electrons diffusing out of the tight layer. In contrast, the Na2SO3/
BiVO4
system
exhibited
obvious
photocatalytic
degradation
performance for various pollutants (Fig. 6a). Wherein, aromatic
compounds with electron-donating substituents (phenol and
aniline) present better degradation rate, while aromatic com-
pounds with electron-withdrawing groups (benzoic acid and
nitrobenzene) are more difficult to be degraded. To further
explore the reasons for the differences in the degradation
performance of various pollutants, a correlation between the
adiabatic ionization potential (AIP) of organic molecules and the
rate constant (k) is constructed. Generally, AIP can be used to Based on above results, an overall model of Na2SO3/BiVO4
system from photoexcitation to surface reaction is proposed. As
depicted
in
Fig. 5d,
the
contact
of
BiVO4
as
an
n-type
semiconductor with water will lead to the formation of a space
charge layer (SCL) and an upward energy band bend. When BiVO4
is excited under visible light, the quasi-Fermi level of holes (EF,P)
drops significantly and generates photovoltage. Under the action
of photovoltage and band bending, holes will grab electrons from
substrate molecules with suitable redox potentials. Meanwhile, the
BiVO4 will be solvated and form double layer (including compact
layer and diffuse layer) mainly composed of solvent molecules
(Fig. 5e). This severely hinders the interfacial interaction and npj Clean Water (2023) 42 X. Gao et al. 6 Fig. 6
Quantitative structure-activity relationship. Degradation performance for a monosubstituted benzenes; b relationship between the k
and AIP value. Experimental condition: [organic] = 4 μM, [Na2SO3] = 0.50 mM, [BiVO4] = 1.0 g L−1, initial pH = 7.0, λ ≥420 nm, and T = 25 °C. The center of the data point is the mean of the two sets of data, and error bars represent the standard deviation of the two sets of data. Fig. 6
Quantitative structure-activity relationship. DISCUSSION In this work, the oxidation kinetics of organic molecular controlled
by the solid-liquid interface in the BiVO4 photocatalytic system is
revealed. Initially, the holes belong to short-range active species
and are localized on the photocatalyst surface. Meanwhile, the
generation of solid–liquid interface in the actual system triggers
the spatial isolation between the photocatalyst and the organic
molecules, which leads to the deactivation of the degradation of
organic molecules on the surface of BiVO4. However, sulfites with
lower AIP and higher adsorption energy on the surface of BiVO4
can accumulate in the compact layer of BiVO4 and trap the
generated photogenerated hole for single-electron oxidation. The
generated sulfite radical undergoes chain transformation under
the induction of oxygen to generate active species (SO4•−) to
degrade SMX. This results in a sulfite-mediated indirect electron
transfer from SMX to BiVO4. A model of Na2SO3/BiVO4 system from
photocatalyst excitation to interface reaction is established. In
addition, there is a good linear correlation between the rate
constant (k) and AIP of various pollutants in the Na2SO3/BiVO4
system, indicating the structure-dependence of organic molecules
degradation. This study provides insight for further understanding
the
effect
of solid-liquid
interface
on
reaction
kinetics
in
heterogeneous photocatalytic system. Preparation of BiVO4 Bismuth vanadate (BiVO4) was synthesized by a hydrothermal
method as reported in the literature35. Typically, 12 mmol
Bi(NO3)3 ∙5H2O was dissolved into 64 mL of 1 M HNO3. After
stirring for 0.5 h, 12 mmol NH4VO3, and 0.1 mol urea were quickly
poured into the above solutions, and the color of the precipitate
gradually changed from blood red to bright yellow. Subsequently,
the beaker was placed in an 80 °C oil bath for 24 h with magnetic
stirring. After natural cooling, the yellow precipitation was Quantitative structure-activity relationships Degradation performance for a monosubstituted benzenes; b relationship between the k
and AIP value. Experimental condition: [organic] = 4 μM, [Na2SO3] = 0.50 mM, [BiVO4] = 1.0 g L−1, initial pH = 7.0, λ ≥420 nm, and T = 25 °C. The center of the data point is the mean of the two sets of data, and error bars represent the standard deviation of the two sets of data. collected by centrifugation, washed with ethanol (EtOH) and
water repeatedly, and then dried at 60 °C overnight. describe the tendency of organic molecules to lose electrons,
which is similar to the electron transfer in the oxidation of organic
compounds by active species. As delineated in Fig. 6b, there is a
good linear correlation between k and the AIP of various
pollutants. In conclusion, Na2SO3/BiVO4 system is more inclined
to the degradation of electron-rich organics, indicating the
structure-dependent degradation of organic molecules. Experimental procedure Batch experiments were carried out in a 100 mL glass beaker at
25 °C under magnetic stirring. In a typical test, 50 mg of BiVO4 was
ultrasonically dispersed into the beaker containing 50 mL of 4 μM
SMX, and then a certain mass of Na2SO3 was introduced under
magnetic stirring. Subsequently, the initial pH was adjusted to 7.0
by using 0.05 M H2SO4 or NaOH. Meanwhile, the Xenon lamp (CEL-
HXF300, Beijing China Education Au-light Co., Ltd) equipped with
420 nm cutoff filter was preheated at least 30 min to ensure the
stability of the light source, and then the light intensity was
adjusted to 400 mW cm−2 using an optical power meter (CEL-
NP2000-10A, Beijing China Education Au-light Co., Ltd). At certain
time intervals, 800 μL of the reacting solution was withdrawn,
mixed with 800 μL of methanol to quench reactive species, filtered
through a 0.22 μm PTFE filter, and then for further analysis. All
experiments were conducted in duplicates at least, and the
averaged values with standard deviations were reported. Analytical methods The concentration of organics was quantified by on a Waters
e2695 equipped with a C18 column and a UV-vis detector with
details shown in Supplementary Table 3. The pH values were
monitored by a Shanghai Leici pH meter with daily calibration. The
concentration of dissolved oxygen (DO) was measured by a JPB-
607A portable meter (Leici, Shanghai, China). ESR signals of radical
spin-trapped with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) were
obtained using a JES-FA200 (JEOL Co., USA) spectrometer
equipped with a 300 W Xe lamp (420 nm filter) as visible light
source. The material characterization techniques, trapping experi-
ments, and photoelectrochemical measurements are given in the
Supplementary Information (Supplementary Method 2–4). Chemicals and materials Sources of chemicals and materials are provided in Supplementary
Method 1. Density functional theory (DFT) calculation analysis
The theoretical calculations of isolated and periodic systems were
performed by Quantum Espresso (QE 6.5), Gaussian 16 C.01, and
CP2K 9.1, respectively36–39. The CP2K 9.1 input file was generated
through Multiwfn 3.8_dev40. Details on DFT calculation can be
found in Supplementary Method 5. REFERENCES 1. Loeb, S. K. et al. The technology horizon for photocatalytic water treatment:
sunrise or sunset? Environ. Sci. Technol. 53, 2937–2947 (2019). 1. Loeb, S. K. et al. The technology horizon for photocatalytic water treatment:
sunrise or sunset? Environ. Sci. Technol. 53, 2937–2947 (2019). 29. Che, H. et al. Rational design of donor-acceptor conjugated polymers with high
performance on peroxydisulfate activation for pollutants degradation. Appl. Catal. B: Environ. 316, 121611 (2022). 2. Hoffmann, M. R., Martin, S. T., Choi, W. & Bahnemann, D. W. Environmental
applications of semiconductor photocatalysis. Chem. Rev. 95, 69–96 (1995). pplications of semiconductor photocatalysis. Chem. Rev. 95, 69–96 30. Wang, D., Chen, J., Gao, X., Ao, Y. & Wang, P. Maximizing the utilization of photo-
generated electrons and holes of g-C3N4 photocatalyst for harmful algae inac-
tivation. Chem. Eng. J. 431, 134105 (2022). 3. Yang, Z. et al. Toward selective oxidation of contaminants in aqueous systems. Environ. Sci. Technol. 55, 14494–14514 (2021). 4. Chen, F. et al. Catalytic degradation of ciprofloxacin by a visible-light-assisted
peroxymonosulfate activation system: performance and mechanism. Water Res. 173, 115559 (2020). 31. Gao, X., Chen, J., Che, H., Ao, Y. & Wang, P. Rationally constructing of a novel
composite photocatalyst with multi charge transfer channels for highly efficient
sulfamethoxazole elimination: Mechanism, degradation pathway and DFT cal-
culation. Chem. Eng. J. 426, 131585 (2021). 5. Jiang, R. et al. Fabrication of Fe3O4 quantum dots modified BiOCl/BiVO4 p-n
heterojunction to enhance photocatalytic activity for removing broad-spectrum
antibiotics under visible light. J. Taiwan Inst. Chem. Eng. 96, 681–690 (2019). 32. Che, H. N. et al. Iodide-induced fragmentation of polymerized hydrophilic carbon
nitride for high-performance quasi-homogeneous photocatalytic H2O2 produc-
tion. Angew. Chem. Int. Ed. 60, 25546–25550 (2021). 6. Long, M. et al. EfficienT PHOTOCATALYTIC DEGRADATION OF PHENOL over
Co3O4/BiVO4 composite under visible light irradiation. J. Phys. Chem. B 110,
20211–20216 (2006). 33. Zhang, Q. et al. Understanding the mechanism of interfacial interaction enhan-
cing photodegradation rate of pollutants at molecular level: Intermolecular pi-pi
interactions favor electrons delivery. J. Hazard. Mater. 430, 128386 (2022). 7. Zhang, Z., Wang, W., Shang, M. & Yin, W. Photocatalytic degradation of rhoda-
mine B and phenol by solution combustion synthesized BiVO4 photocatalyst. Catal. Commun. 11, 982–986 (2010). 34. Zhang, Q. et al. In-depth insight into the mechanism on photocatalytic synergistic
removal of antibiotics and Cr (VI): the decisive effect of antibiotic molecular
structure. Appl. Catal. B Environ. 313, 121443 (2022). 8. DATA AVAILABILITY The data that support the findings of this study are available from the corresponding
author upon reasonable request. Published in partnership with King Fahd University of Petroleum & Minerals npj Clean Water (2023) 42 Published in partnership with King Fahd University of Petroleum & Minerals X. Gao et al. 7 Received: 31 December 2022; Accepted: 2 May 2023; 26. Liang, J. et al. Persulfate oxidation of sulfamethoxazole by magnetic iron-char
composites via nonradical pathways: Fe(IV) versus surface-mediated electron
transfer. Environ. Sci. Technol. 55, 10077–10086 (2021). 27. Wetchakun, N. et al. BiVO4/CeO2 nanocomposites with high visible-light-induced
photocatalytic activity. ACS Appl. Mater. Inter. 4, 3718–3723 (2012). 28. Cooper, J. K. et al. Electronic structure of monoclinic BiVO4. Chem. Mater. 26,
5365–5373 (2014). ACKNOWLEDGEMENTS We are grateful for grants from the Natural Science Foundation of China (51979081,
52100179), Fundamental Research Funds for the Central Universities(B200202103), 14. Wang, Z. et al. Aqueous iron(IV)-oxo complex: an emerging powerful reactive
oxidant formed by iron(ii)-based advanced oxidation processes for oxidative
water treatment. Environ. Sci. Technol. 56, 1492–1509 (2022). We are grateful for grants from the Natural Science Foundation of China (51979081,
52100179), Fundamental Research Funds for the Central Universities(B200202103),
National Science Funds for Creative Research Groups of China (No. 51421006), PAPD,
and Postgraduate Research & Practice Innovation Program of Jiangsu Province
(KYCX22 0680) 52100179), Fundamental Research Funds for the Central Universities(B200202103),
National Science Funds for Creative Research Groups of China (No. 51421006), PAPD,
and Postgraduate Research & Practice Innovation Program of Jiangsu Province National Science Funds for Creative Research Groups of China (No. 51421006), PAPD, water treatment. Environ. Sci. Technol. 56, 1492–1509 (2022). and Postgraduate Research & Practice Innovation Program of Jiangsu Province
(KYCX22_0680). 15. Crespo-Otero, R. & Walsh, A. Variation in surface ionization potentials of pristine
and hydrated BiVO4. J. Phys. Chem. Lett. 6, 2379–2383 (2015). 16. Wang, W. et al. Influence of excess charge on water adsorption on the BiVO4
(010) surface. J. Am. Chem. Soc. 144, 17173–17185 (2022). REFERENCES Tan, H. L., Amal, R. & Ng, Y. H. Alternative strategies in improving the photo-
catalytic and photoelectrochemical activities of visible light-driven BiVO4: a
review. J. Mater. Chem. A 5, 16498–16521 (2017). 35. Gao, X., Ma, C., Liu, Y., Xing, L. & Yan, Y. Self-induced Fenton reaction constructed
by Fe(III) grafted BiVO4 nanosheets with improved photocatalytic performance
and mechanism insight. Appl. Surf. Sci. 467-468, 673–683 (2019). 9. He, S., Chen, Y., Li, X., Zeng, L. & Zhu, M. Heterogeneous photocatalytic activation
of persulfate for the removal of organic contaminants in water: a critical review. ACS EST Eng. 2, 527–546 (2022). and mechanism insight. Appl. Surf. Sci. 467-468, 673–683 (2019 36. Giannozzi, P. et al. QUANTUM ESPRESSO: a modular and open-source software pro-
ject for quantum simulations of materials. J. Phys. Condens. Matter 21, 395502 (2009). 10. Chen, J., Zhou, X., Sun, P., Zhang, Y. & Huang, C. H. Complexation enhancees
Cu(II)-activated peroxydisulfate: a novel activation mechanism and Cu(III) con-
tribution. Environ. Sci. Technol. 53, 11774–11782 (2019). 37. Giannozzi, P. et al. Advanced capabilities for materials modelling with Quantum
ESPRESSO. J. Phys. Condens. Matter 29, 465901 (2017). 38. Frisch, M. J. et al. Gaussian 16 Rev. C.01, Gaussian, Inc. (2016). 11. Li, H., Shan, C. & Pan, B. Fe(III)-Doped g-C3N4 mediated peroxymonosulfate
activation for selective degradation of phenolic compounds via high-valent iron-
oxo species. Environ. Sci. Technol. 52, 2197–2205 (2018). 39. Kühne, T. D. et al. CP2K: an electronic structure and molecular 39. Kühne, T. D. et al. CP2K: an electronic structure and molecular dynamics software
package-Quickstep: Efficient and accurate electronic structure calculations. J. Chem. Phys. 152, 194103 (2020). 12. Zong, Y. et al. Unraveling the overlooked involvement of high-valent cobalt-oxo
species generated from the cobalt(ii)-activated peroxymonosulfate process. Environ. Sci. Technol. 54, 16231–16239 (2020). 40. Lu, T. & Chen, F. Multiwfn: a multifunctional wavefunction analyzer. J. Comput. Chem. 33, 580–592 (2012). 13. Li, Y., Zhang, W., Niu, J. & Chen, Y. Mechanism of photogenerated reactive oxygen
species and correlation with the antibacterial properties of engineered metal-
oxide nanoparticles. ACS Nano 6, 5164–5173 (2012). ADDITIONAL INFORMATION 22. Gao, X. et al. New insight into the mechanism of symmetry-breaking charge
separation induced high-valent iron(IV) for highly efficient photodegradation of
organic pollutants. Appl. Catal. B: Environ. 321, 122066 (2023). Supplementary information The online version contains supplementary material
available at https://doi.org/10.1038/s41545-023-00254-w. 23. Rao, D. et al. New mechanistic insights into the transformation of reactive oxi-
dizing species in an ultraviolet/sulfite system under aerobic conditions: modeling
and the impact of Mn(II). ACS EST Water 1, 1785–1795 (2021). Correspondence and requests for materials should be addressed to Peifang Wang or
Yanhui Ao. 24. Chen, H. et al. Novel solar/sulfite advanced oxidation process for carbamazepine
degradation: radical chemistry, transformation pathways, influence on disinfec-
tion byproducts and toxic changes. Chem. Eng. J. 451, 138634 (2023). Reprints and permission information is available at http://www.nature.com/
reprints Reprints and permission information is available at http://www.nature.com/
reprints AUTHOR CONTRIBUTIONS 17. Wiktor, J. & Pasquarello, A. Electron and hole polarons at the BiVO4–water
interface. ACS Appl. Mater. Inter. 11, 18423–18426 (2019). X.G.: methodology, software, validation, visualization, calculation, writing – original
draft. H.C.: methodology, software, validation, visualization. W.L.: writing – review &
editing, formal analysis. P.W.: formal analysis, writing – review & editing. Y.A.:
conceptualization, writing – review & editing, supervision, funding acquisition,
validation, formal analysis. 18. Wu, S., Shen, L., Lin, Y., Yin, K. & Yang, C. Sulfite-based advanced oxidation and
reduction processes for water treatment. Chem. Eng. J. 414, 128872 (2021). 19. Chong, M. N., Jin, B., Chow, C. W. & Saint, C. Recent developments in photo-
catalytic water treatment technology: a review. Water Res. 44, 2997–3027
(2010). 20. Deng, W. et al. Visible-light-driven photocatalytic degradation of organic water
pollutants promoted by sulfite addition. Environ. Sci. Technol. 51, 13372–13379
(2017). COMPETING INTERESTS 21. Wei, Y. et al. Photocatalytic degradation of organic pollutants in wastewater with
g-C3N4/sulfite system under visible light irradiation. Chemosphere 208, 358–365
(2018). Published in partnership with King Fahd University of Petroleum & Minerals Published in partnership with King Fahd University of Petroleum & Minerals npj Clean Water (2023) 42 © The Author(s) 2023 Reprints and permission information is available at http://www.nature.com/
reprints 25. Wang, Z. et al. Is sulfate radical really generated from peroxydisulfate activated
by iron(II) for environmental decontamination? Environ. Sci. Technol. 52,
11276–11284 (2018). Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims
in published maps and institutional affiliations. Published in partnership with King Fahd University of Petroleum & Minerals npj Clean Water (2023) 42 npj Clean Water (2023) 42 X. Gao et al. 8 Open Access This article is licensed under a Creative Commons
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https://bmcmedresmethodol.biomedcentral.com/counter/pdf/10.1186/1471-2288-13-65
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English
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An R package for analyzing and modeling ranking data
|
BMC Medical research methodology
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© 2013 Lee and Yu; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited. An R package for analyzing and modeling
ranking data Paul H Lee1,2* and Philip LH Yu2 Paul H Lee1,2* and Philip LH Yu2 Lee and Yu BMC Medical Research Methodology 2013, 13:65
http://www.biomedcentral.com/1471-2288/13/65 Lee and Yu BMC Medical Research Methodology 2013, 13:65
http://www.biomedcentral.com/1471-2288/13/65 Open Access * Correspondence: honglee@graduate.hku.hk
1School of Public Health/Department of Community Medicine, The University
of Hong Kong, Room 624-627, Core F, Cyberport 3, 100 Cyberport Road,
Hong Kong, Hong Kong
2Department of Statistics and Actuarial Science, The University of Hong
Kong, Hong Kong, Hong Kong Abstract Background: In medical informatics, psychology, market research and many other fields, researchers often need to
analyze and model ranking data. However, there is no statistical software that provides tools for the comprehensive
analysis of ranking data. Here, we present pmr, an R package for analyzing and modeling ranking data with a
bundle of tools. The pmr package enables descriptive statistics (mean rank, pairwise frequencies, and marginal
matrix), Analytic Hierarchy Process models (with Saaty’s and Koczkodaj’s inconsistencies), probability models
(Luce model, distance-based model, and rank-ordered logit model), and the visualization of ranking data with
multidimensional preference analysis. Results: Examples of the use of package pmr are given using a real ranking dataset from medical informatics, in
which 566 Hong Kong physicians ranked the top five incentives (1: competitive pressures; 2: increased savings; 3:
government regulation; 4: improved efficiency; 5: improved quality care; 6: patient demand; 7: financial incentives)
to the computerization of clinical practice. The mean rank showed that item 4 is the most preferred item and item
3 is the least preferred item, and significance difference was found between physicians’ preferences with respect to
their monthly income. A multidimensional preference analysis identified two dimensions that explain 42% of the
total variance. The first can be interpreted as the overall preference of the seven items (labeled as “internal/
external”), and the second dimension can be interpreted as their overall variance of (labeled as “push/pull factors”). Various statistical models were fitted, and the best were found to be weighted distance-based models with
Spearman’s footrule distance. Conclusions: In this paper, we presented the R package pmr, the first package for analyzing and modeling ranking
data. The package provides insight to users through descriptive statistics of ranking data. Users can also visualize
ranking data by applying a thought multidimensional preference analysis. Various probability models for ranking
data are also included, allowing users to choose that which is most suitable to their specific situations. Keywords: Distance-based model, Luce model, Multidimensional preference analysis, Visualization,
Weighted distance election [1], one’s place of living [2], choice of occupations
[3,4], medical treatment [5], and so on. In analyzing these
data, the focus is on the judges’ perception and preference
of some specific (or all) items. In recent years, this type of
ranking data have also becoming more popular in the
medical literature, particularly in health economics [6-10]
and medical informatics [11]. Lee and Yu BMC Medical Research Methodology 2013, 13:65
http://www.biomedcentral.com/1471-2288/13/65 Lee and Yu BMC Medical Research Methodology 2013, 13:65
http://www.biomedcentral.com/1471-2288/13/65 Indeed, users are free to write/use packages for specific
purposes. Although many packages are highly relevant
to medicine [21,22], there are only a limited number of
packages for analyzing and modeling ranking data. There
are some basic tools for ranking data, for example the
Kendall package and the pspearman package for the com-
putation of Kendall and Spearman rank correlation. None-
theless, to the best of the authors’ knowledge, the only
statistical model currently available in R is the RMallow
package (http://cran.r-project.org/web/packages/RMallow)
for fitting a mixture of Mallows’ models [23]. Here, we
present pmr (probability models for ranking data), an R
package for analyzing and modeling ranking data with a
bundle of statistical tools. A review of statistical analysis
for ranking data is given, prior to demonstrating the im-
plementation of pmr. The current version of pmr and the
user manual can be found in Additional files 1 and 2
respectively. In
addition,
four
ranking
models
are
reviewed, namely the Luce model, distance-based model,
ϕ-component model, and weighted distance-based model. For more details, readers can refer to [15,24,25]. The use
cases diagram of the pmr package is shown in Figure 1. mRNA [13], and consumer behavior in e-commerce ap-
plications [14]. Due to the large number of items, these
ranking datasets often contain missing or tie rankings,
which are impossible to analyze without computers. With the decreasing cost of powerful computers, more
researchers have paid attention to this type of ranking
data, especially those in machine learning and know-
ledge discovery. Analyzing and modeling ranking data is an efficient
way to understand people’s perceptions and preferences
for different items. Over the years, besides statistical
tests for hypothesis testing [15], various models have
been developed for ranking data, including the Luce
model [16], distance-based model [1], ϕ-component
model [17] and weighted distance-based model [18,19]. The maximum likelihood estimator (MLE) of the
aforementioned models does not have a closed form, yet
the MLE can be obtained using iterative algorithms. However, at present, only summary statistics and a
visualization of ranking data are available (partially and
indirectly) in some statistical software (for example, pro-
cedure MDPREF in SAS), not to mention hypothesis
testing and probability models for ranking data. Lee and Yu BMC Medical Research Methodology 2013, 13:65
http://www.biomedcentral.com/1471-2288/13/65 The lack
of software and tools for analyzing ranking data is not a
problem for statisticians who are used to writing pro-
grams for their own means. However many scientists are
not familiar with programming. We believe that a single
package for the analysis of ranking data could offer users
a more complete analysis, allowing them to use a single
program instead of shifting their ranking datasets from
one application to another. Background Ranking data arises when a number of items are to be
ranked. By the nature of the ranking data, they can be di-
vided into two types. The first type is characterized by a
small number of items to be ranked, and they frequently
represent the preference of these items among a group of
judges (individuals). These items can be candidates in an The second type of ranking data is characterized by a
large number of items, and they frequently represent the
ordering of these items in which researchers would like
to determine or predict which items were ranked at the
top positions. Examples of such ranking data include
search engine results [12], integration of microRNA and * Correspondence: honglee@graduate.hku.hk
1School of Public Health/Department of Community Medicine, The University
of Hong Kong, Room 624-627, Core F, Cyberport 3, 100 Cyberport Road,
Hong Kong, Hong Kong
2Department of Statistics and Actuarial Science, The University of Hong
Kong, Hong Kong, Hong Kong Page 2 of 11 Lee and Yu BMC Medical Research Methodology 2013, 13:65
http://www.biomedcentral.com/1471-2288/13/65 Implementation In this section, we give a review of statistical analyses for
ranking data. For a better description of ranking data, some
notations must be defined. For a set of k items, labelled 1,
…, k, a ranking π is a mapping function from 1,…, k to 1,…,
k, where π(i) is the rank given to item i. For example,
π(2) = 3 means that item 2 has a rank of 3. The inverse of
the ranking function (sometimes referred to as ordering)
π-1(i) is defined as the item that has rank i. For example,
π-1(5) = 6 means that the item with rank 5 is item 6. R [20], an open-source program for statistical analysis,
is gaining in popularity because of its high flexibility. Figure 1 Use Case Diagram of the pmr package. Figure 1 Use Case Diagram of the pmr package. Page 3 of 11 Lee and Yu BMC Medical Research Methodology 2013, 13:65
http://www.biomedcentral.com/1471-2288/13/65 Lee and Yu BMC Medical Research Methodology 2013, 13:65
http://www.biomedcentral.com/1471-2288/13/65 Visualizing ranking data: multidimensional preference
analysis Because ranking data often have a high dimension,
visualization is a good first step towards their analysis. Multidimensional preference analysis [28] is a dimension
reduction technique that aims to display ranking data in
a low-dimensional (preferably 2D or 3D) space. It is ap-
plicable to ranking data with five or more items where
the dataset cannot be displayed in a 2D/3D plot. Let X
be an N×k matrix of ranking data such that xij repre-
sents the rank of item j assigned by judge i, centered by
the overall mean rank, i.e., (k + 1)/2. Suppose the singu-
lar value decomposition of X is X = UDV’. A 2D repre-
sentation of the multidimensional preference analysis
denotes the items and judges by the first two columns of
ffiffiffiffiffiffiffiffiffiffi
N−1
p
U and
DV′
ffiffiffiffiffiffiffi
N−1
p
, respectively. Items are usually plotted
as points, whereas judges are plotted as vectors from the
origin. To give a better graphical display, the length of
the ranking vectors can be scaled to fit the position of
the items. It is not difficult to see that the perpendicular
projection of all k item points onto a judge vector will
closely approximate the ranking of the k items by that
judge if the 2D solution fits the data well. Otherwise, we
may look for a higher-dimension solution. Pst ¼
X
k! i¼1
NiI πi sð Þ > πi tð Þ
½
; where I[·] is the indicator function. Note that Pst/N rep-
resents the empirical probability that item s is ranked
higher than item t. In addition to mean ranks and pair-
wise frequencies, one can look more deeply into a rank-
ing
dataset
by
studying
the
so-called
“marginal”
distribution of the items. A marginal matrix, specifically
for this use, is the k×k matrix M in which the (s,t)th
entry equals Mst ¼
X
k! i¼1
NiI πi sð Þ ¼ t
½
: Note that Mst is the frequency of item s being ranked
tth. It is called a marginal matrix because “the ith row
gives the observed marginal distribution of the ranks
assigned to item i, and the jth column gives the marginal
distribution of objects given the rank j.” ([15], page 18). Statistical inferences for ranking data Apart from exploring ranking data using descriptive sta-
tistics and graphs to identify the structure of the data,
statistical inferences can be made to test the significance
of a data structure. The two most commonly used infer-
ences are the test for uniformity in a set of ranking data
and the test for common rank-order preference for two
sets of ranking data. Descriptive statistics for ranking data the (s,t)th entry ast equals the number of times criterion
s is preferred over criterion t, is computed. The weights
are then found as the eigenvalues of the matrix A. The
reliability of these weights depends on the consistency of
the ranking process, which is defined as astatu = asu for s,
t, u = (1,…, k). Therefore, evaluating the consistency of
the ranking data using A is a crucial task in analyzing
ranking data and hence a number of measures have been
developed for this purpose. One popular measure is
Saaty’s index, which is given by Descriptive statistics give an overall picture of the rank-
ing dataset. Not only do descriptive statistics provide a
summary of the ranking dataset, but they also lead us in
an appropriate direction to analyze the dataset. There-
fore, it is suggested that researchers consider descriptive
statistics prior to any sophisticated analysis of ranking
data. We begin with a single measure of the popularity of
an item. It is natural to use the mean rank attributed to
an item to represent the central tendency of the ranks. Mean rank m is defined as the k-dimensional vector in
which the jth entry equals λ max−k
k−1
RIk
; mj ¼
X
k! i¼1
Niπi jð Þ; where λmax is the largest eigenvalue of A, and RIk is the
average value of λ max−k
k−1
for a k×k random matrix. Another
popular measure is Koczkodaj’s index, which equals
max min 1−b
ac
; 1−ac
b
for each triad (a, b, c) in A}. where πi, i = 1, 2, …, k! represents all possible rankings
of the k items, Ni is the observed frequency of ranking i,
and πi(j) is the rank given to item j in ranking i. ac
b
Other consistency indices exist besides these two [27]. Apart from the mean ranks, the pairwise frequencies,
that is, the frequency with which item i is ranked higher
than item j for every possible Ck
2 item pairs (i, j), are also
often used. These pairwise frequencies can be summa-
rized in a k×k matrix called a pair matrix (P) in which
the (s,t)th entry equals Lee and Yu BMC Medical Research Methodology 2013, 13:65
http://www.biomedcentral.com/1471-2288/13/65 Page 4 of 11 When we say that a ranking dataset is uniform, we
mean that all possible rankings have the same probabil-
ity of being observed. Hence, under uniformity, the
expected frequencies of every ranking should be N/k!,
and the standard χ2 test can be applied to test the uni-
formity. However, when k! is too large compare with N,
this is not always applicable, because we may encounter
rankings with fewer than five observation. In such a
case, mean rank, pairs, or marginals can be used to test
the uniformity instead of ranking proportions [15]. Note
that under uniformity, the expected values of mean rank,
pairs,
and
marginals
are
(k + 1)/2,
0.5 N,
and
N/k
respectively. Suppose n judges are asked to rank k items. Luce [29]
proposed a ranking process where independent utilities
V = (V1, V2,..,Vk) ≥0 are assigned to item 1,2, …,k. The
probability of observing ranking πn is P πjV
ð
Þ ¼
Y
k−1
j¼1
V π−1
n
jð Þ
X
k
i¼j
V π−1
n
ið Þ
; and the resulting models is referred to as the Luce
models [16]. The Luce models can be interpreted as a
vase model [15]: imagine there are infinitely many balls
inside a vase, and each ball is labeled j., j = 1, 2, …, k. The proportion of balls labeled with j is proportional to
Vj. Then, the Luce models correspond to the ranking
process whereby the first ball drawn is labeled π-1(1), the
second ball drawn is labeled π-1(2) (with all balls labeled
π-1(1) removed from the vase), and the process con-
tinues until all balls in the vase have the same label. Under
uniformity,
the
test
statistic
when
using
mean rank, pairs, and marginals are ([15], page 58,
Table 3.1) 12N
k k þ 1
ð
Þ
X
k
j¼1
mj−k þ 1
2
2
;
12N
X
k
s>t
Pst−0:5
!2
−
X
k
s>t
mj−k þ 1
2
!2
kþ1
2
66664
3
77775
; and
N K þ 1
ð
Þ
X
k
s>t
Mst−1
k
2
; 12N
k k þ 1
ð
Þ
X
k
j¼1
mj−k þ 1
2
2
; The loglikelihood function is globally concave, and
hence a global maximum exists. The MLE of the param-
eters can thus be obtained using standard methods, e.g.,
the Newton–Raphson algorithm. Statistical models for ranking data: distance-based model Statistical models for ranking data: distance-based model
In what follows, we will introduce the distance-based
model for ranking data. Before doing so, we need to have
a clear definition of the “distance” between two rankings. A distance function is useful in measuring the discrep-
ancy between two rankings. The usual properties of a
distance function between two rankings π and σ are: Inconsistency indices for pairwise comparisons According to the Analytic Hierarchy Process [26], a
group of judges combine the rankings from different cri-
teria to form a final ranking. The Analytic Hierachy
Process has been used to determine the weights of these
criteria. First, a pairwise comparison matrix A, in which Lee and Yu BMC Medical Research Methodology 2013, 13:65
http://www.biomedcentral.com/1471-2288/13/65 Page 4 of 11 Lee and Yu BMC Medical Research Methodology 2013, 13:65
http://www.biomedcentral.com/1471-2288/13/65 Besides MLE, Bayesian
method can also be used for parameter estimation, using
expectation propagation [30], generalized repeated inser-
tion model [31], and random atomic measures [32]. 12N
X
k
s>t
Pst−0:5
!2
−
X
k
s>t
mj−k þ 1
2
!2
kþ1
2
66664
3
77775
; and The Luce model can be extended to incorporate covar-
iates. We can include M covaraites of judge n, xmn, m =
1, 2, …, M, into the utilities, that is, N K þ 1
ð
Þ
X
k
s>t
Mst−1
k
2
; V nj ¼ βj0 þ
X
M
m¼1
βjmxnm; and they follow a χ2 distribution with k-1, Ck
2, and (k-
1)2 degrees of freedom, respectively. 2 where βjm, m = 0, 1, 2, …, M are parameters specific to
item j. This extension of the Luce model is known as the
rank-ordered logit (ROL) model [33-35]. The χ2 test could be used to test for any difference be-
tween two ranking datasets. Before doing so, we align
the matrix (in the case of pairs and marginals) into a
q × 1 vector, for both datasets. We can now use the
standard χ2 test. For comparison between three or
more ranking datasets, MANOVA-like tests can be
used [15]. Statistical models for ranking data: ϕ-component model Some popular right-invariant distances are Spearman’s
rho [36], given by Fligner and Verducci [17] extended the distance-based
models by decomposing the distance metric d(π, σ) into
k-1 distance metrics, R π; σ
ð
Þ ¼
X
k
i¼1
π ið Þ−σ ið Þ
½
2
!0:5
; d π; σ
ð
Þ ¼
X
k−1
i¼1
di π; σ
ð
Þ;
ð1Þ ð1Þ Spearman’s rho square, given by where each di(π, σ)is independent. Both Kendall’s tau
and Cayley’s distance [38] can be decomposed in this
form, and Fligner and Verducci [17] developed two new
classes of ranking models for these, called ϕ-component
models and cyclic structure models, respectively. R2 π; σ
ð
Þ ¼
X
k
i¼1
π ið Þ−σ ið Þ
½
2; Spearman’s footrule, given by Fligner and Verducci [17] showed that Kendall’s tau
satisfies [1]: F π; σ
ð
Þ ¼
X
k
i¼1
π ið Þ−σ ið Þ
j
j; T π; π0
ð
Þ ¼
X
k−1
π0 ið Þ¼1
V π0 ið Þ; where where and Kendall’s tau, given by and Kendall’s tau, given by V π0 ið Þ ¼
X
k
π0 jð Þ¼π0 ið Þþ1
I
π ið Þ−π jð Þ
½
> 0
f
g: T π; σ
ð
Þ ¼ ∑
i<j I
π ið Þ−π jð Þ
½
σ ið Þ−σ jð Þ
½
< 0
f
g; Here, V1 represents the number of adjacent transposi-
tions required to place the best item in π0 in the first
position. V2 is the number of adjacent transpositions re-
quired to place the second best item in π0 in the second
position, and so on. Therefore, the ranking can be de-
scribed as k-1 stages, V1 to Vk-1, where Vi = m can be
interpreted as m mistakes made in stage i. where I() is the indicator function. There are other dis-
tances applicable to ranking data, and readers can refer
to [24] for details. It is reasonable to assume that there is a modal rank-
ing π0, and we expect most of the judges to have rank-
ings close to π0. Statistical models for ranking data: the Luce model
f
d
d
l
f
k After conducting a descriptive analysis for ranking data,
we may have some understanding about the empirical
distribution of the rank-order preferences of different
items and their popularity. To further understand the
data and make inferences about its structure, an effi-
cient method is to establish some statistical models
for ranking data. Over the years, various statistical
models for ranking data have been developed. In this
subsection, we review a commonly used approach,
the Luce model. d π; π
ð
Þ ¼ 0; d π; σ
ð
Þ > 0
if
π≠σ; d π; σ
ð
Þ ¼ d σ; π
ð
Þ: For ranking data, we require that the distance, apart
from having these
usual
properties,
must be
right Lee and Yu BMC Medical Research Methodology 2013, 13:65
http://www.biomedcentral.com/1471-2288/13/65 Page 5 of 11 Lee and Yu BMC Medical Research Methodology 2013, 13:65
http://www.biomedcentral.com/1471-2288/13/65 possible π. The computational time increases exponen-
tially with the number of items [17]. invariant, i.e., d(π, σ) = d(π○γ, σ○γ), where π○γ(i) = π(γ(i)). This requirement ensures that the relabeling of items has
no effect on the distance. Statistical models for ranking data: weighted distance- P π
ð jw; π0Þ ¼ e−dw π;π0
ð
Þ
C w
ð Þ
: Lee and Yu [18,19] proposed an extension of the
distance-based
model
by
replacing
the
(equal-
weighted)
distance
with
a
new
weighted
distance
measure, so that different weights can be assigned to
different ranks. Generally speaking, if wi is large, few people will tend
to disagree that the item ranked i in π0 should not be
ranked i. This is because such disagreement will greatly
increase the distance and hence the probability of ob-
serving it will become very small. If wi is close to zero,
people have little or no preference on how the item
ranked i in π0 is ranked, because a change in its rank
will not affect the distance at all. The extension of
weighted distance-based ranking models can retain the
nature of distance, and at the same time maintain a
greater flexibility. Readers are referred to [19] for the de-
tails of these properties. Motivated by the weighted Kendall’s tau correlation
coefficient [39], Lee and Yu [18,19] defined the weighted
Kendall’s tau distance by Tw π; σ
ð
Þ ¼ ∑
i<j wπ0 ið Þwπ0 jð ÞI
π ið Þ−π jð Þ
½
σ ið Þ−σ jð Þ
½
< 0
f
g: It is important to note that this weighted distance sat-
isfies all the usual distance properties, in particular the
symmetry property, i.e., Tw(π, σ) = Tw(σ, π). Label ranking method using k-nearest neighbor
algorithm Other distance measures can be generalized to a
weighted
distance
in
a
similar
manner
to
this
generalization of Kendall’s tau distance. For examplethe
weighted Spearman’s rho is Label ranking is defined as the problem of classifying a
judge’s ranking over a set of items given the covariate of
this judge and a training dataset. ROL can be used for
this, as it produces utility scores that can generate rank-
ings for the judges. However, when the number of items
and covariate are large, ROL may not be feasible due to
its long computation time. Recently, a local k-nearest
neighbor method has been developed for label rank-
ing [42]. If we want to predict the ranking of judge i,
we can first select the k-nearest neighbor (by Euclid-
ean distance) of i. Second, a statistical model (the
Luce model in [42]) is fitted to these k neighbors and
the parameters will be used to predict the ranking of
judge i. Rw π; σ
ð
Þ ¼
X
k
i¼1
wπ0 ið Þ π ið Þ−σ ið Þ
½
2
!0:5
; The weighted Spearman’s rho square is The weighted Spearman’s rho square is R2
w π; σ
ð
Þ ¼
X
k
i¼1
wπ0 ið Þ π ið Þ−σ ið Þ
½
2; Lee and Yu BMC Medical Research Methodology 2013, 13:65
http://www.biomedcentral.com/1471-2288/13/65 Applying a weighted distance measure dw to the
distance-based model, the probability of observing a
ranking π becomes ϕ-component models in other papers [24]. Mallows’
ϕ -models are special cases of ϕ-component models
when λ1 = … = λk-1. ϕ-component models in other papers [24]. Mallows’
ϕ -models are special cases of ϕ-component models
when λ1 = … = λk-1. Statistical models for ranking data: ϕ-component model According to this framework, Diaconis
[1] developed a class of distance-based models, By applying a dispersion parameter λi to stage Vi, the
Mallows’ ϕ-model is extended to: P π
ð jλ; π0Þ ¼ e−λd π;π0
ð
Þ
C λ
ð Þ
; P π
ð jΛ; π0Þ ¼ e
−
X
k−1
π0 ið Þ¼1
λπ0 ið ÞV π0 ið Þ
C Λ
ð Þ
; where λ > 0 is the dispersion parameter, C(λ) is the pro-
portionality constant, and d(π, π0) is an arbitrary right
invariant distance. When we use Kendall’s tau as the dis-
tance function, the model is called Mallows’ ϕ-model
[37]. In distance-based models, rankings nearer to the
modal ranking π0 have a higher probability of occur-
rence and this is controlled by λ. The distribution of
rankings will be more concentrated around π0 for a
smaller value of λ. where Λ = {λi, i = 1,…, k = 1} and C(Λ) is the proportion-
ality constant, which equals Y
k−1
π0 ið Þ¼1
1−e−k−π0 ið Þþ1
ð
Þλπ0 ið Þ
1−e−λπ0 ið Þ
: A closed form for the proportionality constant C(λ)
only exists for some distances. In principle, it can be
solved numerically by summing
e−λd π; π0
ð
Þ
over all These models were named k-1 parameter models by
Fligner
and
Verducci
[17],
but
were
also
named Page 6 of 11 Lee and Yu BMC Medical Research Methodology 2013, 13:65
http://www.biomedcentral.com/1471-2288/13/65 Results and discussion In this section, we will use a seven-item ranking
dataset q4 [11], in which 566 Hong Kong physicians
ranked the top five incentives (1: competitive pres-
sures; 2: increased savings; 3: government regulation;
4: improved efficiency; 5: improved quality care; 6:
patient
demand;
7:
financial
incentives)
to
the
computerization of clinical practice. Items not ranked
were imputed using the mean rank. The dataset is
not available in the pmr package but is available upon
request. Note that most of the functions in pmr re-
quire the input ranking data to be organized in an
aggregated format, that is, a summary matrix with and the weighted Spearman’s footrule is and the weighted Spearman’s footrule is Fw π; σ
ð
Þ ¼
X
k
i¼1
wπ0 ið Þ π ið Þ−σ ið Þ
j
j: Apart from the weighted Kendall’s tau [39] and
weighted
Spearman’s
rho
square
[40],
many
other
weighted rank correlations have been proposed [41]. Page 7 of 11 Page 7 of 11 de1 <- destat(q4agg); mean <- rep(4,7);
chi <- 12*567*sum((de1$mean.rank - mean)^2 )/7/8;
chi; dchisq(chi,6) Lee and Yu BMC Medical Research Methodology 2013, 13:65
http://www.biomedcentral.com/1471-2288/13/65 Lee and Yu BMC Medical Research Methodology 2013, 13:65
http://www.biomedcentral.com/1471-2288/13/65 and would give the output: rankings
and
their
corresponding
frequencies. To
transform the individual ranking data to an aggre-
gated
format,
the
rankagg
function
can
be
used
(q4agg < - rankagg(q4)). > chi
[1] 524.8747
> dchisq(,6) All analyses of ranking data start from descriptive sta-
tistics. Using the R code destat(q4agg), the destat func-
tion produces the mean rank vector, the pairs matrix,
and the marginal: [1] 524.8747 [1] 524.8747 > dchisq(,6) Descriptive statistics of ranking data: Descriptive statistics of ranking data:
$mean.rank: mean ranks; $pair: pairs; $mar: marginals
$mean.rank
[1] 3.722615 4.070671 5.159011 2.666078 3.307420 4.708481
4.365724 follows:
[2,] 2.0304576 4.6877078
[3,] 7.1972472 3.2369083
[4,] -8.6872015 1.5649170
[5,] -5.9976649 -0.6995201
[6,] 4.2711405 -4.0874211
[7,] 0.9242315 -7.1352958
$ranking
[,1] [,2] [,3] [,4] [,5] [,6] [,7] [,8] [,9] [,10]
[1,] 1 2 3 4 5 6 7 3 0.34578679 4.418398859
[2,] 1 2 3 5 6 7 4 1 1.02368458 2.817632498
[3,] 1 2 4 3 6 7 5 1 -0.28702760 3.436342641
[4,] 1 2 6 3 4 7 5 1 -1.64369628 2.743277371
[5,] 1 2 5 4 3 6 7 1 -1.01088189 3.725333590
…
[397,] 3 5 4 6 7 2 1 1 2.30895882 -2.893356539
[398,] 5 6 3 4 7 2 1 1 1.65446374 -3.173860152
[399,] 6 5 7 3 4 2 1 1 -1.10621436 -4.162134143
[400,] 4 6 5 7 3 2 1 1 1.03438336 -4.189219159
[401,] 5 6 7 4 3 2 1 1 -1.05887363 -4.559990691
$explain
[1] 0.4242463
Multidimensional preference analysis
$item
[,1] [,2]
[1,] 0.2617896 2.4327039 Multidimensional preference analysis
$item $ranking
[,1] [,2] [,3] [,4] [,5] [,6] [,7] [,8] [,9] [,10]
[1,] 1 2 3 4 5 6 7 3 0.34578679 4.418398859
[2,] 1 2 3 5 6 7 4 1 1.02368458 2.817632498
[3,] 1 2 4 3 6 7 5 1 -0.28702760 3.436342641
[4,] 1 2 6 3 4 7 5 1 -1.64369628 2.743277371
[5,] 1 2 5 4 3 6 7 1 -1.01088189 3.725333590
…
[397,] 3 5 4 6 7 2 1 1 2.30895882 -2.893356539
[398,] 5 6 3 4 7 2 1 1 1.65446374 -3.173860152
[399,] 6 5 7 3 4 2 1 1 -1.10621436 -4.162134143
[400,] 4 6 5 7 3 2 1 1 1.03438336 -4.189219159
[401,] 5 6 7 4 3 2 1 1 -1.05887363 -4.559990691
$explain
[1] 0.4242463 Figure 2 Multidimensional preference of the q4 dataset (1:
competitive pressures; 2: increased savings; 3: government
regulation; 4: improved efficiency; 5: improved quality care; 6:
patient demand; 7: financial incentives). Figure 2 Multidimensional preference of the q4 dataset (1:
competitive pressures; 2: increased savings; 3: government
regulation; 4: improved efficiency; 5: improved quality care; 6:
patient demand; 7: financial incentives). can be fitted using the pmr, which requires the stats4
package. We will demonstrate the model fitting proced-
ure. Spearman’s footrule distance usually gives the best
fit [18,19] and hence it will be used in our demonstra-
tion of distance-based models. Descriptive statistics of ranking data:
$mean.rank: mean ranks; $pair: pairs; $mar: marginals
$mean.rank
[1] 3.722615 4.070671 5.159011 2.666078 3.307420 4.708481
4.365724 The parameter estimates of the Luce model can be
obtained using the R code q4.pr <- pl(q4agg); q4.pr@coef,
and the output is as follows: and the 2D plot is given in Figure 2. The coordinates of the items and rankings, and the
proportion of variance explained by the first two dimen-
sions are stored in the values $item, $ranking and $ex-
plain
respectively. The
final
two
columns
of
the
$ranking matrix are the coordinates of the first two col-
umns of
DV′
ffiffiffiffiffiffiffi
N−1
p
. Maximum Likelihood Estimation of the Luce Model
Chi-square residual statistic: 8239.59, df: 5040
Warning messages:
1: In log(pr[i]) : NaNs produced
…
Parameter estimates:
[1] 1.21815 1.01797 0.39800 1.78440 1.40580 0.51181 0.66293 Maximum Likelihood Estimation of the Luce Model
Chi-square residual statistic: 8239.59, df: 5040
Warning messages:
1: In log(pr[i]) : NaNs produced
…
Parameter estimates:
1] 1.21815 1.01797 0.39800 1.78440 1.40580 0.51181 0.66293 Figure 2 shows the multidimensional preference graph. The 2D plot explains around 42 % of the total variance. The first dimension can be interpreted as the overall
preference of the seven items (labeled as “internal/exter-
nal”). The leftmost item (item 4) and rightmost item
(item 3) are the most and the least preferred items, re-
spectively. The second dimension can be interpreted as
the overall variance of the seven items (labeled as “push/
pull factors”). The bottommost item (item 7) has the lar-
gest variance and the topmost item (item 2) has the sec-
ond largest variance among the seven items. Descriptive statistics of ranking data:
$mean.rank: mean ranks; $pair: pairs; $mar: marginals
$mean.rank
[1] 3.722615 4.070671 5.159011 2.666078 3.307420 4.708481
4.365724 [1] 1.82345e-110 The χ2 test statistic equals 524.8747 and the corre-
sponding p-value equals 1.82345 × 10-110. Thus, the
ranking was not uniformly distributed. This example illustrates how to test the uniformity of
a ranking dataset using the destat function, and we will
now explain how to compare two ranking datasets using
the same function. For example, we may wish to test the
hypothesis that physicians with monthly incomes above
and
below
HK$100,000
(rankings
stored in
q4agg. highincome and q4agg.lowincome respectively) have dif-
ferent preferences towards computerization incentives. According to the marginal matrix using the χ2 test, the
following codes: de.highincome <- destat(q4agg.highincome)
de.lowincome <- destat(q4agg.lowincome)
chisq.test(cbind(as.vector(de.highincome$mar),as.vector(de.low
income$mar))) give the output: give the output: > Pearson's Chi-squared test
> data: cbind(de.highincome$mar,de.lowincome$mar)
> X-squared = 66.415, df = 48, p-value = 0.04024 From the descriptive statistics, we can deduce that
item 4, improved efficiency, is the most preferred item,
and item 3, government regulation, is the least preferred
item. > Pearson's Chi-squared test > data: cbind(de.highincome$mar,de.lowincome$mar) > X-squared = 66.415, df = 48, p-value = 0.04024 Statistical
inferences
about
ranking
data
can
be
performed using the destat function. For instance, if we
want to test whether the ranking over seven items is uni-
form using mean rank, the following R code can be
input: The χ2 test statistic equals 66.415 and the correspond-
ing p-value equals 0.04. Thus, we have found a signifi-
cant difference between physicians’ preferences with
respect to their monthly income. Multidimensional preference analysis [28] can help us
understand
more
about
the
physicians’
ranking
process and their preferences over the seven items by
decomposing the rankings into a few dimensions. This can be performed using the mdpref function (R Lee and Yu BMC Medical Research Methodology 2013, 13:65
http://www.biomedcentral.com/1471-2288/13/65 Page 8 of 11 code: mdpref(q4agg,rank.vector = T)). The output is as
follows: code: mdpref(q4agg,rank.vector = T)). The output is as
follows: Figure 2 Multidimensional preference of the q4 dataset (1:
competitive pressures; 2: increased savings; 3: government
regulation; 4: improved efficiency; 5: improved quality care; 6:
patient demand; 7: financial incentives). [1] 1.21815 1.01797 0.39800 1.78440 1.40580 0.51181 0.66293 The warning messages are a result of some of the pre-
dicted probabilities being close to zero. The parameter
estimates of the distance-based model can be obtained
using the R code q4.dbm < - dbm(q4agg); q4.dbm@coef,
and the distance type can be specified using the argu-
ment dtype (default: Kendall’s tau; rho: Spearman’s rho;
rho2: Spearman’s rho square; foot: Spearman’s footrule). Descriptive statistics and plots provide an insight to
the data, but modeling will be more useful if we wish to
have a deeper understanding. The Luce model (pl),
distance-based
model
(dbm),
ϕ-component
model
(phicom) and weighted distance-based model (wdbm) The loglikelihood is a suitable criterion for determin-
ing which model should be used. The model with the
largest loglikelihood is selected. We can compute the
loglikelihood of all models using the minimum value Page 9 of 11 Page 9 of 11 Lee and Yu BMC Medical Research Methodology 2013, 13:65
http://www.biomedcentral.com/1471-2288/13/65 (@min) of the negative loglikelihood function, which is
built-in for maximum likelihood models: be given in the output, together with the corresponding de-
grees of freedom. We can also examine the effect of physicians’ gender
and type (private/public) on their preferences (gender
and type stored in q4cov) using the ROL model. This
can be fitted using the rol function in the pmr package
with the R code q4.rol <- rol(q4,q4cov); q4.rol@coef
where covariate stores the gender and type of every phy-
sicians. The output is as follows: q4.pr <- pl(q4agg); q4.dbm <- dbm(q4agg, dtype=”foot”);
q4.phicom <- phicom(q4agg); q4.wdbm <- wdbm(q4agg,
dtype=”foot”);
q4.pr@min; q4.dbm@min;
q4.phicom@min; q4.wdbm@min q4.pr <- pl(q4agg); q4.dbm <- dbm(q4agg, dtype=”foot”);
q4.phicom <- phicom(q4agg); q4.wdbm <- wdbm(q4agg,
dtype=”foot”);
q4.pr@min; q4.dbm@min;
q4.phicom@min; q4.wdbm@min and the output is as follows: Maximum Likelihood Estimation of the Rank-ordered Logit Model
Beta0item1 Beta0item2 Beta0item3 Beta0item4 Beta0item5
Beta1item6
0.7271744 0.2788784 0.1204306 -0.0927481 0.1671606
1.2260023
Beta1item1 Beta1item2 Beta1item3 Beta1item4 Beta1item5
Beta2item6
0.4346967 0.3648443 0.3145217 0.4642009 0.2825524
0.2187091
Beta2item1 Beta2item2 Beta2item3 Beta2item4 Beta2item5
Beta2item6
-0.4063632 -0.0845522 -0.0058510 0.0126934 0.0029885 -
0.1425022 [1] 4552.1
[1] 4569.9
[1] 4542.4
[1] 4541.1 [1] 4541.1 The
best
model
(with
the
smallest
negative
loglikelihood) is the weighted footrule model. > p_value
[1] 4.458237e-12 5.885750e-03 2.582951e-01 3.893159e-01
1.233049e-01 7.445499e-31 9.236701e-36
[8] 5.797042e-26 7.522277e-19 1.943696e-38 9.250903e-15
5.762506e-10 4.727443e-86 2.893224e-05
[15] 7.788308e-01 5.494515e-01 8.853680e-01 3.384748e-12 References Med Care 2009, 47:634–641. 9. Craig BM, Busschbach JJV, Salomon JA: Modeling ranking, time trade-off,
and visual analog scale values for EQ-5d health states: a review and
comparison of methods. Med Care 2009, 47:634–641. 10. Ratcliffe J, Brazaier J, Tsuchiya A, Symonds T, Brown M: Using DCE and
ranking data to estimate cardinal values for health states for deruving a
preference-based single index from the sexual quality of life
questionnaire. Health Econ 2009, 18:1261–1276. 10. Ratcliffe J, Brazaier J, Tsuchiya A, Symonds T, Brown M: Using DCE and
ranking data to estimate cardinal values for health states for deruving a
preference-based single index from the sexual quality of life
questionnaire. Health Econ 2009, 18:1261–1276. 11. Leung GM, Yu PLH, Wong IOL, Johnston JM, Tin KYK: Incentives and barriers
that influence clinical computerization in Hong Kong: a population-based
physician survey. J Am Med Inform Assoc 2003, 10:201–212. 11. Leung GM, Yu PLH, Wong IOL, Johnston JM, Tin KYK: Incentives and barriers
that influence clinical computerization in Hong Kong: a population-based
physician survey. J Am Med Inform Assoc 2003, 10:201–212. 12. Park ST, Pennock DM: Applying collaborative filtering techniques to
movie search for better ranking and browsing. Proc KDD 2007 2007. 12. Park ST, Pennock DM: Applying collaborative filtering techniques to
movie search for better ranking and browsing. Proc KDD 2007 2007. 13. Lin S, Ding J: Integration of ranked lists via Cross Entropy Monte Carlo with
applications to mRNA and microRNA studies. Biometrics 2009, 65:9–18. 13. Lin S, Ding J: Integration of ranked lists via Cross Entropy Monte Carlo with
applications to mRNA and microRNA studies. Biometrics 2009, 65:9–18. Conclusions In this paper, we presented the pmr R package, the first
package for analyzing and modeling ranking data. The
package provides insight to users through descriptive
statistics of ranking data. Users can also visualize rank-
ing data by applying a thought multidimensional prefer-
ence analysis. Various probability models for ranking
data are also included, allowing users to choose that
which is most suitable to their specific situations. Be-
sides the models introduced in this paper, there are
other functions included in the pmr package that have
not been presented here due to scope limitations, in-
cluding the Analytic Hierarchy Process model (ahp)
[26,43], multidimensional preference analysis (mdpref),
and rank plots (rankplot) [44]. Details of these functions
can be found at http://cran.r-project.org/web/packages/
pmr/pmr.pdf. Future works on developing the package
will include the incorporation of latent class models. References 1. Diaconis P: Group representations in probability and statistics. Hayward:
Institute of Methematical Statistics; 1988. 2. Duncan OD, Brody C: Analyzing rankings of three items. In Social structure
and behavior. Edited by Hauser RM, Mechanic D, Haller AO, Hauser TS. New York: Academic; 1982:269–310. 2. Duncan OD, Brody C: Analyzing rankings of three items. In Social structure
and behavior. Edited by Hauser RM, Mechanic D, Haller AO, Hauser TS. New York: Academic; 1982:269–310. 3. Goldberg AI: The relevance of cosmopolitan/local orientations to
professional values and behavior. Sociol Work Occup 1975, 3:331–356. 4. Yu PLH, Chan LKY: Bayesian analysis of wandering vector models for
displaying ranking data. Stat Sin 2001, 11:445–461. 3. Goldberg AI: The relevance of cosmopolitan/local orientations to
professional values and behavior. Sociol Work Occup 1975, 3:331–356. 4. Yu PLH, Chan LKY: Bayesian analysis of wandering vector models for
displaying ranking data. Stat Sin 2001, 11:445–461. 5. Plumb AAO, Grieve FM, Khan SH: Survey of hospital clinicians’ preferences
regarding the format of radiology reports. Clin Radiol 2009, 64:386–394. 6. Salomon JA: Reconsidering the use of rankings in the valuation of health
states: a model for estimating cardinal values from ordinal data. Popul Health Metrics 2003, 1:1–12. 7. Krabbe PFM, Salomon JA, Murray CJL: Quantificaition of health states with
rank-based nonmetric multidimensional scaling. Med Decis Making 2007,
27:395–405. 7. Krabbe PFM, Salomon JA, Murray CJL: Quantificaition of health states with
rank-based nonmetric multidimensional scaling. Med Decis Making 2007,
27:395–405. 8. McCabe C, Brazier J, Gilks P, Tsuchiya A, Roberts J, O’Hagan A, Stevens K:
Use rank data to estimate health state utility models. J Health Econ 2006,
25:418–431. 8. McCabe C, Brazier J, Gilks P, Tsuchiya A, Roberts J, O’Hagan A, Stevens K:
Use rank data to estimate health state utility models. J Health Econ 2006,
25:418–431. In the pmr package, we aimed at including trad-
itional
ranking
models
like
the
Luce
model
and
distance-based model, and many recently-developed
models for ranking data were not included (examples
included decision tree models for ranking data [18,45,46]
and multistage models [47,48]). Nevertheless, since many
of these models belong to extensions of traditional ranking
models, we believe that the development of new ranking
models can rely on the programming code provided by
package pmr. 9. Craig BM, Busschbach JJV, Salomon JA: Modeling ranking, time trade-off,
and visual analog scale values for EQ-5d health states: a review and
comparison of methods. Authors’ contributions
PHL
h
k PHL wrote the package pmr and drafted the manuscript. PLHY helped in the
development of the package pmr and significantly revised the manuscript. All authors read and approved the final manuscript. Competing interests Competing interests
The authors declare that they have no competing of interests. Acknowledgements The research of Philip L. H. Yu was supported by a grant from the Research
Grants Council of the Hong Kong Special Administrative Region, China
(Project No. HKU 7473/05H). Received: 25 September 2012 Accepted: 25 April 2013
Published: 14 May 2013 Received: 25 September 2012 Accepted: 25 April 2013
Published: 14 May 2013 [1] 1.21815 1.01797 0.39800 1.78440 1.40580 0.51181 0.66293 The pa-
rameters are given by the R code q4.wdbm@coef as
follows: Maximum Likelihood Estimation of the weighted distance-based Maximum Likelihood Estimation of the weighted distance-based
model
Weighted distance type: Spearman’s footrule
Modal ranking: 4562371
Chi-square residual statistic: 7811.15, df: 5040
Call:
NULL
Parameter estimates:
[1] -0.16288 0.20215 0.19890 0.28253 0.27971 0.43962
0.22850 These parameters are difficult to interpret without
their corresponding significance levels. To obtain the
p-values, the following R code can be used: p_value <- rep(1,ncov*(nitem-1))
for (i in 1:3){
for (j in 1:6){
p_value[(i-1)*6+j] <- 2*pnorm(-abs(q4.rol@coef[(i-
1)*6+j]/q4.rol@vcov[(i-1)*6+j, (i-1)*6+j]))
}
} p_value[(i-1)*6+j] <- 2*pnorm(-abs(q4.rol@coef[(i-
1)*6+j]/q4.rol@vcov[(i-1)*6+j, (i-1)*6+j])) From the model parameters, we can conclude that
item 4 is ranked 1st, but the judges preference for this
position is not particularly strong. Note that the modal
ranking in the weighted distance-based model is differ-
ent from that using the mean rank. which gives the output: As the “best” model does not imply that it gives an ad-
equate fit to the data, we need to assess the goodness-of-fit. The sum of squares Pearson residuals (χ2) [18,19] can be
used for this purpose, and is provided in pmr. It is given by > p_value
[1] 4.458237e-12 5.885750e-03 2.582951e-01 3.893159e-01
1.233049e-01 7.445499e-31 9.236701e-36
[8] 5.797042e-26 7.522277e-19 1.943696e-38 9.250903e-15
5.762506e-10 4.727443e-86 2.893224e-05
[15] 7.788308e-01 5.494515e-01 8.853680e-01 3.384748e-12 χ2 ¼
X
k! i
r2
i ; where ri ¼ Oi−Ei
ffiffiffi
Ei
p
is the Pearson residual, and Oi, Ei are the
observed and expected frequencies of ranking i, respect-
ively. The sum of square Pearson residual will automatically where ri ¼ Oi−Ei
ffiffiffi
Ei
p
is the Pearson residual, and Oi, Ei are the
observed and expected frequencies of ranking i, respect-
ively. The sum of square Pearson residual will automatically [15] 7.788308e-01 5.494515e-01 8.853680e-01 3.384748e-12 Page 10 of 11 Lee and Yu BMC Medical Research Methodology 2013, 13:65
http://www.biomedcentral.com/1471-2288/13/65 Lee and Yu BMC Medical Research Methodology 2013, 13:65
http://www.biomedcentral.com/1471-2288/13/65 Lee and Yu BMC Medical Research Methodology 2013, 13:65
http://www.biomedcentral.com/1471-2288/13/65 Additional files According to the results of the ROL model, female
physicians preferred items 1 and 4, and private physi-
cians did not prefer items 1, 2, and 7. Additional file 1: Package source of package pmr. Additional file 2: Reference manual of package pmr. Assume that we want to predict the preference of a list
of physicians with known covariates q4covtest. One pos-
sible method is to assign the utility ranks of the seven
items for these physicians using the parameters obtained
from the ROL model. Another method is to use the local
k-nearest neighbor algorithm with the R code local.knn(q4,
q4covtest,q4cov,knn.k = k). The value of k must be pre-
specified. The pmr package provides the cross-validation
version of the local k-nearest neighbor local.knn.cv(q4,
q4covtest,q4cov). By default this uses 10-fold cross valid-
ation and tests the cross-validation prediction error of k
(defined as the total Kendall’s distance) from 1 to 20. Lee and Yu BMC Medical Research Methodology 2013, 13:65
http://www.biomedcentral.com/1471-2288/13/65 21. Holleczek B, Gondos A, Brenner H: Period R - an R package to calculate
long-term cancer survival estimates using period analysis. Methods Inf
Med 2009, 48(2):123–128. 22. Kreuz M, Rosolowski M, Berger H, Schwaenen C, Wessendorf S, Loeffler M,
Hasenclever D: Development and implementation of an analysis tool for
array-based comparative genomic hybridization. Methods Inf Med 2007,
46(5):608–613. 23. Murphy TB, Martin D: Mixtures of distance-based models for ranking data. Comput Stat Data Anal 2003, 41:645–655. Comput Stat Data Anal 2003, 41:645–655. 24. Critchlow DE, Fligner MA, Verducci JS: Probability models on rankings. J Math Psychol 1991, 35:294–318. J Math Psychol 1991, 35:294–318. 25. Yu PLH: Statistical modelling of ranking data. In Computational
mathematics and modelling. edn. Edited by Lenbury Y, Sanh NV, Wu YH,
Wiwatanapataphee B. ; 2003:319–326. 26. Saaty TL: A scaling methods for priorities in hierarchical structure. J Math
Psychol 1977, 15:234–281. 27. Bozoki S, Rapcsak T: On Saaty’s and Koczkodaj’s inconsistencies of
pairwise comparison matrices. J Global Optim 2008, 42(2):157–175. 28. Carroll JD: Individual differences and multidimensional scaling. In
Multidimensional scaling: theory and applications in the behavioral sciences. Volume 1, edn. Edited by Shepard RN, Romney AK, Nerlove SB. New York:
Seminar Press; 1972. 29. Thurstone LL: A law of comparative judgement. Psychol Rev 1927, 34:273–286. f
f 29. Thurstone LL: A law of comparative judgement. Psychol Rev 1927 30. Guiver J, Snelson E: Bayesian inference for Plackett-Luce ranking models. Proc ICML 2009 2009. 31. Lu T, Boutilier C: Learning mallows models with pairwise preferences. Proc ICML 2011 2011. 32. Caron F, Teh YW: Bayesian nonparametric models for ranked data. Proc NIPS 2012 2012. 33. Chapman RG, Staelin R: Exploiting rank ordered choice set data within
the stochastic utility model. J Market Res 1982, 19:288–301. 33. Chapman RG, Staelin R: Exploiting rank ordered choice set 34. Beggs S, Cardell S, Hausman JA: Assessing the potential demand for
electric cars. J Econ 1981, 16:1–19. 35. Hausman JA, Ruud PA: Specifying and testing econometric models for
ranked-ordered data. J Econ 1987, 34(1-2):82–104. 36. Spearman C: The proof and measurement of association between two
things. Am J Psychol 1904, 15:72–101. g
y
37. Mallows CL: Non-null ranking models. I. Biometrika 1957, 44:114–130. g
38. Cayley A: A note on the theory of permutations. Phil Mag 1849, 34:527–52 39. Shieh GS: A weighted Kendall’s tau statistic. Stat Prob Lett 1998, 39:17–24 40. Availability and requirements 14. Ganesan K, Zhai C: Opinion-based entity ranking. Inf Retr 2012, 15:116–150. 14. Ganesan K, Zhai C: Opinion-based entity ranking. Inf Retr 2012, 15:116–150. 14. Ganesan K, Zhai C: Opinion-based entity ranking. Inf Retr 2012, 15:116–150. 15. Marden JI: Analyzing and modeling rank data. London: Chapman and Hall; 1995. 15. Marden JI: Analyzing and modeling rank data. London: Chapman and Hall; 1995. 15. Marden JI: Analyzing and modeling rank data. London: Chapman and Hall; 1995. Project name: Probability Models for Ranking Data Project name: Probability Models fo Project name: Probability Models for Ranking Data 16. Luce RD: Individual choice behavior. New York: John Wiley and Sons; 1959. 16. Luce RD: Individual choice behavior. New York: John Wiley and Sons; 1959. Project home page: http://cran.r-project.org/web/packages/
pmr/index.html 17. Fligner MA, Verducci JS: Distance based ranking models. J R Stat Soc B
1986, 48(3):359–369. 17. Fligner MA, Verducci JS: Distance based ranking models. J R Stat Soc B
1986, 48(3):359–369. 18. Lee PH, Yu PLH: Distance-based tree models for ranking data. Comput
Stat Data Anal 2010, 54(6):1672–1682. 18. Lee PH, Yu PLH: Distance-based tree models for ranking data. Comput
Stat Data Anal 2010, 54(6):1672–1682. 19. Lee PH, Yu PLH: Mixtures of weighted distance-based models for ranking
data with applications in political studies. Comput Stat Data Anal 2012,
56(8):2486–2500. 19. Lee PH, Yu PLH: Mixtures of weighted distance-based models for ranking
data with applications in political studies. Comput Stat Data Anal 2012,
56(8):2486–2500. 20. R: a language and environment for statistical computating. [http://www.R-
project.org]. Any restrictions to use by non-academics: none Any restrictions to use by non-academics: none Page 11 of 11 Page 11 of 11 Lee and Yu BMC Medical Research Methodology 2013, 13:65
http://www.biomedcentral.com/1471-2288/13/65 Lee and Yu BMC Medical Research Methodology 2013, 13:65
http://www.biomedcentral.com/1471-2288/13/65 Shieh GS, Bai Z, Tsai WY: Rank tests for independence - with a weighted
contamination alternative. Stat Sin 2000, 10:577–593. 41. Tarsitano A: Comparing the effectiveness of rank correlation statistics. In
Working papers, universita della calabria, dipartimento di economia e
statistica, 200906. ; 2009. 42. Cheng W, Dembczynski K, Hullermeier E: Label ranking methods based on
the Plackett-Luce model. Proc ICML 2010 2010. the Plackett-Luce model. Proc ICML 2010 2010 43. Koczkodaj WW, Herman MW, Orlowski M: Using consistency-dri 43. Koczkodaj WW, Herman MW, Orlowski M: Using consistency-driven
pairwise comaprisons in knowledge-based systems. Proc CIKM 1997 1997 pairwise comaprisons in knowledge-based systems. Proc CIKM 44. Thompson GL: Graphical techniques for ranked data. In Probability models
and statistical analyses for ranking data. edn. Edited by Fligner MA, Verducci
JS. New York: Springer; 1993:294–298. 45. Cheng W, Hullermeier E: A new instance-based label ranking approach
using the Mallows model. Proc ISNN 2009 2009. 45. Cheng W, Hullermeier E: A new instance-based label ranking approach
using the Mallows model. Proc ISNN 2009 2009. 46. Yu PLH, Wan WM, Lee PH: Decision tree modelling for ranking data. In
Preference learning. edn. Edited by Furnkranz J, Hullermeier E. Berlin:
Springer-Verlag; 2010:83–106. 46. Yu PLH, Wan WM, Lee PH: Decision tree modelling for ranking data. In
Preference learning. edn. Edited by Furnkranz J, Hullermeier E. Berlin:
Springer-Verlag; 2010:83–106. Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
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Submit your manuscript at
www.biomedcentral.com/submit Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit 47. Fligner MA, Verducci JS: Multi-stage ranking models. Lee and Yu BMC Medical Research Methodology 2013, 13:65
http://www.biomedcentral.com/1471-2288/13/65 J Am Stat Assoc 1988,
83:892–901. 47. Fligner MA, Verducci JS: Multi-stage ranking models. J Am Stat Assoc 1988,
83:892–901. Submit your next manuscript to BioMed Central
and take full advantage of: Submit your next manuscript to BioMed Central
and take full advantage of: 48. Xu L: A multistage ranking model. Psychometrika 2000, 65(2):217–231. doi:10.1186/1471-2288-13-65
Cite this article as: Lee and Yu: An R package for analyzing and
modeling ranking data. BMC Medical Research Methodology 2013 13:65. • Convenient online submission • Thorough peer review
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https://aacr.figshare.com/articles/journal_contribution/Supplementary_Tables_1-3_from_Antitumor_Immunity_Triggered_by_Melphalan_Is_Potentiated_by_Melanoma_Cell_Surface_Associated_Calreticulin/22406952/1/files/39852813.pdf
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Supplementary Tables 1-3 from Antitumor Immunity Triggered by Melphalan Is Potentiated by Melanoma Cell Surface–Associated Calreticulin
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Supplementary Table 1. Meta-data analysis of clinical response to melphalan-based isolated limbtreatment procedures in melanoma patients
Study
Treatment
Number of Overall
Complete Partial
Treatments* Response Response Response
(%)
(%)
(%)
Hoekstra et al. 2014
Mel
18
33
Shetty et al. 2013
Mel
180
28
14
Beasley et al. 2012
Mel
36
28
Rossi et al. 2010
Mel
53
41.5
Beasley et al. 2009
Mel
162
31
33
Cornett et al. 2006
Mel
58
64
25
39
Aloia et al. 2005
Mel
58
88
57
31
Noorda et al. 2004
Mel
40
45
Lienard et al. 1999
Mel
103
77.60
52.40
25.20
Fracker et al. 1996
Mel
43
69
69
Klaase et al. 1994
Mel
45
68
47
20
Klaase et al. 1994
Mel
216
67
42
25
* Isolated Limb Perfusions (ILPs) or Isolated Limb Infusions (ILIs) in Patients with melanoma.
Refs.
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
(11)
(12)
Supplementary Table 2. Meta-data analysis of clinical response to melphalan/TNF or Mel/TNF/IFNγ
combination therapies for isolated limb-treatment procedures in melanoma patients
Study
Treatment
Number of Overall
Complete Partial
Treatments* Response Response Response
(%)
(%)
(%)
Hoekstra et al. 2014
Mel/TNF
39
54
Deroose et al. 2011
Mel/TNF
118
93.2
67.8
25.4
Rossi et. al. 2008
Mel/TNF
12
100
50
50
Hayes et al. 2007
Mel/TNF
27
77
41
37
Cornett et al. 2006
Mel/TNF
58
69
26
43
Grunhagen et al. 2005 Mel/TNF
83
96
69
27
Grunhagen et al. 2004 Mel/TNF
100
95
69
26
Noorda et al. 2004
Mel/TNF
90
59
Rossi et al. 2004
Mel/TNF
20
95
70
25
Feldman et al. 1999
Mel/TNF
6
83
Lienard et al. 1996
Mel/TNF/IFNγ 64
100
78.10
21.90
Mel/TNF
90.70
68.80
21.90
Fracker et al. 1996
Mel/TNF/IFNγ 43
80
80
Vaglini et al. 1994
Mel/TNF
22
77.20
63.60
13.60
Lienard et al. 1994
Mel/TNF/IFNγ 53
100
90
10
Lienard et al. 1992
Mel/TNF/IFNγ 20
100
90
10
* Isolated Limb Perfusions (ILPs) or Isolated Limb Infusions (ILIs) in Patients with melanoma.
Refs.
(1)
(13)
(14)
(15)
(6)
(16)
(17)
(8)
(18)
(19)
(10)
(10)
(20)
(10)
(21)
Supplementary Table 3. Follow up information about the patients whose tumour samples were used
for analysis of infiltration of immune cells and expression levels of cytokines.
Age
Gender
(Years)
Nature of Melanoma
Lesion
Foot and Regional
lymph node
Patient Follow-up Status
61
M
Deceased
62
M
In transit metastasis
Deceased
78
F
In transit metastasis
No Follow-Up
65
F
In transit metastasis
No evaluable disease
65
F
In transit metastasis
No evaluable disease
71
F
In transit metastasis
No evaluable disease
59
F
In Transit and Lymph
Node Metastasis
No Follow-Up
76
F
In transit metastasis
Alive with Lung metastasis
55
F
In transit metastasis
Alive with Brain Metastasis
71
F
In transit metastasis
No Follow-Up
References:
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
Hoekstra HJ, Veerman K, van Ginkel RJ. Isolated limb perfusion for in-transit melanoma
metastases: Melphalan or TNF-melphalan perfusion? Journal of surgical oncology
2014;109(4):338-47.
Shetty G, Beasley GM, Sparks S, Barfield M, Masoud M, Mosca PJ, et al. Plasma cytokine
analysis in patients with advanced extremity melanoma undergoing isolated limb infusion.
Ann Surg Oncol 2013;20(4):1128-35.
Beasley GM, Sharma K, Wong J, Miller M, Turley RS, Lidsky M, et al. A multi-institution
experience comparing the clinical and physiologic differences between upper extremity and
lower extremity melphalan-based isolated limb infusion. Cancer 2012;118(24):6136-43.
Rossi CR, Pasquali S, Mocellin S, Vecchiato A, Campana LG, Pilati P, et al. Long-term
results of melphalan-based isolated limb perfusion with or without low-dose TNF for in-transit
melanoma metastases. Ann Surg Oncol 2010;17(11):3000-7.
Beasley GM, Caudle A, Petersen RP, McMahon NS, Padussis J, Mosca PJ, et al. A multiinstitutional experience of isolated limb infusion: defining response and toxicity in the US. J
Am Coll Surg 2009;208(5):706-15; discussion 15-7.
Cornett WR, McCall LM, Petersen RP, Ross MI, Briele HA, Noyes RD, et al. Randomized
multicenter trial of hyperthermic isolated limb perfusion with melphalan alone compared with
melphalan plus tumor necrosis factor: American College of Surgeons Oncology Group Trial
Z0020. Journal of clinical oncology : official journal of the American Society of Clinical
Oncology 2006;24(25):4196-201.
Aloia TA, Grubbs E, Onaitis M, Mosca PJ, Cheng TY, Seigler H, et al. Predictors of outcome
after hyperthermic isolated limb perfusion: role of tumor response. Arch Surg
2005;140(11):1115-20.
Noorda EM, Vrouenraets BC, Nieweg OE, van Geel BN, Eggermont AM, Kroon BB. Isolated
limb perfusion for unresectable melanoma of the extremities. Arch Surg 2004;139(11):123742.
Lienard D, Eggermont AM, Koops HS, Kroon B, Towse G, Hiemstra S, et al. Isolated limb
perfusion with tumour necrosis factor-alpha and melphalan with or without interferon-gamma
for the treatment of in-transit melanoma metastases: a multicentre randomized phase II study.
Melanoma Res 1999;9(5):491-502.
Lienard D, Eggermont AM, Kroon BB, Schraffordt Koops H, Lejeune FJ. Isolated limb
perfusion in primary and recurrent melanoma: indications and results. Seminars in surgical
oncology 1998;14(3):202-9.
Klaase JM, Kroon BB, van Geel AN, Eggermont AM, Franklin HR, van Dongen JA. Is there
an indication for a double perfusion schedule with melphalan for patients with recurrent
melanoma of the limbs? Melanoma Res 1994;4 Suppl 1:13-6.
Klaase JM, Kroon BB, van Geel AN, van Wijk J, Franklin HR, Eggermont AM, et al. Limb
recurrence-free interval and survival in patients with recurrent melanoma of the extremities
treated with normothermic isolated perfusion. J Am Coll Surg 1994;178(6):564-72.
Deroose JP, Grunhagen DJ, van Geel AN, de Wilt JH, Eggermont AM, Verhoef C. Long-term
outcome of isolated limb perfusion with tumour necrosis factor-alpha for patients with
melanoma in-transit metastases. The British journal of surgery 2011;98(11):1573-80.
Rossi CR, Russano F, Mocellin S, Chiarion-Sileni V, Foletto M, Pilati P, et al. TNF-based
isolated limb perfusion followed by consolidation biotherapy with systemic low-dose
interferon alpha 2b in patients with in-transit melanoma metastases: a pilot trial. Ann Surg
Oncol 2008;15(4):1218-23.
Hayes AJ, Neuhaus SJ, Clark MA, Thomas JM. Isolated limb perfusion with melphalan and
tumor necrosis factor alpha for advanced melanoma and soft-tissue sarcoma. Ann Surg Oncol
2007;14(1):230-8.
Grunhagen DJ, de Wilt JH, van Geel AN, Graveland WJ, Verhoef C, Eggermont AM. TNF
dose reduction in isolated limb perfusion. Eur J Surg Oncol 2005;31(9):1011-9.
Grunhagen DJ, Brunstein F, Graveland WJ, van Geel AN, de Wilt JH, Eggermont AM. One
hundred consecutive isolated limb perfusions with TNF-alpha and melphalan in melanoma
18.
19.
20.
21.
patients with multiple in-transit metastases. Annals of surgery 2004;240(6):939-47; discussion
47-8.
Rossi CR, Foletto M, Mocellin S, Pilati P, Lise M. Hyperthermic isolated limb perfusion with
low-dose tumor necrosis factor-alpha and melphalan for bulky in-transit melanoma
metastases. Ann Surg Oncol 2004;11(2):173-7.
Feldman AL, Alexander HR, Jr., Bartlett DL, Fraker DL, Libutti SK. Management of
extremity recurrences after complete responses to isolated limb perfusion in patients with
melanoma. Ann Surg Oncol 1999;6(6):562-7.
Vaglini M, Santinami M, Manzi R, Inglese MG, Santoro N, Persiani L, et al. Treatment of intransit metastases from cutaneous melanoma by isolation perfusion with tumour necrosis
factor-alpha (TNF-alpha), melphalan and interferon-gamma (IFN-gamma). Dose-finding
experience at the National Cancer Institute of Milan. Melanoma Res 1994;4 Suppl 1:35-8.
Lienard D, Ewalenko P, Delmotte JJ, Renard N, Lejeune FJ. High-dose recombinant tumor
necrosis factor alpha in combination with interferon gamma and melphalan in isolation
perfusion of the limbs for melanoma and sarcoma. Journal of clinical oncology : official
journal of the American Society of Clinical Oncology 1992;10(1):52-60.
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Supplementary Figure S2 from The p53/p21 Complex Regulates Cancer Cell Invasion and Apoptosis by Targeting Bcl-2 Family Proteins
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Figure S2. Bcl-XL is a target of the cooperative action of p53 and p21. A, In vitro
translation mixtures of p21 and Bcl-XL (top) or H1299 cell lysates (bottom) were
immunoprecipitated with an anti-p21 antibody or control IgG. Precipitates and input
controls were analyzed by western blotting with anti-p21 and anti-Bcl-XL antibodies. B,
p53, p53K305N, and p21 proteins were generated in the indicated combinations by in vitro
translation. The products were immunoprecipitated with an anti-Bcl-XL antibody. Bax
and p53 levels in the precipitates were analyzed by western blotting. C, H1299 cells
were transfected with pcDNA3, pcDNA3-p53K305N, and pcDNA3-p21 in the indicated
combinations (left). Control and p53K305N transfectants were treated with control or p21
siRNA (right). Cell lysates were prepared and immunoprecipitated with an anti-Bcl-XL
antibody or control IgG. Precipitates and inputs were analyzed by western blotting using
the indicated antibodies. D, The indicated transfectants were treated with control or p21 Figure S2. Bcl-XL is a target of the cooperative action of p53 and p21. A, In vitro
translation mixtures of p21 and Bcl-XL (top) or H1299 cell lysates (bottom) were
immunoprecipitated with an anti-p21 antibody or control IgG. Precipitates and input
controls were analyzed by western blotting with anti-p21 and anti-Bcl-XL antibodies. B,
p53, p53K305N, and p21 proteins were generated in the indicated combinations by in vitro
translation. The products were immunoprecipitated with an anti-Bcl-XL antibody. Bax
and p53 levels in the precipitates were analyzed by western blotting. C, H1299 cells
were transfected with pcDNA3, pcDNA3-p53K305N, and pcDNA3-p21 in the indicated
combinations (left). Control and p53K305N transfectants were treated with control or p21
siRNA (right). Cell lysates were prepared and immunoprecipitated with an anti-Bcl-XL
antibody or control IgG. Precipitates and inputs were analyzed by western blotting using
the indicated antibodies. D, The indicated transfectants were treated with control or p21 Figure S2. Bcl-XL is a target of the cooperative action of p53 and p21. A, In vitro
translation mixtures of p21 and Bcl-XL (top) or H1299 cell lysates (bottom) were
immunoprecipitated with an anti-p21 antibody or control IgG. Precipitates and input
controls were analyzed by western blotting with anti-p21 and anti-Bcl-XL antibodies. B,
p53, p53K305N, and p21 proteins were generated in the indicated combinations by in vitro Figure S2. Bcl-XL is a target of the cooperative action of p53 and p21. A, In vitro
translation mixtures of p21 and Bcl-XL (top) or H1299 cell lysates (bottom) were
immunoprecipitated with an anti-p21 antibody or control IgG. Precipitates and input
controls were analyzed by western blotting with anti-p21 and anti-Bcl-XL antibodies. B,
p53, p53K305N, and p21 proteins were generated in the indicated combinations by in vitro
translation. The products were immunoprecipitated with an anti-Bcl-XL antibody. Bax
and p53 levels in the precipitates were analyzed by western blotting. C, H1299 cells
were transfected with pcDNA3, pcDNA3-p53K305N, and pcDNA3-p21 in the indicated
combinations (left). Control and p53K305N transfectants were treated with control or p21
siRNA (right). Cell lysates were prepared and immunoprecipitated with an anti-Bcl-XL
antibody or control IgG. Precipitates and inputs were analyzed by western blotting using
the indicated antibodies. D, The indicated transfectants were treated with control or p21 Figure S2. Bcl-XL is a target of the cooperative action of p53 and p21. Figure S2. Bcl-XL is a target of the cooperative action of p53 and p21. A, In vitro
translation mixtures of p21 and Bcl-XL (top) or H1299 cell lysates (bottom) were
immunoprecipitated with an anti-p21 antibody or control IgG. Precipitates and input
controls were analyzed by western blotting with anti-p21 and anti-Bcl-XL antibodies. B,
p53, p53K305N, and p21 proteins were generated in the indicated combinations by in vitro
translation. The products were immunoprecipitated with an anti-Bcl-XL antibody. Bax
and p53 levels in the precipitates were analyzed by western blotting. C, H1299 cells
were transfected with pcDNA3, pcDNA3-p53K305N, and pcDNA3-p21 in the indicated
combinations (left). Control and p53K305N transfectants were treated with control or p21
siRNA (right). Cell lysates were prepared and immunoprecipitated with an anti-Bcl-XL
antibody or control IgG. Precipitates and inputs were analyzed by western blotting using
the indicated antibodies. D, The indicated transfectants were treated with control or p21 A, In vitro
translation mixtures of p21 and Bcl-XL (top) or H1299 cell lysates (bottom) were
immunoprecipitated with an anti-p21 antibody or control IgG. Precipitates and input
controls were analyzed by western blotting with anti-p21 and anti-Bcl-XL antibodies. B,
p53, p53K305N, and p21 proteins were generated in the indicated combinations by in vitro
translation. The products were immunoprecipitated with an anti-Bcl-XL antibody. Bax
and p53 levels in the precipitates were analyzed by western blotting. C, H1299 cells
were transfected with pcDNA3, pcDNA3-p53K305N, and pcDNA3-p21 in the indicated
combinations (left). Control and p53K305N transfectants were treated with control or p21
siRNA (right). Cell lysates were prepared and immunoprecipitated with an anti-Bcl-XL
antibody or control IgG. Precipitates and inputs were analyzed by western blotting using
the indicated antibodies. D, The indicated transfectants were treated with control or p21 1 1 siRNA and then subjected to 20 Gy -irradiation. After 24 h of irradiation, cell lysates
were prepared and analyzed for Bcl-XL/Bax and Bcl-XL/p53 interactions by co-
immunoprecipitation. 2 2
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The incidence of experimental smoking in school children: an 8-year follow-up of the child and adolescent behaviors in long-term evolution (CABLE) study
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RESEARCH ARTICLE Open Access Abstract Background: Studies have established that most regular adult smokers become addicted in their adolescent years. We investigated the incidence of and risk factors associated with initial experimental smoking among a group of
school children who were followed for 8 years. Methods: We used cohort data collected as part of the Child and Adolescent Behaviors in Long-term Evolution
(CABLE) study, which selected nine elementary schools each from an urban area (Taipei City) and a rural area
(Hsingchu county) in northern Taiwan. From 2002 to 2008, children were asked annually whether they had smoked
in the previous year. An accelerated lifetime model with Weibull distribution was used to examine the factors
associated with experimental smoking. Results: In 2001, 2686 4th-graders participated in the study. For each year from 2002 to 2008, their incidences of
trial smoking were 3.1%, 4.0%, 2.8%, 6.0%, 5.3%, 5.0% and 6.0%, respectively. There was an increase from 7th to 8th
grade (6.0%). Children who were males, lived in rural areas, came from single-parent families, had parents who
smoked, and had peers who smoked were more likely to try smoking earlier. The influence of parents and peers
on experimental smoking demonstrated gradient effects. Conclusions: This study used a cohort to examine incidence and multiple influences, including individual factors,
familial factors, and community factors, on experimental smoking in adolescents. The findings fit the social
ecological model, highlighting the influences of family and friends. School and community attachment were
associated with experimental smoking in teenagers. Keywords: cigarette smoking, adolescent behavior, epidemiological factors, socioeconomic factors prominent public health issues and is a leading cause of
premature death worldwide [3,4]. A high prevalence of
smoking has been observed in Chinese societies [5]. Sev-
eral studies have established that most regular adult
smokers become addicted to nicotine in their adolescent
years [6-8]. Public health researchers have conducted
extensive research on adolescent smoking and its rela-
tionship with factors such as parental and peer relations,
academic performance, socioeconomic factors, and
environmental factors [9]. Due to the specific targeting
of advertising by the tobacco industry, growing popula-
tions of adolescents in both developing and developed
countries are taking up smoking [10]. If not effectively Chang et al. BMC Public Health 2011, 11:844
http://www.biomedcentral.com/1471-2458/11/844 Chang et al. BMC Public Health 2011, 11:844
http://www.biomedcentral.com/1471-2458/11/844 The incidence of experimental smoking in school
children: an 8-year follow-up of the child and
adolescent behaviors in long-term evolution
(CABLE) study Hsing-Yi Chang1*, Wen-Chi Wu2, Chi-Chen Wu1, Jennifer Y Cheng1, Baai-Shyun Hurng3 and Lee-Lan Yen1,4 Hsing-Yi Chang1*, Wen-Chi Wu2, Chi-Chen Wu1, Jennifer Y Cheng1, Baai-Shyun Hurng3 and © 2011 Chang et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited. * Correspondence: hsingyi@nhri.org.tw
1Division of Preventive Medicine and Health Service Research, Institute of
Population Health Sciences, National Health Research Institutes, Zhunan
Town, Miaoli County, Taiwan, ROC
Full list of author information is available at the end of the article Background Smoking is one of the most popular forms of drug use
and is a major risk factor for lung cancer and cardiovas-
cular disease [1]. In a systematic review of 17 studies,
smoking was found to be associated with peripheral
arterial disease not only among current smokers but
also among former smokers [2]. Due to its public acces-
sibility, smoking has become one of the world’s most * Correspondence: hsingyi@nhri.org.tw
1Division of Preventive Medicine and Health Service Research, Institute of
Population Health Sciences, National Health Research Institutes, Zhunan
Town, Miaoli County, Taiwan, ROC
Full list of author information is available at the end of the article Chang et al. BMC Public Health 2011, 11:844
http://www.biomedcentral.com/1471-2458/11/844 Page 2 of 9 Page 2 of 9 each from an urban area (Taipei City) and a rural area
(Hsingchu County) in northern Taiwan were selected. The 1st and 4th graders in each school (representing the
1st and 2nd cohorts) were selected as the baseline
cohorts to be followed annually. The CABLE study was
approved by the Internal Review Board of the National
Health Research Institutes (approval code: EC9009003). All parents of students in the study signed a consent
form allowing their children to participate. We used
data from the initial cohort of fourth-graders in this
study because they were entering adolescence. Complete
data on all 8 years of follow-up were available for more
than two-thirds of the children. addressed, the world will see generations of young smo-
kers suffer from highly preventable cardiovascular dis-
eases and risk premature death. The reasons behind
adolescent smoking are complex and involve multiple
influences, including individual factors, socioeconomic
factors, familial factors, peer influences, school and
environmental factors. It has been reported that the
majority of youth experimental smokers become daily
smokers [7,8]. An understanding of the age of experi-
mental smoking and related risk factors is important for
designing early intervention programs targeting high-
risk groups. Many studies have monitored the smoking status of
youth worldwide. The Global Youth Tobacco Survey
(GYTS) is the largest surveillance system, which con-
ducts periodic surveys on tobacco use in a wide range
of countries [11]. Many longitudinal studies have also
explored the factors predicting regular smoking in teens
[8]. However, these studies, especially longitudinal stu-
dies of experimental smoking in school children, have
rarely been conducted in Asia. Background Previous studies exam-
ined either current smoking behavior or the initiation
age of smoking. As mentioned above, a large proportion
of experimental youth smokers continue to smoke in
adulthood. It is important to understand when these
youth first tried smoking and the factors that are asso-
ciated with these trials. The outcome variable was experimental smoking from
2002 to 2008. Each year, children were asked whether
they had smoked in the past year. We asked the ques-
tion, ‘Have you smoked cigarettes, even one puff?’ The
answers included (1) never; (2) not this year, but the
year before; (3) not this month, but this year; (4) one or
two times this month; (5) many times this month; and
(6) almost every day this month. This set of questions
was similar to the Global Youth Tobacco Survey [14]. Students who had not answered ‘yes’ in previous years
and who answered ‘yes’ in a subsequent year were con-
sidered new cases. Because we were studying experi-
mental smoking in children aged approximately 10
years, we considered one inhalation of tobacco to be an
incidence of experimental smoking. In addition to indi-
vidual factors, we were interested in the effects of family
and community on the initiation of smoking. The Child and Adolescent Behaviors in Long-term
Evolution (CABLE) study [12] was initiated to examine
the behavioral development of school children in Tai-
wan. Children were visited annually and were asked
questions related to their behaviors. The study has accu-
mulated long-term information on these children’s
behaviors and risk factors. Information concerning when
children first tried smoking and the factors associated
with smoking experimentation can be extracted from
these data. The purpose of this study was to investigate
the incidence of experimental smoking and its risk fac-
tors in a group of school children who were followed
for 8 years. Individual factors included gender, residential area,
self-perceived school performance, depressive symptoms
and self-competence. Depressive symptoms were mea-
sured by 7 questions, which asked about loss of appetite,
feeling sad, crying for no reason, getting upset over
nothing, feeling frightened, difficulty sleeping, and lack
of motivation to do anything in the previous two weeks. A three-point-scale was used with 1 point given for not
at all, 2 for sometimes, and 3 for often. The sum of the
scores for all items was used. A higher score implied
more depressive symptoms. Background The questions were based
on the depressive symptoms of the Center for Epidemio-
logical Studies Depression Scale for Children (CES-DC)
[15], which has been widely used in Taiwanese surveys. Cronbach’s a, which measures how well each individual
item in a scale correlates with the sum of the remaining
items, was used for the internal consistency or validity
of the scales. The survey of 8th graders was used to esti-
mate Cronbach’s a. The widely accepted social science
cut-off is an alpha of .70 or higher for a set of items to
be considered a scale [16]. The Cronbach’s a was 0.74. Methods Data for this study came from the study of Child and
Adolescent Behaviors in Long-term Evolution (CABLE)
[12,13]. The study was designed to observe the develop-
ment of children based on the ecological model, which
emphasizes that individual, interpersonal, organizational,
community and public policy factors shape the develop-
ment of a child. Additional information was collected
during the yearly follow-ups to reflect problems faced
by the children at that age, but the changes on questions
were minor. Students from urban and rural areas were
sampled to identify the developmental differences
between these two groups [13]. Nine elementary schools Self-competence was measured by 6 items, including
optimism, feeling happy, willingness to try new things,
working hard, facing problems positively, and perceiving Chang et al. BMC Public Health 2011, 11:844
http://www.biomedcentral.com/1471-2458/11/844 Page 3 of 9 Page 3 of 9 for each item were summed to obtain the score for par-
ental supervision. Cronbach’s a was 0.77. Details of the
questions can be found in Appendix 1. oneself as being as good as others. The concept was
based on Tafarodi and Swann [17]. The Chinese version
was evaluated by Song et al. [18]. A 5-point Likert scale
was used, with scores ranging from 1 (never) to 5
(always). The scores of the 8th graders were used to cal-
culate Cronbach’s a, which was 0.79. Peer smoking, school attachment and neighbors were
considered as community factors. Children were asked
whether none, a few, about half, most or all of their
peers smoked. School attachment was assessed by
whether children liked their current school, teachers,
and classmates. Responses to each item had five options
ranging from 1 (do not like it/them at all) to 5 (like it/
them very much). These questions were included in the
questionnaire from 2003. The average of scores from
2003 to 2008 was used in the analysis. Eight items were
used to assess community attachment: (1) Do you or
your family visit or talk to your neighbors? (2) When
you go away, does your family ask neighbors to house
sit, including checking the mail, watering the garden,
and feeding the dog? (3) Do you think your neighbors
are trustworthy? (4) Do you think your neighbors are
kind and friendly? (5) Do you and your family partici-
pate in community activities? (6) Do you like where you
live? (7) Do you think your living environment is safe? Methods (8) Are there ever strange people wandering around the
neighborhood? A 5-point scale was used for each item,
with 1 point indicating never and 5 points indicating all
the time. A score in the lowest quartile (22 points) was
considered low, and a score in the upper quartile (29
points) was considered high. Details of the questions
used in all scales are given in Appendix 1 (Additional
file 1, appendix 1). Family factors included socio-economic status, coming
from a single-parent family and the degree of parental
support and supervision. Parental smoking was also
assessed. Family socioeconomic status was measured by
family income and father’s education level. The cate-
gories of monthly family income were (1) less than 19,
999NTD (new Taiwan dollars, 1 NTD ≈0.03 USD); (2)
20,000~39, 999NTD; (3) 40, 000NTD~59, 999NTD; (4)
60,000~79, 999NTD; (5) 80,000~99, 999NTD; (6)
100,000~119, 999NTD; (7) 120,000~139, 999NTD; and
(8) ≥140, 000NTD. Education levels were (1) elemen-
tary school or less; (2) junior high; (3) senior high; (4)
vocational school; (5) college; (6) university; and (7)
graduate school or above. We added together the levels
of family income and education and then divided them
into 3 categories: low < 8 (1st quartile); medium 8-13;
and high 13 (4th quartile). Parental smoking status was assessed for both parents. The combination of answers resulted in three categories:
(1) both parents smoke; (2) only one parent smokes; and
(3) neither parent smokes. The questions on parental
support and supervision were part of family interactions
[19]. They were developed by a panel of experts specia-
lizing in behavioral sciences and education. They
adapted the concepts from various researchers [20,21]. Once the experts reached consensus, the questions were
examined by the fieldworkers to evaluate their feasibility. Then, the CABLE team has extracted factors on family
interactions. The family interactions consisted of 6
aspects, including parental support, family activities, psy-
chological control, parental discipline, behavioral super-
vision, and family conflict [19]. We only used two
aspects in this study, which were parental support and
supervision. Parental support was assessed by 6 ques-
tions: providing encouragement in difficult times, prais-
ing good performance, providing comfort when you are
upset, taking care of you when you are sick, listening,
and taking an interest in your school life. Each response
had four options ranging from 1 (never) to 4 (every
time). Methods Scores for each item were added together to
obtain the score for parental support. The Cronbach’s a
was 0.91. Parental supervision was assessed by four
questions, which asked whether parents were aware of
what children did in their spare time, what they did on
their way home from school, who they spent time with,
and how they used their allowance. A 4-point scale was
used, with 1 indicating that parents did not know any-
thing and 4 indicating that they knew everything. Scores The event time was 4th grade to the year of experi-
mental smoking. Because experimental smoking could
occur before the first interview, between the two inter-
view years, or after 2008, the data contained all types of
censoring: left censoring, interval censoring and right
censoring. The survival time was the time that students
never smoked. A parametric model is appropriate for
this type of data. A Weibull distribution consists of two
parameters describing the shape and scale of the distri-
bution curve [22]. Therefore, an accelerated lifetime
model (ALT), which models the survival time assuming
Weibull distribution, was used. All analyses were con-
ducted using SAS version 10 (SAS Inc., Cary, USA). Results Table 1 shows the characteristics of the study subjects. There were 2071 subjects who participated in the study
in 2001. In 2002, we sent invitations to those who did
not consent in 2001, and 615 of these subjects re-joined
the study. Therefore, the total number of children at the
start of the study was 2686 4th-graders. Over two-thirds
of the participants were followed for 8 years. More boys
than girls had tried smoking at least once during the 8-
year follow-up. More ever-smokers lived in a rural area Chang et al. BMC Public Health 2011, 11:844
http://www.biomedcentral.com/1471-2458/11/844 Page 4 of 9 Page 4 of 9 Table 1 Characteristics of the study sample
Never
smoked
Smoked
Characteristic
n
(%)
n
(%)
c2
Gender (n = 2686)
78.21 ***
Boys
831
(60.13)
551
(39.87)
Girls
992
(76.07)
312
(23.93)
Residential area (n = 2686)
43.88 ***
Taipei (urban)
1062
(73.39)
385
(26.61)
Hsinchu (rural)
761
(61.42)
478
(38.58)
Perceived school performance (n = 2341)
6.46 *
Poor
861
(67.21)
420
(32.79)
Fair
575
(64.90)
311
(35.10)
Good
130
(74.71)
44
(25.29)
Single-parent family (n =
2668)
54.92 ***
No
1559
(71.03)
636
(28.97)
Yes
253
(53.49)
220
(46.51)
SES (n = 2611)
54.18 ***
Low
341
(57.02)
257
(42.98)
Medium
1037
(69.41)
457
(30.59)
High
400
(77.07)
119
(22.93)
Parental smoking (n = 2418)
84.48 ***
Neither
743
(78.05)
209
(21.95)
One
815
(64.43)
450
(35.57)
Both
99
(49.25)
102
(50.75)
Peer smoking (n = 2540)
463.45 ***
None
556
(90.11)
61
(9.89)
Few
900
(71.60)
357
(28.40)
Over half
234
(35.14)
432
(64.86)
Community attachment
(n = 2686)
12.71 **
Low
591
(66.18)
302
(33.82)
Medium
893
(66.59)
448
(33.41)
High
339
(75.00)
113
(25.00)
Scores
Mean
(SD)
Mean
(SD)
t
Depressive symptoms
11.06
(2.76)
11.57
(2.79)
-4.46 ***
Self-competence
21.15
(4.01)
20.28
(3.97)
5.13 ***
Parental support
18.89
(4.32)
17.29
(4.71)
8.41 ***
Parental supervision
13.22
(2.82)
12.21
(3.19)
7.74 ***
School attachment
10.86
(1.43)
10.22
(1.54)
10.18 ***
*: p < 0.05; **: p < 0.01; ***: p < 0.001 Table 1 Characteristics of the study sample Children who started smoking had significantly higher
depressive symptom scores, lower self-competence,
lower parental support, less parental supervision, and
lower school attachment scores. The smoking incidence
rate is shown graphically in Figure 1. There was a drop
in incidence in the 7th grade followed by a sharp
increase in the 8th grade. Results Boys were consistently more
likely to take up smoking than girls. Table 2 shows the results of survival analysis. We used
an accelerated failure time model assuming a Weibull
distribution. The scale was 0.57, which indicated that
the hazard was increasing at a decreasing rate. A nega-
tive value indicated an early initiation. Boys and stu-
dents who lived in a rural area, came from a single-
parent family, had parents who smoked, had peers who
smoked, had low school attachment, or had median
community attachment started smoking earlier than did
their counterparts. Discussion This study followed a group of 4th-graders annually and
assessed the incidence of experimental smoking and
related risk factors. The results showed that the inci-
dence of experimental smoking increased with age. There was a drop in incidence in the 7th grade followed
by an increase in the 8th grade. Individual factors, such
as gender and residential area, familial factors such as a
single-parent family and parental smoking status, and
community factors, such as peer smoking status, school
attachment and community cohesion, were risk factors
for smoking initiation in school children. The reasons for adolescent smoking are complex and
multifaceted. However, researchers have been able to
detect general trends related to the commencement of
smoking in adolescence. In Tyas and Pederson’s review
[23], age was associated with an increase in smoking
prevalence and initiation. Chen and colleagues reported
that the mean age of initiation of use any of tobacco,
alcohol, or illicit drugs was 16 to 18 years [24]. We
observed a similar pattern, except for the decrease in
the 7th grade, which may have been a result of the tran-
sition involved in attending a new school. In Taiwan, six
years of education was compulsory until 1968 after
which compulsory education was extended to 9 years. All students attend elementary school for 6 years after
which they move to junior high school for the next 3
years. During the first year in a new school, students
might go through a period of adjustment. This is also
the time when students are entering adolescence and
when they begin to explore and experiment with new
things. Once they have adjusted to the new environ-
ment, the students may begin experimenting. This phe-
nomenon
could
be
the
reason
for
a
drop
in
experimental smoking incidence in the 7th grade (Hsinchu) than in an urban area (Taipei city). Children
who had poorer perceived school performance were
more likely to start smoking during the follow-up. Chil-
dren with medium to low socioeconomic status were
more likely to smoke than those with high socioeco-
nomic status. Children with one or both parents who
were smokers were more likely to be smokers them-
selves. Socioeconomic status was inversely associated
with smoking initiation, as was community attachment. All of these factors reached statistical significance. Chang et al. Discussion BMC Public Health 2011, 11:844
http://www.biomedcentral.com/1471-2458/11/844 Page 5 of 9 Grade
Overall1
3.12 3.99 2.84 5.98 5.25 5.00 5.98
Boys1
4.63 4.45 3.36 7.05 7.01 6.81 7.97
Girls1
1.58 3.54 2.34 4.96 3.60 3.36 4.25
Figure 1 Incidence (1/100) of smoking in school children from 2002 to 2008. 1. Incidence rate (1/100)
Chang et al. BMC Public Health 2011, 11:844
http://www.biomedcentral.com/1471-2458/11/844
Page 5 of 9 followed by a sharp increase in the 8th grade. One of the
advantages of our study was the examination of changes
over time. influence in boys and an association between smoking
and self-control problems in girls. Societal influences
are another possible contributing factor. Chinese culture
still believes that males should be strong and adventur-
ous, whereas female smoking is not socially acceptable. Our results are consistent with other observations in
Taiwan [25,26]. We plotted the mean scores of depression, family sup-
port, family supervision, self-competence, and school
attachment (Figure 2). The time effect for each of these
repeated measurements was significant. From the plots,
we see that the depression score increased over time. Family support dropped sharply between the 7th and 8th
grades, as did family supervision. The differences
between the self-competence and school attachment
scores of 7th and 8th graders were not as significant. We
speculate that family support or supervision played an
important role in the sharp increase in experimental
smoking. This is a difficult stage for families with ado-
lescents. On one hand, adolescents ask for indepen-
dence; on the other hand, they may not have good self-
control or make good behavioral choices. Family structure and socioeconomic factors both play
pivotal roles in the smoking status of adolescents. We
found that children from single-parent families experi-
mented with smoking earlier than their counterparts. Lin et al. reported that parents’ marital status was sig-
nificantly associated with adolescent smoking behavior
in southern Taiwan [27]. Griesbach et al. [28] found
that single-parent families and stepfamilies had a
higher probability of having smokers in the household,
and Covey and Tan [29] confirmed that two-parent
families were protective against adolescent smoking. Painful disruptions to normal family life, such as par-
ental separation, can trigger depressive symptoms in
adolescents, leading to problem behaviors, such as
smoking [29]. A gender difference was observed in this study. Gen-
der differences in adolescent smoking have been dis-
cussed previously. Simons-Morton et al. Discussion [9] proposed an
independent association between smoking and peer Chang et al. BMC Public Health 2011, 11:844
http://www.biomedcentral.com/1471-2458/11/844 Page 6 of 9 Page 6 of 9 Table 2 Relative risks for smoking initiation in school children
Variable
Estimate
s. e
95% C.I. c2
Pr > c2
Intercept
2.56
0.34
(1.89, 3.22)
56.80
< .0001
Individual factors
Boys vs. girls
-0.31
0.05
(-0.41, -0.20)
34.06
< .0001
Taipei vs. Hsinchu
0.23
0.05
(0.13, 0.34)
19.41
< .0001
School achievement:
Poor vs. good
0.07
0.11
(-0.14, 0.28)
0.39
0.53
Fair vs. good
0.03
0.11
(-0.18, 0.24)
0.08
0.78
Depressive symptoms score
0.00
0.01
(-0.02, 0.02)
0.05
0.82
Self-competence
0.00
0.01
(-0.01, 0.01)
0.02
0.90
Familial factors
Single-parent family
-0.20
0.06
(-0.32, -0.08)
10.04
0.00
SES:
Low vs. high
-0.09
0.08
(-0.26, 0.07)
1.23
0.27
Medium vs. high
0.06
0.07
(-0.08, 0.21)
0.73
0.39
Parental smoking:
One vs. none
-0.24
0.06
(-0.36, -0.13)
17.82
< .0001
Both vs. none
-0.37
0.09
(-0.55, -0.20)
18.07
< .0001
Parental support
0.01
0.01
(-0.00, 0.02)
1.35
0.25
Parental supervision
0.01
0.01
(0.00, 0.03)
2.22
0.14
Community factors
Peer smoking:
Few vs. none
-0.78
0.11
(-1.00, -0.56)
48.64
< .0001
Over half vs. none
-1.35
0.12
(-1.58, -1.12)
131.87
< .0001
School attachment
0.09
0.02
(0.05, 0.12)
20.01
< .0001
Community attachment:
Low vs. high
-0.14
0.08
(-0.30, 0.03)
2.69
0.10
Medium vs. high
-0.15
0.07
(-0.29, -0.01)
4.15
0.04
Scale
0.57
0.02
(0.53, 0.62)
Weibull Shape
1.75
0.07
(1.62, 1.89) Table 2 Relative risks for smoking initiation in school children result explains our findings of the effect of parental
smoking and lack of supervision on children’s experi-
mental smoking. However, a comparison of the influ-
ence of fathers and mothers was not the focus of our
study. We found that parental smoking was a risk factor for
smoking initiation in adolescents. Similar results were
reported from Hong Kong [30]. There was also a possi-
ble gradient effect: children who had two parents who
smoked were more likely to initiate smoking than were
those with only one parent who smoked. Parental influ-
ence may play a role in adolescents’ “preparatory period”
during which their opinions and beliefs towards smok-
ing are formed. This period determines their “suscept-
ibility” to smoking, with “non-susceptible” defined as
“strong negative intentions against future smoking” and
“susceptible” defined as “one or more questions without
strong negative intentions against future smoking” [31]. Discussion Never-smokers who believed that their parents would
disapprove of their smoking in the future were less sus-
ceptible to initiating smoking. Distefan and colleagues
[31] found that maternal smoking strongly encourages
the progression of both male and female experimenters
to become established smokers. These authors also
showed that parents may serve as an impediment to
adolescents’ choice of friends who smoke [31]. This The influence of peers is one of the most determina-
tive factors in adolescents’ experimental smoking. A
similar factor was reported in a cross-sectional study in
southern Taiwan [27]. We observed a gradient effect for
peer influences. Students with a few peers who smoked
were likely to experiment with smoking early but not as
early as students who had a majority of peers who
smoked (-0.78 vs. -1.35). All of these results reached sta-
tistical significance. However, students who smoke may
over report the number of their smoking peers. In a
study of Chinese adolescents and their perceived smok-
ing norms, Chen et al. found that adolescent smokers
were inclined to overestimate the number of smokers
among their peers and surrounding adults [32]. The
results found a positive association between perceived
smoking norms and an increased risk of smoking Chang et al. BMC Public Health 2011, 11:844
http://www.biomedcentral.com/1471-2458/11/844 Chang et al. BMC Public Health 2011, 11:844 Page 7 of 9 Page 7 of 9 Figure 2 Plots of (a) mean scores of depression; (b) mean scores of family support; (c) mean scores of family supervision; (d) mean
scores of self-competence; and (e) mean scores of school attachment over time. All values reached statistical significance at the 5% level
for testing time trend. Figure 2 Plots of (a) mean scores of depression; (b) mean scores of family support; (c) mean scores of family supervision; (d) mean
scores of self-competence; and (e) mean scores of school attachment over time. All values reached statistical significance at the 5% level
for testing time trend. the community. Further investigation on the quality of
the community would provide useful information to
explain these findings. initiation, experimentation, and establishment. In addi-
tion to educating youth about the negative conse-
quences of smoking, it has been suggested that future
anti-smoking health programs should be geared toward
teaching adolescents how to overcome peer pressure to
conform [33]. The main limitation of this study was that students
were recruited from only two areas in Taiwan. Discussion There-
fore, the results should be generalized with caution. Nevertheless, we obtained results similar to other stu-
dies, suggesting that the risk factors for experimental
smoking are universal. Our findings should be taken
into account when designing anti-smoking policies for
teenagers. Another limitation of this study was the use
of self-reported status. Our study was conducted in
schools, and it is possible that students did not respond
truthfully regarding their smoking behavior for fear of
being identified or punished. Many steps were taken to
ensure the students’ comfort and willingness to respond
truthfully. We stressed that their answers during the
survey would not be provided to school teachers, and
we sent our own interviewers to distribute and collect
the questionnaires to ensure that teachers would not
obtain the results. School is part of adolescents’ community. We found
that as students’ attachment to school increased, they
were less likely to start smoking. Adolescents spend
the majority of their time at school. In addition to tak-
ing classes, adolescents develop relationships with their
classmates and establish peer groups that greatly
impact their decisions and beliefs. Many studies have
shown that students who excel and adhere to high aca-
demic standards are less likely to engage in smoking
behaviors [6,23]. In addition to students’ attitudes
toward studying, the school atmosphere can influence
adolescent smoking status. Alexander et al. found an
increase in smoking rates among popular students in
schools with a higher smoking prevalence, whereas a
decrease in smoking rates was evident among popular
students in schools with a lower smoking prevalence
[34]. We found that students with median community
attachment experimented with smoking earlier than
those with high attachment. However, students with
low attachment did not differ from those with high
attachment. It is likely that students who do not attach
to the community are less likely to be influenced by References 1. Lawlor DA, Song YM, Sung J, Ebrahim S, Smith GD: The association of
smoking and cardiovascular disease in a population with low cholesterol
levels: a study of 648,346 men from the Korean national health system
prospective cohort study. Stroke 2008, 39(3):760-767. 1. Lawlor DA, Song YM, Sung J, Ebrahim S, Smith GD: The association of
smoking and cardiovascular disease in a population with low cholesterol
levels: a study of 648,346 men from the Korean national health system
prospective cohort study. Stroke 2008, 39(3):760-767. 25. Hsu CC, Levy DT, Wen CP, Cheng TY, Tsai SP, Chen T, Eriksen MP, Shu CC:
The effect of the market opening on trends in smoking rates in Taiwan. Health policy (Amsterdam, Netherlands) 2005, 74(1):69-76. 2. Willigendael EM, Teijink JA, Bartelink ML, Kuiken BW, Boiten J, Moll FL,
Buller HR, Prins MH: Influence of smoking on incidence and prevalence
of peripheral arterial disease. J Vasc Surg 2004, 40(6):1158-1165. 26. Wen CP, Levy DT, Cheng TY, Hsu CC, Tsai SP: Smoking behaviour in
Taiwan, 2001. Tobacco control 2005, 14(Suppl 1):i51-55. Taiwan, 2001. Tobacco control 2005, 14(Suppl 1):i51-55. 27. Lin PL, Tzeng LC, Lu KY, Hsu HM: Factors related to tobacco use among
adolescents in southern Taiwan. J Nurs Res 2008, 16(4):243-251. 27. Lin PL, Tzeng LC, Lu KY, Hsu HM: Factors related to tobacco use among
adolescents in southern Taiwan. J Nurs Res 2008, 16(4):243-251. p
p
g
3. Ezzati M, Henley SJ, Lopez AD, Thun MJ: Role of smoking in global and
regional cancer epidemiology: current patterns and data needs. Int J
Cancer 2005, 116(6):963-971. 28. Griesbach D, Amos A, Currie C: Adolescent smoking and family structure
in Europe. Social Science & Medicine 2003, 56:41-52. 4. Leistikow BN, Martin DC, Jacobs J, Rocke DM, Noderer K: Smoking as a risk
factor for accident death: a meta-analysis of cohort studies. Accid Anal
Prev 2000, 32(3):397-405. 4. Leistikow BN, Martin DC, Jacobs J, Rocke DM, Noderer K: Smoking as a risk
factor for accident death: a meta-analysis of cohort studies. Accid Anal
Prev 2000, 32(3):397-405. 29. Covey L, Tam D: Depressive Mood, the Single-Parent Home, and
Adolescent Cigarette Smoking. American journal of public health 1990,
80(11):1330-1333. 5. Cheng Y, Jin Y, Gu H, Zhao C: [Prevalence survey of smoking pattern
among peasants in China]. Wei Sheng Yan Jiu 2003, 32(4):366-368, 406. 30. Conclusions This study used a cohort to examine the incidence and
multiple influences, including individual factors, famil-
ial factors, and community factors, on experimental
smoking in adolescents. The findings presented in this
study are consistent with those of other studies, Chang et al. BMC Public Health 2011, 11:844
http://www.biomedcentral.com/1471-2458/11/844 Chang et al. BMC Public Health 2011, 11:844
http://www.biomedcentral.com/1471-2458/11/844 Chang et al. BMC Public Health 2011, 11:844
http://www.biomedcentral.com/1471-2458/11/844 Page 8 of 9 Page 8 of 9 8. Park S, Weaver TE, Romer D: Predictors of the transition from
experimental to daily smoking among adolescents in the United States. J Spec Pediatr Nurs 2009, 14(2):102-111. particularly in terms of the association between the
influences of family and friends and school and com-
munity attachment with experimental smoking in teen-
agers. We found gradient effects for the influence of
parents and friends on the decision to experiment with
smoking. We observed a sharp increase in incidence
from 7th to 8th grades. Possible reasons were curiosity
on smoking and decreasing parental support and
supervision. 9. Simons-Morton B, Crump A, Haynie D, Saylor K, Eitel P, Yu K: Psychosocial,
school, and parent factors associated with recent smoking among early-
adolescent boys and girls. Preventive medicine 1999, 28(2):138-148. 10. King G, Flisher A, Mallet R, Graham J, Lombard C, Rawson T, Morojele N,
Muller M: Smoking in Cape Town: Community Influences on Adolescent
Tobacco Use. Preventive medicine 2003, 36:114-123. 11. Tobacco use among youth: a cross country comparison. Tobacco control
2002, 11(3):252-270. 12. Wu WC, Chang HY, Yen LL, Lee TS: Changes in health risk behaviors of
elementary school students in northern Taiwan from 2001 to 2003:
results from the child and adolescent behaviors in long-term evolution
study. BMC public health 2007, 7:323. Competing interests p y
22. Lee ET: Statistical Methods for Survival Data Analysis New York: Wiley-
Interscience Publication; 2003. The authors declare that they have no competing interests. 23. Tyas SL, Pederson LL: Psychosocial factors related to adolescent smoking:
a critical review of the literature. Tobacco control 1998, 7:409-420. Received: 23 June 2011 Accepted: 3 November 2011
Published: 3 November 2011 Received: 23 June 2011 Accepted: 3 November 2011
Published: 3 November 2011 Received: 23 June 2011 Accepted: 3 November 2011
Published: 3 November 2011 24. Chen KT, Chen CJ, Fagot-Campagna A, Narayan KM: Tobacco, betel quid,
alcohol, and illicit drug use among 13- to 35-year-olds in I-Lan, rural
Taiwan: prevalence and risk factors. American journal of public health 2001,
91(7):1130-1134. Author details
1 1Division of Preventive Medicine and Health Service Research, Institute of
Population Health Sciences, National Health Research Institutes, Zhunan
Town, Miaoli County, Taiwan, ROC. 2School of Health Care Management,
Department of Nutrition and Health Sciences, Kainan University, Taiwan,
ROC. 3Center for Population and Health Survey Research, Bureau of Health
Promotion, Department of Health, Taichung, Taiwan, ROC. 4Institute of
Health Policy and Management, College of Public Health, National Taiwan
University. Rm. 623, No. 17, Xu-Zhou Road, Taipei 100, Taiwan, ROC. j
p y
y
16. Streiner DL, Norman GR: Health Measurement Scales: A Practical Guide to
Their Development and Use New York: Oxford University Press, Inc.; 2003. 17. Tafarodi RW, Swann WB Jr: Self-linking and self-competence as
dimensions of global self-esteem: initial validation of a measure. Journal
of personality assessment 1995, 65(2):322-342. p
y
18. Song H, Thompson RA, Ferrer E: Attachment and self-evaluation in
Chinese adolescents: age and gender differences. Journal of adolescence
2009, 32(5):1267-1286. Authors’ contributions 19. Wu WC, Kao CH, Yen LL, Lee TS: Comparison of children’s self-reports of
depressive symptoms among different family interaction types in
northern Taiwan. BMC public health 2007, 7:116. HYC constructed the study idea, wrote the manuscript, and revised it. WCW
analyzed the data and contributed intensive discussion on the study. CCW
collected, edited and managed the data. JYC reviewed the literature and
drafted part of the manuscript. BSH was responsible for the fieldwork and
contributed intensive discussion on the study. LLY designed and initiated
the CABLE study and contributed intensive discussion on the manuscript. All
authors read and approved the final manuscript. 20. Cole DA, Rehm LP: Family interaction patterns and childhood depression. Journal of abnormal child psychology 1986, 14(2):297-314. 21. Seguin M, Manion I, Cloutier P, McEvoy L, Cappelli M: Adolescent
depression, family psychopathology and parent/child relations: a case
control study. The Canadian child and adolescent psychiatry review = La
revue canadienne de psychiatrie de l’enfant et de l’adolescent 2003, 12(1):2-9. Acknowledgements
P
f h
d g
Part of the study was sponsored by the National Health Research Institutes. 15. Faulstich ME, Carey MP, Ruggiero L, Enyart P, Gresham F: Assessment of
depression in childhood and adolescence: an evaluation of the Center
for Epidemiological Studies Depression Scale for Children (CES-DC). The
American journal of psychiatry 1986, 143(8):1024-1027. Additional file 1: Appendix 1. Definition of explanatory variables. pp
14. Chen PL, Chiou HY, Chen YH: Chinese version of the Global Youth
Tobacco Survey: cross-cultural instrument adaptation. BMC public health
2008, 8:144. Additional material 13. Yen LL, Chen L, Lee SH, Hsiao C, Pan LY: Child and adolescent behaviour
in long-term evolution (CABLE): a school-based health lifestyle study. Promotion & education 2002, , Suppl 1: 33-40. Additional file 1: Appendix 1. Definition of explanatory variables. References Wang MP, Ho SY, Lam TH: Parental Smoking, Exposure to Secondhand
Smoke at Home, and Smoking Initiation Among Young Children. Nicotine Tob Res 2011. 6. Karp I, O’Louglin J, Paradis G, Hanley J, Difranza J: Smoking trajectories of
adolescent novice smokers in a longitudinal study of tobacco use. Annual Epidemiology 2005, 15:445-452. 31. Distefan J, Gilpin E, Choi W, Pierce J: Parental Influences Predict
Adolescent Smoking in the United States, 1989-1993. Journal of
Adolescent Health 1998, 22:466-474. 7. Kelder S, Perry C, Klepp K-I, Lytle L: Longitudinal Tracking of Adolescent
Smoking, Physical Activity, and Food Choice Behaviors. American journal
of public health 1994, 84(7):1121-1126. 32. Chen X, Stanton B, Fang X, Li X, Lin D, Zhang J, Liu H, Yang H: Perceived
smoking norms, socio- environmental factors, personal attitudes and Page 9 of 9 Page 9 of 9 Chang et al. BMC Public Health 2011, 11:844
http://www.biomedcentral.com/1471-2458/11/844 adolescent smoking in China: a mediation analysis with longitudinal
data. Journal of Adolescent Health 2006, 38(4):359-368. 33. Kobus K: Peers and adolescent smoking. Addiction (Abingdon, England)
2003, 98(Suppl 1):37-55. 34. Alexander C, Piazza M, Mekos D, Valente T: Peers, schools, and adolescent
cigarette smoking. Journal of Adolescent Health 2001, 29:22-30. Pre-publication history
The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1471-2458/11/844/prepub
doi:10.1186/1471-2458-11-844
Cite this article as: Chang et al.: The incidence of experimental smoking
in school children: an 8-year follow-up of the child and adolescent
behaviors in long-term evolution (CABLE) study. BMC Public Health 2011
11:844. adolescent smoking in China: a mediation analysis with longitudinal
data. Journal of Adolescent Health 2006, 38(4):359-368. 33. Kobus K: Peers and adolescent smoking. Addiction (Abingdon, England)
2003, 98(Suppl 1):37-55. 34. Alexander C, Piazza M, Mekos D, Valente T: Peers, schools, and adolescent
cigarette smoking. Journal of Adolescent Health 2001, 29:22-30. Pre-publication history The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1471-2458/11/844/prepub doi:10.1186/1471-2458-11-844
Cite this article as: Chang et al.: The incidence of experimental smoking
in school children: an 8-year follow-up of the child and adolescent
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English
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Drawbacks of Dialysis Procedures for Removal of EDTA
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PloS one
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cc-by
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RESEARCH ARTICLE Introduction Copyright: © 2017 Mo´nico et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. Metal chelators, such as EDTA, are widely used for inhibition of proteases during protein puri-
fication or during preparation of cell or tissue extracts [1]. EDTA is also employed in proce-
dures to eliminate endotoxin from certain protein preparations [2] or to prevent oxidation by
metals [3]. However, the presence of EDTA in biological samples, even at low concentrations,
may interfere with assays like those aimed at assessing the effects of divalent cations on protein
or cellular functions and hence, it is often necessary to remove the chelator. Dialysis or gel fil-
tration are routinely used in research articles [2, 4] and recommended in commercial technical
application notes for exchange or removal of low molecular weight buffer components, includ-
ing EDTA, from cell or tissue extracts or protein preparations. Dialysis is also preferred when
the volume of protein available or its concentration are too low for size exclusion chromatog-
raphy with columns long enough to render a good separation. Moreover, dialysis is usually the
method of choice when the protein is purified from bacterial inclusion bodies and the dena-
tured protein needs to be refolded prior to the experiments by this procedure [5, 6]. This is the
case of intermediate filament proteins, which are typically purified using buffers containing
EDTA. In addition, they require solubilization either from inclusion bodies, in the case of Andreia Mo´nico1, Eva Martı´nez-Senra1, F. Javier Cañada1, Silvia Zorrilla2, Dolores Pe´rez-
Sala1* 1 Department of Chemical and Physical Biology, Centro de Investigaciones Biolo´gicas, C.S.I.C., Madrid,
Spain, 2 Department of Cellular and Molecular Biology, Centro de Investigaciones Biolo´gicas, C.S.I.C.,
Madrid, Spain * dperezsala@cib.csic.es * dperezsala@cib.csic.es a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 Editor: Eugene A. Permyakov, Russian Academy of
Medical Sciences, RUSSIAN FEDERATION Editor: Eugene A. Permyakov, Russian Academy of
Medical Sciences, RUSSIAN FEDERATION Abstract Ethylenediaminetetraacetic acid (EDTA) is a chelating agent commonly used in protein puri-
fication, both to eliminate contaminating divalent cations and to inhibit protease activity. For
a number of subsequent applications EDTA needs to be exhaustively removed. Most purifi-
cation methods rely in extensive dialysis and/or gel filtration in order to exchange or remove
protein buffer components, including metal chelators. We report here that dialysis protocols,
even as extensive as those typically employed for protein refolding, may not effectively
remove EDTA, which is reduced only by approximately two-fold and it also persists after
spin-column gel filtration, as determined by NMR and by colorimetric methods. Remarkably,
the most efficient removal was achieved by ultrafiltration, after which EDTA became virtually
undetectable. These results highlight a potentially widespread source of experimental vari-
ability affecting free divalent cation concentrations in protein applications. OPEN ACCESS
Citation: Mo´nico A, Martı´nez-Senra E, Cañada FJ,
Zorrilla S, Pe´rez-Sala D (2017) Drawbacks of
Dialysis Procedures for Removal of EDTA. PLoS
ONE 12(1): e0169843. doi:10.1371/journal. pone.0169843 Drawbacks of Dialysis Procedures for
Removal of EDTA Andreia Mo´nico1, Eva Martı´nez-Senra1, F. Javier Cañada1, Silvia Zorrilla2, Dolores Pe´rez-
Sala1* OPEN ACCESS Citation: Mo´nico A, Martı´nez-Senra E, Cañada FJ,
Zorrilla S, Pe´rez-Sala D (2017) Drawbacks of
Dialysis Procedures for Removal of EDTA. PLoS
ONE 12(1): e0169843. doi:10.1371/journal. pone.0169843 Citation: Mo´nico A, Martı´nez-Senra E, Cañada FJ,
Zorrilla S, Pe´rez-Sala D (2017) Drawbacks of
Dialysis Procedures for Removal of EDTA. PLoS
ONE 12(1): e0169843. doi:10.1371/journal. pone.0169843 Editor: Eugene A. Permyakov, Russian Academy of
Medical Sciences, RUSSIAN FEDERATION Editor: Eugene A. Permyakov, Russian Academy of
Medical Sciences, RUSSIAN FEDERATION
Received: November 10, 2016
Accepted: December 23, 2016
Published: January 18, 2017 Protein dialysis Bovine serum albumin (BSA) at 1 mg/ml in 5 mM Pipes, pH 7.0 containing 1 mM EDTA was
dialyzed against 5 mM Pipes, 1 mM DTT, pH 7.0 with four buffer changes and, subsequently,
into 5 mM Pipes, 0.25 mM DTT, pH 7.0 with two changes. Dialysis of vimentin followed the
standard refolding protocol [8]. Briefly, the human recombinant protein at 1 mg/ml in 5 mM
Tris-HCl, pH 7.6, containing 8 M urea, 1 mM EDTA, 10 mM β-mercaptoethanol, 0.4 mM
PMSF and approximately 150 mM KCl, was subjected to step-wise dialysis against 5 mM Pipes
pH 7.0, 1 mM DTT containing 6 M urea, then 4 M urea, 2 M urea and no urea at r.t., and
finally, to two additional steps against 5 mM Pipes, pH 7.0, 0.25 mM DTT, the last one for 16 h
at 16˚C. Spin-column gel filtration and ultrafiltration Gel filtration was conducted using PD SpinTrap G-25 1 ml columns equilibrated with 5 mM
Pipes, 0.1 mM DTT, pH 7.0, before loading 140 μl samples of vimentin or BSA and eluting
according to the instructions of the manufacturer. For ultrafiltration, BSA or vimentin samples
(250 μl) containing 1 mM EDTA were diluted 10-fold with EDTA-free buffer, applied to Milli-
pore Amicon Ultra filter units (10 K pore size) and centrifuged at 3000xg for 15 min at 16˚C,
which concentrated the samples down to their original volume. Then, samples were diluted
again 10-fold with buffer without EDTA and the procedure was repeated. Materials Amicon ultrafiltration devices (10K cut off) were from Millipore. PD-SpinTrap G-25 columns
were from GE Healthcare. Slide-A-Lyzer MINI Dialysis devices (20K cut off) were from
Thermo. 96-well plates were from Falcon. Recombinant hamster vimentin was from Cytoskel-
eton, Inc. Recombinant human vimentin was from Biomedal (Sevilla, Spain). Other reagents
were of the highest quality from Sigma. Data Availability Statement: All relevant data are
within the paper. Data Availability Statement: All relevant data are
within the paper. Funding: This work has been funded by the
European Union’s Horizon 2020 research and
innovation programme under the Marie
Sklowdowska-Curie grant agreement number
675132 (http://cordis.europa.eu/project/rcn/
198275_en.html), and by grants from the Spanish
Ministerio de Economı´a y Competitividad
(MINECO/FEDER, http://www.mineco.gob.es/
portal/site/mineco/idi) SAF2015-68590R to DPS
and CTQ2015-64597-C2-2-P to FJC. The funders PLOS ONE | DOI:10.1371/journal.pone.0169843
January 18, 2017 1 / 9 Incomplete Removal of EDTA by Dialysis recombinant proteins expressed in bacteria, or from insoluble eukaryotic cell cytoskeletal frac-
tions, for which high urea concentrations, up to 9.5 M, are used [7–9]. Later, the denatured
proteins are refolded by step-wise dialysis to gradually remove urea, usually in low salt buffer
to keep the protein unassembled, as high ionic strength triggers polymerization [8]. In fact,
intermediate filament proteins such as desmin, vimentin or glial fibrillary acidic protein
(GFAP) purified through these methods, have been widely studied to assess polymerization or
association changes induced by increasing ionic strength or by divalent cations, usually at mil-
limolar concentrations [9, 10]. had no role in study design, data collection and
analysis, decision to publish, or preparation of the
manuscript. Competing Interests: The authors have declared
that no competing interests exist. Nevertheless, confirmation of adequate elimination of additives used during purification or
refolding is necessary for subsequent applications, above all, when studying the impact of low
concentrations of various ligands on protein structure or function. Here we show that EDTA
can be carried on unnoticed during protein dialysis. Moreover, we provide simple methods for
its detection and improved removal. NMR analysis NMR spectra were acquired in a 500 MHz Bruker AVANCE equipped with a SEF 19F-1H
probe or a 600MHz Bruker AVANCE equipped with a cryogenic triple resonance TXI probe. In the case of samples dissolved in deuterated buffer (20 mM deuterated Tris, Cambridge Iso-
tope, UK), spectra were acquired with a simple 90˚ pulse sequence (zg Bruker pulse sequence)
and with 32K data points and 2 second recovery delay and 10 ppm of spectral width centered
at 4.7 ppm (chemical shift of residual HDO signal). In the case of samples dissolved in non-
deuterated buffers, a 10% volume of deuterated water was added for locking deuterium signal
and the standard Bruker pulse sequence “zgesgp” using excitation sculpting gradients for
water signal suppression was used. The spectra were acquired at 25˚C with 32K points, 2 sec-
ond recovery delay and 14 ppm of spectral width centered at 4.7 ppm (chemical shift of water). The spectra were acquired using from 8 to 2048 scans depending on the sample concentration;
no line broadening was applied in the processing. Bruker TOPSPIN software was used for
acquisition and processing the spectra. For testing the pH dependency of chemical shift signals
of EDTA, samples of 1 to 3 mM EDTA, with and without cation (Ca2+, Zn2+, Mg2+ or La3+), in
20 mM deuterated Tris were prepared at different pH (between 5 and 9) by acidification with
deuterated hydrochloric acid. The actual pH was measured after the addition of the corre-
sponding dichloride salt of each cation. EDTA and ZnCl2 solutions were prepared in deuter-
ated water. Colorimetric estimation of EDTA concentration The concentration of EDTA in the protein solutions was estimated through a colorimetric
assay by monitoring its competition with 4-(2-pyridylazo)-resorcinol (PAR) for Zn binding. The binding of zinc to PAR forms a colored complex with absorbance at 492 nm [11]. The
presence of EDTA induces a decrease in the formation of this complex. A calibration curve PLOS ONE | DOI:10.1371/journal.pone.0169843
January 18, 2017 2 / 9 Incomplete Removal of EDTA by Dialysis was obtained by titration of samples containing 100 μM PAR and 10 μM Zn with known con-
centrations of EDTA and measuring the absorbance at 492 nm, using a Varioskan Flash
(Thermo) microplate reader. The amount of EDTA in the protein preparations was deter-
mined from their absorbance at 492 nm after incubation with 100 μM PAR and 10 μM Zn,
and extrapolation using the calibration curve. Sample volumes of 1 to 10 μl were used in a typi-
cal total assay volume of 100 μl. Measurements were performed 5 min after mixing of reagents. PLOS ONE | DOI:10.1371/journal.pone.0169843
January 18, 2017 Results and Discussion We have recently explored protein modification by electrophilic lipids using both cultured
cells and commercially available protein preparations, including the IF protein vimentin [12,
13]. In order to study the effect of micromolar concentrations of divalent cations on these pro-
cesses we set out to rule out the presence of metal chelators in the protein preparation. For this
we first conducted NMR experiments. Proton NMR has been previously used for detecting
either EDTA or divalent cations as their EDTA chelates in blood samples obtained with EDTA
as anticoagulant [14, 15]. In addition, NMR methods have been employed to quantitate metals
in human serum upon addition of exogenous EDTA [16]. As shown in Fig 1, free EDTA gives
two singlet peaks, the one at low field corresponding to the 8 methylene protons of acetyl moi-
eties, and the one at high field to the 4 protons of the ethylenediamine moiety. The shifts of
these peaks are strongly dependent on the pH of the sample and their signals show a significant
broadening at low pH (Fig 1). Moreover, complexation of EDTA with different metals gives
rise to characteristic patterns, where the methylene protons of acetyl moieties become un-
equivalent, resulting in differentiated signals due to the characteristic structures of the chelates,
as depicted in Fig 1 for the EDTA-calcium complex. This ensures the specificity of the detec-
tion and minimizes pH dependence, since the shifts of the signals of the complexed EDTA
form appear constant in this pH range (Fig 1 and [16]). Therefore, protein samples were analyzed after adding known amounts of a divalent cation
such as calcium or zinc, the latter case being illustrated in Fig 2. This revealed the presence of a
substantial amount of EDTA in a commercial sample, which is presented as a lyophilized PLOS ONE | DOI:10.1371/journal.pone.0169843
January 18, 2017 3 / 9 Incomplete Removal of EDTA by Dialysis Fig 1. Proton NMR spectra of EDTA and its Ca2+-complexed forms at different pH. EDTA (3 mM) and
CaCl2 (1.5 mM) were mixed in deuterated Tris buffer to yield equimolecular amounts of free and Ca2+-
complexed EDTA. Proton NMR spectra of the mixture were acquired at different pH: (A) pH 8.2, (B) pH 7.0,
(C) pH 5.9. The signals corresponding to protons of the free and Ca2+-complexed forms of EDTA are
indicated. The NMR spectrum of EDTA alone is identical to that of free EDTA presented. Results and Discussion The moieties
responsible for each of the components of the signals are depicted in bold. The signal corresponding to Tris
buffer is labeled with *. doi:10 1371/journal pone 0169843 g001 Fig 1. Proton NMR spectra of EDTA and its Ca2+-complexed forms at different pH. EDTA (3 mM) and
CaCl2 (1.5 mM) were mixed in deuterated Tris buffer to yield equimolecular amounts of free and Ca2+-
complexed EDTA. Proton NMR spectra of the mixture were acquired at different pH: (A) pH 8.2, (B) pH 7.0,
(C) pH 5.9. The signals corresponding to protons of the free and Ca2+-complexed forms of EDTA are
indicated. The NMR spectrum of EDTA alone is identical to that of free EDTA presented. The moieties
responsible for each of the components of the signals are depicted in bold. The signal corresponding to Tris
buffer is labeled with *. doi:10.1371/journal.pone.0169843.g001 doi:10.1371/journal.pone.0169843.g001 preparation that after reconstitution in water should yield a solution of folded protein in 5
mM Pipes pH 7.0, 1 mM DTT, 5% (w/v) sucrose and 1% (w/v) dextran. In order to obtain a
preparation more suitable for our studies we subjected a human vimentin sample in buffer
containing 8 M urea, 5 mM Tris-HCl pH 7.6, 1 mM EDTA, 10 mM β-mercaptoethanol, 0.4
mM PMSF and approximately 150 mM KCl to the typical renaturation dialysis procedure,
which included four sequential steps with at least six changes against EDTA-free buffers [8]. NMR analysis of the protein solutions thus obtained showed the retention of EDTA after dialy-
sis (Fig 2A). g
A complementary colorimetric assay was also used to further confirm the presence of
EDTA and to estimate its levels in the protein preparations after dialysis. For this we took
advantage of the ability of the compound 4-(2-pyridylazo)-resorcinol (PAR) to form a colored
2:1 complex with zinc with high affinity (effective dissociation constant of the Zn(PAR)2 com-
plex, at pH 7.0, 2.1 x 10−12 M2 [17]), which presents an absorption maximum at 492 nm and
can be measured spectrophotometrically. In turn, EDTA forms a 1:1 complex with zinc of
extremely high affinity: 6 x 10−14 M at pH 7.0 [18]. Therefore, solutions containing EDTA
should compete with PAR for zinc binding, in such a way that the decrease in the absorption
at 492 nm measured at the equilibrium can be used to infer the concentration of EDTA present
in the sample. PLOS ONE | DOI:10.1371/journal.pone.0169843
January 18, 2017 Results and Discussion Middle panel: reference proton spectrum of EDTA in presence of Zn2+,
the two coupled doublets corresponding to the AB spin system of the methylene protons of the four acetyl
groups, non-equivalent due to the structure of the metal chelate, and the singlet corresponding to the four
equivalent protons of the ethylenediamine moiety are apparent. Lower panel: reference proton spectrum of
EDTA at pH 7.2, only the two characteristic singlets of uncomplexed EDTA appear. (B) Detection of EDTA
by colorimetric analysis. Upper panel: Calibration curve showing the dependence on EDTA concentration of
the absorbance at 492 nm of mixtures containing 100 μM PAR and 10 μM ZnCl2. Lower panel: Amount of
EDTA remaining in samples from vimentin and BSA subjected to extensive dialysis as determined from the
absorbance at 492 nm after incubation with PAR and ZnCl2, using the calibration curve. Initial EDTA
concentration in the samples was 1 mM. Data shown are mean ± SD of 4 assays. quantitation of EDTA in protein samples. (A) NMR analysis. Upper panel: 500MHz Fig 2. Detection and quantitation of EDTA in protein samples. (A) NMR analysis. Upper panel: 500MHz
1D-proton NMR spectrum of a commercial sample of vimentin (0.7 μM) with added 150 μM ZnCl2. Signals for
the protons of sucrose, a stabilizer present in the sample as indicated in specifications, and those of PIPES
buffer are easily identified. An additional singlet at 2.79 ppm and two coupled doublets at 3.25 and 3.32 ppm
(2JHH = 17.3 Hz) are also observed. Middle panel: reference proton spectrum of EDTA in presence of Zn2+,
the two coupled doublets corresponding to the AB spin system of the methylene protons of the four acetyl
groups, non-equivalent due to the structure of the metal chelate, and the singlet corresponding to the four
equivalent protons of the ethylenediamine moiety are apparent. Lower panel: reference proton spectrum of
EDTA at pH 7.2, only the two characteristic singlets of uncomplexed EDTA appear. (B) Detection of EDTA
by colorimetric analysis. Upper panel: Calibration curve showing the dependence on EDTA concentration of
the absorbance at 492 nm of mixtures containing 100 μM PAR and 10 μM ZnCl2. Lower panel: Amount of
EDTA remaining in samples from vimentin and BSA subjected to extensive dialysis as determined from the
absorbance at 492 nm after incubation with PAR and ZnCl2, using the calibration curve. Initial EDTA
concentration in the samples was 1 mM. Results and Discussion For this, we titrated known amounts of EDTA into 100 μM PAR and 10 μM
zinc assay mixtures and measured the absorbance at 492 nm to build a calibration curve (Fig
2B, upper panel). We then assayed the EDTA present in the dialysates from their absorbance PLOS ONE | DOI:10.1371/journal.pone.0169843
January 18, 2017 4 / 9 Incomplete Removal of EDTA by Dialysis Fig 2. Detection and quantitation of EDTA in protein samples. (A) NMR analysis. Upper panel: 500MHz
1D-proton NMR spectrum of a commercial sample of vimentin (0.7 μM) with added 150 μM ZnCl2. Signals for
the protons of sucrose, a stabilizer present in the sample as indicated in specifications, and those of PIPES
buffer are easily identified. An additional singlet at 2.79 ppm and two coupled doublets at 3.25 and 3.32 ppm
(2JHH = 17.3 Hz) are also observed. Middle panel: reference proton spectrum of EDTA in presence of Zn2+,
the two coupled doublets corresponding to the AB spin system of the methylene protons of the four acetyl
groups, non-equivalent due to the structure of the metal chelate, and the singlet corresponding to the four
equivalent protons of the ethylenediamine moiety are apparent. Lower panel: reference proton spectrum of
EDTA at pH 7.2, only the two characteristic singlets of uncomplexed EDTA appear. (B) Detection of EDTA
by colorimetric analysis. Upper panel: Calibration curve showing the dependence on EDTA concentration of
the absorbance at 492 nm of mixtures containing 100 μM PAR and 10 μM ZnCl2. Lower panel: Amount of
EDTA remaining in samples from vimentin and BSA subjected to extensive dialysis as determined from the
absorbance at 492 nm after incubation with PAR and ZnCl2, using the calibration curve. Initial EDTA
concentration in the samples was 1 mM. Data shown are mean ± SD of 4 assays. Fig 2. Detection and quantitation of EDTA in protein samples. (A) NMR analysis. Upper panel: 500MHz Fig 2. Detection and quantitation of EDTA in protein samples. (A) NMR analysis. Upper panel: 500MHz
1D-proton NMR spectrum of a commercial sample of vimentin (0.7 μM) with added 150 μM ZnCl2. Signals for
the protons of sucrose, a stabilizer present in the sample as indicated in specifications, and those of PIPES
buffer are easily identified. An additional singlet at 2.79 ppm and two coupled doublets at 3.25 and 3.32 ppm
(2JHH = 17.3 Hz) are also observed. PLOS ONE | DOI:10.1371/journal.pone.0169843
January 18, 2017 Results and Discussion Data shown are mean ± SD of 4 assays. doi:10.1371/journal.pone.0169843.g002 doi:10.1371/journal.pone.0169843.g002 when mixed with the above specified concentrations of PAR and zinc. These measurements
evidenced that samples of vimentin or bovine serum albumin (BSA) originally containing 1
mM EDTA still retained ~450 μM EDTA after dialysis, in good agreement with parallel deter-
minations by NMR (Fig 2A). Therefore, EDTA removal can be easily and conveniently moni-
tored by this procedure. Nevertheless, it should be taken into account that other metal-binding
compounds or proteins present in the sample may also compete with PAR for zinc binding,
for which detection of EDTA by several methods is advisable. EDTA was also not removed
from solutions of other intermediate filament proteins such as GFAP or desmin treated under
similar conditions (unpublished observations). As the amount of EDTA remaining in the samples was incompatible with many protein
studies, we attempted to setup a procedure to effectively remove this chelator. Bearing in mind
that the quantity of protein available for biological assays is often limited, we used protocols
for small sample volumes, namely, spin column gel filtration and ultrafiltration (Fig 3A). We
started by processing the protein solutions already subjected to dialysis by spin-column gel fil-
tration. Although EDTA was further reduced by this treatment, the protein solutions still con-
tained ~200 μM EDTA, as determined by the PAR competition assay (Fig 3B). As an
alternative, we turned to ultrafiltration to remove the EDTA. BSA or vimentin samples con-
taining 1 mM EDTA were diluted 10-fold with EDTA-free buffer and ultrafiltrated as indi-
cated in Fig 3A, which concentrated the samples down to their original volume. Then, samples
were diluted again 10-fold with buffer without EDTA and the procedure was repeated. Finally,
assessment of EDTA content by the colorimetric assay confirmed that EDTA was more 5 / 9 PLOS ONE | DOI:10.1371/journal.pone.0169843
January 18, 2017 Incomplete Removal of EDTA by Dialysis Fig 3. Procedures for EDTA removal from protein samples and detection of EDTA. (A) The protein
samples containing 1 mM EDTA were subjected to the indicated purification procedures. Left panel: Samples
of BSA or vimentin were subjected to spin column gel filtration as detailed in methods. Right panel: protein
samples were applied to Millipore Amicon Ultra filter units (10 K pore size) and subjected to two rounds of
ultrafiltration, as described in the text. Results and Discussion The signal pattern of the AB system at 3.32 and 3.25 ppm and the singlet at
2.79 ppm corresponding to the Zn2+-EDTA chelate are clearly visible. Fig 3. Procedures for EDTA removal from protein samples and detection of EDTA. (A) The protein
samples containing 1 mM EDTA were subjected to the indicated purification procedures. Left panel: Samples
of BSA or vimentin were subjected to spin column gel filtration as detailed in methods. Right panel: protein
samples were applied to Millipore Amicon Ultra filter units (10 K pore size) and subjected to two rounds of
ultrafiltration, as described in the text. (B) Colorimetric determination of EDTA present in protein samples after
diverse purification procedures using the PAR competition assay. Results shown are mean ± SD of 4 (dialysis
plus gel filtration), 2 to 7 (ultrafiltration), or 3 to 7 (ultrafiltration plus dialysis) assays. (C) NMR analysis. Upper
panel: 600MHz 1D-proton NMR spectrum of an ultrafiltrated sample of vimentin (1.8 μM final concentration). Signals of buffer and additives used in the purification (glycerol, from the ultrafiltration filters, and DTT) are
observed. Protein signals appear at baseline noise level and are not recognizable. Middle panel: The same
sample analyzed in the upper panel monitored after addition of 150 μM of ZnCl2. A quadruplet that appears at
3.92 ppm corresponds to trifluoroethanol added for referencing. Lower panel: monitorization of the sample
after addition of 20 μM EDTA. The signal pattern of the AB system at 3.32 and 3.25 ppm and the singlet at
2.79 ppm corresponding to the Zn2+-EDTA chelate are clearly visible. doi:10.1371/journal.pone.0169843.g003 effectively removed by this procedure (Fig 3B). At this point, EDTA was not detectable by
NMR, limiting its possible concentration to 10 μM or lower in the undiluted protein sample
(Fig 3C). A further advantage of this method is that protein dilution can be minimized by
monitoring the final retentate volume. Lastly, combination of ultrafiltration and dialysis led to
the reduction of EDTA in the protein samples below 5 μM according to the colorimetric assay
(Fig 3B). We have found numerous examples in the literature and in technical notes and protocols
where dialysis is employed to remove EDTA. In contrast, evidences on the limitations of this
method to completely remove the chelator are scarce and not readily found in bibliographic
searches. Results and Discussion (B) Colorimetric determination of EDTA present in protein samples afte
diverse purification procedures using the PAR competition assay. Results shown are mean ± SD of 4 (dialysis
plus gel filtration), 2 to 7 (ultrafiltration), or 3 to 7 (ultrafiltration plus dialysis) assays. (C) NMR analysis. Upper
panel: 600MHz 1D-proton NMR spectrum of an ultrafiltrated sample of vimentin (1.8 μM final concentration). Signals of buffer and additives used in the purification (glycerol, from the ultrafiltration filters, and DTT) are
observed. Protein signals appear at baseline noise level and are not recognizable. Middle panel: The same
sample analyzed in the upper panel monitored after addition of 150 μM of ZnCl2. A quadruplet that appears at
3.92 ppm corresponds to trifluoroethanol added for referencing. Lower panel: monitorization of the sample
after addition of 20 μM EDTA. The signal pattern of the AB system at 3.32 and 3.25 ppm and the singlet at
2.79 ppm corresponding to the Zn2+-EDTA chelate are clearly visible. Fig 3. Procedures for EDTA removal from protein samples and detection of EDTA. (A) The protein Fig 3. Procedures for EDTA removal from protein samples and detection of EDTA. (A) The protein
samples containing 1 mM EDTA were subjected to the indicated purification procedures. Left panel: Samples
of BSA or vimentin were subjected to spin column gel filtration as detailed in methods. Right panel: protein
samples were applied to Millipore Amicon Ultra filter units (10 K pore size) and subjected to two rounds of
ultrafiltration, as described in the text. (B) Colorimetric determination of EDTA present in protein samples after
diverse purification procedures using the PAR competition assay. Results shown are mean ± SD of 4 (dialysis
plus gel filtration), 2 to 7 (ultrafiltration), or 3 to 7 (ultrafiltration plus dialysis) assays. (C) NMR analysis. Upper
panel: 600MHz 1D-proton NMR spectrum of an ultrafiltrated sample of vimentin (1.8 μM final concentration). Signals of buffer and additives used in the purification (glycerol, from the ultrafiltration filters, and DTT) are
observed. Protein signals appear at baseline noise level and are not recognizable. Middle panel: The same
sample analyzed in the upper panel monitored after addition of 150 μM of ZnCl2. A quadruplet that appears at
3.92 ppm corresponds to trifluoroethanol added for referencing. Lower panel: monitorization of the sample
after addition of 20 μM EDTA. PLOS ONE | DOI:10.1371/journal.pone.0169843
January 18, 2017 Results and Discussion Our observations together with these previous works [19, 20] indicate that it should
not be assumed that EDTA is thoroughly removed by dialysis procedures. Thus, complete
removal of EDTA should be assessed either by the procedures described herein or by other
available methods, like HPLC or spectrometric procedures [21, 22], as in [19]. The efficiency
of EDTA removal by dialysis may vary with buffer composition. In particular, Kuzmenko
et al., showed that EDTA removal was poorer when dialysing against buffers with low ionic
strength [19], with incomplete removal even after six buffer changes over three days dialysis. To test the influence of ionic strength on the effectiveness of dialysis under our experimental
conditions, we have assessed the removal of EDTA (1 mM initial concentration) from a BSA PLOS ONE | DOI:10.1371/journal.pone.0169843
January 18, 2017 6 / 9 Incomplete Removal of EDTA by Dialysis sample by dialysis performed as in Fig 2, except that 150 mM NaCl was included in all the dial-
ysis buffers and the procedure was carried out at r.t. Under these conditions, EDTA remaining
in the protein sample was only 33.5 ± 35.0 μM (average value ± SD of 7 determinations), com-
pared to 290.5 ± 23.4 μM EDTA remaining in the absence of NaCl (mean ± SD of 5 determina-
tions). Therefore, these results confirm the previous observations [19] and show the superior
effectiveness of EDTA removal by dialysis at physiological ionic strength. The concentration of EDTA remaining after dialysis will likely not affect the results of stud-
ies employing millimolar concentrations of divalent cations [23], which in some cases are even
performed in the presence of 1 mM EDTA [9]. Nevertheless, they should be taken into account
when performing assays with micromolar concentrations of metals. The presence of EDTA
can also affect various protein parameters, like thermal denaturation [20], or induce pH alter-
ations upon addition of divalent cations [24]. Also, in studies assessing the biological effects of
dialyzed proteins or cellular or plasma fractions, residual EDTA could be responsible for some
of the effects observed. Potential reasons for the lack of effectiveness of dialysis procedures could include the
reported ability of EDTA to form supramolecular aggregates in solution, as well as its capacity
to associate with proteins ([25] and references therein). Conclusions Results presented here show that, despite its low molecular mass (292.24 Da), EDTA is often
retained inside dialysis bags with pore-sizes over a hundred times larger, and extending the
dialysis time and the number of buffer changes does not seem to allow successful removal of
the chelator in those cases. Therefore, determination of the level of EDTA in the dialysates is
needed whenever interference of this compound with the intended assays is suspected. For an
initial assessment of the potential presence of EDTA, we suggest a fast and cost-efficient
method, suitable for small sample amounts, which is based on the competition of EDTA and
the zinc-binding compound PAR for complexation of this divalent cation. After testing proce-
dures other than dialysis routinely employed to separate macromolecules from small com-
pounds, we concluded that ultracentrifugation could be a good choice to remove EDTA when
necessary. Moreover, when the protein needs to be subjected to dialysis as part of a refolding
protocol, the combination of dialysis and ultrafiltration in no particular order renders opti-
mum results in terms of chelator elimination. Results and Discussion For instance, EDTA has been reported
to bind to α-lactalbumin altering the equilibrium between different conformers of the protein
and its apparent thermal stability [26]. Size exclusion chromatography using spin desalting columns was among the methods we
evaluated to diminish the concentration of EDTA and we found this procedure less effective
compared with ultrafiltration. However, we cannot rule out that the use of longer columns
with higher resolution may improve the species separation. In addition, care should be exer-
cised with other chromatographic procedures employed in protein purification since EDTA
has been reported to be abnormally retained in ion exchange chromatography under certain
conditions [24]. Author Contributions Resources: DPS FJC SZ. Supervision: DPS SZ FJC. Validation: DPS SZ FJC. Validation: DPS SZ FJC. Visualization: AM SZ FJC DPS. Visualization: AM SZ FJC DPS. Writing – original draft: DPS SZ FJC. Writing – original draft: DPS SZ FJC. Writing – review & editing: AM EMS FJC SZ DPS. Writing – review & editing: AM EMS FJC SZ DPS. Writing – review & editing: AM EMS FJC SZ DPS. Acknowledgments We thank Prof. Germa´n Rivas (CIB, CSIC) for insightful comments. The technical assistance
of MJ Carrasco is gratefully appreciated. 7 / 9 PLOS ONE | DOI:10.1371/journal.pone.0169843
January 18, 2017 Incomplete Removal of EDTA by Dialysis PLOS ONE | DOI:10.1371/journal.pone.0169843
January 18, 2017 References 1. Grabski AC. Advances in preparation of biological extracts for protein purification. Methods Enzymol. 2009; 463: 285–303. doi: 10.1016/S0076-6879(09)63018-4 PMID: 19892178 2. Wright JR, Zlogar DF, Taylor JC, Zlogar TM, Restrepo CI. Effects of endotoxin on surfactant protein A
and D stimulation of NO production by alveolar macrophages. Am J Physiol. 1999; 276: L650–8. PMID:
10198363 3. Stadtman ER. Metal ion-catalyzed oxidation of proteins: biochemical mechanism and biological conse-
quences. Free Radic Biol Med. 1990; 9: 315–25. PMID: 2283087 4. Wingender E, Dilloo D, Seifart KH. Zinc ions are differentially required for the transcription of ribosomal
5S RNA and tRNA in a HeLa-cell extract. Nucleic Acids Res. 1984; 12: 8971–85. PMID: 6440120 5. Rudolph R, Lilie H. In vitro folding of inclusion body proteins. FASEB J. 1996; 10: 49–56. PMID:
8566547 6. Tsumoto K, Ejima D, Kumagai I, Arakawa T. Practical considerations in refolding proteins from inclusion
bodies. Protein Expr Purif. 2003; 28: 1–8. PMID: 12651100 7. Quinlan RA, Moir RD, Stewart M. Expression in Escherichia coli of fragments of glial fibrillary acidic pro-
tein: characterization, assembly properties and paracrystal formation. J Cell Sci. 1989; 93: 71–83. PMID: 2693466 8. Herrmann H, Hofmann I, Franke WW. Identification of a nonapeptide motif in the vimentin head domain
involved in intermediate filament assembly. J Mol Biol. 1992; 223: 637–50. PMID: 1542111 9. Lin YC, Broedersz CP, Rowat AC, Wedig T, Herrmann H, Mackintosh FC, et al. Divalent cations cross-
link vimentin intermediate filament tail domains to regulate network mechanics. J Mol Biol. 2010; 399:
637–44. doi: 10.1016/j.jmb.2010.04.054 PMID: 20447406 10. Bar H, Sharma S, Kleiner H, Mucke N, Zentgraf H, Katus HA, et al. Interference of amino-terminal des-
min fragments with desmin filament formation. Cell Motil Cytoskeleton. 2009; 66: 986–99. doi: 10.1002/
cm.20396 PMID: 19530175 11. Koch M, Bhattacharya S, Kehl T, Gimona M, Vasak M, Chazin W, et al. Implications on zinc binding to
S100A2. Biochim Biophys Acta. 2007; 1773: 457–70. doi: 10.1016/j.bbamcr.2006.12.006 PMID:
17239974 12. Gharbi S, Garzo´n B, Gayarre J, Timms J, Pe´rez-Sala D. Study of protein targets for covalent modifica-
tion by the antitumoral and anti-inflammatory prostaglandin PGA1: focus on vimentin. J Mass Spectrom. 2007; 42:1474–84. doi: 10.1002/jms.1291 PMID: 17960581 PLOS ONE | DOI:10.1371/journal.pone.0169843
January 18, 2017 8 / 9 Incomplete Removal of EDTA by Dialysis 13. Pe´rez-Sala D, Oeste CL, Martı´nez AE, Garzo´n B, Carrasco MJ, Cañada FJ. References Vimentin filament organiza-
tion and stress sensing depend on its single cysteine residue and zinc binding. Nat Commun. 2015; 6:
7287. doi: 10.1038/ncomms8287 PMID: 26031447 14. Barton RH, Waterman D, Bonner FW, Holmes E, Clarke R, Nicholson JK, et al. The influence of EDTA
and citrate anticoagulant addition to human plasma on information recovery from NMR-based metabolic
profiling studies. Mol BioSyst. 2010; 6: 215–24. doi: 10.1039/b907021d PMID: 20024083 15. Nicholson JK, Buckingham MJ, Sadler PJ. High resolution 1H n.m.r. studies of vertebrate blood and
plasma. Biochem J. 1983; 211: 605–15. PMID: 6411064 16. Somashekar BS, Ijare OB, Nagana Gowda GA, Ramesh V, Gupta S, Khetrapal CL. Simple pulse-
acquire NMR methods for the quantitative analysis of calcium, magnesium and sodium in human
serum. Spectrochimica Acta A, Mol Biomol Spectrosc. 2006; 65: 254–60. 17. Kocyla A, Pomorski A, Krezel A. Molar absorption coefficients and stability constants of metal com-
plexes of 4-(2-pyridylazo)resorcinol (PAR): Revisiting common chelating probe for the study of metallo-
proteins. Journal Inorg Biochem. 2015; 152: 82–92. 18. Krezel A, Maret W. The biological inorganic chemistry of zinc ions. Arch Biochem Biophys. 2016; 611:
3–19. doi: 10.1016/j.abb.2016.04.010 PMID: 27117234 19. Kuzmenko AI, Wu H, Bridges JP, McCormack FX. Surfactant lipid peroxidation damages surfactant pro-
tein A and inhibits interactions with phospholipid vesicles. J Lipid Res. 2004; 45: 1061–8. doi: 10.1194/
jlr.M300360-JLR200 PMID: 15026426 20. Searcy DG, Greif L. Apparent thermal destabilization of Escherichia coli nucleoprotein due to the incom-
plete dialysis of EDTA. Biochim Biophys Acta. 1976; 418: 133–6. PMID: 812553 21. Kord AS, Tumanova I, Matier WL. A novel HPLC method for determination of EDTA in a cataract inhibit-
ing ophthalmic drug. J Pharm Biomed Anal. 1995; 13: 575–80. PMID: 9696572 22. Kratochvil B, White MC. Spectrophotometric Determination of Microgram Quantities of (Ethylenedini-
trilo)Tetraacetic Acid with Bis(2,4,6-Tripyridyl-S-Triazine)Iron(Ii). Anal Chem. 1965; 37: 111–3. PMID:
14230701 23. Dammann C, Koster S. Dynamics of counterion-induced attraction between vimentin filaments followed
in microfluidic drops. Lab Chip. 2014; 14: 2681–7. doi: 10.1039/c3lc51418h PMID: 24834442 24. Sharpe JC, London E. Inadvertent concentrating of EDTA by ion exchange chromatography: avoiding
artifacts that can interfere with protein purification. Anal Biochem. 1997; 250: 124–5. doi: 10.1006/abio. 1997.2210 PMID: 9234908 25. Muller M, Haeberli A. pH-dependent formation of ethylenediaminetetraacetic acid supramolecular
aggregates. FEBS Lett. 1994; 340: 17–21. PMID: 8119401 26. Kronman MJ, Bratcher SC. An experimental artifact in the use of chelating metal ion buffers. PLOS ONE | DOI:10.1371/journal.pone.0169843
January 18, 2017 References Binding of
chelators to bovine alpha-lactalbumin. J Biol Chem. 1983; 258: 5707–9. PMID: 6406505 9 / 9
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Interaction between susceptibility loci in cGAS-STING pathway, MHC gene and HPV infection on the risk of cervical precancerous lesions in Chinese population
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Oncotarget
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cc-by
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Di Xiao1,*, Weihuang Huang1,*, Meiling Ou1, Congcong Guo1, Xingguang Ye1, Yang Liu1,
Man Wang1, Baohuan Zhang1, Na Zhang1, Shiqi Huang1, Jiankun Zang5, Zixing Zhou1,
Zihao Wen1, Chengli Zeng1, Chenfei Wu1, Chuican Huang1, Xiangcai Wei1,4, Guang
Yang2,3, Chunxia Jing1,3
1Department of Epidemiology, School of Medicine, Jinan University, Guangzhou, Guangdong, China
2Department of Parasitology, School of Medicine, Jinan University, Guangzhou, Guangdong, China
3Key Laboratory of environmental exposure and health in Guangzhou, Jinan University, Guangzhou, Guangdong, China
4Family Planning Research Institute of Guangdong, Guangzhou, Guangdong Province, China
5Department of Pathophysiology, School of Medicine, Jinan University, Guangzhou, Guangdong, China
*These authors are contibuted equally to this work Di Xiao1,*, Weihuang Huang1,*, Meiling Ou1, Congcong Guo1, Xingguang Ye1, Yang Liu1,
Man Wang1, Baohuan Zhang1, Na Zhang1, Shiqi Huang1, Jiankun Zang5, Zixing Zhou1,
Zihao Wen1, Chengli Zeng1, Chenfei Wu1, Chuican Huang1, Xiangcai Wei1,4, Guang
Yang2,3, Chunxia Jing1,3
1Department of Epidemiology, School of Medicine, Jinan University, Guangzhou, Guangdong, China
2Department of Parasitology, School of Medicine, Jinan University, Guangzhou, Guangdong, China
3Key Laboratory of environmental exposure and health in Guangzhou, Jinan University, Guangzhou, Guangdong, China
4Family Planning Research Institute of Guangdong, Guangzhou, Guangdong Province, China
5Department of Pathophysiology, School of Medicine, Jinan University, Guangzhou, Guangdong, China
*These authors are contibuted equally to this work Correspondence to: Guang Yang, email: guangyangphd@gmail.com
Chunxia Jing, email: jcxphd@gmail.com Keywords: cGAS-STING, MHC, SNP, interaction, cervical precancerous lesions
Received: May 16, 2016 Accepted: September 25, 2016 Published: October 01, 2016 ABSTRACT Human papillomavirus (HPV) infection is a definite risk factor for cervical cancer. Nevertheless, only some infected individuals actually develop cervical cancer. The cGAS-
STING pathway in innate immunity plays an important role in protecting against HPV
infection. Chen et al. described that the rs2516448 SNP in the MHC locus may affect
susceptibility to cervical cancer, a finding that we attempted to replicate in a Chinese
population. To investigate the effects of cGAS, STING and MHC polymorphisms on
susceptibility to cervical precancerous lesions, 9 SNPs were analyzed in 164 cervical
precancerous lesion cases and 428 controls. Gene-gene and gene-environment
interactions were also evaluated. We found a significantly decreased risk of cervical
precancerous lesions for the GG genotype of rs311678 in the cGAS gene (ORadjusted
= 0.40, 95% CI: 0.16−0.98). Moreover, MDR analysis identified a significant three-locus
interaction model, involving HPV infection, age at menarche and rs311678 in cGAS. Additionally, a significant antagonistic interaction between HPV infection and rs311678
was found on an additive scale. In conclusion, our results indicate that the rs311678
polymorphism in the cGAS gene confers genetic susceptibility to cervical precancerous
lesions. Moreover, the three-way gene-environment interactions further demonstrate
that the rs311678 polymorphism in cGAS can significantly decrease the risk of HPV
infection and the elder at menarche. Oncotarget, 2016, Vol. 7, (No. 51), pp: 84228-84238 Oncotarget, 2016, Vol. 7, (No. 51), pp: 84228-84238 www.impactjournals.com/oncotarget/ INTRODUCTION countries [2, 3]. Cervical cancer develops through a
multistep process, with three cervical intraepithelial
neoplasia grades, 1 to 3 (CIN1-3) [4]. However, it will take
several years, even decades, from pre-cancer to invasive
cervical cancer, which offers us many opportunities
for intervention. Therefore, early detection of cervical
precancerous lesions and their causes likely contribute to
reducing the incidence and mortality of cervical cancer. Cervical cancer is the third most common cancer
among women, with approximately 530,000 new cases
annually worldwide [1]. Moreover, with more than half
a million new cases and 275,000 deaths per year, cervical
cancer continues to constitute a major public health problem
and particularly affects young women in developing www.impactjournals.com/oncotarget Population characteristics The general characteristics are detailed in Table 1. Significant differences were found in age and HPV
infection between the SIL cases and controls. No
significant differences in the body mass index (BMI),
number of term births, age at menarche, family history
of cancer or genital cleaning after each intercourse were
observed between the two groups. A recent study reported that one way that HPV may
cause cancer is by targeting tumor suppressor proteins in
the host [10, 20]. Oncogenes from HPV bind to the protein
STING, allowing the virus to subvert the host’s antiviral
immunity and initiate infection, which, for an unlucky
few, eventually causes cancer [20]. However, IFN-β serves
as a signal linking innate and adaptive immunity that can
defend against high-risk HPV activity [21, 22]. Analysis
of SNP data from the 1000 Genomes Project revealed
that there are four SNP-derived isoforms in hSTING,
with R71H-G230A-R293Q (HAQ) in 20.4%, R232H in
13.7%, G230A-R293Q (AQ) in 5.2%, and R293Q in 1.5%
of human population [23]. Furthermore, hSTING variation
can affect innate immune signaling and HAQ STING is
defective in stimulation of INFβ production [23, 24]. Association between cGAS, STING and MHC
polymorphisms and cervical precancerous
lesions As shown in Table 2, the association between
rs311678 in cGAS and cervical precancerous lesions was
significant under the allelic model (G vs A, P = 0.048),
while the other eight SNPs were not found to be associated
with the risk of cervical precancerous lesions. The association of 9 SNPs in the cGAS-STING
signaling pathway and MHC gene with cervical
precancerous lesion risk is shown in Supplementary Table
S4. We found that individuals with the GG genotype in
cGAS rs311678 had a 60% decreased risk of developing
cervical precancerous lesions (ORadjusted = 0.40, 95% CI =
0.16–0.98, P = 0.045) compared with those with the AA
wild-type, while the other 8 SNPs were not observed to be
relevant to the risk of cervical precancerous lesions. Thus, we hypothesized that individual genetic
differences in the cGAS-STING pathway could influence
the expression of IFN-I, which might influence the host
antiviral response, and thus affect the susceptibility
to developing cervical cancer [25, 26]. Furthermore,
recent studies on the T allele of rs2516448 in the major
histocompatibility complex (MHC) region, which
increased the susceptibility to cervical cancer in a Swedish
population, were published [27, 28]. The risk allele of
rs2516448 in the first locus is in perfect LD with A5.1,
a frameshift mutation of the MHC class I polypeptide-
related sequence A gene(MICA). Chen et al. [28] reported
that the MICA-A5.1 variant results in less membrane-
bound MICA, which may affect immune activation and
immune surveillance against HPV-infected cells, and www.impactjournals.com/oncotarget Oncotarget 84228 increase the risk of cervical cancer development. Our
study aimed to confirm this finding in Chinese women. Human papilloma virus (HPV) appears to be
a necessary factor in the development of almost all
cases (> 90%) of cervical cancer [5]. Molecular and
epidemiologic studies have shown that persistent infection
with high-risk HPV plays a role in the development of
both cervical cancer and cervical precancerous lesions
[6–8]. However, only some of the infected individuals
develop cervical cancer. Although HPV is an essential
factor for the transformation of cervical epithelial cells, it
is not sufficient, and a variety of environmental and host
factors influence the development of cervical cancer. i Investigating
genetic
differences
and
their
interactions with the host’s innate immune system could
lead us to understand the precancerous lesions more
comprehensively. In this study, nine polymorphisms
in three candidate genes, including the cGAS gene
(rs610913, rs311678, rs4032697, rs311675, rs9352000,
rs7761170), the STING gene (rs1131769, rs7380824) and
the MHC (rs2516448), were genotyped, and the possible
associations of these SNPs with cervical precancerous
lesions were investigated. Furthermore, we sought to
explore the potential interactions between these SNPs
and environmental factors in the etiology of cervical
precancerous lesions in the Chinese population. As a first line of antiviral defense, innate immune
cells express cytokines such as type I interferons (IFN-I)
that can activate and recruit innate and adaptive immune
cells [9]. cGAMP synthase (cGAS) detects intracellular
DNA and signals through the adapter protein stimulator
of interferon genes (STING) to induce IFN-I, initiating
the antiviral response to DNA viruses [10–12]. In
principle, the microorganisms that can carry DNA into
the host cytoplasm, such as DNA viruses (e.g., HSV-1,
KSHV and adenovirus) [13–16], bacteria(e.g., Group B
Streptococcus) [17], and retroviruses(e.g., HIV) [18] could
potentially trigger the cGAS-STING pathway [12]. While
it is still unknown how the activity of cGAS is regulated
during host defense [19]. MDR cGAS detects cytosolic DNA in a sequence-
independent manner, which elicits the cGAS-STING
pathway to prime innate immune responses to various
DNA viruses [19, 29]. Anghelina D et al. [30] have
identified that cGAS deficiency resulted in a reduced early
antiviral innate response to viral infection. Therefore, with
the capacity for DNA sensing, cGAS plays an important
role in IFN-I responses against DNA viruses [12, 14, 31]. Notably, cGAS can in principle detect any DNA that
invades the cytoplasm, bind to DNA and is activated
by DNA, irrespective of its sequence [29]. Hence, HPV
infection was considered rather than high-risk HPV or
low-risk HPV type. rs311678 is located in the intron 3–4
region of the cGAS gene. In our study, the minor allele in
cGAS rs311678 reduced the risk of cervical precancerous
lesions and had an antagonistic interaction with HPV
infection. Furthermore, the results of qPCR and western
blotting were basically consistent with our work, in which
G allele in cGAS rs311678 might reduce the risk of
cervical precancerous lesions in functional analysis. It has
been demonstrated that introns contribute to the regulation
of gene expression [32, 33] and transcript processing [34]. Matsushita et al. [35] reported that rs4376531 at intron 17
of the ARHGEF10 gene affected its transcriptional activity,
which ultimately resulted in an increased incidence of
atherothrombotic stroke. Similarly, we hypothesized
that the rs311678 variant in the cGAS gene might lead
to a higher level of cGAS transcripts and higher cGAS-
STING pathway activity, which may interfere with the
process whereby HPV encodes antagonists for the evasion
of innate immune detection of HPV viral DNA [10, 36],
thereby leading to HPV clearance. Secondly, the structure
of mouse cGAS bound to an 18bp dsDNA through two
binding sites, forming a 2:2 complex, therefore, we
hypothesized that G of rs311678 might influence these
two DNA binding sites that increase the affinity with HPV
DNA, resulting in the activation of cGAS and thereby
enhancing IFN-β induction and ultimately contributing to
a decreased risk of cervical precancerous lesions [37, 38]. It is noteworthy that the cGAS/STING cascade has been
viewed as an adjuvant target for vaccine applications
[16].On the other hand, better understanding of the battle
between the virus and the human host will most certainly
improve the prevention and treatment of all viral diseases
[31]. MDR We could expect to target the activation of the cGAS-
STING pathway for the effective prevention of cervical
precancerous lesions. According to the MDR selection model, a three-factor
interaction model, containing HPV infection, age at menarche
and rs311678 was optimal, with maximum CVC (10/10) and
the highest TBA (71.64%) (significance test P = 0.001, and P
for permutation test = 0.000–0.001) (Table 4). Three-way interaction between HPV infection,
rs311678 status, and age at menarche with risk
for cervical precancerous lesions Based on the MDR model, we further performed
a risk analysis of different combinations among the 3
factors (Table 5). The combination without any risk factors
(including non-HPV-infected, wild-type for rs311678
and lower age at menarche: < 15 years) was used as a
reference group. The individuals with a combination of 3
factors had a 4.27-fold greater risk of developing cervical
precancerous lesions (OR = 4.27, 95% CI 1.81–10.04,
P = 0.001), while the OR for cervical precancerous
lesions in the presence of HPV infection, wild-type for
rs311678 and increased age at menarche (≥ 15 years) was
the highest (OR, 7.72; 95% CI, 3.39–17.57). Functional studies To further study the functions of rs311678 in cGAS
gene, thirty samples involved in 14 cases and 16 controls
were randomly selected from our samples. As showed in
Figure 1A, the AG and GG genotype in cGAS rs311678
had the lower mRNA expression levels in the SIL group,
compared with control group (for AG genotype, P = 0.010;
for GG genotype, P = 0.029). However, there was no
difference in cGAS mRNA expression between SIL cases
and controls among AA genotype in rs311678 (P = 0.925). Figure 1B revealed that the expression of cGAS protein
in rs311678 AG was higher in control group than in SIL
group (P = 0.008). However, there were no differences
in cGAS protein expression in AA and GG genotypes
between SIL cases and controls (for AA genotype,
P = 0.667; for GG genotype, P = 0.095). Haplotype analysis The associations between cGAS and STING
haplotypes and the risk of cervical precancerous lesions
were estimated. No haplotypes in cGAS or STING gene
was found to be significantly associated with risk of
cervical precancerous lesions (Supplementary Table S5). www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 84229 Biological interaction of HPV infection and
cGAS rs311678 for cervical precancerous lesions along with STING and HPV infection on the risk of
cervical precancerous lesions. The roles of the cGAS-
STING pathway and the MHC genes in the pathogenesis
of cervical precancerous lesions are still unclear. To
investigate whether SNPs in these molecules affected the
occurrence of cervical precancerous lesions, our study
comprehensively evaluated the associations between
variants of the cGAS-STING pathway, the MHC and
cervical precancerous lesions. We evaluated the interactive effects of HPV infection
and each SNP based on an additive scale. According to the
S index, a significant antagonistic interaction was found
between cGAS rs311678 and HPV infection (S = 0.55,
95% CI = 0.32–0.96, P = 0.033) (Table 3). DISCUSSION To our knowledge, this is the first study that
describes the interactive effects of SNPs in cGAS and www.impactjournals.com/oncotarget Oncotarget 84230 Table 1: The distribution of demographic characteristics
Characteristics
Cases No = 164
Controls No = 428
Pa
Age (years)
41.49 ± 7.97
42.93 ± 7.85
0.046
BMI (kg/m2)b
22.14 ± 2.82
22.36 ± 3.09
0.420
HPV infection
< 0.001
No
32 (19.5%)
251 (58.6%)
Yes
132 (80.5%)
177 (41.4%)
Number of term births
2.09 ± 1.07
2.04 ± 0.97
0.555
Age at menarche (years)
15.12 ± 1.97
14.93 ± 1.67
0.257
Family history of cancer
0.372
No
164 (100.0%)
421 (98.8%)
Yes
0 (0%)
5 (1.2%)
Genital cleaning after each intercourse
0.064
never
57 (36.5%)
178 (42.6%)
occasional
47 (30.1%)
141 (33.7%)
frequently
52 (33.3%)
99 (23.7%)
The initial pregnancy of age (years)
23.94 ± 3.52
24.11 ± 3.31
0.572
Data are shown using means ± standard deviation for continuous factors.a The χ2 test for categorical variables and student t-test
for continuous variables. bBMI (kg/m2), body mass index. Bold values are statistically significant. Data are shown using means ± standard deviation for continuous factors.a The χ2 test for categorical variables and student t-test
for continuous variables. bBMI (kg/m2), body mass index. Bold values are statistically significant. The present study statistically validated an interaction
between HPV and each SNP in the cGAS-STING pathway
in relation to the risk of cervical precancerous lesions,
which is not enough. The MDR model showed that the
three-locus model (i.e., HPV infection, age at menarche
and rs311678) is the best model for the prediction of the
risk of cervical precancerous lesions in our population. DISCUSSION Notably, in our further study, the interaction among HPV
Table 2: The association of the allele in cGAS, STING and MHC genes with the risk of cervical
precancerous lesions
Gene
SNPs
Allele
Cases
Controls
OR (95% CI)
P
(n = 164)
(n = 428)
cGAS
rs610913
C
82
190
1 (Ref)
A
166
442
0.87 (0.64–1.19)
0.386
rs311678
A
190
450
1 (Ref)
G
72
234
0.73 (0.53–1.00)
0.048
rs4032697
A
306
778
1 (Ref)
G
8
29
0.70 (0.32–1.55)
0.379
rs311675
T
300
760
1 (Ref)
A
14
43
0.83 (0.45–1.53)
0.541
rs9352000
T
294
762
1 (Ref)
G
16
45
0.92 (0.51–1.66)
0.785
rs7761170
G
218
556
1 (Ref)
T
65
164
1.01 (0.73–1.40)
0.948
STING
rs1131769
G
264
694
1 (Ref)
A
34
80
1.12 (0.73–1.71)
0.610
rs7380824
C
98
284
1 (Ref)
T
142
364
1.13 (0.84–1.53)
0.424
MHC
rs2516448
G
208
532
1 (Ref)
A
67
168
1.02 (0.74–1.41)
0.905
Bold values are statistically significant. Table 2: The association of the allele in cGAS, STING and MHC genes with the risk of cervical
precancerous lesions iation of the allele in cGAS, STING and MHC genes with the risk of cervica Table 2: The association of the allele in cGAS, STING and MHC genes with the risk of cervical
precancerous lesions Bold values are statistically significant. three-locus model (i.e., HPV infection, age at menarche
and rs311678) is the best model for the prediction of the
risk of cervical precancerous lesions in our population. Notably, in our further study, the interaction among HPV three-locus model (i.e., HPV infection, age at menarche
and rs311678) is the best model for the prediction of the
risk of cervical precancerous lesions in our population. Notably, in our further study, the interaction among HPV The present study statistically validated an interaction
between HPV and each SNP in the cGAS-STING pathway
in relation to the risk of cervical precancerous lesions,
which is not enough. The MDR model showed that the www.impactjournals.com/oncotarget Oncotarget 84231 Table 3: Results for gene-environment interaction analysis for each candidate SNP and HPV
infection
Interaction group
Deviation from additive model
Gene
S (95% CI)
Pa
1
rs610913*HPV infection
2.08 (0.41–10.59)
0.377
2
rs311678*HPV infection
0.55 (0.32–0.96)
0.033
3
rs4032697*HPV infection
0.57 (0.15–2.23)
0.414
4
rs311675*HPV infection
1.26 (0.46–3.45)
0.663
5
rs9352000*HPV infection
0.59 (0.22–1.59)
0.296
6
rs7761170*HPV infection
1.03 (0.56–1.90)
0.924
7
rs1131769*HPV infection
1.27 (0.62–2.63)
0.517
8
rs7380824*HPV infection
1.11 (0.63–1.95)
0.717
9
rs2516448*HPV infection
0.71 (0.43–1.17)
0.180
CI = confidence interval. aAdjusted for age(years) and the initial pregnancy of age(< 24 and ≥ 24 years). Bold values are
statistically significant. Table 3: Results for gene-environment interaction analysis for each candidate SNP and HPV
infection Table 3: Results for gene-environment interaction analysis for each candidate SNP and HPV
infection CI = confidence interval. aAdjusted for age(years) and the initial pregnancy of age(< 24 and ≥ 24 years). Bold values are
statistically significant. Table 4: MDR models of GAS, STING and MHC gene and environmental factors of cervical
precancerous lesions
Best Model
Training balanced
accuracy
Testing balanced
accuracy
Cross-validation
Consistency
P valuea
HPV infection
0.6957
0.6962
10/10
0.000–0.001
HPV infection, age at menarche
0.7088
0.6902
7/10
0.000–0.001
HPV infection, age at menarche,rs311678
0.7451
0.7164
10/10
0.000–0.001
HPV infection,age at menarche, genital
cleansing after each intercourse,number of
term birth
0.7989
0.5347
4/10
0.487
a1000-fold permutation test. The best model(see text for details) are in bold. Table 2: The association of the allele in cGAS, STING and MHC genes with the risk of cervical
precancerous lesions odels of GAS, STING and MHC gene and environmental factors of cervica Table 4: MDR models of GAS, STING and MHC gene and environmental factors of cervical
precancerous lesions MDR models of GAS, STING and MHC gene and environmental factors of
us lesions Oncotarget
84232
www.impactjournals.com/oncotarget
infection, age at menarche and rs311678 was important
at the population level; among HPV-infected individuals,
women with age at menarche over 15 years and, carrying
the minor G allele in cGAS rs311678 had a reduced risk
of cervical precancerous lesions by 45% compared with
individuals who were wild type at rs311678 (Figure 2). Table 5: Risk group analysis with 3 risk factors: HPV infection, age at menarche and rs311678
HPV
infection
Age at
menarcheb
rs311678
Cases
Controls
OR (95% CI)
Pa
−
−
−
10
82
1
−
+
−
5
57
0.69 (0.22,2.12)
0.512
−
−
+
13
53
2.02 (0.82–4.95)
0.124
−
+
+
3
56
0.43 (0.11–1.65)
0.219
+
-
−
43
51
7.11 (3.27–15.44)
< 0.001
+
+
−
36
35
7.72 (3.39–17.57)
< 0.001
+
−
+
29
47
5.06 (2.26–11.33)
< 0.001
+
+
+
21
40
4.27 (1.81–10.04)
0.001
aAdjusted for age(years) and the initial pregnancy of age (< 24 and ≥ 24 years). bAge at menarche(−, < 15years; +: ≥ 15 years). OR = odds
ratio, CI = confidence interval. Bold values are statistically significant. Table 5: Risk group analysis with 3 risk factors: HPV infection, age at menarche and rs311678
HPV
A
t Table 5: Risk group analysis with 3 risk factors: HPV infection, age at menarche and rs311678 Table 5: Risk group analysis with 3 risk factors: HPV infection, age at me aAdjusted for age(years) and the initial pregnancy of age (< 24 and ≥ 24 years). bAge at menarche(−, < 15years; +: ≥ 15 years). OR = odds
ratio, CI = confidence interval. Bold values are statistically significant. infection, age at menarche and rs311678 was important
at the population level; among HPV-infected individuals,
women with age at menarche over 15 years and, carrying the minor G allele in cGAS rs311678 had a reduced risk
of cervical precancerous lesions by 45% compared with
individuals who were wild type at rs311678 (Figure 2). www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 84232 rs2516448 is located 7.32 kb downstream of the
MHC genes. HPV testing Total DNA from cervical cells was extracted using a
commercial magnetic beads kit (Chemagen; PekinElmer,
Waltham, MA), according to the manufacturer’s
instructions. Then, 16 HPV types (6, 11, 16, 18, 31, 33, 35,
39, 45, 51, 52, 56, 58, 59, 66 and 68) were detected with
the MassARRAY (Sequenom, San Diego, CA) technique
based on matrix-assisted laser desorption/ionization time-
of flight mass spectrometry [42]. All of the procedures
were performed in the clinical standard laboratory of
Beijing Genomics Institute. Table 2: The association of the allele in cGAS, STING and MHC genes with the risk of cervical
precancerous lesions Although it has been reported that the minor
allele of rs2516448 increased the susceptibility to cervical
cancer in Swedish women [27, 28], we did not find this
association in Chinese women. One possible explanation
could be that this association is relevant to only Caucasian
populations and not to other ethnic groups. Further studies
are needed to test the association between MHC rs2516448 and cervical precancerous lesions among those with non-
Caucasian backgrounds [39]. In addition, variability in the
frequency of HPV types, intratype variation, or host-virus
interactions in the Chinese population may be responsible
for the differences in association [38]. There are some potential limitations of this study. The main limitation of our study was the small number of
SIL cases and controls, which might limit the likelihood of Figure 1: qRT-PCR, western blot analyses of cGAS rs311678. The results were expressed as the mean ± S.D. *P < 0.05. (A) The
cGAS mRNA expression in SIL cases and controls by cGAS rs311678. (B) Western blotting analysis for cGAS rs311678 in human PBMCs. Figure 1: qRT-PCR, western blot analyses of cGAS rs311678. The results were expressed as the mean ± S.D. *P < 0.05. (A) The
cGAS mRNA expression in SIL cases and controls by cGAS rs311678. (B) Western blotting analysis for cGAS rs311678 in human PBMCs. Figure 1: qRT-PCR, western blot analyses of cGAS rs311678. The results were expressed as the mean ± S.D. *P < 0.05. (A) The
cGAS mRNA expression in SIL cases and controls by cGAS rs311678. (B) Western blotting analysis for cGAS rs311678 in human PBMCs. Figure 2: Risk analysis with 3 factors. HPV infection, age at menarche and rs311678. Figure 2: Risk analysis with 3 factors. HPV infection, age at menarche and rs311678. Figure 2: Risk analysis with 3 factors. HPV infection, age at menarche and rs311678. www.impactjournals.com/oncotarget Oncotarget 84233 Study sample Genomic DNA from peripheral blood was extracted
with phenol-chloroform DNA extraction methods. The samples were stored at −20°C until they were
used. The DNA concentration was determined using a
spectrophotometer (Nano Drop ND-1000, PerkinElmer,
USA). Samples with a mean OD260 nm/OD280 nm of
1.8–2.0 and DNA concentration > 20 ng/μl were considered
to be free of contamination. The study was approved by the Ethics Committee
of School of Medicine in Jinan University. Cervical
specimens were collected with a broom-like device
(Qiagen, Valencia) and placed into a ThinPrep Pap test vial
containing PreservCyt Solution. Referral Pap specimens
were processed locally using the ThinPrep 2000 System
(Hologic) and evaluated for routine screening cytology. A pap smear was positive for SIL if low (LSIL) and high
(HSIL) grade squamous intraepithelial lesion, as classified
according to the Bethesda Classification System, was
detected[41]. Excluding other types of uterine diseases
and no history of hysterectomy, a total of 164 cases were
composed of 120 LSIL (73.2%) and 44 HSIL (26.8%). A
total of 428 control samples without intraepithelial lesion
or malignancy were recruited from the area of residence
of the cases. Nine candidate SNPs were genotyped using a
matrix-assisted laser desorption/ionization time of flight
mass spectrometry (MALDI-TOF MS) method. Detail
primer and amplified length for the determination of the
9 SNP genotypes is provided in Supplementary Table
S2. The reaction mixture was desalted by adding resin,
mixed, and resuspended in 16 μL of water. Once the
primer extension reaction was completed, the samples
were spotted onto target samples using a RS1000
MassARRAY Nanodispenser and genotyped using
MALDI-TOF. Genotyping was performed in real time
with Typer software version 4.0. Genotype sequencing
was performed by Invitrogen Trading (Shanghai) Co., Ltd. The distribution of SNPs in the cGAS, STING and MHC
genes is shown in Supplementary Table S3. Genotype analysis detecting weaker associations between SNPs and cervical
precancerous lesions. A larger study would be required
to address this issue. Also, we’d like to mention that the
limited sample size of functional studies depended on
availability of patients’ PBMCs. Another limitation is
that the methods of calculating interactions based on the
additive scale only apply to two factors at two levels, while
the 95% confidence intervals of S were not calculated by
the Excel spreadsheet when the variable factors were
multiply variable [40]. Tag single-nucleotide polymorphisms (SNPs) in
the cGAS and STING genes were selected from the
HapMap database (http://www.hapmap.org) according
to the following selection strategy [43]. The screened
region extended 10 kilobases upstream of the annotated
transcription start site and downstream at the end of
the last exon of each gene, which covered most of the
genetic information in the Han Chinese in Beijing (CHB)
population from the HapMap database (HapMap data rel
27 Phase + III, Feb09, on NCBI B36 assembly, dbSNP
b126) [44]. tagSNPs were selected using a pairwise
tagging algorithm setting the Hardy-Weinberg P-value,
minor allele frequency (MAF) and r2 thresholds at 0.01,
0.01 and 0.8, respectively. The linkage disequilibrium
(LD) pattern of each gene in the CHB population exhibited
strong LD in several groups of tagSNPs (r2 ≥ 0.8),
indicating that the most common SNPs can be captured by
a subset of tagSNPs. Furthermore, we also selected SNPs
based on previous findings from the literature. Eventually,
9 candidate SNPs were selected, including rs610913,
rs311678, rs4032697, rs311675, rs9352000, rs7761170 in
cGAS, rs1131769, rs7380824 in STING and rs2516448
in the MHC. In conclusion, our study provides evidence that
the cGAS rs311678 polymorphism influences the risk
of cervical precancerous lesions. Moreover, our study
also finds that two-way and three-way gene-environment
interactions exist in the etiology of cervical precancerous
lesions. This study provides new epidemiological clues
about the protective role of the cGAS-STING pathway
in cervical precancerous lesions and further insights into
such interactions in the etiology of cervical precancerous
lesions. Furthermore, our results provide further evidence
that introns should no longer be considered nonsense. Quantitative real-time reverse transcription
PCR (qRT-PCR) analysis The peripheral blood mononuclear cells (PBMCs)
were isolated from EDTA-anticoagulated blood with
Ficoll–Hypaque
density-gradient
centrifugation. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 84234 Total RNA was extracted from PBMCs using TRIzol
(Invitrogen, Carlsbad, CA), followed by reverse
transcription using a transcriptase cDNA kit (Takara-
PrimeScript RT Master Mix kit, Otsu, Japan). Then
we finished qRT-RCR analysis to quantify the mRNA
expression of cGAS with the SYBR PrimerScript RT-PCR
kit (TaKaRa, Otsu, Japan) normalized to mRNA β-actin. The assays were performed on a Bio-Rad’s CFX96 real-
time system (Bio-Rad Laboratories). Cycle conditions
were 95°C for 30s followed by 45 cycles at 95°C for
5s and 60°C for 30s. Relative expression levels were
calculated using the 2−∆∆Ct method. The data of the two
independent analyses for each sample and parameters were
averaged and relative expression levels were presented
as the relative fold change analyzed using unpaired two-
tailed t test. or two copies of the minor allele). The risk for cervical
precancerous lesions for a given SNP and HPV infection
status was expressed by ORi,,j where the first index (i)
indicated the HPV infection status coded as 0 for non-
infected and 1 for infected subjects, and the second index
(j) indicated the SNP genotype, coded as 0 for subjects
homozygous for the major allele and 1 for subjects bearing
one or two copies of the minor allele. Subjects who were
HPV-uninfected and homozygous for the major allele were
considered the reference group, thus coding their cervical
precancerous lesions risk as OR00 = 1. The relative ORs
were obtained by logistic regression. The confidence
intervals were calculated by the regression coefficients and
the corresponding covariance matrix [47]. Deviation from
an additive model was calculated as the relative excess
risk due to S (the synergy index). Biological interactions
in the regression models were tested as departure from
additivity. Based on the adjusted odds ratios obtained
in the logistic regression models, an Excel spreadsheet
(www.epinet.se) was used to calculate S on an additive
scale and its corresponding confidence intervals [47]. An S
value (95% CI) that does not cross 1 indicates a biological
interaction [48, 49]. In addition, S > 1 indicates synergetic
effects and S < 1 indicates antagonistic effects [50, 51]. Statistical analysis Logistic regression was used to calculate odds ratios
(ORs) and their relative 95% confidence intervals (CIs)
for risk estimation. A chi-square test was used to evaluate
the Hardy-Weinberg equilibrium and the dependence of
the allele frequencies between the cases and controls. In
addition, a t-test or chi-square test was used to examine
differences in the demographic characteristics and HPV
infection status between the cases and controls. MDR software v.3.0.2 and MDR permutation testing
software (version 1.0 beta 2) were used in this study and
were freely available online (www.epistasis.org). All other
statistical analyses were performed using SPSS software
v.16.0 (SPSS, Inc.). Significant associations were defined
as P < 0.05. Based on the observed genotypes, haplotype
frequencies and effects were examined using the SHEsis,
a powerful software platform (http://analysis.bio-x.cn/
myAnalysis.php) for analyses of haplotype construction
[45]. The global score test was used to estimate the overall
differences in haplotype frequencies between cases and
controls. The estimated ORs and 95% CI were also used
to estimate the effects of individual haplotypes on cervical
precancerous lesions. Western blot PBMCs were homogenized in RIPA Lysis Buffer
at 4°C, and then centrifuged 10 min in 13,000 × g at
4°C. Protein concentrations of the homogenates were
determined by BCA kit. The proteins were separated
by 8% sodium dodecy1 sulface polyacrylamide gel
electrophoresis (SDS-PAGE) and electrotransferred to
Immobilon-P membrane (Millipore, Bedford, MA, USA). The primary antibody (Supplementary Table S1) at 4°C
overnight and then with a corresponding anti-rabbit IgG
conjugated to horseradish peroxidase (1 : 5000) at 37°C
for 1 h. Immunoreactive bands were visualized with a
chemiluminescent substrate (ECL) kit. Finally, the multifactor dimensionally reduction
(MDR) method [52–55] was applied to analyze high-
order gene-gene and gene-environment interactions. To
search for the best n-factor model, the data were divided
into 10 sets: 1 for testing and 9 for training. In brief, the
multilocus genotypes were pooled into high-risk and low-
risk groups, effectively reducing the genotype predictors
to one dimension. The result was a set of models, and the
Testing Balanced Accuracy (TBA) and cross-validation
consistency (CVC) indexes were used to determine the
overall best model. An MDR-pt procedure was used
to evaluate the significance of the selected models via
calculating empirical 1000-fold permutation tests .The
model with the maximum TBA and CVC and a P value
for TBA in MDR-pt results less than 0.05 was considered
the best model. REFERENCES 13. Christensen MH, Jensen SB, Miettinen JJ, Luecke S,
Prabakaran T, Reinert LS, Mettenleiter T, Chen ZJ,
Knipe DM, Sandri-Goldin RM, Enquist LW, Hartmann R,
Mogensen TH, et al. HSV-1 ICP27 targets the TBK1-
activated STING signalsome to inhibit virus-induced type I
IFN expression. EMBO J. 2016; 35:1385–1399. 1. Steenbergen RD, Snijders PJ, Heideman DA, Meijer CJ. Clinical implications of (epi)genetic changes in HPV-
induced cervical precancerous lesions. Nat Rev Cancer. 2014; 14:395–405. 1. Steenbergen RD, Snijders PJ, Heideman DA, Meijer CJ. Clinical implications of (epi)genetic changes in HPV-
induced cervical precancerous lesions. Nat Rev Cancer. 2014; 14:395–405. 2. Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM. Estimates of worldwide burden of cancer in 2008:
GLOBOCAN 2008. Int J Cancer. 2010; 127:2893–2917. 14. Ma Z, Jacobs SR, West JA, Stopford C, Zhang Z, Davis Z,
Barber GN, Glaunsinger BA, Dittmer DP, Damania B. Modulation of the cGAS-STING DNA sensing pathway
by gammaherpesviruses. Proc Natl Acad Sci USA. 2015;
112:E4306–4315. 3. Arbyn M, Castellsague X, de Sanjose S, Bruni L,
Saraiya M, Bray F, Ferlay J. Worldwide burden of cervical
cancer in 2008. Ann Oncol. 2011; 22:2675–2686. 15. Lam E, Stein S, Falck-Pedersen E. Adenovirus detection
by the cGAS/STING/TBK1 DNA sensing cascade. J Virol. 2014; 88:974–981. 4. Milosevic-Djordjevic O, Stosic I, Grujicic D, Bankovic D,
Arsenijevic S. Cervical precancerous lesions—chromosomal
instability in peripheral blood lymphocytes in relation to
lesion stage, age and smoking habits. Acta Obstet Gynecol
Scand. 2011; 90:1082–1087. 16. Li XD, Wu J, Gao D, Wang H, Sun L, Chen ZJ. Pivotal
roles of cGAS-cGAMP signaling in antiviral defense and
immune adjuvant effects. Science. 2013; 341:1390–1394. 5. Walboomers JM, Jacobs MV, Manos MM, Bosch FX,
Kummer JA, Shah KV, Snijders PJ, Peto J, Meijer CJ,
Munoz N. Human papillomavirus is a necessary cause
of invasive cervical cancer worldwide. J Pathol. 1999;
189:12–19. 17. Andrade WA, Firon A, Schmidt T, Hornung V,
Fitzgerald KA, Kurt-Jones EA, Trieu-Cuot P, Golenbock DT,
Kaminski PA. Group B Streptococcus Degrades Cyclic-di-
AMP to Modulate STING-Dependent Type I Interferon
Production. Cell Host Microbe. 2016; 20:49–59. 6. Schiffman MH, Bauer HM, Hoover RN, Glass AG,
Cadell DM, Rush BB, Scott DR, Sherman ME,
Kurman RJ, Wacholder S, et al. Epidemiologic evidence
showing that human papillomavirus infection causes most
cervical intraepithelial neoplasia. J Natl Cancer Inst. 1993;
85:958–964. 18. ACKNOWLEDGMENTS AND FUNDING This work was supported in part by the Major
Research Plan of the National Natural Science Foundation
of China (91543132), National Natural Science
Foundation of China (Grant Nos: 81541070; 30901249;
81101267), the Guangdong Natural Science Foundation
(Grant Nos: 10151063201000036; S2011010002526;
2016A030313089),
Guangdong
Province
Medical We explored the additive interaction between the
factors according to the following strategy [46]. Among
cases and controls, a binary classification was used both
for HPV infection (infection vs. non-infection) and for
genotypes (homozygous for the major allele vs. one www.impactjournals.com/oncotarget Oncotarget 84235 11. Bode C, Fox M, Tewary P, Steinhagen A, Ellerkmann RK,
Klinman D, Baumgarten G, Hornung V, Steinhagen F. Human plasmacytoid dentritic cells elicit a Type I Interferon
response by sensing DNA via the cGAS-STING signaling
pathway. Eur J Immunol. 2016. Research Foundation (Grant Nos: A2014374; A2015310)
and Project from Jinan university (Grant Nos: 21612426,
21615426, JNUPHPM2016001, and JNUPHPM2016002). The authors have no conflicts of interest to report. The authors have no conflicts of interest to report. CONFLICTS OF INTEREST 12. Sun L, Wu J, Du F, Chen X, Chen ZJ. Cyclic GMP-AMP
synthase is a cytosolic DNA sensor that activates the type I
interferon pathway. Science. 2013; 339:786–791. REFERENCES BMC Med. 2014; 12:7. 26. Horng JT, Hu KC, Wu LC, Huang HD, Lin FM, Huang SL,
Lai HC, Chu TY. Identifying the combination of genetic
factors that determine susceptibility to cervical cancer. IEEE Trans Inf Technol Biomed. 2004; 8:59–66. 40. Xiao D, Ye X, Zhang N, Ou M, Guo C, Zhang B, Liu Y,
Wang M, Yang G, Jing C. A meta-analysis of interaction
between Epstein-Barr virus and HLA-DRB1*1501 on risk
of multiple sclerosis. Sci Rep. 2015; 5:18083. 27. Chen D, Hammer J, Lindquist D, Idahl A, Gyllensten U. A variant upstream of HLA-DRB1 and multiple variants
in MICA influence susceptibility to cervical cancer in a
Swedish population. Cancer Med. 2014; 3:190–198. 41. Solomon D, Davey D, Kurman R, Moriarty A, O’Connor D,
Prey M, Raab S, Sherman M, Wilbur D, Wright T, Jr. and Young N. The 2001 Bethesda System: terminology
for reporting results of cervical cytology. Jama. 2002;
287:2114–2119. 28. Chen D, Juko-Pecirep I, Hammer J, Ivansson E, Enroth S,
Gustavsson I, Feuk L, Magnusson PK, McKay JD,
Wilander E, Gyllensten U. Genome-wide association study
of susceptibility loci for cervical cancer. J Natl Cancer Inst. 2013; 105:624–633. 42. Jing L, Zhong X, Zhong Z, Huang W, Liu Y, Yang G,
Zhang X, Zou J, Jing C, Wei X. Prevalence of human
papillomavirus infection in Guangdong Province, China:
a population-based survey of 78,355 women. Sex Transm
Dis. 2014; 41:732–738. 29. Cai X, Chiu YH, Chen Z. The cGAS-cGAMP-STING
Pathway of Cytosolic DNA Sensing and Signaling. Mol
Cell. 2014; 54:289–296. 30. Anghelina D, Lam E, Falck-Pedersen E. Diminished
antiviral innate response to Adenovirus vectors in cGAS/
STING deficient mice minimally impacts adaptive
immunity. J Virol. 2016. 43. Guo C, Zhang L, Nie L, Zhang N, Xiao D, Ye X, Ou M,
Liu Y, Zhang B, Wang M, Lin H, Yang G, Jing C. Association of polymorphisms in the MyD88, IRAK4 and
TRAF6 genes and susceptibility to type 2 diabetes mellitus
and diabetic nephropathy in a southern Han Chinese
population. Mol Cell Endocrinol. 2016. 31. Ma Z, Damania B. The cGAS-STING Defense Pathway
and Its Counteraction by Viruses. Cell Host Microbe. 2016;
19:150–158. 44. A haplotype map of the human genome. Nature. 2005;
437:1299–1320. 32. Lareau LF, Inada M, Green RE, Wengrod JC, Brenner SE. Unproductive splicing of SR genes associated with highly
conserved and ultraconserved DNA elements. Nature. 2007;
446:926–929. 45. Shi YY, He L. REFERENCES Gao D, Wu J, Wu YT, Du F, Aroh C, Yan N, Sun L, Chen ZJ. Cyclic GMP-AMP synthase is an innate immune sensor of
HIV and other retroviruses. Science. 2013; 341:903–906. 19. Xia P, Ye B, Wang S, Zhu X, Du Y, Xiong Z, Tian Y,
Fan Z. Glutamylation of the DNA sensor cGAS regulates
its binding and synthase activity in antiviral immunity. Nat
Immunol. 2016; 17:369–378. 7. Bosch FX, Manos MM, Munoz N, Sherman M, Jansen AM,
Peto J, Schiffman MH, Moreno V, Kurman R, Shah KV. Prevalence of human papillomavirus in cervical cancer: a
worldwide perspective. International biological study on
cervical cancer (IBSCC) Study Group. J Natl Cancer Inst. 1995; 87:796–802. 20. Mueller KL. Viral oncogenes remove the host’s STING. J Acoust Soc Am. 2015; 350:2334. 21. Le Bon A, Tough DF. Links between innate and adaptive
immunity via type I interferon. Curr Opin Immunol. 2002;
14:432–436. 8. Lehtinen M, Luukkaala T, Wallin KL, Paavonen J,
Thoresen S, Dillner J, Hakama M. Human papillomavirus
infection, risk for subsequent development of cervical
neoplasia and associated population attributable fraction. J Clin Virol. 2001; 22:117–124. 22. Habiger C, Jager G, Walter M, Iftner T, Stubenrauch F. IFN-kappa inhibits HPV31 transcription by inducing Sp100
proteins. J Virol. 2015. 23. Yi G, Brendel VP, Shu C, Li P, Palanathan S, Cheng Kao C. Single nucleotide polymorphisms of human STING can
affect innate immune response to cyclic dinucleotides. PloS
one. 2013; 8:e77846. 9. Paijo J, Doring M, Spanier J, Grabski E, Nooruzzaman M,
Schmidt T, Witte G, Messerle M, Hornung V, Kaever V,
Kalinke U. cGAS Senses Human Cytomegalovirus and
Induces Type I Interferon Responses in Human Monocyte-
Derived Cells. PLoS Pathog. 2016; 12:e1005546. 24. Jin L, Xu LG, Yang IV, Davidson EJ, Schwartz DA,
Wurfel MM, Cambier JC. Identification and characterization
of a loss-of-function human MPYS variant. Genes Immun. 2011; 12:263–269. 10. Lau L, Gray EE, Brunette RL, Stetson DB. DNA tumor
virus oncogenes antagonize the cGAS-STING DNA-sensing
pathway. Science. 2015; 350:568–571. www.impactjournals.com/oncotarget Oncotarget 84236 25. Hemminki K, Dong C, Vaittinen P. Familial risks in cervical
cancer: is there a hereditary component? Int J Cancer. 1999;
82:775–781. association studies of cervical intraepithelial neoplasia 3
(CIN3) identifies a new susceptibility locus. Oncotarget. 2016; 7:42216–42224. doi: 10.18632/oncotarget.9916. 39. Hosang GM, Shiles C, Tansey KE, McGuffin P, Uher R. Interaction between stress and the BDNF Val66Met
polymorphism in depression: a systematic review and meta-
analysis. REFERENCES SHEsis, a powerful software platform for
analyses of linkage disequilibrium, haplotype construction,
and genetic association at polymorphism loci. Cell Res. 2005; 15:97–98. 33. Wong JJ, Ritchie W, Ebner OA, Selbach M, Wong JW,
Huang Y, Gao D, Pinello N, Gonzalez M, Baidya K,
Thoeng A, Khoo TL, Bailey CG, et al. Orchestrated intron
retention regulates normal granulocyte differentiation. Cell. 2013; 154:583–595. 46. Tunesi S, Ferrante D, Mirabelli D, Andorno S, Betti M,
Fiorito G, Guarrera S, Casalone E, Neri M, Ugolini D,
Bonassi S, Matullo G, Dianzani I, et al. Gene-asbestos
interaction in malignant pleural mesothelioma susceptibility. Carcinogenesis. 2015. 34. Sjakste T, Paramonova N, Grislis Z, Trapina I, Kairisa D. Analysis of the single-nucleotide polymorphism in the
5′UTR and part of intron I of the sheep MSTN gene. DNA
Cell Biol. 2011; 30:433–444. 47. Andersson T, Alfredsson L, Kallberg H, Zdravkovic S,
Ahlbom A. Calculating measures of biological interaction. Eur J Epidemiol. 2005; 20:575–579. 35. Matsushita T, Ashikawa K, Yonemoto K, Hirakawa Y,
Hata J, Amitani H, Doi Y, Ninomiya T, Kitazono T, Ibayashi S,
Iida M, Nakamura Y, Kiyohara Y, et al. Functional SNP of
ARHGEF10 confers risk of atherothrombotic stroke. Hum
Mol Genet. 2010; 19:1137–1146. 48. Yang X, So WY, Ma RC, Ko GT, Kong AP, Zhao H,
Luk AO, Lam CW, Ho CS, Tong PC, Chan JC. Low LDL
cholesterol, albuminuria, and statins for the risk of cancer in
type 2 diabetes: the Hong Kong diabetes registry. Diabetes
care. 2009; 32:1826–1832. 36. Goubau D, Deddouche S, Reis e Sousa C. Cytosolic sensing
of viruses. Immunity. 2013; 38:855–869. 49. Chaumont A, Voisin C, Sardella A, Bernard A. Interactions
between domestic water hardness, infant swimming and
atopy in the development of childhood eczema. Environ
Res. 2012; 116:52–57. 37. Li X, Shu C, Yi G, Chaton CT, Shelton CL, Diao J,
Zuo X, Kao CC, Herr AB, Li P. Cyclic GMP-AMP
synthase is activated by double-stranded DNA-induced
oligomerization. Immunity. 2013; 39:1019–1031. 50. Rothman KJ. The estimation of synergy or antagonism. American journal of epidemiology. 1976; 103:506–511. 38. Chen D, Enroth S, Liu H, Sun Y, Wang H, Yu M, Deng L,
Xu S, Gyllensten U. Pooled analysis of genome-wide 51. Foraita R. A conditional synergy index to assess biological
interaction. Eur J Epidemiol. 2009; 24:485–494. www.impactjournals.com/oncotarget Oncotarget 84237 52. Hahn LW, Ritchie MD, Moore JH. Multifactor
dimensionality reduction software for detecting gene-gene
and gene-environment interactions. Bioinformatics. 2003;
19:376–382. www.impactjournals.com/oncotarget REFERENCES interactions in the presence of genotyping error, missing
data, phenocopy, and genetic heterogeneity. Genet
Epidemiol. 2003; 24:150–157. 55. Heidema AG, Feskens EJ, Doevendans PA, Ruven HJ,
van Houwelingen HC, Mariman EC, Boer JM. Analysis of
multiple SNPs in genetic association studies: comparison
of three multi-locus methods to prioritize and select SNPs. Genet Epidemiol. 2007; 31:910–921. 53. Ritchie MD, Hahn LW, Roodi N, Bailey LR, Dupont WD,
Parl FF, Moore JH. Multifactor-dimensionality reduction
reveals high-order interactions among estrogen-metabolism
genes in sporadic breast cancer. Am J Hum Genet. 2001;
69:138–147. 54. Ritchie MD, Hahn LW, Moore JH. Power of multifactor
dimensionality
reduction
for
detecting
gene-gene www.impactjournals.com/oncotarget Oncotarget 84238
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Literatur · Besprechungen
gebots auswirken könnte. Mit 64 Hypothesen,
verdichtet auf sechs Erfolgskonstrukte mit
branchentypischen messbaren Erfolgsindikatoren, ist das Raster für die Befragung festgelegt.
Ausgewählt werden die Verlage und die damit verbundenen Experten durch die Verbandslisten von Bundesverband Deutscher Zeitungsverleger und Verband Deutscher Zeitschriftenverleger. Mit einer Rücklaufquote von
knapp 20 Prozent auf Basis der angeschriebenen einzelnen Entscheider kann Illenberger
aufgrund der Mehrfachadressierung unterschiedlicher Entscheider pro Verlag in einzelnen Segmenten sehr hohe Repräsentativität erzielen (41 Prozent im Markt der Zeitungen).
Damit besitzt die Studie eine solide Grundlage
für die anschließende Bewertung der Ergebnisse. Dennoch sind bei manchen Aussagen die
Stichprobengrößen grenzwertig. Dies zeigt
wieder einmal das generelle Problem bei komplexen Modellen zur empirischen Überprüfung
von Kausalitäten, das die Ergebnisse und Erkenntnisse von Illenberger jedoch in keiner
Weise schmälert. Was sind nun also die zentralen Erfolgsfaktoren der einzelnen Printgattungen im Online-Markt?
Zeitungen sind der Studie zufolge vor allem
dann erfolgreich, wenn sie die „User Experience“ verbessern. Um hier erfolgreich zu sein,
wird neben anderen Maßnahmen in der Arbeit
als zentrales Element die Verringerung von
kostenpflichtigen Inhalten empfohlen. Als weitere Maßnahmen wären der Einsatz von Newslettern, die Verbesserung der Website oder die
Ausweitung der Personalisierung zu nennen.
Die Erhöhung der Umsetzungskompetenz erweist sich als weiterer Erfolgsfaktor, z. B. durch
eine stärkere Berücksichtigung der Wünsche
und Bedürfnisse der Nutzer.
Im Zeitschriftensektor sind für den Erfolg
bei Reichweiten und Marktstellung die zwei
Erfolgsfaktoren Optimierung der Markenbekanntheit (durch SEM/SEO) und die Steigerung der Umsetzungskompetenz relevant. Der
Marktstellungserfolg erweist sich dabei auch als
zentraler Erfolgsfaktor für den ökonomischen
Erfolg, was der Geschäftsmodelllogik dieses
Mediums entspricht.
Fachzeitschriften haben aufgrund der Heterogenität ein weniger einheitliches Bild bei den
zentralen Erfolgsfaktoren. Die Verbesserung
der User Experience lässt sich aber auch hier als
Erfolgsfaktor nachweisen.
Die Arbeit von Illenberger behandelt ein
zentrales Problem der Verlags- und Medienbranche und besitzt daher eine hohe thematische Relevanz. Sie ist gut fundiert aufgebaut,
gut lesbar geschrieben und so strukturiert, dass
man sich schnell über Ziel, Methodik und Ergebnisse der Studie informieren kann. Sie leistet
einen wichtigen Beitrag zur Versachlichung der
eher emotionalen Diskussion über Erfolgsfaktoren, die nicht nur in der Praxis gerne aus Erfahrung und Evidenz abgeleitet wird. Durch die
unklare Abgrenzung der Untersuchungsobjekte im Hinblick auf die Markenführung im Verhältnis der Print- und Online-Angebote kann
das „professionelle Markenmanagement“
(S. 242) nur als Postulat formuliert werden.
Dieser Punkt stellt jedoch auch in seiner Komplexität ein eigenständiges Forschungsthema
dar. Wie immer bei empirischen Arbeiten kann
man über die Hintergründe der Zusammenhänge an der einen oder anderen Stelle diskutieren.
Wenn Reichweite bei der Erfolgsobjektivierung eine entscheidende Rolle spielt, sind die
Ergebnisse mit einem hohen Stellenwert für die
Verbesserung der User Experience kaum verwunderlich. Die hierbei zugeordnete Maßnahme der Verringerung kostenpflichtiger Inhalte
trägt zur Steigerung, bzw. zum Erhalt der
Reichweite bei, während die Erhöhung des
Paid-Content-Anteils die Reichweite limitiert
bzw. senkt. Für eine weitergehende Diskussion
wäre zu Beginn der Untersuchung ggf. noch eine umfangreichere Analyse der Paid-ContentVarianten (metered access, freemium, pay-wall
etc.) notwendig. Dies ist jedoch auch vor dem
Hintergrund der Entstehungszeit im Jahr 2009
zu Recht nicht Gegenstand der Untersuchung
geworden.
Thomas Breyer-Mayländer
Dimitri Liebsch / Nicola Mößner (Hrsg.)
Visualisierung und Erkenntnis
Bildverstehen und Bildverwenden in Naturund Geisteswissenschaften
Köln: Halem, 2012. – 382 S.
ISBN 978-3-86962-053-4
Was unterscheidet eine Nudelwerbung vom
Röntgenbild? Wie stiften Bilder eine Form von
Wissen? In dem von Dimitri Liebsch und Nicola Mößner herausgegebenen Band werden
grundsätzliche und nebensächliche Fragen zum
Bildverständnis in 14 Beiträgen diskutiert. Dabei geht dieser interdisziplinäre Sammelband
vor allem von philosophischen und naturwissenschaftlichen Schnittstellen aus und spannt
einen großen Schirm auf, von „Bildern in der
Wissenschaft“ (Klaus Sachs-Hombach) über
„Nützliche Bilder“ (Rolf F. Nohr) zu der Frage
„Gedankenlesen mittels Neuroimaging?“ (Pe-
https://doi.org/10.5771/1615-634x-2013-4-603, am 29.06.2024, 13:53:29
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- https://www.nomos-elibrary.de/agb
603
M&K 61. Jahrgang 4/2013
ter Hucklenbroich) bis hin zum Beitrag von
Jörg R. J. Schirra „Sind Bilder ein Gegenstand
der Informatik?“.
Die Themen und Ansätze sind weit gestreut
und enthalten überwiegend spannende und
manchmal auch überraschende Kombinationen
und Thesen. Einigen Beiträgen ist jedoch gemein, dass sie der attestierten „Bilderflut“ habhaft werden wollen, indem sie ästhetische Bewertungen zur Kategorisierung anführen, wie
etwa in dem Beitrag von Andreas Müller, Jochen Kuhn, Alwine Lenzner und Wolfgang
Schnotz, „Schöne Bilder in den Naturwissenschaften: motivierend, anregend oder doch nur
schmückendes Beiwerk?“. Hier wird zwischen
„dekorativen“ und „instruktionalen“ Bildern
unterschieden. In ihrer aufwendigen Untersuchung des didaktischen Wertes solcher Bilder
für die gymnasiale Oberstufe messen sie die
„Wirkung“ der Vergleichsbilder auf eine Expertengruppe und auf eine Gruppe von Schülern und kommen zu dem Zwischenfazit, dass
die Kontextualisierung der Bilder eine entscheidende Rolle bei ihrer Bewertung spielt und drei
Arten von Erwartungen entscheidend sind: Die
„Situations-Ergebnis-Erwartung“, die „Handlungs-Ergebnis-Erwartung“ sowie die „Ergebnis-Folgen-Erwartung“, alle drei stammen aus
der kognitiven Motivationspsychologie. Im
Zwischenergebnis fragen die Autoren, warum
die affektive Wirkung dekorativer Bilder nicht
auf Motivation und Lernen durchschlägt
(S. 224) und kommen zu dem Schluss, dass der
Kontext bzw. die Einbettung der Bilder ausschlaggebend für ihre Wirkung sei. Die Bildwirkung besteht aus der Interaktion zwischen
Kognition und Emotion.
Auch Rolf F. Nohr unterscheidet im Rekurs
auf den Baseler Bildwissenschaftler Gottfried
Böhm zwischen „starken“ und „schwachen“
Bildern, wobei Kunstwerke zu den starken und
Wissenschaftsbilder zu den schwachen zählen.
Dieser, für die Medien- und Kommunikationswissenschaft wohl interessanteste Beitrag des
Sammelbandes, läuft jedoch – wie bereits der
zuvor erwähnte Artikel – Gefahr, eine bipolare
Unterscheidung subjektiv zu setzen, ohne dies
empirisch überprüft zu haben. Ob stark oder
schwach, dekorativ oder instruktional, sollte
nicht vorab unterstellt, sondern durch einen
Pre-Test überprüft werden. Wenn die Valenz
von Bildtypen bereits im Untersuchungsdesign
postuliert wird, ist es schwierig, später eine tatsächliche „Wirkung“ messen zu können. Oder,
wie dies im Beitrag „Klassifikation von wissenschaftlichen Darstellungen“ von Martin Lemke, Tobias Breidenmoser, Manfred Drack und
Fynn Ole Engler zutreffend formuliert wird:
604
„Wir sind völlig frei darin zu klassifizieren. Allerdings ist es nicht klug, diese Freiheit ganz
auszunutzen“ (S. 180).
Für die Medien- und Kommunikationswissenschaft interessant ist Nohrs Exkurs zur Geschichte der Informationsgrafik, von der biblia
pauperum des Spätmittelalters über Comenius
bis hin zu den „Klassikern“ der Bilddidaktik
und Isotype, zum dem einflussreichen „Wiener
Kreis“ angehörenden Otto Neurath sowie Fritz
Kahn. Insgesamt enthält der Band mehrere interessante, interdisziplinäre Beiträge und ist sicherlich für die naturwissenschaftliche Fachdidaktik und die interdisziplinäre Bildwissenschaft relevant. Allerdings fehlt eine sozialwissenschaftliche Perspektive, und weder der Medienbegriff noch der omnipräsente Kommunikationsbegriff sind durch einen Beitrag, etwa
zur visuellen Kommunikation, vertreten. Für
die Medien- und Kommunikationswissenschaft
ist der Sammelband somit kaum relevant, zumal
wichtige Termini wie „Rezeption“ und „Wirkung“ von Bildern nicht reflektiert werden. Der
Band enthält zahlreiche Abbildungen, einige
davon in Farbe, sowie ein Personen- und Sachregister. Die Beantwortung der eingangs gestellten Fragen zu Bildverstehen und Bildverwenden bleiben die meisten Beiträge schuldig,
vielleicht auch, weil diese „Ergebnis-Erwartung“ an einen gut redigierten Sammelband
doch zu hoch gegriffen ist?
Marion G. Müller
Werner A. Meier / Heinz Bonfadelli / Josef
Trappel (Hrsg.)
Gehen in den Leuchttürmen die Lichter
aus?
Was aus den Schweizer Leitmedien wird
Wien/Münster: Lit, 2012. –336 S.
ISBN 978-3-643-80136-4
Die Medien in der Schweiz befinden sich offenbar in einem kritischen Zustand. Darin sind
sich die Autorinnen und Autoren dieses Bandes
einig. Eine Medienkrise, eine Tageszeitungskrise, eine Werbewirtschaftskrise, eine Publikumskrise, eine Finanzkrise, eine Krise des Informationsjournalismus, eine Krise des professionellen Journalismus, eine Krise der Leitmedien – all diese Krisen werden schon in den
Überschriften der 15 Beiträge dieses Sammelbandes annonciert. In den Texten selbst werden
weitere Krisen festgestellt.
Genau genommen sind es vor allem die Tageszeitungen als traditionelle regionale Leitmedien in der Schweiz, deren Funktionsfähigkeit
https://doi.org/10.5771/1615-634x-2013-4-603, am 29.06.2024, 13:53:29
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Composition and conservation of the mRNA-degrading machinery in bacteria
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Journal of biomedical science
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Abstract RNA synthesis and decay counteract each other and therefore inversely regulate gene expression in pro- and
eukaryotic cells by controlling the steady-state level of individual transcripts. Genetic and biochemical data
together with recent in depth annotation of bacterial genomes indicate that many components of the bacterial
RNA decay machinery are evolutionarily conserved and that their functional analogues exist in organisms
belonging to all kingdoms of life. Here we briefly review biological functions of essential enzymes, their
evolutionary conservation and multienzyme complexes that are involved in mRNA decay in Escherichia coli and
discuss their conservation in evolutionarily distant bacteria. 1. mRNA turnover and its role in gene expression
In contrast to metabolically stable DNA serving as a
storehouse of genetic information, the fraction of total
RNA that delivers coding information to the protein-
synthesizing machinery (i.e. mRNA) is intrinsically labile
and continuously synthesized. The steady-state level of
mRNA is tightly controlled enabling bacteria to selec-
tively copy (transcribe) and decode genetic information
pertinent to a particular physiological state (Figure 1). Since the steady-state level of mRNA can vary and is a
function of RNA synthesis and decay, the control of
mRNA stability plays an essential role in the regulation
of gene expression. As transcription and translation are
coupled in bacteria, the degree of their coupling can
control the access of individual transcripts to the RNA
decay machinery, thus influencing the rate of mRNA
turnover. For more information about the crosstalk
between translation and mRNA decay in bacteria and its
regulation by environmental factors, we recommend
some recent reviews (see [1-5]). of the putative organization and composition of bacterial
mRNA decay machineries that belong to phylogeneti-
cally distant species should enable us to gain critical
insights into the evolution of RNA decay pathways and
their conservation in bacteria. The main objective of
this review was therefore to assess the evolutionary con-
servation of RNases and ancillary factors that are
involved in mRNA turnover and briefly discuss their
specific roles in this process. * Correspondence: vladimir_kaberdin@ehu.es; mbsue@gate.sinica.edu.tw
† Contributed equally
1Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan
Full list of author information is available at the end of the article © 2011 Kaberdin et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited. Kaberdin et al. Journal of Biomedical Science 2011, 18:23
http://www.jbiomedsci.com/content/18/1/23 Kaberdin et al. Journal of Biomedical Science 2011, 18:23
http://www.jbiomedsci.com/content/18/1/23 Composition and conservation of the
mRNA-degrading machinery in bacteria Vladimir R Kaberdin1,2,3*†, Dharam Singh1† and Sue Lin-Chao1* 2. Enzymes with major and ancillary functions in
mRNA turnover and their phylogenetic
conservation in bacteria Early studies on RNA processing and decay in E. coli, a
Gram-negative bacterium that belongs to the gamma
division of proteobacteria, revealed several endoribonu-
cleases (cleave RNA internally), exoribonucleases
(sequentially remove mononucleotides from either the 5’
or the 3’-end of RNA) and other RNA-modifying
enzymes with important functions in mRNA turnover
(Table 1). The specific roles of these enzymes as well as
their functional homologues found in another model
organism, the Gram-positive bacterium Bacillus subtilis,
have been reviewed recently [5]. Here, we focus on the
phylogenetic conservation of the major RNases (e.g.,
RNase E, polynucleotide phosphorylase, RNase II) and
ancillary RNA-modifying enzymes (RNA pyrophospho-
hydrolase (RppH), poly(A) polymerase I (PAPI) and
RNA helicase B (RhlB)) involved in the turnover of
mRNAs in bacteria. Previous bioinformatic approaches The ability of bacteria to rely on remarkably diverse
metabolic pathways in order to adopt and strive in dif-
ferent environmental niches suggests that the nature
and number of enzymatic activities involved in specific
metabolic pathways including mRNA turnover can
greatly vary from species to species. Hence, an analysis * Correspondence: vladimir_kaberdin@ehu.es; mbsue@gate.sinica.edu.tw
† Contributed equally
1Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan
Full list of author information is available at the end of the article Page 2 of 12 Kaberdin et al. Journal of Biomedical Science 2011, 18:23
http://www.jbiomedsci.com/content/18/1/23 Kaberdin et al. Journal of Biomedical Science 2011, 18:23
http://www.jbiomedsci.com/content/18/1/23 Figure 1 RNA synthesis and turnover as part of the gene
expression network in bacteria. Different types of RNA (mRNAs,
ribosomal and transfer RNA pre-cursors and various non-coding
RNAs) either can directly be involved in translation (e.g. mRNAs) or
undergo further processing (pre-cursors of stable RNA) or
degradation (untranslated or poorly translated mRNAs) by the RNA
decay machinery. The final products of RNA turnover,
mononucleotides, are used for the next cycles of RNA synthesis
(recycling). absent in some phyla of bacteria (Figure 2). The lack of
genes coding for this endoribonuclease suggests that
either (i) the main functions of RNase E/G are occasion-
ally taken over by other endoribonucleases or that (ii)
RNase E/G is redundant for RNA processing and decay
in some species. The first possibility is supported by a recent analysis of
RNA processing and decays in B. subtilis (class Firmi-
cutes) [12-14]. Despite the discovery of RNase E-like clea-
vages in this bacterium [15], they were subsequently
attributed to the action of two B. 2. Enzymes with major and ancillary functions in
mRNA turnover and their phylogenetic
conservation in bacteria The potential presence of
mRNA degrading and mRNA-modifying ancillary
enzymes was examined in all classes of bacteria by
searching for the corresponding annotated genes and
protein sequences available in the NCBI database http://
www.ncbi.nlm.nih.gov/. The result of this analysis leads
to several important conclusions regarding the nature
and occurrence of RNases, ancillary enzymes (see 2.1
and 2.2) and their multienzyme assemblies (see 2.3) in
evolutionarily distant species. In contrast to Firmicutes, Actinomycetes and other
phylas of bacteria whose members can apparently sur-
vive without RNase E/G by using its functional homolo-
gues, RNase Y and/or RNases J1/J2, some bacterial
species seem to be able to carry out RNA processing
and decay even in the absence of all these endoribonu-
clases (i.e., RNase E/G, RNase Y, and RNases J1/J2). Examples are some pathogenic bacteria that belong to
the clades of Deinococcus, Dictyoglomy, Spirochaetales
and Tenericutes. Many of these pathogens lack genes
encoding not only the above endoribonucleases but also
many exonucleases (see also 2.2). Several studies revealed that the 5’-phosphorylation
status of mRNA can control the efficiency of cleavages
by RNase E/G homologues [17-21] as well as by RNases
J1/J2 [12] and RNase Y [16]. As the E. coli pyropho-
sphohydrolase RppH (initially designated NudH/YgdP)
is able to facilitate RNase E cleavage of primary tran-
scripts by 5’ pyrophosphate removal [22], we examined
the presence of nudH/ygdP genes in genomes of phylo-
genetically distant bacteria. Despite the apparent
absence of these genes in many classes of bacteria
(Figure 2), their homologues that belong to the same
family of Nudix hydrolases are known to be widely
present in all three domains of life (reviewed in [23]). Therefore, it seems likely that the RNA pyrophosphohy-
drolase-mediated stimulation of mRNA decay in some
bacterial species involves other members of the Nudix
family of hydrolases. 2. Enzymes with major and ancillary functions in
mRNA turnover and their phylogenetic
conservation in bacteria subtilis endoribonu-
cleases (RNases J1 and J2) that bear primarily functional
rather than sequence homology to their E. coli counter-
part. Both RNase J1 and J2 were suggested to functionally
represent RNase E/G in B. subtilis by mimicking the abil-
ity of RNase E to make endoribonucleolytic cuts in a 5’-
end-dependent manner [12] as well as its property to
form multienzyme complexes [13,14]. Interestingly, one
recent study reported the existence and characterization
of another B. subtilis endoribonuclease, RNase Y, and
suggested that this enzyme is also functionally related to
RNase E/G, in particular with regard to its role in mRNA
turnover [16]. Consistent with this suggestion, we found
that RNase Y appears to occur more frequently than
RNases J1/J2 in the phyla that lack RNase E/G (Figure 2). Figure 1 RNA synthesis and turnover as part of the gene
expression network in bacteria. Different types of RNA (mRNAs,
ribosomal and transfer RNA pre-cursors and various non-coding
RNAs) either can directly be involved in translation (e.g. mRNAs) or
undergo further processing (pre-cursors of stable RNA) or
degradation (untranslated or poorly translated mRNAs) by the RNA
decay machinery. The final products of RNA turnover,
mononucleotides, are used for the next cycles of RNA synthesis
(recycling). have revealed that several mRNA-degrading enzymes are
not strictly conserved and can be absent in some classes
of bacteria [6,7]. The availability of new genomic data
and discovery of novel RNases in bacteria prompted us
to re-assess the phylogenetic conservation of these
enzymes in bacterial species for which the sequence of
the entire genome is available. The potential presence of
mRNA degrading and mRNA-modifying ancillary
enzymes was examined in all classes of bacteria by
searching for the corresponding annotated genes and
protein sequences available in the NCBI database http://
www.ncbi.nlm.nih.gov/. The result of this analysis leads
to several important conclusions regarding the nature
and occurrence of RNases, ancillary enzymes (see 2.1
and 2.2) and their multienzyme assemblies (see 2.3) in
evolutionarily distant species. have revealed that several mRNA-degrading enzymes are
not strictly conserved and can be absent in some classes
of bacteria [6,7]. The availability of new genomic data
and discovery of novel RNases in bacteria prompted us
to re-assess the phylogenetic conservation of these
enzymes in bacterial species for which the sequence of
the entire genome is available. 2.1 Conservation and diversity of major enzymes
controlling the endoribonucleolytic decay of mRNA Despite their indispensable functions in the processing
of ribosomal and transfer RNA in E. coli, three major
endoribonucleases, RNase E, RNase III and RNase P
unequally contribute to mRNA decay. With few excep-
tions [8,9], the endoribonucleolytic decay of E. coli tran-
scripts primarily involves RNase E and sometimes
RNase III (reviewed in [10]). Moreover, previous studies
of mRNA decay pathways in E. coli demonstrated the
key role of RNase E, a member of the RNase E/G family
of ribonucleases, in carrying out the first endoribonu-
cleolytic cleavages initiating the ribonucleolytic decay of
E. coli transcripts (reviewed in [11]). Although homolo-
gues of RNase E/G are predicted to be present in many
bacterial species, they are either partially or completely Page 3 of 12 Page 3 of 12 Kaberdin et al. Journal of Biomedical Science 2011, 18:23
http://www.jbiomedsci.com/content/18/1/23 Table 1 Major ribonucleases acting on single-stranded (ss) or double-stranded (ds) regions of RNA and ancillary
RNA-modifying enzymes (pyrophosphohydrolase, RppH; poly(A) polymerase I, PAPI; and DEAD-box RNA helicases)
involved in RNA turnover in bacteria
Endoribonucleases
Name
Essential for
cell survival
Description of the reaction
catalyzed
Specific functions in vivo
RNase E/G
Yes
Cleavage of A/U-rich ss regions of RNA yielding
5’-monophosphorylated products; 5’-end-dependent hydrolase
Ribosomal and transfer RNA processing,
initiation of decay of non-coding and mRNAs,
turnover of messenger, non-coding and stable
RNA decay intermediates
RNase III
Yes
Endonucleolytic cleavage of ds regions of RNA yielding
5’-monophosphorylated products
Ribosomal and transfer RNA processing and
mRNA processing and decay
RNases J1/J2*
RNaseJ1/Yes
Endonucleolytic cleavage of ss regions of RNA yielding
5’-monophosphorylated products; 5’-end-dependent hydrolase
RNA processing and decay in
B. subtilis
RNase Y
Yes
Endonucleolytic cleavage of ss regions of RNA yielding
5’-monophosphorylated products; 5’-end-dependent hydrolase
Degradation of B. subtilis transcripts containing
SAM-dependent riboswitches
Exoribonucleases
Name
Essential for
cell survival
Description of the reaction
catalyzed
Specific functions in vivo
RNase PH
No
tRNA nucleotidyltransferase
Exonucleolytic trimming of the 3’-termini of
tRNA precursors
PNPase
No
(i) Phosphorolytic 3’ to 5’ exoribonuclease and
(ii) 3’-terminal oligonucleotide polymerase activities
3’ to 5’ decay of ssRNA
RNase II
Yes
Exonucleolytic cleavage in the 3’ to 5’ direction to yield
ribonucleoside 5’-monophosphates
Removal of 3’-terminal nucleotides in
monomeric tRNA precursors, 3’ to 5’
exonucleolytic decay of unstructured RNAs
RNase R
No
Exonucleolytic cleavage in the 3’ to 5’ direction to yield
ribonucleoside 5’-monophosphates
3’ to 5’ exonucleolytic decay of structured RNAs
(e.g. 2.1 Conservation and diversity of major enzymes
controlling the endoribonucleolytic decay of mRNA mRNA and rRNA)
RNase J1/J2*
Yes
Exonucleolytic cleavage in the 5’ to 3’ direction to yield
nucleoside 5’-monophosphates
5’ to 3’ exonucleolytic decay of B. subtilis RNAs
Oligoribo-
nuclease
yes
Exonucleolytic cleavage of short oligonucleotides to yield
nucleoside 5’-phosphates
Completion of the last steps of RNA decay
Ancillary RNA-modifying enzymes
Name
Essential for
cell survival
Description of the reaction
catalyzed
Specific functions in vivo
RppH
No
Removal of pyrophosphate groups from the 5’-end of
triphosphorylated RNAs
Facilitation of endoribonucleolytic cleavages of
primary transcripts by RNase E/G
PAPI
No
Addition of adenosines to the 3’-end of RNA
Facilitation of 3’ to 5’ exonuclolytic decay of
structured RNAs by adding 3’ poly(A) tails
DEAD-box
helicases
No
ATP-dependent unwinding of
ds regions of RNAs
Facilitation of the PNPase- dependent decay of
structured RNAs
The presented classification of the enzymes and their functions in vivo were adopted from several enzyme databases (KEGG, http://www.genome.jp; EXPASY,
http://us.expasy.org/enzyme/; and IntEnz, http://www.ebi.ac.uk/intenz/.*RNases J1/J2 possess both exo- and endoribonucleolytic activities. The presented classification of the enzymes and their functions in vivo were adopted from several enzyme databases (KEGG, http://w
http://us.expasy.org/enzyme/; and IntEnz, http://www.ebi.ac.uk/intenz/.*RNases J1/J2 possess both exo- and endoribonucleolytic activ 2.2 Conservation and diversity of major enzymes
controlling exoribonucleolytic decay of mRNA revealed that, in contrast to apparently similar roles of
RNase II and PNPase in the degradation of E. coli
mRNA [24], only PNPase plays a central role in the 3’-
exonucleolytic decay of B. subtilis mRNA [25] with
apparently less significant contribution of other exoribo-
nucleases [25] including RNase PH [26], RNase R [27]
and YhaM [28]. This is consistent with the previous
finding that the 3’-to-5’ exonuleolytic mRNA decay in B. subtilis, contrary to RNA turnover in E. coli, primarily
proceeds through an “energy-saving” phosphorolytic
pathway [29] mediated by PNPase. Further studies will
be necessary to address systematically how phylogeneti-
cally distant bacteria combine different sets of exoribo-
nucleases to carry out mRNA decay. Finally, given the revealed that, in contrast to apparently similar roles of
RNase II and PNPase in the degradation of E. coli
mRNA [24], only PNPase plays a central role in the 3’-
exonucleolytic decay of B. subtilis mRNA [25] with
apparently less significant contribution of other exoribo-
nucleases [25] including RNase PH [26], RNase R [27]
and YhaM [28]. This is consistent with the previous
finding that the 3’-to-5’ exonuleolytic mRNA decay in B. subtilis, contrary to RNA turnover in E. coli, primarily
proceeds through an “energy-saving” phosphorolytic
pathway [29] mediated by PNPase. Further studies will
be necessary to address systematically how phylogeneti-
cally distant bacteria combine different sets of exoribo-
nucleases to carry out mRNA decay. Finally, given the Kaberdin et al. Journal of Biomedical Science 2011, 18:23
http://www.jbiomedsci.com/content/18/1/23 high degree of phylogenetic conservation of PNPase and
RNase II, it seems reasonable that one of the key ancil-
lary enzymes, PAPI, which assists PNPase and RNase II
in the degradation of structured RNAs, is likewise pre-
sent in most of the bacteria, as shown in Figure 2. Kaberdin et al. Journal of Biomedical Science 2011, 18:23
http://www.jbiomedsci.com/content/18/1/23 Kaberdin et al. Journal of Biomedical Science 2011, 18:23
http://www.jbiomedsci.com/content/18/1/23 [36,37]. Consistent with these reports, the existence of
functional interactions between the major components
of the degradosome was confirmed in vivo [38-43] and
in vitro [33,44]. Apart from binding to RNase E, two
major components of the E. coli degradosome, PNPase
and RhlB helicase, were shown to form a complex
resembling the eukaryotic exosome, a multienzyme
assembly with RNA-hydrolyzing and RNA-unwinding
activities (reviewed in [35]). The formation and func-
tions of this complex in E. coli may not be unusual as
both enzymes appear to exist in excess to RNase E in
vivo and therefore can be involved in alternative pro-
tein-protein interactions. However, the actual contribu-
tion of this complex to RNA metabolism in bacteria
remains to be determined. mRNA molecules that are
degraded by these multiprotein assemblies (i.e., degrado-
some and exosome) are simultaneously exposed to sev-
eral ribonucleolytic and other RNA-modifying activities
and therefore undergo fast and coordinated decay with-
out accumulation of detectable amounts of intermediate
products. [36,37]. Consistent with these reports, the existence of
functional interactions between the major components
of the degradosome was confirmed in vivo [38-43] and
in vitro [33,44]. Apart from binding to RNase E, two
major components of the E. coli degradosome, PNPase
and RhlB helicase, were shown to form a complex
resembling the eukaryotic exosome, a multienzyme
assembly with RNA-hydrolyzing and RNA-unwinding
activities (reviewed in [35]). The formation and func-
tions of this complex in E. coli may not be unusual as
both enzymes appear to exist in excess to RNase E in
vivo and therefore can be involved in alternative pro-
tein-protein interactions. However, the actual contribu-
tion of this complex to RNA metabolism in bacteria
remains to be determined. mRNA molecules that are
degraded by these multiprotein assemblies (i.e., degrado-
some and exosome) are simultaneously exposed to sev-
eral ribonucleolytic and other RNA-modifying activities
and therefore undergo fast and coordinated decay with-
out accumulation of detectable amounts of intermediate
products. high degree of phylogenetic conservation of PNPase and
RNase II, it seems reasonable that one of the key ancil-
lary enzymes, PAPI, which assists PNPase and RNase II
in the degradation of structured RNAs, is likewise pre-
sent in most of the bacteria, as shown in Figure 2. controlling exoribonucleolytic decay of mRNA A search for putative homologues of the three major
mRNA-degrading exoribonucleases of E. coli (polynu-
cleotide phosphorylase (PNPase), RNase II and RNase
R) in other bacteria revealed that the corresponding
genes can be found in nearly every class of bacteria
(Figure 2). Although these observations suggest that
mRNA decay in the majority of bacteria could be
dependent on all three exoribonucleases, the actual con-
tribution of each exoribonuclease to mRNA decay in
these species may differ, as anticipated from previous
studies of exonucleolytic decay of mRNA in B. subtilis
(Firmicutes) and E. coli (Proteobacteria). These studies Kaberdin et al. Journal of Biomedical Science 2011, 18:23
http://www.jbiomedsci.com/content/18/1/23 Kaberdin et al. Journal of Biomedical Science 2011, 18:23
http://www.jbiomedsci.com/content/18/1/23 Page 4 of 12 Figure 2 The phylogenetic distribution of main ribonucleases (RNase E/G, RNase III, RNases J1/J2, RNase Y, RNase PH, PNPase, RNase
R, RNase II, Oligoribonuclease) and ancillary RNA modifying enzymes (RppH, PAPI, DEAD-box helicases) involved in the disintegration
and turnover of bacterial transcripts are indicated by colored filled circles (from ‘a’ to ‘l’, respectively). The percentage of organisms in
each phylum/class of bacteria for which the presence of each particular enzyme has been predicted by searching the NCBI database is indicated
by differentially colored circles. The data are compiled based on analysis of completely sequenced genomes (1217 complete genome sequences
available by 4 November 2010). Draft assemblies of genomes and hypothetical proteins were excluded from the analysis. Figure 2 The phylogenetic distribution of main ribonucleases (RNase E/G, RNase III, RNases J1/J2, RNase Y, RNase PH, PNPase, RNase
R, RNase II, Oligoribonuclease) and ancillary RNA modifying enzymes (RppH, PAPI, DEAD-box helicases) involved in the disintegration
and turnover of bacterial transcripts are indicated by colored filled circles (from ‘a’ to ‘l’, respectively). The percentage of organisms in
each phylum/class of bacteria for which the presence of each particular enzyme has been predicted by searching the NCBI database is indicated
by differentially colored circles. The data are compiled based on analysis of completely sequenced genomes (1217 complete genome sequences
available by 4 November 2010). Draft assemblies of genomes and hypothetical proteins were excluded from the analysis. Page 5 of 12 2.3. Conservation of mRNA-degrading multienzyme
complexes coli degradosome, is poorly conserved among
RNase E/G homologues [36]. Despite the overall lack of
conservation, the PNPase-binding site of E. coli RNase E
(residues 1021-1061, see [37]) is known to possess high
similarity to a short amino acid sequence found in H. influenza Rd RNase E (residues 896-927, [36]). More-
over, this sequence is highly conserved among RNase E/
G homologues of certain g-proteobacteria (e.g., Erwinia,
Shigella, and Citrobacter) and therefore is presently
annotated in the NCBI database as the PNPase-binding
domain. The conservation of this domain (although pri-
marily in enterobacterial species) is also supported by a
recent analysis of Vibrio angustum S14 RNase E [46]. This study defined the last 80 amino acids at the C-ter-
minus of Vibrio angustum S14 RNase E as the potential
site for PNPase binding and revealed the putative eno-
lase-binding domain, a region also highly conserved
amongst enterobacteria [47,48]. Collectively, the above
findings and genomic data suggest that degradosome-
like complexes are widespread in enterobacteria and
organized in a similar manner. therefore can potentially modulate the efficiency and/or
specificity of the degradosome-mediated RNA decay. More significant differences in the composition of degra-
dosomes can be found in other a-proteobacteria. It has
been shown that RNase E of Rhodobacter capsulatis
forms a degradosome-like complex with two DEAD-box
RNA helicases of 74 and 65 kDa and the transcription
termination factor Rho [54]. Thus, the degradosome-
dependent mRNA decay appears to involve different
combinations of enzymatic activities even within the
same class of bacteria. In addition to analyzing the composition of degrado-
some complexes in Proteobacteria, some efforts were
dedicated to identify degradosome-like complexes in
Actinobacteria. These studies revealed that, similar to
their E. coli counterpart, RNase E/G homologues can
interact with PNPase in Streptomyces [55] and are able
to co-purify with GroEL and metabolic enzymes in
Mycobacteria [56]. The specific role of these polypep-
tides in RNA metabolism and the degree, to which their
interaction with RNase E/G is conserved in Actinobac-
teria, remains to be established. Aside from degradosome complexes that are believed
to function in Proteobacteria and Actinobacteria, the
existence of RNase E-based degradosomes in other
classes of bacteria remains questionable. The small size
(ca. 2.3. Conservation of mRNA-degrading multienzyme
complexes 450-600 a.a., see Table 2) of RNase E/G homologues
in many other classes of bacteria indicate that they pri-
marily contain the evolutionarily conserved catalytic
core of the enzyme and appear to lack regions serving
as scaffolds for degradosome assembly [36,57]. g
In contrast to the apparently similar organization of
enterobacterial degradosomes, their counterparts in
other subclasses of g-proteobacteria are less conserved. For instance, an analysis of the degradosome composi-
tion in the psychrotolerant g-proteobacterium Pseu-
doalteromonas haloplanktis revealed that RNase E
associates with PNPase and RhlB but not with enolase
[49]. Moreover, a different degradosome-like complex
consisting of RNase E, the hydrolytic exoribonuclease
RNase R, and the DEAD-box helicase RhlE was puri-
fied from another psychrotrophic g-proteobacterium,
Pseudomonas syringae Lz4W [50]. As RNA structures
are more stable at low temperatures and RNase R can
degrade structural RNAs more efficiently than PNPase
[51], the presence of RNase R (rather than PNPase) in
this complex may be more advantageous for the degra-
dosome-mediated decay in this psychrotrophic bacter-
ium. RNase E-based degradosomes have also been
isolated from other subclasses of proteobacteria. Hard-
wick and co-workers have recently isolated and charac-
terized an RNase E-containing complex from the
Gram-negative a-proteobacterium Caulobacter crescen-
tus [52]. Apart from RNase E, this complex was found
to contain PNPase, a DEAD-box RNA helicase and
aconitase, an iron-dependent enzyme involved in the
tricarboxylic acid cycle. One can envisage that, similar
to its mycobacterial counterpart [53], C. crescentus
aconitase may possess RNA-binding properties, and Interestingly, recent studies demonstrated that the
Gram-positive bacterium B. subtilis (Firmicutes) possesses
degradosome-like complexes, in which RNase E is repre-
sented by its functional homologues, RNases J1/J2 and
RNase Y, interacting with PNPase, phosphofructokinase
and enolase [13]. Further characterization of these com-
plexes and elucidation of their specific roles in mRNA
decay in B. subtilis and related species can offer many
important insights into the mechanisms underlying
mRNA decay in Firmicutes, the largest group of Gram-
positive bacteria that have been studied so far [58]. 2.3. Conservation of mRNA-degrading multienzyme
complexes Many E. coli mRNAs have relatively short half-lives (2-4
min) and are normally degraded in vivo without accu-
mulation of intermediate products (reviewed in [30]), a
phenomenon frequently referred to as the ‘all-or-noth-
ing’ mechanism of mRNA turnover. The high processiv-
ity of mRNA decay is often discussed with reference to
the coordinated action of ribonucleolytic enzymes and
ancillary proteins that can associate with each other to
form multienzyme ribonucleolytic complexes such as
the E. coli degradosome (Figure 3A, [31-33]) and the
bacterial exosome-like complex (Figure 3B) [34,35]. Analyses of the E. coli degradosome revealed that RNase
E serves as a “scaffolding” protein, through the C-term-
inal part of which other interacting protein partners
such as PNPase (exoribonuclease), RhlB (DEAD-box
helicase) and enolase (glycolytic enzyme) are bound Although significant progress has been achieved in the
characterization of the E. coli degradosome (reviewed in
[45]), our current knowledge of the composition and Figure 3 Bacterial mRNA decay machineries. (A) The RNA degradosome is a multicomponent ribonucleolytic complex that includes an
endoribonuclease (RNase E), a 3’®5’ exoribonuclease (polynucleotide phosphorylase (PNPase)), a DEAD-box RNA helicase (RhlB helicase), and the
glycolytic enzyme enolase [31-33]). (B) In E. coli, PNPase is associated with the RhlB independently of the RNA degradosome to form an
evolutionarily conserved RNA-degradation machine termed as the “bacterial exosome” [34,35]. This complex was shown to catalyze the 3’® 5’
exonucleolytic degradation of RNA using RhlB as cofactor to unwind structured RNA in an ATP-dependent manner. Figure 3 Bacterial mRNA decay machineries. (A) The RNA degradosome is a multicomponent ribonucleolytic complex that includes an
endoribonuclease (RNase E), a 3’®5’ exoribonuclease (polynucleotide phosphorylase (PNPase)), a DEAD-box RNA helicase (RhlB helicase), and the
glycolytic enzyme enolase [31-33]). (B) In E. coli, PNPase is associated with the RhlB independently of the RNA degradosome to form an
evolutionarily conserved RNA-degradation machine termed as the “bacterial exosome” [34,35]. This complex was shown to catalyze the 3’® 5’
exonucleolytic degradation of RNA using RhlB as cofactor to unwind structured RNA in an ATP-dependent manner. Kaberdin et al. Journal of Biomedical Science 2011, 18:23
http://www.jbiomedsci.com/content/18/1/23 Page 6 of 12 properties of similar complexes in other bacteria is still
very limited. A previous comparison of RNase E/G
sequences revealed that the C-terminal half of E. coli
RNase E (residues 499-1061), which is involved in pro-
tein-protein interactions with other major components
of the E. 3.2 Ancillary enzymes facilitate mRNA turnover by
assisting ribonucleases to this model (Figure 4A), conversion of E. coli
mRNAs into their primary decay intermediates is fre-
quently initiated by endoribonucleolytic cuts catalyzed
by endoribonucleases specific for single- (e.g., RNase
E/G) or double-stranded (RNase III) RNA. This step
can be preceded (but not always, see [59]) by pyropho-
sphate removal (see below). During the initial endori-
bonucleolytic step, bacterial RNase E/G (or its
functional homologues, RNases J1/J2 or RNase Y)
attacks the full-length monophosphorylated (or some-
times triphosphorylated [59]) mRNAs to generate pri-
mary decay intermediates that are further degraded
cooperatively by the combined action of endo- and
exoribonucleases (Figure 4A). In E coli, the later steps
of mRNA decay were shown to involve PNPase and
RNase II, or occasionally RNase R [51,60], which
further degrade mRNA decay intermediates to yield
short oligonucleotides that are, in turn, converted to
mononucleotides by oligoribonuclease [61]. to this model (Figure 4A), conversion of E. coli
mRNAs into their primary decay intermediates is fre-
quently initiated by endoribonucleolytic cuts catalyzed
by endoribonucleases specific for single- (e.g., RNase
E/G) or double-stranded (RNase III) RNA. This step
can be preceded (but not always, see [59]) by pyropho-
sphate removal (see below). During the initial endori-
bonucleolytic step, bacterial RNase E/G (or its
functional homologues, RNases J1/J2 or RNase Y)
attacks the full-length monophosphorylated (or some-
times triphosphorylated [59]) mRNAs to generate pri-
mary decay intermediates that are further degraded
cooperatively by the combined action of endo- and
exoribonucleases (Figure 4A). In E coli, the later steps
of mRNA decay were shown to involve PNPase and
RNase II, or occasionally RNase R [51,60], which
further degrade mRNA decay intermediates to yield
short oligonucleotides that are, in turn, converted to
mononucleotides by oligoribonuclease [61]. In addition to the major degrading enzymes, a number
of ancillary mRNA-modifying enzymes can facilitate
mRNA turnover (Table 1). In fact, pyrophosphate
removal at the 5’-end and addition of a single-stranded,
poly(A) extension at the 3’-end are two critical steps in
the mRNA decay pathway promoting mRNA cleavage in
E. coli and presumably in other proteobacteria. In gen-
eral, however, the participation of these enzymes in
mRNA decay in some bacterial species or organelles is
not required (see section 2). 3.1 Both endo- and exoribonucleases act cooperatively
to control mRNA decay Despite phylogenetic conservation (Figure 2) and their
apparent diversity (for a review, see [10]), mRNA
decay pathways in E. coli are believed to include a
number of common enzymatic steps catalyzed by ribo-
nucleases and several ancillary mRNA-modifying
enzymes. To discuss the role of each enzyme, we will
refer to a unified model of mRNA turnover. According Kaberdin et al. Journal of Biomedical Science 2011, 18:23
http://www.jbiomedsci.com/content/18/1/23 Page 7 of 12 Page 7 of 12 Table 2 Bacterial RNase E/G homologues represented in the NCBI protein database
Phylum/Class
Length
(aa)
Potential to form degradosome- like complex
Organisms tested for the presence of
degradosome-like complexes/Reference
Predicted based on the
size of the protein
Experimentally
verified
Actinobacteria
463-1373
+
+
S. coelicolor /[55]
M. tuberculosis; M. bovis /[56]
Aquificae
466-470
-
-
Bacteroidetes/Chlorobi
503-570
-
-
Chlamydiae/Verrucomicrobia
group
510-554
-
-
Cyanobacteria
602-808
-
-
Deferribacteres
507
-
-
Elusimicrobia
488
-
-
Fibrobacteres/Acidobacteria
group
511
-
-
Firmicutes
Bacilli
441-615
-
-
Clostridia
393-571
-
-
Fusobacteria
432-458
-
-
Gemmatimonadetes
520
-
-
Nitrospirae
514-522
-
-
Planctomycetes
509-588
-
-
Proteobacteria
Alpha
411-1123
+
+
R. capsulatus/[54]
C. crescentus [52]
Beta
394-1125
+
-
Gamma
410-1302
+
+
E. coli/[32,33]
P. syringe/[50]
V. angustum S14 RNase E [46]
P. haloplanktis [49]
Delta
486-926
+
-
Synergistetes
495-547
-
-
Thermotogae
454-481
-
- Table 2 Bacterial RNase E/G homologues represented in the NCBI protein database 3.2 Ancillary enzymes facilitate mRNA turnover by
assisting ribonucleases 3.2 Ancillary enzymes facilitate mRNA turnover by
assisting ribonucleases 3.2 Ancillary enzymes facilitate mRNA turnover by
assisting ribonucleases One of these enzymes,
RppH, was shown to accelerate mRNA decay by con-
verting the 5’-triphosphate group of primary transcripts
to 5’ monophosphate, thereby rendering mRNA species
that are more efficiently recognized and cleaved by
RNase E [17,18] and RNase G [19]. Unlike RppH, whose action promotes endoribonucleo-
lytic cleavages, some mRNA-modifying enzymes can Kaberdin et al. Journal of Biomedical Science 2011, 18:23
http://www.jbiomedsci.com/content/18/1/23 Kaberdin et al. Journal of Biomedical Science 2011, 18:23
http://www.jbiomedsci.com/content/18/1/23 Page 8 of 12 Figure 4 Current unified model of mRNA decay pathways in Escherichia coli. (A) Schematic representation of major enzymatic steps
involved in the disintegration and complete turnover of primary transcripts in E. coli. The decay of a regular transcript is usually initiated by
endonucleolytic cleavage to generate primary decay intermediates that are further converted to short oligoribonucleotides by the combined
action of exo- and endoribonucleases. The oligoribonucleotides are further degraded into mononucleotides by oligoribonuclease. (B) Ancillary
enzymes facilitating mRNA turnover and their modes of action. Degradation of mRNA can be stimulated via pyrophosphate removal by RppH,
which converts 5’-triphosporylated primary transcripts into their monophosphorylated variants, thus facilitating their endoribonucleolytic cleavage
by RNase E [22,76] or by RNases J1/J2 [12] or by RNase Y [16] in B. subtilis. As the action of exoribonucleases can be inhibited by 3’-terminal
stem-loop structures, two groups of ancillary RNA-modifying enzymes, PAPI and RhlB, help exonucleases to overcome this inhibitory effect. PAPI
exerts its action by adding short stretches of adenosine residues, thereby facilitating exonuclease binding and subsequent cleavage of structured
RNAs [10] Enzymes of the second group DEAD-box helicases such as E coli RhlB increase the efficiency of the exonuclease-dependent decay Figure 4 Current unified model of mRNA decay pathways in Escherichia coli. (A) Schematic representation of major enzymatic steps
involved in the disintegration and complete turnover of primary transcripts in E. coli. The decay of a regular transcript is usually initiated by
endonucleolytic cleavage to generate primary decay intermediates that are further converted to short oligoribonucleotides by the combined
action of exo- and endoribonucleases. The oligoribonucleotides are further degraded into mononucleotides by oligoribonuclease. (B) Ancillary
enzymes facilitating mRNA turnover and their modes of action. Degradation of mRNA can be stimulated via pyrophosphate removal by RppH,
which converts 5’-triphosporylated primary transcripts into their monophosphorylated variants, thus facilitating their endoribonucleolytic cleavage
by RNase E [22,76] or by RNases J1/J2 [12] or by RNase Y [16] in B. subtilis. Kaberdin et al. Journal of Biomedical Science 2011, 18:23
http://www.jbiomedsci.com/content/18/1/23 coli and appar-
ently other bacteria employ a mechanism that increases
the susceptibility of an mRNA decay intermediate to exo-
nucleases by adding a poly(A) tail to its 3’ end (Figure
4B). Consequently, repetitive cycles of poly(A) addition
carried out by PAPI combined with exonuclease-catalyzed
trimming was shown to result in the complete digestion
of structured RNAs by either PNPase or RNase II in vitro
[65]. Consistent with these findings, mRNA decay in a
mutant lacking functional PAPI results in the accumula-
tion of intermediate products of mRNA decay [64,66-69],
thus indicating that the addition of poly(A) tails is indeed
required for the normal mRNA turnover in E. coli. Because several aspects of poly(A)-assisted mRNA turn-
over including its role in the decay of stable RNA fall
beyond the scope of this review, the interested reader is
referred to other work covering this topic [70]. functional (but not sequence) conservation. Although
they were found only in particular classes of bacteria, at
least one of them is present in nearly every species. Thus, although RNA processing/decay in phylogeneti-
cally distant bacterial species is not necessarily carried
out by the same set of ribonucleolytic enzymes (see pre-
vious sections), the minimal set of enzymatic activities
(at least one functional homologue of RNase E/G and
one 3’ to 5’ exoribonuclease) required for mRNA turn-
over in prokaryotic organisms is likely conserved in a
vast majority of bacterial species. Surprisingly, the number of enzymes with potential
roles in RNA processing and decay is dramatically
reduced in several intracellular pathogens possessing
relatively small (less than 1 Mbp) genomes (e.g., Myco-
plasma (Tenericutes), Rickettsia (a-Proteobacteria) and
Chlamydia (Chlamydiae/Verrucomicrobia group)). In
contrast to the presence of seven distinct exoribonu-
cleases in E. coli, only one of them can be found in
Mycoplasma (subclass Mollicutes (Tenericutes)). Analy-
sis of RNA metabolism in Mycoplasma genitalium sug-
gests that exonucleolytic decay in this bacterium can
be accomplished by a single exoribonuclease, RNase R
[71]. Another prominent feature of Mycoplasma is the
lack of genomic sequences potentially encoding a
homologues of E. coli PAPI known to catalyze the
addition of poly(A) to the 3’ end of E. coli transcripts
[72]. The lack of this enzyme is consistent with the
recent finding that demonstrated the absence of polya-
denylated RNA in Mycoplasma [73]. Kaberdin et al. Journal of Biomedical Science 2011, 18:23
http://www.jbiomedsci.com/content/18/1/23 Although the poly
(A)-dependent enhancement of mRNA decay is likely
redundant for some intracellular pathogens, it seems to
be more important in some Proteobacteria and Firmi-
cutes, as it can offer an additional mean to control the
efficiency of mRNA turnover. In other words, unlike
pathogens that continuously reside in host cells, bac-
teria that strive in highly diverse and continuously
changing environments (e.g., Escherichia coli) use a
large number of ribonucleases and ancillary mRNA-
modifying enzymes such as poly(A) polymerases to effi-
ciently regulate mRNA stability in response to environ-
mental signals. Future studies addressing the main
differences between the mechanisms of mRNA decay
of intracellular pathogens and the currently used
model organisms (E. coli and B. subtilis) may lead to
important insights concerning the evolution of the
mRNA decay machinery in bacteria. In E. coli, the exonucleolytic decay of highly structured
RNAs can also be assisted by the RhlB (Figure 4B). This
enzyme unwinds RNA structures in an ATP-dependent
manner and therefore facilitates their degradation by exo-
nucleases in vivo [39] and in vitro [33,34]. Moreover, RhlB
is an integral part of the multienzyme RNA degradosome
and exosome-like complexes and believed to exert its
functions primarily as component of the mRNA decay
machinery. Kaberdin et al. Journal of Biomedical Science 2011, 18:23
http://www.jbiomedsci.com/content/18/1/23 Page 9 of 12 stimulate degradation by 3’ to 5’ exonucleases (reviewed
in [62,63]). Previous work has shown that the 3’ to 5’
degradation of transcripts by PNPase and RNase II in E. coli proceeds only efficiently on unstructured mRNAs
and is impeded by stable stem-loop structures occurring
internally (e.g., in intergenic regions of polycistronic tran-
scripts such as REP stabilizers found in the malEFG and
many other intergenic regions [39]) or at the 3’ end of
bacterial transcripts (i.e., transcription terminators [64]). These structures typically cause exoribonuclease stalling
and subsequent dissociation of exoribonucleases from
decay intermediates (reviewed in [62,63]). To prevent
accumulation of decay intermediates that are resistant to
3’ to 5’ degradation by exoribonucleses, E. coli and appar-
ently other bacteria employ a mechanism that increases
the susceptibility of an mRNA decay intermediate to exo-
nucleases by adding a poly(A) tail to its 3’ end (Figure
4B). Consequently, repetitive cycles of poly(A) addition
carried out by PAPI combined with exonuclease-catalyzed
trimming was shown to result in the complete digestion
of structured RNAs by either PNPase or RNase II in vitro
[65]. Consistent with these findings, mRNA decay in a
mutant lacking functional PAPI results in the accumula-
tion of intermediate products of mRNA decay [64,66-69],
thus indicating that the addition of poly(A) tails is indeed
required for the normal mRNA turnover in E. coli. Because several aspects of poly(A)-assisted mRNA turn-
over including its role in the decay of stable RNA fall
beyond the scope of this review, the interested reader is
referred to other work covering this topic [70]. stimulate degradation by 3’ to 5’ exonucleases (reviewed
in [62,63]). Previous work has shown that the 3’ to 5’
degradation of transcripts by PNPase and RNase II in E. coli proceeds only efficiently on unstructured mRNAs
and is impeded by stable stem-loop structures occurring
internally (e.g., in intergenic regions of polycistronic tran-
scripts such as REP stabilizers found in the malEFG and
many other intergenic regions [39]) or at the 3’ end of
bacterial transcripts (i.e., transcription terminators [64]). These structures typically cause exoribonuclease stalling
and subsequent dissociation of exoribonucleases from
decay intermediates (reviewed in [62,63]). To prevent
accumulation of decay intermediates that are resistant to
3’ to 5’ degradation by exoribonucleses, E. 3.2 Ancillary enzymes facilitate mRNA turnover by
assisting ribonucleases As the action of exoribonucleases can be inhibited by 3’-terminal
stem-loop structures, two groups of ancillary RNA-modifying enzymes, PAPI and RhlB, help exonucleases to overcome this inhibitory effect. PAPI
exerts its action by adding short stretches of adenosine residues, thereby facilitating exonuclease binding and subsequent cleavage of structured
RNAs [10]. Enzymes of the second group, DEAD-box helicases such as E. coli RhlB, increase the efficiency of the exonuclease-dependent decay
by unwinding double-stranded RNA regions in an ATP-dependent fashion. Figure 4 Current unified model of mRNA decay pathways in Escherichia coli Figure 4 Current unified model of mRNA decay pathways in Escherichia coli. (A) Schematic representation o Figure 4 Current unified model of mRNA decay pathways in Escherichia coli. (A) Schematic representation of major enzymatic steps
involved in the disintegration and complete turnover of primary transcripts in E. coli. The decay of a regular transcript is usually initiated by
endonucleolytic cleavage to generate primary decay intermediates that are further converted to short oligoribonucleotides by the combined
action of exo- and endoribonucleases. The oligoribonucleotides are further degraded into mononucleotides by oligoribonuclease. (B) Ancillary
enzymes facilitating mRNA turnover and their modes of action. Degradation of mRNA can be stimulated via pyrophosphate removal by RppH,
which converts 5’-triphosporylated primary transcripts into their monophosphorylated variants, thus facilitating their endoribonucleolytic cleavage
by RNase E [22,76] or by RNases J1/J2 [12] or by RNase Y [16] in B. subtilis. As the action of exoribonucleases can be inhibited by 3’-terminal
stem-loop structures, two groups of ancillary RNA-modifying enzymes, PAPI and RhlB, help exonucleases to overcome this inhibitory effect. PAPI
exerts its action by adding short stretches of adenosine residues, thereby facilitating exonuclease binding and subsequent cleavage of structured
RNAs [10]. Enzymes of the second group, DEAD-box helicases such as E. coli RhlB, increase the efficiency of the exonuclease-dependent decay
by unwinding double-stranded RNA regions in an ATP-dependent fashion. Kaberdin et al. Journal of Biomedical Science 2011, 18:23
http://www.jbiomedsci.com/content/18/1/23 Competing interests 24. Donovan WP, Kushner SR: Polynucleotide phosphorylase and
ribonuclease II are required for cell viability and mRNA turnover in
Escherichia coli K-12. Proc Natl Acad Sci USA 1986, 83(1):120-124. Received: 3 March 2011 Accepted: 22 March 2011
Published: 22 March 2011 cherichia coli K-12. Proc Natl Acad Sci USA 1986, 83(1):120-124. 25. Oussenko IA, Abe T, Ujiie H, Muto A, Bechhofer DH: Participation of 3’-to-5’
exoribonucleases in the turnover of Bacillus subtilis mRNA. J Bacteriol
2005, 187(8):2758-2767. Acknowledgements
W
h
k D H K h
f We thank Dr H Kuhn for editing of the manuscript. VRK was supported by
IKERBASQUE (Basque Foundation for Science) and the Thematic Research
Program of Academia Sinica (AS 97-23-22). DS was supported by Academia
Sinica, Distinguished Postdoctoral Fellowship program. This work was also
supported by grants from the National Science Council, Taiwan (NSC 98-
2321-B-001-009; NSC 99-2321-B-001-004) and by an intramural fund from
Academia Sinica to S L-C. We apologize to those authors whose work could
not be cited due to space constraints. 17. Mackie GA: Ribonuclease E is a 5’-end-dependent endonuclease. Nature
1998, 395(6703):720-723. 18. Mackie GA: Stabilization of circular rpsT mRNA demonstrates the 5’-end
dependence of RNase E action in vivo. J Biol Chem 2000,
275(33):25069-25072. 19. Jourdan SS, McDowall KJ: Sensing of 5’ monophosphate by Escherichia
coli RNase G can significantly enhance association with RNA and
stimulate the decay of functional mRNA transcripts in vivo. Mol Microbiol
2008, 67(1):102-115. Author details
1
f
l 20. Kaberdin VR, Bizebard T: Characterization of Aquifex aeolicus RNase E/G. Biochem Biophys Res Commun 2005, 327(2):382-392. 20. Kaberdin VR, Bizebard T: Characterization of Aquifex aeolicus RNase E/G. Biochem Biophys Res Commun 2005, 327(2):382-392. 1Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan. 2Department of Immunology, Microbiology and Parasitology, University of
the Basque Country, UPV/EHU, Leioa, Spain. 3IKERBASQUE, Basque
Foundation for Science, 48011, Bilbao, Spain. 21. Zeller ME, Csanadi A, Miczak A, Rose T, Bizebard T, Kaberdin V: Quaternary
structure and biochemical properties of mycobacterial RNase E/G. Biochem J 2007, 403:207-215. Authors’ contributions 22. Deana A, Celesnik H, Belasco JG: The bacterial enzyme RppH triggers
messenger RNA degradation by 5’ pyrophosphate removal. Nature 2008,
451(7176):355-358. The manuscript was prepared by VRK, DS and SL-C. All authors read and
approved the final manuscript. The manuscript was prepared by VRK, DS and SL-C. All authors read and
approved the final manuscript. 23. McLennan AG: The Nudix hydrolase superfamily. Cell Mol Life Sci 2006,
63(2):123-143. Competing interests
The authors declare that they have no competing interests. Kaberdin et al. Journal of Biomedical Science 2011, 18:23
http://www.jbiomedsci.com/content/18/1/23 Prog Nucleic Acids Res Mol Biol 1999, 62:55-108. 10. Coburn GA, Mackie GA: Degradation of mRNA in Escherichia coli: An old
problem with some new twists. Prog Nucleic Acids Res Mol Biol 1999, 62:55-108. problem with some new twists. Prog Nucleic Acids Res Mol Biol 1999, 62:55-108. 11. Carpousis AJ, Luisi BF, McDowall KJ: Endonucleolytic initiation of mRNA
decay in Escherichia coli. Prog Mol Biol Transl Sci 2009, 85:91-135. 11. Carpousis AJ, Luisi BF, McDowall KJ: Endonucleolytic initiation of mRNA
decay in Escherichia coli. Prog Mol Biol Transl Sci 2009, 85:91-135. 12. Even S, Pellegrini O, Zig L, Labas V, Vinh J, Brechemmier-Baey D, Putzer H:
Ribonucleases J1 and J2: two novel endoribonucleases in B. subtilis with
functional homology to E. coli RNase E. Nucleic Acids Res 2005,
33(7):2141-2152. 13. Commichau FM, Rothe FM, Herzberg C, Wagner E, Hellwig D, Lehnik-
Habrink M, Hammer E, Volker U, Stulke J: Novel activities of glycolytic
enzymes in Bacillus subtilis: interactions with essential proteins involved
in mRNA processing. Mol Cell Proteomics 2009, 8(6):1350-1360. 14. Mathy N, Hebert A, Mervelet P, Benard L, Dorleans A, de la Sierra-Gallay IL,
Noirot P, Putzer H, Condon C: Bacillus subtilis ribonucleases J1 and J2 form a
complex with altered enzyme behaviour. Mol Microbiol 2010, 75(2):489-498. 15. Condon C, Putzer H, Luo D, Grunberg-Manago M: Processing of the
Bacillus subtilis thrS leader mRNA is RNase E-dependent in Escherichia
coli. J Mol Biol 1997, 268(2):235-242. 16. Shahbabian K, Jamalli A, Zig L, Putzer H: RNase Y, a novel
endoribonuclease, initiates riboswitch turnover in Bacillus subtilis. EMBO J
2009, 28(22):3523-3533. 16. Shahbabian K, Jamalli A, Zig L, Putzer H: RNase Y, a nove Kaberdin et al. Journal of Biomedical Science 2011, 18:23
http://www.jbiomedsci.com/content/18/1/23 coli oligor-
ibonuclease led to the discovery of B. subtilis Ytq1 [74]. This enzyme possesses an oligoribonuclease-like activity
and is able to complement the E. coli orn mutant; homo-
logues of its gene are present in many bacteria [75]. Although Ytq1 can be considered as a functional homo-
logue of oligoribonuclease, further efforts are needed to
disclose the nature and distribution of functional homolo-
gues that may exist in bacterial species lacking both oli-
goribonuclease and Ytq1. 3. Richards J, Sundermeier T, Svetlanov A, Karzai AW: Quality control of
bacterial mRNA decoding and decay. Biochim Biophys Acta 2008,
1779(9):574-582. 4. Dreyfus M: Killer and protective ribosomes. Prog Mol Biol Transl Sci 2009,
85:423-466. 5. Arraiano CM, Andrade JM, Domingues S, Guinote IB, Malecki M, Matos RG,
Moreira RN, Pobre V, Reis FP, Saramago M, et al: The critical role of RNA
processing and degradation in the control of gene expression. FEMS
Microbiol Rev 2010, 34(5):883-923. Microbiol Rev 2010, 34(5):883 923. 6. Condon C, Putzer H: The phylogenetic distribution of bacterial
ribonucleases. Nucleic Acids Res 2002, 30(24):5339-5346. 7. Zuo Y, Deutscher MP: Exoribonuclease superfamilies: structural analysis
and phylogenetic distribution. Nucleic Acids Res 2001, 29(5):1017-1026. 8. Alifano P, Rivellini F, Piscitelli C, Arraiano CM, Bruni CB, Carlomagno MS:
Ribonuclease E provides substrates for ribonuclease P-dependent
processing of a polycistronic messenger RNA. Genes Dev 1994,
8(24):3021-3031. 6. Condon C, Putzer H: The phylogenetic distribution of bacterial
ribonucleases. Nucleic Acids Res 2002, 30(24):5339-5346. 7. Zuo Y, Deutscher MP: Exoribonuclease superfamilies: structural analysis
and phylogenetic distribution. Nucleic Acids Res 2001, 29(5):1017-1026. 8
Alif
P Ri
lli i F Pi it lli C A
i
CM B
i CB C l
MS 8. Alifano P, Rivellini F, Piscitelli C, Arraiano CM, Bruni CB, Carlomagno MS:
Ribonuclease E provides substrates for ribonuclease P-dependent
processing of a polycistronic messenger RNA. Genes Dev 1994,
8(24):3021-3031. 9. Li Y, Altman S: A specific endoribonuclease, RNase P, affects gene
expression of polycistronic operon mRNAs. Proc Natl Acad Sci USA 2003,
100(23):13213-13218. 10. Coburn GA, Mackie GA: Degradation of mRNA in Escherichia coli: An old
problem with some new twists. Prog Nucleic Acids Res Mol Biol 1999, 62:55-108. 11. Carpousis AJ, Luisi BF, McDowall KJ: Endonucleolytic initiation of mRNA
decay in Escherichia coli. Prog Mol Biol Transl Sci 2009, 85:91-135. 10. Coburn GA, Mackie GA: Degradation of mRNA in Escherichia coli: An old
problem with some new twists. Kaberdin et al. Journal of Biomedical Science 2011, 18:23
http://www.jbiomedsci.com/content/18/1/23 the major components of the E. coli degradosome and
their impact on mRNA turnover [38-43] suggest that
multienzyme complexes (instead of a pull of non-inter-
acting enzymes) are favorable for attaining a higher effi-
ciency of mRNA decay. The role of similar complexes in
other bacteria is still poorly defined. Moreover, we do not
know to which degree RNase E/G-based degradosomes
resemble their counterparts containing RNases J1/J2 or
RNase Y existing in many other classes of bacteria. Like-
wise, the mechanisms modulating the composition, activ-
ity and specificity of these multienzyme assemblies in
response to changing physiological conditions remain lar-
gely unknown and merit further analysis. Finally,
although the last step of mRNA decay in E. coli has been
shown to be accomplished by oligoribonuclease encoded
by the orn gene [61], this gene is apparently absent in
many other bacterial species (Figure 2). A search for
activities that can degrade RNA oligoribonucleotides in
Firmicutes lacking sequence homologues of E. coli oligor-
ibonuclease led to the discovery of B. subtilis Ytq1 [74]. This enzyme possesses an oligoribonuclease-like activity
and is able to complement the E. coli orn mutant; homo-
logues of its gene are present in many bacteria [75]. Although Ytq1 can be considered as a functional homo-
logue of oligoribonuclease, further efforts are needed to
disclose the nature and distribution of functional homolo-
gues that may exist in bacterial species lacking both oli-
goribonuclease and Ytq1. the major components of the E. coli degradosome and
their impact on mRNA turnover [38-43] suggest that
multienzyme complexes (instead of a pull of non-inter-
acting enzymes) are favorable for attaining a higher effi-
ciency of mRNA decay. The role of similar complexes in
other bacteria is still poorly defined. Moreover, we do not
know to which degree RNase E/G-based degradosomes
resemble their counterparts containing RNases J1/J2 or
RNase Y existing in many other classes of bacteria. Like-
wise, the mechanisms modulating the composition, activ-
ity and specificity of these multienzyme assemblies in
response to changing physiological conditions remain lar-
gely unknown and merit further analysis. Finally,
although the last step of mRNA decay in E. coli has been
shown to be accomplished by oligoribonuclease encoded
by the orn gene [61], this gene is apparently absent in
many other bacterial species (Figure 2). A search for
activities that can degrade RNA oligoribonucleotides in
Firmicutes lacking sequence homologues of E. 4. Conclusion and perspectives A previous analysis of RNA processing/decay pathways
in several distantly-related bacterial species including
the two major model organisms, E. coli (Proteobacteria)
and B. subtilis (Firmicutes) has identified the key ribo-
nucleases involved in mRNA turnover in bacteria
(reviewed in [5]). Herein, a search for their homologues
in bacteria with completely sequenced genomes revealed
that many components of the bacterial mRNA decay
machinery (RNase III and three major exoribonucleases,
PNPase, RNase II and RNase R) as well as PAPI and
RhlB) are highly conserved across the bacterial kingdom
(see Figure 2). In contrast, the major endoribonucleases
RNase E/G, RNases J1/J2, and RNase Y possess only Similar to other essential cellular processes controlling
inheritance and expression of genetic information (i.e.,
DNA replication, transcription and translation), mRNA
decay was found to be carried out by multienzyme com-
plexes, several of which have been isolated from Proteo-
bacteria, Actinobacteria and Firmicutes over the last two
decades. The existence of functional interactions between Page 10 of 12 Kaberdin et al. Journal of Biomedical Science 2011, 18:23
http://www.jbiomedsci.com/content/18/1/23 Kaberdin et al. Journal of Biomedical Science 2011, 18:23
http://www.jbiomedsci.com/content/18/1/23 Kaberdin et al. Journal of Biomedical Science 2011, 18:23
http://www.jbiomedsci.com/content/18/1/23 Kaberdin et al. Journal of Biomedical Science 2011, 18:23
http://www.jbiomedsci.com/content/18/1/23 Lin PH, Lin-Chao S: RhlB helicase rather than enolase is the beta-subunit
of the Escherichia coli polynucleotide phosphorylase (PNPase)-
exoribonucleolytic complex. Proc Natl Acad Sci USA 2005,
102(46):16590-16595. 56. Kovacs L, Csanadi A, Megyeri K, Kaberdin VR, Miczak A: Mycobacterial
RNase E-associated proteins. Microbiol Immunol 2005, 49(11):1003-1007. 35. Lin-Chao S, Chiou NT, Schuster G: The PNPase, exosome and RNA
helicases as the building components of evolutionarily-conserved RNA
degradation machines. J Biomed Sci 2007, 14(4):523-532. 57. McDowall KJ, Cohen SN: The N-terminal domain of the rne gene product
has RNase E activity and is non-overlapping with the arginine-rich RNA-
binding site. J Mol Biol 1996, 255(3):349-355. 36. Kaberdin VR, Miczak A, Jakobsen JS, Lin-Chao S, McDowall KJ, von Gabain A:
The endoribonucleolytic N-terminal half of Escherichia coli RNase E is
evolutionarily conserved in Synechocystis sp. and other bacteria but not
the C-terminal half, which is sufficient for degradosome assembly. Proc
Natl Acad Sci USA 1998, 95(20):11637-11642. 58. Condon C: What is the role of RNase J in mRNA turnover? RNA Biol 2010
7(3). 59. Kime L, Jourdan SS, Stead JA, Hidalgo-Sastre A, McDowall KJ: Rapid
cleavage of RNA by RNase E in the absence of 5’ monophosphate
stimulation. Mol Microbiol 2010, 76(3):590-604. cleavage of RNA by RNase E in the absence of 5’ monophosphate
stimulation. Mol Microbiol 2010, 76(3):590-604. 60. Andrade JM, Hajnsdorf E, Regnier P, Arraiano CM: The poly(A)-dependent
degradation pathway of rpsO mRNA is primarily mediated by RNase R. RNA 2009, 15(2):316-326. 37. Vanzo NF, Li YS, Py B, Blum E, Higgins CF, Raynal LC, Krisch HM,
Carpousis AJ: Ribonuclease E organizes the protein interactions in the
Escherichia coli RNA degradosome. Genes Dev 1998, 12(17):2770-2781. 61. Ghosh S, Deutscher MP: Oligoribonuclease is an essential component of
the mRNA decay pathway. Proc Natl Acad Sci USA 1999, 96(8):4372-4377. 38. Bernstein JA, Lin PH, Cohen SN, Lin-Chao S: Global analysis of Escherichia
coli RNA degradosome function using DNA microarrays. Proc Natl Acad
Sci USA 2004, 101(9):2758-2763. 62. Cohen SN: Surprises at the 3’ end of prokaryotic RNA. Cell 1995,
80(6):829-832. 39. Khemici V, Carpousis AJ: The RNA degradosome and poly(A) polymerase
of Escherichia coli are required in vivo for the degradation of small
mRNA decay intermediates containing REP-stabilizers. Mol Microbiol 2004,
51(3):777-790. 63. Dreyfus M, Regnier P: The poly(A) tail of mRNAs: bodyguard in
eukaryotes, scavenger in bacteria. Cell 2002, 111(5):611-613. 64. Kaberdin et al. Journal of Biomedical Science 2011, 18:23
http://www.jbiomedsci.com/content/18/1/23 Xu F, Lin-Chao S, Cohen SN: The Escherichia coli pcnB gene promotes
adenylation of antisense RNAI of ColE1-type plasmids in vivo and
degradation of RNAI decay intermediates. Proc Natl Acad Sci USA 1993,
90:6756-6760. 40. Liou GG, Jane WN, Cohen SN, Lin NS, Lin-Chao S: RNA degradosomes exist
in vivo in Escherichia coli as multicomponent complexes associated with
the cytoplasmic membrane via the N-terminal region of ribonuclease E. Proc Natl Acad Sci USA 2001, 98(1):63-68. 65. Coburn GA, Mackie GA: Differential sensitivities of portions of the mRNA
for ribosomal protein S20 to 3’-exonucleases dependent on
oligoadenylation and RNA secondary structure. J Biol Chem 1996,
271(26):15776-15781. 41. Leroy A, Vanzo NF, Sousa S, Dreyfus M, Carpousis AJ: Function in
Escherichia coli of the non-catalytic part of RNase E: role in the
degradation of ribosome-free mRNA. Mol Microbiol 2002, 45(5):1231-1243. 66. Coburn GA, Mackie GA: Overexpression, purification, and properties of
Escherichia coli ribonuclease II. J Biol Chem 1996, 271(2):1048-1053. 42. Lopez PJ, Marchand I, Joyce SA, Dreyfus M: The C-terminal half of RNase
E, which organizes the Escherichia coli degradosome, participates in
mRNA degradation but not rRNA processing in vivo. Mol Microbiol 1999,
33(1):188-199. 67. Hajnsdorf E, Regnier P: E. coli rpsO mRNA decay: RNase E processing at
the beginning of the coding sequence stimulates poly(A)-dependent
degradation of the mRNA. J Mol Biol 1999, 286(4):1033-1043. 43. Xu F, Cohen SN: RNA degradation in Escherichia coli regulated by 3’
adenylation and 5’ phosphorylation. Nature 1995, 374(6518):180-183. 68. Haugel-Nielsen J, Hajnsdorf E, Regnier P: The rpsO mRNA of Escherichia
coli is polyadenylated at multiple sites resulting from endonucleolytic
processing and exonucleolytic degradation. EMBO J 1996,
15(12):3144-3152. 44. Coburn GA, Mackie GA: Reconstitution of the degradation of the mRNA
for ribosomal protein S20 with purified enzymes. J Mol Biol 1998,
279(5):1061-1074. 45. Carpousis AJ: The RNA degradosome of Escherichia coli: an mRNA-
degrading machine assembled on RNase E. Annu Rev Microbiol 2007,
61:71-87. 69. Kaberdin VR, Chao YH, Lin-Chao S: RNase E cleaves at multiple sites in
bubble regions of RNA I stem loops yielding products that dissociate
differentially from the enzyme. J Biol Chem 1996, 271(22):13103-13109. y
y
70. Regnier P, Hajnsdorf E: Poly(A)-assisted RNA decay and modulators of
RNA stability. Prog Mol Biol Transl Sci 2009, 85:137-185. 46. Kaberdin et al. Journal of Biomedical Science 2011, 18:23
http://www.jbiomedsci.com/content/18/1/23 27. Oussenko IA, Bechhofer DH: The yvaJ gene of Bacillus subtilis encodes a
3’-to-5’ exoribonuclease and is not essential in a strain lacking
polynucleotide phosphorylase. J Bacteriol 2000, 182(9):2639-2642. 49. Ait-Bara S, Carpousis AJ: Characterization of the RNA degradosome of
Pseudoalteromonas haloplanktis: conservation of the RNase E-RhlB
interaction in the {gamma}-Proteobacteria. J Bacteriol 2010,
192(20):5413-5423. 28. Oussenko IA, Sanchez R, Bechhofer DH: Bacillus subtilis YhaM, a member
of a new family of 3’-to-5’ exonucleases in gram-positive bacteria. J
Bacteriol 2002, 184(22):6250-6259. 50. Purusharth RI, Klein F, Sulthana S, Jager S, Jagannadham MV, Evguenieva-
Hackenberg E, Ray MK, Klug G: Exoribonuclease R interacts with
endoribonuclease E and an RNA helicase in the psychrotrophic
bacterium Pseudomonas syringae Lz4W. J Biol Chem 2005,
280(15):14572-14578. 29. Deutscher MP, Reuven NB: Enzymatic basis for hydrolytic versus
phosphorolytic mRNA degradation in Escherichia coli and Bacillus subtilis. Proc Natl Acad Sci USA 1991, 88(8):3277-3280. 30. Belasco JG, Higgins CF: Mechanisms of mRNA decay in bacteria: a
perspective. Gene 1988, 72(1-2):15-23. 51. Cheng ZF, Deutscher MP: An important role for RNase R in mRNA decay. Mol Cell 2005, 17(2):313-318. 31. Carpousis AJ, Van Houwe G, Ehretsmann C, Krisch HM: Copurification of E. coli RNase E and PNPase: evidence for a specific association between
two enzymes important in RNA processing and degradation. Cell 1994,
76(5):889-900. 52. Hardwick SW, Chan VS, Broadhurst RW, Luisi BF: An RNA degradosome
assembly in Caulobacter crescentus. Nucleic Acids Res 2010. y
53. Banerjee S, Nandyala AK, Raviprasad P, Ahmed N, Hasnain SE: Iron- 53. Banerjee S, Nandyala AK, Raviprasad P, Ahmed N, Hasnain SE: Iron-
dependent RNA-binding activity of Mycobacterium tuberculosis aconitase. J Bacteriol 2007, 189(11):4046-4052. dependent RNA-binding activity of Mycobacterium tuberculosis aconitase. J Bacteriol 2007, 189(11):4046-4052. 32. Miczak A, Kaberdin VR, Wei CL, Lin-Chao S: Proteins associated with RNase
E in a multicomponent ribonucleolytic complex. Proc Natl Acad Sci USA
1996, 93(9):3865-3869. 54. Jager S, Fuhrmann O, Heck C, Hebermehl M, Schiltz E, Rauhut R, Klug G: An
mRNA degrading complex in Rhodobacter capsulatus. Nucl Acids Res 2001,
29(22):4581-4588. 33. Py B, Higgins CF, Krisch HM, Carpousis AJ: A DEAD-box RNA helicase in
the Escherichia coli RNA degradosome. Nature 1996, 381(6578):169-172. 55. Lee K, Cohen SN: A Streptomyces coelicolor functional orthologue of
Escherichia coli RNase E shows shuffling of catalytic and PNPase-binding
domains. Mol Microbiol 2003, 48(2):349-360. 34. References 1. Deana A, Belasco JG: Lost in translation: the influence of ribosomes on
bacterial mRNA decay. Genes Dev 2005, 19(21):2526-2533. 26. Craven MG, Henner DJ, Alessi D, Schauer AT, Ost KA, Deutscher MP,
Friedman DI: Identification of the rph (RNase PH) gene of Bacillus subtilis:
evidence for suppression of cold-sensitive mutations in Escherichia coli. J
Bacteriol 1992, 174(14):4727-4735. 26. Craven MG, Henner DJ, Alessi D, Schauer AT, Ost KA, Deutscher MP,
Friedman DI: Identification of the rph (RNase PH) gene of Bacillus subtilis:
evidence for suppression of cold-sensitive mutations in Escherichia coli. J
Bacteriol 1992, 174(14):4727-4735. bacterial mRNA decay. Genes Dev 2005, 19(21):2526-2533. 2. Kaberdin VR, Bläsi U: Translation initiation and the fate of bacterial
mRNAs. FEMS Microbiol Reviews 2006, 30:967-979. Page 11 of 12 Kaberdin et al. Journal of Biomedical Science 2011, 18:23
http://www.jbiomedsci.com/content/18/1/23 Erce MA, Low JK, March PE, Wilkins MR, Takayama KM: Identification and
functional analysis of RNase E of Vibrio angustum S14 and two-hybrid
analysis of its interaction partners. Biochim Biophys Acta 2009,
1794(8):1107-1114. 71. Lalonde MS, Zuo Y, Zhang J, Gong X, Wu S, Malhotra A, Li Z:
Exoribonuclease R in Mycoplasma genitalium can carry out both RNA
processing and degradative functions and is sensitive to RNA ribose
methylation. RNA 2007, 13(11):1957-1968. 47. Chandran V, Luisi BF: Recognition of enolase in the Escherichia coli RNA
degradosome. J Mol Biol 2006, 358(1):8-15. 72. Cao GJ, Sarkar N: Identification of the gene for an Escherichia coli poly(A)
polymerase. Proc Natl Acad Sci USA 1992, 89(21):10380-10384. 48. Nurmohamed S, McKay AR, Robinson CV, Luisi BF: Molecular recognition
between Escherichia coli enolase and ribonuclease E. Acta Crystallogr D
Biol Crystallogr 2010, 66(Pt 9):1036-1040. Page 12 of 12 Page 12 of 12 Kaberdin et al. Journal of Biomedical Science 2011, 18:23
http://www.jbiomedsci.com/content/18/1/23 73. Portnoy V, Schuster G: Mycoplasma gallisepticum as the first analyzed
bacterium in which RNA is not polyadenylated. FEMS Microbiol Lett 2008,
283(1):97-103. 74. Mechold U, Fang G, Ngo S, Ogryzko V, Danchin A: YtqI from Bacillus
subtilis has both oligoribonuclease and pAp-phosphatase activity. Nucleic
Acids Res 2007, 35(13):4552-4561. 75. Fang M, Zeisberg WM, Condon C, Ogryzko V, Danchin A, Mechold U:
Degradation of nanoRNA is performed by multiple redundant RNases in
Bacillus subtilis. Nucleic Acids Res 2009, 37(15):5114-5125. 76. Celesnik H, Deana A, Belasco JG: Initiation of RNA decay in Escherichia coli
by 5’ pyrophosphate removal. Mol Cell 2007, 27(1):79-90. doi:10.1186/1423-0127-18-23
Cite this article as: Kaberdin et al.: Composition and conservation of the
mRNA-degrading machinery in bacteria. Journal of Biomedical Science
2011 18:23. 73. Portnoy V, Schuster G: Mycoplasma gallisepticum as the first analyzed
bacterium in which RNA is not polyadenylated. FEMS Microbiol Lett 2008,
283(1):97-103. 74. Mechold U, Fang G, Ngo S, Ogryzko V, Danchin A: YtqI from Bacillus
subtilis has both oligoribonuclease and pAp-phosphatase activity. Nucleic
Acids Res 2007, 35(13):4552-4561. 75. Fang M, Zeisberg WM, Condon C, Ogryzko V, Danchin A, Mechold U:
Degradation of nanoRNA is performed by multiple redundant RNases in
Bacillus subtilis. Nucleic Acids Res 2009, 37(15):5114-5125. 76. Celesnik H, Deana A, Belasco JG: Initiation of RNA decay in Escherichia coli
by 5’ pyrophosphate removal. Mol Cell 2007, 27(1):79-90. doi:10.1186/1423-0127-18-23
Cite this article as: Kaberdin et al.: Composition and conservation of the
mRNA-degrading machinery in bacteria. Journal of Biomedical Science
2011 18:23. 73. Portnoy V, Schuster G: Mycoplasma gallisepticum as the first analyzed
bacterium in which RNA is not polyadenylated. FEMS Microbiol Lett 2008,
283(1):97-103. 74. Mechold U, Fang G, Ngo S, Ogryzko V, Danchin A: YtqI from Bacillus
subtilis has both oligoribonuclease and pAp-phosphatase activity. Nucleic
Acids Res 2007, 35(13):4552-4561. 75. Fang M, Zeisberg WM, Condon C, Ogryzko V, Danchin A, Mechold U:
Degradation of nanoRNA is performed by multiple redundant RNases in
Bacillus subtilis. Nucleic Acids Res 2009, 37(15):5114-5125. 76. Celesnik H, Deana A, Belasco JG: Initiation of RNA decay in Escherichia coli
by 5’ pyrophosphate removal. Mol Cell 2007, 27(1):79-90. doi:10.1186/1423-0127-18-23
Cite this article as: Kaberdin et al.: Composition and conservation of the
mRNA-degrading machinery in bacteria. Journal of Biomedical Science
2011 18:23. Kaberdin et al. Journal of Biomedical Science 2011, 18:23
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Isolation and Identification of Porcine Deltacoronavirus and Alteration of Immunoglobulin Transport Receptors in the Intestinal Mucosa of PDCoV-Infected Piglets
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Viruses
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cc-by
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Received: 9 November 2019; Accepted: 7 January 2020; Published: 9 January 2020 Abstract: Porcine deltacoronavirus (PDCoV) is a porcine enteropathogenic coronavirus that causes
watery diarrhea, vomiting, and frequently death in piglets, causing serious economic losses to the pig
industry. The strain CHN-JS-2017 was isolated and identified by cytopathology, immunofluorescence
assays, transmission electron microscopy, and sequence analysis. A nucleotide sequence alignment
showed that the whole genome of CHN-JS-2017 is 97.4%–99.6% identical to other PDCoV strains. The pathogenicity of the CHN-JS-2017 strain was investigated in orally inoculated five-day-old
piglets; the piglets developed acute, watery diarrhea, but all recovered and survived. CHN-JS-2017
infection-induced microscopic lesions were observed, and viral antigens were detected mainly by
immunohistochemical staining in the small intestine. The neonatal Fc receptor (FcRn) and polymeric
immunoglobulin receptor (pIgR) are crucial immunoglobulin (Ig) receptors for the transcytosis
ofimmunoglobulin G (IgG), IgA, or IgM. Importantly, CHN-JS-2017 infected five-day-old piglets
could significantly down-regulate the expression of FcRn, pIgR, and nuclear factor-kappa B (NF-κB)in
the intestinal mucosa. Note that the level of FcRn mRNA in the intestinal mucosa of normal piglets is
positively correlated with pIgR and NF-κB. At the same time, the expressions of FcRn, pIgR, and
NF-κB mRNA are also positively correlated in infected piglets. These results may help explain the
immunological and pathological changes associated with porcine deltacorononirus infection. ywords: Porcine deltacoronavirus; Neonatal Fc receptor; polymeric immunoglobulin receptor; NF-κB viruses viruses Article Shaoju Qian 1, Xiangchao Jia 1, Zitong Gao 1, Weida Zhang 1, Qingrong Xu 1,2,3 and Zili Li 1,2, Shaoju Qian , Xiangchao Jia , Zitong Gao , Weida Zhang , Qingrong Xu
and Zili Li
1
State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine,
Huazhong Agricultural University, Wuhan 430070, China; qianshaoju@webmail.hzau.edu.cn (S.Q.);
jiaxianghcao@webmail.hzau.edu.cn (X.J.); zitongGao@webmail.hzau.edu.cn (Z.G.);
ZhangWD171215@webmail.hzau.edu.cn (W.Z.); XuQingRong@mail.hzau.edu.cn (Q.X.)
2
Key Laboratory of Preventive Veterinary Medicine in Hubei Province, Wuhan 430070, China
3
Key Laboratory of Development of Veterinary Diagnostic Products,
Ministry of Agriculture of the People’s Republic of China, Wuhan 430070, China
*
Correspondence: lizili@mail.hzau.edu.cn 1
State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine,
Huazhong Agricultural University, Wuhan 430070, China; qianshaoju@webmail.hzau.edu.cn (S.Q.);
jiaxianghcao@webmail.hzau.edu.cn (X.J.); zitongGao@webmail.hzau.edu.cn (Z.G.);
ZhangWD171215@webmail.hzau.edu.cn (W.Z.); XuQingRong@mail.hzau.edu.cn (Q.X.)
2
Key Laboratory of Preventive Veterinary Medicine in Hubei Province, Wuhan 430070, China 3
Key Laboratory of Development of Veterinary Diagnostic Products,
Ministry of Agriculture of the People’s Republic of China, Wuhan 430070, China *
Correspondence: lizili@mail.hzau.edu.cn www.mdpi.com/journal/viruses 1. Introduction Porcine deltacoronavirus (PDCoV) is an enveloped, single-stranded positive-sense (+ssRNA)
virus belonging to the family Coronaviridae. PDCoV was first identified in Hong Kong, China in 2012,
and subsequently detected on swine farms in the United States, Canada, South Korea, mainland China,
Thailand, Laos, and Vietnam; molecular monitoring studies have shown that PDCoV was a common
viral pathogen of pigs worldwide [1–5]. The clinical symptoms of PDCoV-infected piglets are like that of other porcine enteric pathogens,
such as porcine epidemic diarrhea virus (PEDV) or transmissible gastroenteritis virus (TGEV), including
diarrhea, vomiting, dehydration, and mortality [5]. TGEV and PEDV replicate in enterocytes of the Viruses 2020, 12, 79; doi:10.3390/v12010079 www.mdpi.com/journal/viruses www.mdpi.com/journal/viruses 2 of 13 Viruses 2020, 12, 79 small intestine. All PDCoV-infected pigs had PEDV-like lesions characterized by thin and transparent
intestinal walls and an accumulation of large amounts of yellow fluid in the intestinal lumen [6]. Further, histology analysis exhibited acute diffuse, severe atrophic enteritis, and mild vacuolation of
superficial epithelial cells in the cecum and colon [6]. The PDCoV strain OH-FD22 causes thinning of
the intestinal wall, resulting in yellow fluid accumulation in the intestinal cavity [7]. Subsequently,
several studies have reproduced similar clinically diarrheal symptoms in gnotobiotic and conventional
pigs with isolated PDCoV strains [8–11]. PDCoV is enteropathogenic in pigs, and lesions are similar
but relatively milder than PEDV [12]. The secretory IgA plays a major role in the mucosal anti-infection immunity and is transported by
the polymeric immunoglobulin receptor (pIgR). However, recent studies have found that IgG also
plays an important role in pathogen infection, which is transported by neonatal Fc receptor (FcRn) [13]. PDCoV is an intestinal pathogen, but little information exists about the regulation of host immune
responses due to PDCoV infection. PDCoV infection suppresses interferon (IFN)-β production by
inhibiting the retinoic acid-inducible gene I (RIG-I) signaling pathway in Lilly Laboratories Cell–Porcine
Kidney (LLC-PK) cells [14]. PDCoV nonstructural protein 5 (nsp5) inhibits IFN-β production by
the cleavage of nuclear factor-kappa B (NF-κB) Essential Modulator (NEMO) and cleavage of signal
transducer and activator of transcription 2 genes (STAT2) in porcine kidney epithelial (PK-15) cells. In addition, PDCoV N and the accessory protein NS6 were found to antagonize the production of
IFN-β in Human Embryonic Kidney (HEK)293T cells [15–17]. 1. Introduction Although research into these cells has
provided invaluable information about the interaction between enteric coronaviruses and their hosts,
the research may not yield relevant biological information consistent with in vivo studies. PDCoV
induced toll-like receptor TLR3, IL-12, IFN-α, IFN-β, and protein kinase R (PKR) messenger ribonucleic
acid (mRNA) expression in infected Peyer’s patches from weaned piglets [18]. However, little is known
about the innate immune response to PDCoV infection in vivo. In this study, we successfully isolated PDCoV strain CHN-JS-2017 and performed a histological
examination and an immunohistochemistry staining analysis to study its pathogenicity in five-day-old
piglets. In addition, to better understand in vivo immunological changes after PDCoV infection, we
evaluated the effects of PDCoV on the pig intestinal gut pIgR, FcRn, and NF-κB and their correlation
by RT-qPCR, which may be related to the pathogenesis of PDCoV. 2.1. Clinical Samples and Virus Isolation Contents from the intestines of seven PDCoV-positive piglets were collected from a commercial
pig farm in Jiangsu Province and analyzed by N-gene based RT-qPCR [4]. These samples were,
respectively, homogenized in 10 mL of Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Grand
Island, NY, USA), and the supernatant was collected by centrifugation (4000 g at 4 ◦C for 10 min). The
separated supernatant was filtered through a 0.22-µm filter (Millipore, MA) and stored at −80 ◦C for
PDCoV isolation. Cells were obtained from the China Center for Type Culture Collection (Wuhan, China). LLC-PK
cells were cultured in DMEM (Hyclone, USA) containing 10% fetal bovine serum (FBS) (Gibco) and
the maintenance medium for PDCoV propagation was DMEM supplemented with 6 µg/mL trypsin
(Gibco, USA) in a 5% CO2 incubator. LLC-PK cells were cultured in six-well plates to 90% of the
cell monolayer, washed three times, and maintained with 0.5 mL filtered inoculum in 1.5 mL of
maintenance medium for 2 h. After virus adsorption, the cells were then washed three times with
PBS and cultured continuously in 2 mL maintenance medium at 37 ◦C in 5% CO2. At 24 hpi, an
obvious cytopathic effect (CPE) was observed in the treated cells; the infected cells were lysed using
a freeze-thaw method, and centrifuged (4000 g at 4 ◦C for 10 min). The supernatants were stored at
−80 ◦C. The virus was serially propagated for three passages and titrated in LLC-PK cells. The isolate 3 of 13 Viruses 2020, 12, 79 (designated as CHN-JS-2017) was purified by plaque purification and tested with the median tissue
culture infectious dose (TCID50) assay protocol, as described previously [7]. (designated as CHN-JS-2017) was purified by plaque purification and tested with the median tissue
culture infectious dose (TCID50) assay protocol, as described previously [7]. 2.4. Electron Microscopy The purified PDCoV-infected LLC-PK cells in six well-plates, harvested at 24 hpi, were negatively
stained. The virions were stained with 2% phosphotungstic acid (pH 6.8) for 1.5 min and examined
using a Hitachi Model H-7650 transmission electron microscope (TEM). The virus samples were
observed according to the methods described in earlier studies [20]. 2.3. Western Blot LLC-PK cells were infected with PDCoV (MOI 0.01) in 24 well-plates at 24 hpi; the cell lysates
were prepared for 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE),
and proteins were electroblotted onto a polyvinylidene difluoride membrane (Bio-Rad, USA). FcRn
polyclonal antibodies and PDCoV-N polyclonal antibodies were prepared in our laboratory. Mouse
mAbs against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and rabbit polyclonal against
phospho-NF-κB and pIgR were purchased from Abclona (China). PDCoV-N, phospho-NF-κB, FcRn,
and the pIgR antibody (1:2000) were used as primary antibodies, followed by horseradish peroxidase
(HRP)-conjugated goat anti-rabbit immunoglobulin-G (IgG) or anti-mouse IgG (ABclonal, China) as
secondary antibodies (1:5000). Proteins were visualized with enhanced chemiluminescence (ECL), as
described previously [19]. 2.2. Immunofluorescence Assay (IFA) Immunofluorescence assays (IFA) were performed, as described previously, to observe
PDCoV-infected LLC-PK cells [9]. LLC-PK cells infected with PDCoV at a multiplicity of infection (MOI)
of 0.01 at 24 hpi were fixed with 4% paraformaldehyde in 24-well plates. Subsequently, PDCoV N-protein
polyclonal antibody (1:100) (prepared in our laboratory) was used as the primary antibody, followed
by fluorescent isothiocyanate (FITC)-labeled goat anti-rabbit secondary antibody (1:500) (Abclona,
WuhanChina), and was then counterstained at room temperature with 4′,6-diamidino-2-phenylindole
(DAPI). Fluorescence was examined using a fluorescence microscope (Olympus IX73, Tokyo, Japan). 2.5. Phylogenetic Analysis Viral RNA was extracted from the isolated third-passage PDCoV strain CHN-JS-2017 with
TRIzol™reagent (Thermo Fisher Scientific, Waltham, MA, USA), and complementary deoxyribonucleic
acid (cDNA) was synthesized by using a reverse transcription-polymerase chain reaction (RT-PCR)
kit (TaKaRa, Dalian, China). The primers used for the amplification of the genomic fragments of
CHN-JS-2017 were described previously (Table S1) [21]. The 5′ and 3′ termini of the genomic sequence
were synthesized using rapid amplification of the cDNA ends (RACE) (Vazyme, Nanjing, China). The PCR products were cloned into pMD18-T (TaKaRa, Dalian, China), and sequenced by Sanger
sequencing. The genomic fragments were assembled using Lasergene 7.0 (DNASTAR, Inc., Madison,
Wisconsin USA) and the assembled genomic sequences were submitted to the GenBank database
under accession number MN249445. A phylogenetic tree was performed for the whole genome of the
26 PDCoV strains from different countries using the contiguous method with MEGA 7 software [22]. 2.8. Statistical Analysis Data were analyzed as mean ± SEM. Differences among groups were performed by one-way
ANOVA using Prism (GraphPad Software Inc., San Diego, CA, USA). Pearson’s test was used to assess
the correlations between the mRNA levels of different genes in the results. The significance level for all
analyses was set as p < 0.05 (∗) and p < 0.01 (∗∗). 2.7. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Analysis Fecal swabs and small intestine tissues from every piglet were homogenized respectively and
centrifuged at 6000 g for 5 min. Total RNA was extracted from the supernatant with TRIzol@
reagent (Thermo Fisher Scientific, USA); cDNA was synthesized from the extracted RNA by reverse
transcription using an RT-PCR kit (Takara, Dalian), and real-time RT-qPCR was performed using the
SYBR Green Real-Time PCR Mix (Takara, Dalian). The N gene amplified from the PDCoV CHN-JS-2017
strain was cloned into the pMD18-T cloning vector (Takara, Dalian). The pMD18-N plasmid was
serially diluted 10-fold to generate a standard curve in each plate. The viral RNA in the sample
was calculated based on the results for the standard curve. Virus titers were detected by Tissue
culture infective dose (TCID50), as previously described [8]. The specific pig pIgR, NF-κB, FcRn, and
GAPDH gene primers were synthesized by GenScript Corporation (Nanjing, China) as described
previously (Table S2) [19,24]. The expression level of the gene was calculated relative to the expression
of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using the delta-delta cycle to threshold
(2−∆∆CT) method. 2.6. Inoculation of Piglets with Porcine Deltacoronavirus (PDCoV) Strain CHN-JS-2017 The animal study was approved under the guidance of the Scientific Ethics Committee of Huazhong
Agricultural University (HZAUSW-2018-011) and performed in accordance with the committee’s
regulations and guidelines. The piglets were randomly divided into two groups (six piglets per group)
in separate rooms. The piglets were fed milk powder. After one day of adaptation, the challenged
group of piglets were orally inoculated with PDCoV CHN-JS-2017 (5 × 106 TCID50/mL, 3 mL/head), Viruses 2020, 12, 79 4 of 13 while the control group of piglets received 3 mL of maintenance medium orally. Every piglet in both
groups was observed daily for lethargy, vomiting, and diarrhea. Fecal swabs for viral RNA detection
were collected at 0, 1, 2, 3, 4, 7, 14, 18, and 21 dpi from all piglets. Three of six piglets in each group were randomly selected for necropsy at four days post-inoculation
(dpi). During the necropsies, fresh jejuna were collected and fixed with 10% formalin for 36 h, and
then dehydrated, embedded, sectioned, and mounted onto glass slides. After they were stained
with hematoxylin and eosin (H&E), the slides were examined and analyzed with conventional
microscopy. The fixed tissue sections were evaluated for PDCoV antigen by immunohistochemistry
(IHC) using a PDCoV N-protein polyclonal antibody (1:100 dilution), produced in our laboratory. The
immunohistochemistry slides were evaluated by a veterinary pathologist according to the evaluation
system of histology and immunohistochemistry, as described previously [23]. RT-qPCR was used to
detect the expression of FcRn, pIgR, and NF-κB genes in the porcine duodenum proximal or distal,
jejunum proximal or distal, ileum proximal or distal. 3.1. Virus Isolation and Characterization Seven PDCoV-positive samples were inoculated onto LLC-PK cells; only one of the isolates showed
visible CPE (enlarged, rounded, and aggregated) at 36 h post-infection and was identified as PDCoV
CHN-JS-2017(Figure 1A). The PDCoV isolate was confirmed by IFA and Western blot with a PDCoV
N-protein polyclonal antibody in the LLC-PK cells. PDCoV N-protein-specific immunofluorescence
was detected (Figure 1A), but not in the uninfected group. Western blot analysis also detected N
protein in PDCoV-infected LLC-PK cells (Figure 1B). The PDCoV CHN-JS-2017 infected LLC-PK cells
were examined by transmission electron microscopy (TEM). Typical crown particles with spiky surface
protrusions were observed with negative staining on TEM (Figure 1C). The plaque-purified virus was
titrated to 106.25 TCID50/mL. 5 of 13
5 of 14 Viruses 2020, 12, 79 Figure 1. Isolation and characterization of porcine deltacoronavirus (PDCoV) strain CHN-JS-2017. (A)
The cytopathic effect (CPE) and immunofluorescence staining (IFA) of LLC-PK cells infected with
PDCoV. (a) CPE of LLC-PK cells at 24 h after PDCoV infection. (b) Morphology of the mock-treated
LLC-PK cells at 24 h. (c–h) PDCoV-infected or mock-treated LLC-PK cells were examined by IFA at
24 hpi using polyclonal against PDCoV N protein. (c and d) IFA with PDCoV N protein (green), (e
and f) IFA with 4′,6-diamidino-2-phenylindole (DAPI)(blue), all views were visualized by using a
fluorescence microscope. (B) PDCoV-infected or mock-treated LLC-PK cells were subjected to
analysis with polyclonal antibodies against PDCoV N protein by Western blot at 24 hpi. (C) Electron
i
i i
f PDC V
ti l
Th
d
i t t th
i
ti l
Figure 1. Isolation and characterization of porcine deltacoronavirus (PDCoV) strain CHN-JS-2017. (A) The cytopathic effect (CPE) and immunofluorescence staining (IFA) of LLC-PK cells infected with
PDCoV. (a) CPE of LLC-PK cells at 24 h after PDCoV infection. (b) Morphology of the mock-treated
LLC-PK cells at 24 h. (c–h) PDCoV-infected or mock-treated LLC-PK cells were examined by IFA
at 24 hpi using polyclonal against PDCoV N protein. (c and d) IFA with PDCoV N protein (green),
(e and f) IFA with 4′,6-diamidino-2-phenylindole (DAPI)(blue), all views were visualized by using a
fluorescence microscope. (B) PDCoV-infected or mock-treated LLC-PK cells were subjected to analysis
with polyclonal antibodies against PDCoV N protein by Western blot at 24 hpi. (C) Electron microscopic
image of PDCoV particles. The red arrow points to the virus particle. Figure 1. Isolation and characterization of porcine deltacoronavirus (PDCoV) strain CHN-JS-2017. 3.1. Virus Isolation and Characterization (A)
The cytopathic effect (CPE) and immunofluorescence staining (IFA) of LLC-PK cells infected with
PDCoV. (a) CPE of LLC-PK cells at 24 h after PDCoV infection. (b) Morphology of the mock-treated
LLC-PK cells at 24 h. (c–h) PDCoV-infected or mock-treated LLC-PK cells were examined by IFA at
24 hpi using polyclonal against PDCoV N protein. (c and d) IFA with PDCoV N protein (green), (e
and f) IFA with 4′,6-diamidino-2-phenylindole (DAPI)(blue), all views were visualized by using a
fluorescence microscope. (B) PDCoV-infected or mock-treated LLC-PK cells were subjected to
analysis with polyclonal antibodies against PDCoV N protein by Western blot at 24 hpi. (C) Electron
Figure 1. Isolation and characterization of porcine deltacoronavirus (PDCoV) strain CHN-JS-2017. (A) The cytopathic effect (CPE) and immunofluorescence staining (IFA) of LLC-PK cells infected with
PDCoV. (a) CPE of LLC-PK cells at 24 h after PDCoV infection. (b) Morphology of the mock-treated
LLC-PK cells at 24 h. (c–h) PDCoV-infected or mock-treated LLC-PK cells were examined by IFA
at 24 hpi using polyclonal against PDCoV N protein. (c and d) IFA with PDCoV N protein (green),
(e and f) IFA with 4′,6-diamidino-2-phenylindole (DAPI)(blue), all views were visualized by using a
fluorescence microscope. (B) PDCoV-infected or mock-treated LLC-PK cells were subjected to analysis
with polyclonal antibodies against PDCoV N protein by Western blot at 24 hpi. (C) Electron microscopic
image of PDCoV particles. The red arrow points to the virus particle. microscopic image of PDCoV particles. The red arrow points to th
3.2. Characterization of the PDCoV CHN-JS-2017 Genomic Sequence microscopic image of PDCoV particles. The red arrow points to the
3.2. Characterization of the PDCoV CHN-JS-2017 Genomic Sequence The PDCoV strain CHN-JS-2017 is indicated in bold and highlighted with a box. Figure 2. A phylogenetic tree constructed on the basis of the complete genome (A) of the PDCoV
strain from different countries and local areas. CHN-JS-2017 is represented by a triangle. The
phylogenetic tree was constructed by the adjacency method in MEGA 7 (http://www.megasoftware. net). Bootstrap analysis was performed using 1000 replicates. (B) Three main deletions or insertions
in the complete genome alignment. A multiple sequence alignment was constructed with ClustalW
g
p y
g
p
g
( )
from different countries and local areas. CHN-JS-2017 is represented by a triangle. The phylogenetic
tree was constructed by the adjacency method in MEGA 7 (http://www.megasoftware.net). Bootstrap
analysis was performed using 1000 replicates. (B) Three main deletions or insertions in the complete
genome alignment. A multiple sequence alignment was constructed with ClustalW in the DNAStar
software. The PDCoV strain CHN-JS-2017 is indicated in bold and highlighted with a box. in the DNAStar software. The PDCoV strain CHN-JS-2017 is indicated in bold and hi
a box
3.3. Clinical Observations and Fecal Shedding in Piglets Challenged with CHN-JS-2017 in the DNAStar software. The PDCoV strain CHN-JS-2017 is indicated in bold and h
a box
3.3. Clinical Observations and Fecal Shedding in Piglets Challenged with CHN-JS-2017 3.3. Clinical Observations and Fecal Shedding in Piglets Challenged with CHN-JS-2017
To assess the pathogenicity of the PDCoV strain CHN-JS-2017 to piglets, five-day-old piglets
were challenged with PDCoV CHN-JS-2017. The piglets in the control group were all in a healthy
state. Two of the six piglets in the challenge group developed diarrhea at 1 dpi, and the remaining
piglets showed lethargy, anorexia, and watery diarrhea at 2–6 dpi, and showed acute diarrhea at 3–
6 dpi (Table 1). Severe watery diarrhea gradually recovered afterward. Clinical symptoms were
scored according to their level of diarrhea; severe diarrhea was observed in piglets at 3–6 dpi (Figure
To assess the pathogenicity of the PDCoV strain CHN-JS-2017 to piglets, five-day-old piglets were
challenged with PDCoV CHN-JS-2017. The piglets in the control group were all in a healthy state. Two
of the six piglets in the challenge group developed diarrhea at 1 dpi, and the remaining piglets showed
lethargy, anorexia, and watery diarrhea at 2–6 dpi, and showed acute diarrhea at 3–6 dpi (Table 1). Severe watery diarrhea gradually recovered afterward. microscopic image of PDCoV particles. The red arrow points to the
3.2. Characterization of the PDCoV CHN-JS-2017 Genomic Sequence 3.2. Characterization of the PDCoV CHN-JS-2017 Genomic Sequence
A nucleotide sequence alignment showed that the whole genome of CHN-JS-2017(GenBank no:
MN249445) is 97.4%–99.6% identical to other PDCoV strains. Phylogenetic analysis indicated that the
PDCoV strains from the United States and South Korea clustered into a large clade, whereas CHN-
JS-2017 clustered with other PDCoV strains detected in China since 2014 (Figure 2A). These data
imply that the Chinese strains might share a more recent common ancestor with the US and South
Korean strains than the ancestor of the strains from Thailand and Vietnam. A sequence analysis
showed a 6-nt (TTTGAA) deletion at positions 1732 to 1737 in nsp2 (corresponding to the HKU15-44
sequence), 3-nt (AAT) deletion at positions 19461 to 19463 in the S gene, and 10-nt (ACAAAAGTTG)
A nucleotide sequence alignment showed that the whole genome of CHN-JS-2017(GenBank no:
MN249445) is 97.4%–99.6% identical to other PDCoV strains. Phylogenetic analysis indicated that
the PDCoV strains from the United States and South Korea clustered into a large clade, whereas
CHN-JS-2017 clustered with other PDCoV strains detected in China since 2014 (Figure 2A). These
data imply that the Chinese strains might share a more recent common ancestor with the US and
South Korean strains than the ancestor of the strains from Thailand and Vietnam. A sequence analysis
showed a 6-nt (TTTGAA) deletion at positions 1732 to 1737 in nsp2 (corresponding to the HKU15-44
sequence), 3-nt (AAT) deletion at positions 19461 to 19463 in the S gene, and 10-nt (ACAAAAGTTG)
insertion at positions 25360 to 25369 in the 3′-UTR (Figure 2B). 6 of 13
6 of 14 Viruses 2020, 12, 79 Figure 2. A phylogenetic tree constructed on the basis of the complete genome (A) of the PDCoV
strain from different countries and local areas. CHN-JS-2017 is represented by a triangle. The
phylogenetic tree was constructed by the adjacency method in MEGA 7 (http://www.megasoftware. net). Bootstrap analysis was performed using 1000 replicates. (B) Three main deletions or insertions
in the complete genome alignment. A multiple sequence alignment was constructed with ClustalW
i
h DNAS
f
Th PDC V
i
CHN JS 2017 i i di
d i
b ld
d hi hli h
d
i h
Figure 2. A phylogenetic tree constructed on the basis of the complete genome (A) of the PDCoV strain
from different countries and local areas. CHN-JS-2017 is represented by a triangle. microscopic image of PDCoV particles. The red arrow points to the
3.2. Characterization of the PDCoV CHN-JS-2017 Genomic Sequence The phylogenetic
tree was constructed by the adjacency method in MEGA 7 (http://www.megasoftware.net). Bootstrap
analysis was performed using 1000 replicates. (B) Three main deletions or insertions in the complete
genome alignment. A multiple sequence alignment was constructed with ClustalW in the DNAStar
software. The PDCoV strain CHN-JS-2017 is indicated in bold and highlighted with a box. Figure 2. A phylogenetic tree constructed on the basis of the complete genome (A) of the PDCoV
strain from different countries and local areas. CHN-JS-2017 is represented by a triangle. The
phylogenetic tree was constructed by the adjacency method in MEGA 7 (http://www.megasoftware. net). Bootstrap analysis was performed using 1000 replicates. (B) Three main deletions or insertions
in the complete genome alignment. A multiple sequence alignment was constructed with ClustalW
h
AS
f
h
C
C
S
d
d
b ld
d h
hl
h
d
h
Figure 2. A phylogenetic tree constructed on the basis of the complete genome (A) of the PDCoV strain
from different countries and local areas. CHN-JS-2017 is represented by a triangle. The phylogenetic
tree was constructed by the adjacency method in MEGA 7 (http://www.megasoftware.net). Bootstrap
analysis was performed using 1000 replicates. (B) Three main deletions or insertions in the complete
genome alignment. A multiple sequence alignment was constructed with ClustalW in the DNAStar
software. The PDCoV strain CHN-JS-2017 is indicated in bold and highlighted with a box. Figure 2. A phylogenetic tree constructed on the basis of the complete genome (A) of the PDCoV strain Figure 2. A phylogenetic tree constructed on the basis of the complete genome (A) of the PDCoV
strain from different countries and local areas. CHN-JS-2017 is represented by a triangle. The
phylogenetic tree was constructed by the adjacency method in MEGA 7 (http://www.megasoftware. net). Bootstrap analysis was performed using 1000 replicates. (B) Three main deletions or insertions
in the complete genome alignment. A multiple sequence alignment was constructed with ClustalW
Figure 2. A phylogenetic tree constructed on the basis of the complete genome (A) of the PDCoV strain
from different countries and local areas. CHN-JS-2017 is represented by a triangle. The phylogenetic
tree was constructed by the adjacency method in MEGA 7 (http://www.megasoftware.net). Bootstrap
analysis was performed using 1000 replicates. (B) Three main deletions or insertions in the complete
genome alignment. A multiple sequence alignment was constructed with ClustalW in the DNAStar
software. microscopic image of PDCoV particles. The red arrow points to the
3.2. Characterization of the PDCoV CHN-JS-2017 Genomic Sequence Clinical symptoms were scored according to
their level of diarrhea; severe diarrhea was observed in piglets at 3–6 dpi (Figure 3A). Despite watery
diarrhea with vomiting, lethargy, and anorexia, no deaths occurred during the study. These results
showed that PDCoV CHN-JS-2017 was pathogenic to newborn piglets. 3A). Despite watery diarrhea with vomiting, lethargy, and anorexia, no deaths occurred during the
study. These results showed that PDCoV CHN-JS-2017 was pathogenic to newborn piglets. Table 1. Clinical observation records of five-day-old pigs challenged with PDCoV. dpi(d)
Lethargy, Vomiting and
Anorexia
Fecal Consistency
Normal
Mild
Diarrhea
Watery
Diarrhea
0
0/6
6/6
0/6
0/6
1
2/6
4/6
1/6
1/6
2
3/6
3/6
1/6
2/6
3
4/6
2/6
1/6
3/6
4*
3/3
0/3
2/3
1/3
Table 1. Clinical observation records of five-day-old pigs challenged with PDCoV. dpi(d)
Lethargy, Vomiting and
Anorexia
Fecal Consistency
Normal
Mild Diarrhea
Watery Diarrhea
0
0/6
6/6
0/6
0/6
1
2/6
4/6
1/6
1/6
2
3/6
3/6
1/6
2/6
3
4/6
2/6
1/6
3/6
4*
3/3
0/3
2/3
1/3
7
2/3
1/3
1/3
1/3
8-21
0/3
3/3
0/3
0/3
* Three pigs were necropsied at four days post-inoculation. e watery diarrhea with vomiting, lethargy, and anorexia, no deaths occurred
e results showed that PDCoV CHN-JS-2017 was pathogenic to newborn pigle
Table 1. Clinical observation records of five-day-old pigs challenged with PDCoV. 7
2/3
1/3
1/3
1/3
8-21
0/3
3/3
0/3
0/3
* Three pigs were necropsied at four days post-inoculation. PDCoV RNA was detected by RT-qPCR in feces collected from piglets in the oral challenge group,
nd all piglets were found to exhibit peak virus shedding of 105–108 copies/mL or 104–106 TCID50/mL 7
2/3
1/3
1/3
1/3
8-21
0/3
3/3
0/3
0/3
* Three pigs were necropsied at four days post-inoculation. PDCoV RNA was detected by RT-qPCR in feces collected from piglets in the oral challenge
group, and all piglets were found to exhibit peak virus shedding of 105–108 copies/mL or 104–106
PDCoV RNA was detected by RT-qPCR in feces collected from piglets in the oral challenge group,
and all piglets were found to exhibit peak virus shedding of 105–108 copies/mL or 104–106 TCID50/mL
in feces at 2–6 dpi and high levels in the feces. microscopic image of PDCoV particles. The red arrow points to the
3.2. Characterization of the PDCoV CHN-JS-2017 Genomic Sequence Virus shedding occurred for about a week; after that, the
virus shedding was gradually reduced, reaching a level which was undetectable by RT-PCR at 18 dpi 7
2/3
1/3
1/3
1/3
8-21
0/3
3/3
0/3
0/3
PDCoV RNA was detected by RT-qPCR in feces collected from piglets in the oral challenge group, 8-21
0/3
3/3
0/3
0/3
* Three pigs were necropsied at four days post-inoculation. PDCoV RNA was detected by RT-qPCR in feces collected from piglets in the oral challenge
group, and all piglets were found to exhibit peak virus shedding of 105–108 copies/mL or 104–106
TCID /
L i
f
t 2 6 d i
d hi h l
l i
th f
Vi
h ddi
d f
b
t
k
PDCoV RNA was detected by RT-qPCR in feces collected from piglets in the oral challenge group,
and all piglets were found to exhibit peak virus shedding of 105–108 copies/mL or 104–106 TCID50/mL
in feces at 2–6 dpi and high levels in the feces. Virus shedding occurred for about a week; after that, the
virus shedding was gradually reduced, reaching a level which was undetectable by RT-PCR at 18 dpi 8 21
0/3
3/3
0/3
0/3
* Three pigs were necropsied at four days post-inoculation. PDCoV RNA was detected by RT-qPCR in feces collected from piglets in the oral challenge
group, and all piglets were found to exhibit peak virus shedding of 105–108 copies/mL or 104–106
and all piglets were found to exhibit peak virus shedding of 105–108 copies/mL or 104–106 TCID50/mL
in feces at 2–6 dpi and high levels in the feces. Virus shedding occurred for about a week; after that, the
virus shedding was gradually reduced, reaching a level which was undetectable by RT-PCR at 18 dpi 7 of 13
7 of 14 Viruses 2020, 12, 79 (Figure 3B). The distribution of CHN-JS-2017 in various tissues was also examined by quantitative
real-time RT-qPCR in three pigs from each group sacrificed at 4 dpi. Viral RNA was detected in
duodenums (average 107.64 copies/mL or 105.79 TCID50/mL), jejunum (average 108.03 copies/mL or
106.18 TCID50/mL), ileum (average 108.16 copies/mL or 106.32 TCID50/mL) (Figure 3C). No PDCoV RNA
was detected in the negative control piglets. a te t at, t e vi us s eddi g was g adua y educed, eac i g a eve w ic was u detectab e by
PCR at 18 dpi (Figure 3B). microscopic image of PDCoV particles. The red arrow points to the
3.2. Characterization of the PDCoV CHN-JS-2017 Genomic Sequence (C) Virus distribution in the small intestine of piglets challenged with PDCoV. DU-P: Duodenum
proximal. DU-D: Duodenum distal. JE-P: jejunum proximal. JE-D: jejunum distal. IL-P: ileum proximal. IL-D: ileum distal. Figure 3. Fecal viral shedding and virus distribution in piglets challenged with PDCoV-JS-2017. Figure 3. Fecal viral shedding and virus distribution in piglets challenged with PDCoV-JS-2017. (A)
Diarrhea of piglets in different groups. The severity of diarrhea was scored based on visual
examination; 0 = normal and no diarrhea; 1 = mild and fluidic diarrhea; 2 = severe watery diarrhea;
scores of 1 or more were considered diarrheic. (B) Fecal virus shedding in PDCoV-challenged piglets. (C) Virus distribution in the small intestine of piglets challenged with PDCoV. DU-P: Duodenum
Figure 3. Fecal viral shedding and virus distribution in piglets challenged with PDCoV-JS-2017. (A) Diarrhea of piglets in different groups. The severity of diarrhea was scored based on visual
examination; 0 = normal and no diarrhea; 1 = mild and fluidic diarrhea; 2 = severe watery diarrhea;
scores of 1 or more were considered diarrheic. (B) Fecal virus shedding in PDCoV-challenged piglets. (C) Virus distribution in the small intestine of piglets challenged with PDCoV. DU-P: Duodenum
proximal. DU-D: Duodenum distal. JE-P: jejunum proximal. JE-D: jejunum distal. IL-P: ileum proximal. IL-D: ileum distal. proximal. DU-D: Duodenum distal
proximal. IL-D: ileum distal. 3.4. Gross Pathology and Histopathology microscopic image of PDCoV particles. The red arrow points to the
3.2. Characterization of the PDCoV CHN-JS-2017 Genomic Sequence The distribution of CHN-JS-2017 in various tissues was also examined by
quantitative real-time RT-qPCR in three pigs from each group sacrificed at 4 dpi. Viral RNA was
detected in duodenums (average 107.64 copies/mL or 105.79 TCID50/mL), jejunum (average 108.03
copies/mL or 106.18 TCID50/mL), ileum (average 108.16 copies/mL or 106.32 TCID50/mL) (Figure 3C). No
PDCoV RNA was detected in the negative control piglets. Figure 3. Fecal viral shedding and virus distribution in piglets challenged with PDCoV-JS-2017. (A)
Diarrhea of piglets in different groups. The severity of diarrhea was scored based on visual
examination; 0 = normal and no diarrhea; 1 = mild and fluidic diarrhea; 2 = severe watery diarrhea;
scores of 1 or more were considered diarrheic. (B) Fecal virus shedding in PDCoV-challenged piglets. (C) Virus distribution in the small intestine of piglets challenged with PDCoV. DU-P: Duodenum
i
l
DU D
D
d
di
l
JE P
j j
i
l
JE D
j j
di
l
IL P
il
Figure 3. Fecal viral shedding and virus distribution in piglets challenged with PDCoV-JS-2017. (A) Diarrhea of piglets in different groups. The severity of diarrhea was scored based on visual
examination; 0 = normal and no diarrhea; 1 = mild and fluidic diarrhea; 2 = severe watery diarrhea;
scores of 1 or more were considered diarrheic. (B) Fecal virus shedding in PDCoV-challenged piglets. (C) Virus distribution in the small intestine of piglets challenged with PDCoV. DU-P: Duodenum
proximal. DU-D: Duodenum distal. JE-P: jejunum proximal. JE-D: jejunum distal. IL-P: ileum proximal. IL-D: ileum distal. Figure 3. Fecal viral shedding and virus distribution in piglets challenged with PDCoV-JS-2017. (A)
Diarrhea of piglets in different groups. The severity of diarrhea was scored based on visual
examination; 0 = normal and no diarrhea; 1 = mild and fluidic diarrhea; 2 = severe watery diarrhea;
scores of 1 or more were considered diarrheic. (B) Fecal virus shedding in PDCoV-challenged piglets. (C) Virus distribution in the small intestine of piglets challenged with PDCoV. DU-P: Duodenum
Figure 3. Fecal viral shedding and virus distribution in piglets challenged with PDCoV-JS-2017. (A) Diarrhea of piglets in different groups. The severity of diarrhea was scored based on visual
examination; 0 = normal and no diarrhea; 1 = mild and fluidic diarrhea; 2 = severe watery diarrhea;
scores of 1 or more were considered diarrheic. (B) Fecal virus shedding in PDCoV-challenged piglets. proximal. DU-D: Duodenum dista
proximal. IL-D: ileum distal.
3.4. Gross Pathology and Histopathology Intestinal changes of infected and uninfected piglets challenged with PDCoV CHN-JS-2017. (A) Macroscopic damage of piglets challenged with PDCoV at 4 dpi. (B) Macroscopic view of 4 dpi
negative control piglets. (C,D) Hematoxylin and eosin (H&E)-stained jejunal tissue sections of piglets
challenged with PDCoV (C) or negative control piglets (D). (E,F) Immunohistochemically stained
jejunal tissue sections of challenged piglets (E) or negative control piglets (F). Figure 4. Intestinal changes of infected and uninfected piglets challenged with PDCoV CHN-JS-2017. (A) Macroscopic damage of piglets challenged with PDCoV at 4 dpi. (B) Macroscopic view of 4 dpi
negative control piglets. (C,D) Hematoxylin and eosin (H&E)-stained jejunal tissue sections of piglets
challenged with PDCoV (C) or negative control piglets (D). (E,F) Immunohistochemically stained
jejunal tissue sections of challenged piglets (E) or negative control piglets (F). Figure 4. Intestinal changes of infected and uninfected piglets challenged with PDCoV CHN-JS-2017. (A) Macroscopic damage of piglets challenged with PDCoV at 4 dpi. (B) Macroscopic view of 4 dpi
negative control piglets. (C,D) Hematoxylin and eosin (H&E)-stained jejunal tissue sections of piglets
challenged with PDCoV (C) or negative control piglets (D). (E,F) Immunohistochemically stained
jejunal tissue sections of challenged piglets (E) or negative control piglets (F). Figure 4. Intestinal changes of infected and uninfected piglets challenged with PDCoV CHN-JS-2017. (A) Macroscopic damage of piglets challenged with PDCoV at 4 dpi. (B) Macroscopic view of 4 dpi
negative control piglets. (C,D) Hematoxylin and eosin (H&E)-stained jejunal tissue sections of piglets
challenged with PDCoV (C) or negative control piglets (D). (E,F) Immunohistochemically stained
jejunal tissue sections of challenged piglets (E) or negative control piglets (F). 3 5 Alteration of NF-κB FcRn and pIgR mRNA in the Small Intestinal Mucosa after PDCoV Infection
3.5. Alteration of NF-κB FcRn and pIgR mRNA in the Small Intestinal Mucosa after PDCoV Infection f
p g
f
f
To characterize the effects of PDCoV infection, the levels of NF-κB FcRn, and pIgR in the
intestinal mucosa of infected or non-infected control piglets were examined. The mRNA levels of NF-
κB, FcRn, and pIgR mRNA in the intestinal mucosa of piglets were statistically significantly reduced
(p < 0.01) compared to the control group, especially in the jejunum (Figure 5A–C). Moreover, the
levels of NF-κB, FcRn, and pIgR in the jejunum proximal (JE-P) levels of mRNA were significantly
lower than in the jejunum distal (JE-D). f
p g
f
f
To characterize the effects of PDCoV infection, the levels of NF-κB FcRn, and pIgR in the
intestinal mucosa of infected or non-infected control piglets were examined. The mRNA levels of NF-
κB, FcRn, and pIgR mRNA in the intestinal mucosa of piglets were statistically significantly reduced
(p < 0.01) compared to the control group, especially in the jejunum (Figure 5A–C). Moreover, the
levels of NF-κB, FcRn, and pIgR in the jejunum proximal (JE-P) levels of mRNA were significantly
lower than in the jejunum distal (JE-D). Similar, the protein expression of NF-κB, FcRn, and pIgR
were found to be consistent with the results of RT-PCR (Figure 5D)
To characterize the effects of PDCoV infection, the levels of NF-κB FcRn, and pIgR in the intestinal
mucosa of infected or non-infected control piglets were examined. The mRNA levels of NF-κB, FcRn,
and pIgR mRNA in the intestinal mucosa of piglets were statistically significantly reduced (p < 0.01)
compared to the control group, especially in the jejunum (Figure 5A–C). Moreover, the levels of NF-κB,
FcRn, and pIgR in the jejunum proximal (JE-P) levels of mRNA were significantly lower than in the
jejunum distal (JE-D). Similar, the protein expression of NF-κB, FcRn, and pIgR were found to be
consistent with the results of RT-PCR (Figure 5D). proximal. DU-D: Duodenum dista
proximal. IL-D: ileum distal.
3.4. Gross Pathology and Histopathology 3.4. Gross Pathology and Histopathology
The overall pathological and histological changes for piglets infected with the PDCoV CHN-JS-
2017 strain were determined, and a necropsy performed at 4 dpi. The small intestine (accumulation
of large amounts of yellow fluid) was transparent, thin-walled, and gas-swelled (Figure 4A). The
control group was normal, and no lesions were observed (Figure 4B). Histopathological examination
showed necrosis of small intestinal epithelial cells, atrophy of intestinal villi, and vacuolation in
infected pigs compared with the control group (Figure 4C,D). Consistent with the histopathological
results, the PDCoV antigen was detected by immunohistochemical staining in the cytoplasm of the
The overall pathological and histological changes for piglets infected with the PDCoV CHN-JS-2017
strain were determined, and a necropsy performed at 4 dpi. The small intestine (accumulation of large
amounts of yellow fluid) was transparent, thin-walled, and gas-swelled (Figure 4A). The control group
was normal, and no lesions were observed (Figure 4B). Histopathological examination showed necrosis
of small intestinal epithelial cells, atrophy of intestinal villi, and vacuolation in infected pigs compared
with the control group (Figure 4C,D). Consistent with the histopathological results, the PDCoV antigen
was detected by immunohistochemical staining in the cytoplasm of the infected villous enterocytes
compared with the control group (Figure 4E,F). Taken together, these results indicate that PDCoV
CHN-JS-2017 may cause intestinal lesions in piglets. 8 of 13
8 of 14 Viruses 2020, 12, 79 Figure 4. Intestinal changes of infected and uninfected piglets challenged with PDCoV CHN-JS-2017. (A) Macroscopic damage of piglets challenged with PDCoV at 4 dpi. (B) Macroscopic view of 4 dpi
negative control piglets. (C,D) Hematoxylin and eosin (H&E)-stained jejunal tissue sections of piglets
challenged with PDCoV (C) or negative control piglets (D). (E,F) Immunohistochemically stained
jejunal tissue sections of challenged piglets (E) or negative control piglets (F). Figure 4. Intestinal changes of infected and uninfected piglets challenged with PDCoV CHN-JS-2017. (A) Macroscopic damage of piglets challenged with PDCoV at 4 dpi. (B) Macroscopic view of 4 dpi
negative control piglets. (C,D) Hematoxylin and eosin (H&E)-stained jejunal tissue sections of piglets
challenged with PDCoV (C) or negative control piglets (D). (E,F) Immunohistochemically stained
jejunal tissue sections of challenged piglets (E) or negative control piglets (F). 5 Alt
ti
f NF
B F R
d I R
RNA i
th S
ll I t ti
l M
ft
PDC V I f ti Figure 4. proximal. DU-D: Duodenum dista
proximal. IL-D: ileum distal.
3.4. Gross Pathology and Histopathology Similar, the protein expression of NF-κB, FcRn, and pIgR
were found to be consistent with the results of RT-PCR (Figure 5D)
To characterize the effects of PDCoV infection, the levels of NF-κB FcRn, and pIgR in the intestinal
mucosa of infected or non-infected control piglets were examined. The mRNA levels of NF-κB, FcRn,
and pIgR mRNA in the intestinal mucosa of piglets were statistically significantly reduced (p < 0.01)
compared to the control group, especially in the jejunum (Figure 5A–C). Moreover, the levels of NF-κB,
FcRn, and pIgR in the jejunum proximal (JE-P) levels of mRNA were significantly lower than in the
jejunum distal (JE-D). Similar, the protein expression of NF-κB, FcRn, and pIgR were found to be
consistent with the results of RT-PCR (Figure 5D). 9 of 13
9 of 14 Viruses 2020, 12, 79 Figure 5. Expressions of nuclear factor-kappa B(NF-kB), neonatal Fc receptor (FcRn) and polymeric
immunoglobulin receptor (pIgR) mRNA expression in the small intestinal mucosa of piglets induced
by PDCoV CHN-JS-2017 at 4 dpi. The mRNA levels of NF-kB (A), FcRn (B), and pIgR (C) in the small
intestinal mucosa were determined individually in each animal by RT-qPCR. All data are expressed
as the mean ± SEM of three independent experiments. * p < 0.05, ** p < 0.01, relative to the mock-treated
piglets. Relative amplification of the target genes expression levels was normalized to GAPDH
expression. (D) The protein levels of NF-kB, FcRn, and pIgR at the small intestinal mucosa were
determined individually in each animal by Western blot. DU-P: Duodenum proximal. DU-D:
Duodenum distal. JE-P: jejunum proximal. JE-D: jejunum distal. IL-P: ileum proximal. IL-D: ileum
distal
Figure 5. Expressions of nuclear factor-kappa B(NF-kB), neonatal Fc receptor (FcRn) and polymeric
immunoglobulin receptor (pIgR) mRNA expression in the small intestinal mucosa of piglets induced
by PDCoV CHN-JS-2017 at 4 dpi. The mRNA levels of NF-kB (A), FcRn (B), and pIgR (C) in the
small intestinal mucosa were determined individually in each animal by RT-qPCR. All data are
expressed as the mean ± SEM of three independent experiments. * p < 0.05, ** p < 0.01, relative to
the mock-treated piglets. Relative amplification of the target genes expression levels was normalized
to GAPDH expression. (D) The protein levels of NF-kB, FcRn, and pIgR at the small intestinal
mucosa were determined individually in each animal by Western blot. DU-P: Duodenum proximal. DU-D: Duodenum distal. JE-P: jejunum proximal. proximal. DU-D: Duodenum dista
proximal. IL-D: ileum distal.
3.4. Gross Pathology and Histopathology JE-D: jejunum distal. IL-P: ileum proximal. IL-D:
ileum distal. Figure 5. Expressions of nuclear factor-kappa B(NF-kB), neonatal Fc receptor (FcRn) and polymeric
immunoglobulin receptor (pIgR) mRNA expression in the small intestinal mucosa of piglets induced
by PDCoV CHN-JS-2017 at 4 dpi. The mRNA levels of NF-kB (A), FcRn (B), and pIgR (C) in the small
intestinal mucosa were determined individually in each animal by RT-qPCR. All data are expressed
as the mean ± SEM of three independent experiments. * p < 0.05, ** p < 0.01, relative to the mock-treated
piglets. Relative amplification of the target genes expression levels was normalized to GAPDH
expression. (D) The protein levels of NF-kB, FcRn, and pIgR at the small intestinal mucosa were
determined individually in each animal by Western blot. DU-P: Duodenum proximal. DU-D:
Duodenum distal. JE-P: jejunum proximal. JE-D: jejunum distal. IL-P: ileum proximal. IL-D: ileum
Figure 5. Expressions of nuclear factor-kappa B(NF-kB), neonatal Fc receptor (FcRn) and polymeric
immunoglobulin receptor (pIgR) mRNA expression in the small intestinal mucosa of piglets induced
by PDCoV CHN-JS-2017 at 4 dpi. The mRNA levels of NF-kB (A), FcRn (B), and pIgR (C) in the
small intestinal mucosa were determined individually in each animal by RT-qPCR. All data are
expressed as the mean ± SEM of three independent experiments. * p < 0.05, ** p < 0.01, relative to
the mock-treated piglets. Relative amplification of the target genes expression levels was normalized
to GAPDH expression. (D) The protein levels of NF-kB, FcRn, and pIgR at the small intestinal
mucosa were determined individually in each animal by Western blot. DU-P: Duodenum proximal. DU-D: Duodenum distal. JE-P: jejunum proximal. JE-D: jejunum distal. IL-P: ileum proximal. IL-D:
ileum distal. distal. 3.6. Correlation between NF-κB and FcRn or pIgR in the Small Intestine Mucosa in Infected or Non-Infected
3.6. Correlation between NF-κB and FcRn or pIgR in the Small Intestine Mucosa in Infected or Non-Infected
Control Piglets p g
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Control Piglets
As shown above, the mRNA levels of FcRn, pIgR, and NF-κB genes tend to be lower after PDCoV
infection It has been reported that NF κB is involved in the regulation of FcRn and pIgR expression
As shown above, the mRNA levels of FcRn, pIgR, and NF-κB genes tend to be lower after PDCoV
infection. It has been reported that NF-κB is involved in the regulation of FcRn and pIgR expression in
mice. proximal. DU-D: Duodenum dista
proximal. IL-D: ileum distal.
3.4. Gross Pathology and Histopathology Therefore, the correlation between the mRNA levels of these genes was examined. infection. It has been reported that NF κB is involved in the regulation of FcRn and pIgR expression
in mice. Therefore, the correlation between the mRNA levels of these genes was examined. The levels of NF-κB mRNA were positively correlated with FcRn mRNA levels in uninfected
piglets (p < 0.05) (Figure 6A), while no significant correlation was observed between NF-κB and pIgR
mRNA levels in uninfected piglets (p > 0.05) (Figure 6B). In addition, the mRNA levels of FcRn were
positively correlated with those of pIgR in uninfected piglets (p < 0.05) (Figure 6C). Notably, the levels
of NF-κB and pIgR or FcRn mRNA were positively correlated with each other in infected piglets (p <
0.05) (Figure 6D,E). The levels of FcRn mRNA were positively correlated with pIgR mRNA in the
The levels of NF-κB mRNA were positively correlated with FcRn mRNA levels in uninfected
piglets (p < 0.05) (Figure 6A), while no significant correlation was observed between NF-κB and pIgR
mRNA levels in uninfected piglets (p > 0.05) (Figure 6B). In addition, the mRNA levels of FcRn were
positively correlated with those of pIgR in uninfected piglets (p < 0.05) (Figure 6C). Notably, the levels
of NF-κB and pIgR or FcRn mRNA were positively correlated with each other in infected piglets
(p < 0.05) (Figure 6D,E). The levels of FcRn mRNA were positively correlated with pIgR mRNA in the
small intestine mucosa of infected piglets (Figure 6F). 10 of 13
10 of 14 10 of 13
10 of 14 Viruses 2020, 12, 79 Figure 6. Correlations between the mRNA levels of NF-kB, pIgR and FcRn in the intestinal mucosa of
infected piglets. Significant positive correlations were found between NF-kB and FcRn (A) and FcRn
and pIgR (B), but not between NF-kB and pIgR (C) in uninfected piglets; however, significant positive
correlations were found between NF-kB and FcRn (D), FcRn and pIgR (E), and NF-kB and pIgR (F) in
infected piglets. FcRn ΔCt = Ct FcRn − CtGAPDH, NF-kB ΔCt = Ct NF-kB – Ct GAPDH, pIgR ΔCt = Ct pIgR – Ct
GAPDH
Figure 6. Correlations between the mRNA levels of NF-kB, pIgR and FcRn in the intestinal mucosa of
infected piglets. GAPDH.
4. Discussion Many
researchers have tried to isolate PDCoV, but the relatively low success rate may be related to the type
of disease, the freshness of the sample, the amount of trypsin added to the cells, and the preservation
conditions of the sample. o
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conditions of the sample. To characterize viral isolates, genome-wide sequencing and multi-sequence alignment and
phylogenetic analysis are needed. All known PDCoV strains have high levels of nucleotide sequence
identity. It is worth noting that the PDCoV CHN-JS-2017 strain can be clustered into a clade with
other strains from mainland China, indicating that the current PDCoV strains are prevalent in many
provinces in China. The PDCoV strain CHN-JS-2017 genome is more like the strains from China, the
United States, and South Korea than the Thailand strain, while the PDCoV strains from Thailand and
Vietnam are clustered in another clade. However, whether these unique variants contribute to the
efficiency and virulence of viral replication requires further investigation. Pending further validation,
mutations in the nsp2 and S gene imply that these unique variations contribute to the enhanced
virulence of this strain. Additionally, a 10-nt insertion in this strain was found in the 3′UTR, which is
different to the other PDCoV strain; the PDCoV strain HKU21-8295 from the common-moorhen also
contained this 7-nt insertion, and HKU13-3514 from munia also contained this 1-nt insertion,
To characterize viral isolates, genome-wide sequencing and multi-sequence alignment and
phylogenetic analysis are needed. All known PDCoV strains have high levels of nucleotide sequence
identity. It is worth noting that the PDCoV CHN-JS-2017 strain can be clustered into a clade with
other strains from mainland China, indicating that the current PDCoV strains are prevalent in many
provinces in China. The PDCoV strain CHN-JS-2017 genome is more like the strains from China, the
United States, and South Korea than the Thailand strain, while the PDCoV strains from Thailand and
Vietnam are clustered in another clade. However, whether these unique variants contribute to the
efficiency and virulence of viral replication requires further investigation. Pending further validation,
mutations in the nsp2 and S gene imply that these unique variations contribute to the enhanced
virulence of this strain. GAPDH.
4. Discussion 4. Discussion
Since the first detection of PDCoV in piglet diarrhea in the United States in 2014—the first
confirmed pathogenicity in pigs [4,23]—this new type of porcine intestinal coronavirus has attracted
attention worldwide. In mainland China, the prevalence of PDCoV in pigs is 33.71% in Jiangxi
Province [25], 23.4% in Guangdong Province [26], and 10.2% in Hebei Province [27]. Although the
prevalence of PDCoV worldwide is confirmed, the isolation rate of PDCoV is low. In this study, we
successfully isolated the CHN-JS-2017 strain of PDCoV (which is highly pathogenic to piglets) and
Since the first detection of PDCoV in piglet diarrhea in the United States in 2014—the first
confirmed pathogenicity in pigs [4,23]—this new type of porcine intestinal coronavirus has attracted
attention worldwide. In mainland China, the prevalence of PDCoV in pigs is 33.71% in Jiangxi
Province [25], 23.4% in Guangdong Province [26], and 10.2% in Hebei Province [27]. Although the
prevalence of PDCoV worldwide is confirmed, the isolation rate of PDCoV is low. In this study, we
successfully isolated the CHN-JS-2017 strain of PDCoV (which is highly pathogenic to piglets) and
conducted the preliminary exploration of its pathogenic characteristics. y
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)
conducted the preliminary exploration of its pathogenic characteristics. The isolation of PDCoV is difficult, and the addition of trypsin is essential for the isolation of the
virus. We attempted to isolate the virus from the intestinal contents of seven PDCoV-positive pigs
using LLC-PK cells. Obvious lesions were observed 24 hours after inoculation in the challenged
group and confirmed by IFA and TEM. Only PDCoV CHN-JS-2017 was successfully isolated. Many
researchers have tried to isolate PDCoV, but the relatively low success rate may be related to the type
of disease, the freshness of the sample, the amount of trypsin added to the cells, and the preservation
The isolation of PDCoV is difficult, and the addition of trypsin is essential for the isolation of
the virus. We attempted to isolate the virus from the intestinal contents of seven PDCoV-positive
pigs using LLC-PK cells. Obvious lesions were observed 24 hours after inoculation in the challenged
group and confirmed by IFA and TEM. Only PDCoV CHN-JS-2017 was successfully isolated. proximal. DU-D: Duodenum dista
proximal. IL-D: ileum distal.
3.4. Gross Pathology and Histopathology Significant positive correlations were found between NF-kB and FcRn (A) and FcRn
and pIgR (B), but not between NF-kB and pIgR (C) in uninfected piglets; however, significant positive
correlations were found between NF-kB and FcRn (D), FcRn and pIgR (E), and NF-kB and pIgR (F) in
infected piglets. FcRn ∆Ct = Ct FcRn −CtGAPDH, NF-kB ∆Ct = Ct NF-kB – Ct GAPDH, pIgR ∆Ct = Ct pIgR
– Ct GAPDH. Figure 6. Correlations between the mRNA levels of NF-kB, pIgR and FcRn in the intestinal mucosa of
infected piglets. Significant positive correlations were found between NF-kB and FcRn (A) and FcRn
and pIgR (B), but not between NF-kB and pIgR (C) in uninfected piglets; however, significant positive
correlations were found between NF-kB and FcRn (D), FcRn and pIgR (E), and NF-kB and pIgR (F) in
infected piglets. FcRn ΔCt = Ct FcRn − CtGAPDH, NF-kB ΔCt = Ct NF-kB – Ct GAPDH, pIgR ΔCt = Ct pIgR – Ct
Figure 6. Correlations between the mRNA levels of NF-kB, pIgR and FcRn in the intestinal mucosa of
infected piglets. Significant positive correlations were found between NF-kB and FcRn (A) and FcRn
and pIgR (B), but not between NF-kB and pIgR (C) in uninfected piglets; however, significant positive
correlations were found between NF-kB and FcRn (D), FcRn and pIgR (E), and NF-kB and pIgR (F) in
infected piglets. FcRn ∆Ct = Ct FcRn −CtGAPDH, NF-kB ∆Ct = Ct NF-kB – Ct GAPDH, pIgR ∆Ct = Ct pIgR
– Ct GAPDH. GAPDH.
4. Discussion Additionally, a 10-nt insertion in this strain was found in the 3′UTR, which is
different to the other PDCoV strain; the PDCoV strain HKU21-8295 from the common-moorhen also
contained this 7-nt insertion, and HKU13-3514 from munia also contained this 1-nt insertion, indicating
that these insertions in the 3′UTR may be related to cross-species transmissibility. 11 of 13 Viruses 2020, 12, 79 Viruses 2020, 12, 79 It is worth noting that several groups have confirmed the pathogenicity of conventional pig
PDCoV strains [8–11]. Overall, these results confirm that PDCoV causes severe diarrhea and vomiting
in pigs aged 5–21 days. We used the PDCoV CHN-JS-2017 strain to infect five-day-old newborn piglets
by oral feeding. Our success in inducing severe diarrhea and vomiting in piglets through oral infections
strongly suggests that PDCoV poses a significant threat to newborn piglets on pig farms. In addition,
we collected fecal swabs from piglets challenged with PDCoV and detected viral fecal shedding with
real-time PCR. PDCoV RNA was detected from 1–14 dpi, and no RNA were detected in the control
group piglets during the study period. This indicates that PDCoV infection by fecal-oral contamination
may be the main route of transmission for piglets. In addition, during the necropsy on the seventh
day, large lesions were clearly observed in the small intestine of the piglets [9]. In an earlier report,
microscopic lesions were observed in all parts of the small intestine infected with PEDV [28]. In the
current study, we observed microscopic lesions of the jejuna of CHN-JS-2017 infected piglets, indicative
of PDCoV. The disease caused by PDCoV is milder than the disease caused by PEDV. In addition, the
PDCoV antigen in the cytoplasm of the villous intestinal cells of the piglets challenged with PDCoV
was detected by immunohistochemical analysis. Taken together, these results confirm that the PDCoV
CHN-JS-2017 strain isolated in this study may cause intestinal disease in newborn piglets. IgA and IgG play a major role in mucosal infection immunity; some cytokines or pathogens have
also evolved ways to regulate pIgR and FcRn to facilitate infection [29,30]. Reovirus, LPS (a ligand for
Toll-like receptor 4 (TLR4) TLR4), IFN-γ, and tumor necrosis factor-alpha (TNF-α) all regulate pIgR and
FcRn expression, mainly by the activation of the NF-κB or Janus kinase/signal transducers and activators
of transcription (JAK-STAT) JAK-STAT pathway [31–34]. Author Contributions: S.Q. and Z.L. conceived and designed the experiments and wrote the manuscript; S.Q., X.J.,
Z.G. and W.Z. performed the experiments; S.Q., X.J. and Z.G. analyzed the data; S.Q., Q.X. and Z.L. writing—review
and editing; Z.L. Funding acquisition. All authors have read and agreed to the published version of the manuscript. Conflicts of Interest: The authors declare no conflict of interest. Supplementary Materials: The following are available online at http://www.mdpi.com/1999-4915/12/1/79/s1.
Table S1. Primers for amplification of the PDCoV genomic fragments by RT-qPCR. Table S2. Primers used for
RT-qPCR analysis of the gene expression of piglet intestinal mucosa. Acknowledgments: We would like to thank Pei Zhang at Wuhan Institute of Virology for TEM technical assistance
and Wanpo Zhang at the College of Veterinary Medicine, Huazhong Agricultural University for histopathology
technical assistance. Funding: This work was supported by the National Natural Science Foundation of China (31572500 to Z.L.). pp
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Table S1. Primers for amplification of the PDCoV genomic fragments by RT-qPCR. Table S2. Primers used for
RT-qPCR analysis of the gene expression of piglet intestinal mucosa.
Author Contributions: S.Q. and Z.L. conceived and designed the experiments and wrote the manuscript; S.Q., X.J.,
Z.G. and W.Z. performed the experiments; S.Q., X.J. and Z.G. analyzed the data; S.Q., Q.X. and Z.L. writing—review
and editing; Z.L. Funding acquisition. All authors have read and agreed to the published version of the manuscript.
Funding: This work was supported by the National Natural Science Foundation of China (31572500 to Z.L.).
Acknowledgments: We would like to thank Pei Zhang at Wuhan Institute of Virology for TEM technical assistance
and Wanpo Zhang at the College of Veterinary Medicine, Huazhong Agricultural University for histopathology References [CrossRef] 7. Hu, H.; Jung, K.; Vlasova, A.N.; Saif, L.J. Experimental infection of gnotobiotic pigs with the
cell-culture-adapted porcine deltacoronavirus strain OH-FD22. Arch. Virol. 2016, 161, 3421–3434. [CrossRef] 8. Chen, Q.; Gauger, P.; Stafne, M.; Thomas, J.; Arruda, P.; Burrough, E.; Madson, D.; Brodie, J.; Magstadt, D.;
Derscheid, R.; et al. Pathogenicity and pathogenesis of a United States porcine deltacoronavirus cell culture
isolate in 5-day-old neonatal piglets. Virology 2015, 482, 51–59. [CrossRef] 9. Dong, N.; Fang, L.; Yang, H.; Liu, H.; Du, T.; Fang, P.; Wang, D.; Chen, H.; Xiao, S. Isolation, genomic
characterization, and pathogenicity of a Chinese porcine deltacoronavirus strain CHN-HN-2014. Vet. Microbiol. 2016, 196, 98–106. [CrossRef] 10. Vitosh-Sillman, S.; Loy, J.D.; Brodersen, B.; Kelling, C.; Doster, A.; Topliff, C.; Nelson, E.; Bai, J.; Schirtzinger, E.;
Poulsen, E.; et al. Experimental infection of conventional nursing pigs and their dams with Porcine
deltacoronavirus. J. Vet. Diagn. Investig. Off. Publ. Am. Assoc. Vet. Lab. Diagn. Inc 2016, 28, 486–497. [CrossRef] 11. Xu, Z.; Zhong, H.; Zhou, Q.; Du, Y.; Chen, L.; Zhang, Y.; Xue, C.; Cao, Y. A Highly Pathogenic Strain of Porcine
Deltacoronavirus Caused Watery Diarrhea in Newborn Piglets. Virol. Sin. 2018, 33, 131–141. [CrossRef]
[PubMed] 12. Wang, Y.W.; Yue, H.; Fang, W.; Huang, Y.W. Complete Genome Sequence of Porcine Deltacoronavirus Strain
CH/Sichuan/S27/2012 from Mainland China. Genome Announc. 2015, 3, e00945. [CrossRef] [PubMed] 13. Li, Z.; Palaniyandi, S.; Zeng, R.; Tuo, W.; Roopenian, D.C.; Zhu, X. Transfer of IgG in the female genital tract
by MHC class I-related neonatal Fc receptor (FcRn) confers protective immunity to vaginal infection. Proc. Natl. Acad. Sci. USA 2011, 108, 4388–4393. [CrossRef] [PubMed] 14. Luo, J.; Fang, L.; Dong, N.; Fang, P.; Ding, Z.; Wang, D.; Chen, H.; Xiao, S. Porcine deltacoronavirus (PDCoV)
infection suppresses RIG-I-mediated interferon-beta production. Virology 2016, 495, 10–17. [CrossRef] 15. Fang, P.; Fang, L.; Ren J; Hong Y; Liu X; Zhao Y; Wang D; Peng G; Xiao S. Porcine Deltacoronavirus Accessory
Protein NS6 Antagonizes Interferon Beta Production by Interfering with the Binding of RIG-I/MDA5 to
Double-Stranded RNA. J. Virol. 2018, 92, e00712-18. [CrossRef] 16. Chen, J.; Fang, P.; Wang, M.; Peng, Q.; Ren, J.; Wang, D.; Peng, G.; Fang, L.; Xiao, S.; Ding, Z. Porcine
deltacoronavirus nucleocapsid protein antagonizes IFN-beta production by impairing dsRNA and PACT
binding to RIG-I. Virus Genes 2019, 55, 520–531. [CrossRef] 17. Likai, J.; Shasha, L.; Wenxian, Z.; Jingjiao, M.; Jianhe, S.; Hengan, W.; Yaxian, Y. GAPDH.
4. Discussion 12 of 13 12 of 13 Viruses 2020, 12, 79 References 1. Woo, P.C.; Lau, S.K.; Lam, C.S.; Lau, C.C.; Tsang, A.K.; Lau, J.H.; Bai, R.; Teng, J.L.; Tsang, C.C.; Wang, M.;
et al. Discovery of seven novel Mammalian and avian coronaviruses in the genus deltacoronavirus supports
bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as
the gene source of gammacoronavirus and deltacoronavirus. J. Virol. 2012, 86, 3995–4008. [PubMed] 1. Woo, P.C.; Lau, S.K.; Lam, C.S.; Lau, C.C.; Tsang, A.K.; Lau, J.H.; Bai, R.; Teng, J.L.; Tsang, C.C.; Wang, M.;
et al. Discovery of seven novel Mammalian and avian coronaviruses in the genus deltacoronavirus supports
bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as
the gene source of gammacoronavirus and deltacoronavirus. J. Virol. 2012, 86, 3995–4008. [PubMed] 2. Marthaler, D.; Raymond, L.; Jiang, Y.; Collins, J.; Rossow, K.; Rovira, A. Rapid detection, complete genome
sequencing, and phylogenetic analysis of porcine deltacoronavirus. Emerg. Infect. Dis. 2014, 20, 1347–1350. [CrossRef] [PubMed] 2. Marthaler, D.; Raymond, L.; Jiang, Y.; Collins, J.; Rossow, K.; Rovira, A. Rapid detection, complete genome
sequencing, and phylogenetic analysis of porcine deltacoronavirus. Emerg. Infect. Dis. 2014, 20, 1347–1350. [CrossRef] [PubMed] 3. Saeng-Chuto, K.; Lorsirigool, A.; Temeeyasen, G.; Vui, D.T.; Stott, C.J.; Madapong, A.; Tripipat, T.; Wegner, M.;
Intrakamhaeng, M.; Chongcharoen, W.; et al. Different Lineage of Porcine Deltacoronavirus in Thailand,
Vietnam and Lao PDR in 2015. Transbound. Emerg. Dis. 2017, 64, 3–10. [CrossRef] [PubMed] 3. Saeng-Chuto, K.; Lorsirigool, A.; Temeeyasen, G.; Vui, D.T.; Stott, C.J.; Madapong, A.; Tripipat, T.; Wegner, M.;
Intrakamhaeng, M.; Chongcharoen, W.; et al. Different Lineage of Porcine Deltacoronavirus in Thailand,
Vietnam and Lao PDR in 2015. Transbound. Emerg. Dis. 2017, 64, 3–10. [CrossRef] [PubMed] 4. Wang, L.; Byrum, B.; Zhang, Y. Detection and genetic characterization of deltacoronavirus in pigs, Ohio,
USA, 2014. Emerg. Infect. Dis. 2014, 20, 1227–1230. [CrossRef] [PubMed] 4. Wang, L.; Byrum, B.; Zhang, Y. Detection and genetic characterization of deltacoronavirus in pigs, Ohio,
USA, 2014. Emerg. Infect. Dis. 2014, 20, 1227–1230. [CrossRef] [PubMed] 5. Zhang, J. Porcine deltacoronavirus: Overview of infection dynamics, diagnostic methods, prevalence and
genetic evolution. Virus Res. 2016, 226, 71–84. [CrossRef] 5. Zhang, J. Porcine deltacoronavirus: Overview of infection dynamics, diagnostic methods, prevalence and
genetic evolution. Virus Res. 2016, 226, 71–84. [CrossRef] 6. Jung, K.; Hu, H.; Eyerly, B.; Lu, Z.; Chepngeno, J.; Saif, L.J. Pathogenicity of 2 porcine deltacoronavirus
strains in gnotobiotic pigs. Emerg. Infect. Dis. 2015, 21, 650–654. GAPDH.
4. Discussion The down-regulation of FcRn and pIgR by
reovirus have been shown in the tracheal mucosa of simian-human immunodeficiency virus/simian
immunodeficiency virus (SHIV/SIV)-infected rhesus monkeys [35,36]. Human cytomegalovirus evades
humoral immunity by the degradation of FcRn [30]. TGEV-induced FcRn expression by activating NF-κB
signaling in porcine small intestinal epithelial (IPEC-J2) cells [19]. However, PDCoV CHN-JS-2017
significantly down-regulated FcRn, pIgR, and NF-κB expressions, and they were positively related
to each other in infected piglets. Therefore, the reduced levels of FcRn and pIgR mRNA might be
attributed to the reduced levels of NF-κB. In addition, PDCoV might reduce the ability of FcRn and
pIgR to transport PDCoV-specific IgG and IgA across the mucosal epithelium, resulting in impaired
intestinal immunity in the mucosa, reducing the host’s anti-infection ability. These data demonstrated
the altered expression of FcRn and pIgR in coronavirus, and by extension, coronavirus infections,
which might have contributed to providing another insight into the immune escape strategy of PDCoV. g
p
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gy
In summary, we successfully isolated and characterized PDCoV CHN-JS-2017 in cultured cells,
and CHN-JS-2017 is pathogenic in piglets. Furthermore, the inoculated piglets showed down-regulated
FcRn and pIgR expression by NF-κB mRNA. At the same time, we found that the expression of FcRn
and pIgR of piglets infected with PDCoV is positively correlated with NF-κB. The results help us to
understand the molecular epidemiology and pathogenesis of PDCoV and also provide insights into
the development of effective vaccines against this significant pathogen. Supplementary Materials: The following are available online at http://www.mdpi.com/1999-4915/12/1/79/s1. Table S1. Primers for amplification of the PDCoV genomic fragments by RT-qPCR. Table S2. Primers used for
RT-qPCR analysis of the gene expression of piglet intestinal mucosa. Author Contributions: S.Q. and Z.L. conceived and designed the experiments and wrote the manuscript; S.Q., X.J.,
Z.G. and W.Z. performed the experiments; S.Q., X.J. and Z.G. analyzed the data; S.Q., Q.X. and Z.L. writing—review
and editing; Z.L. Funding acquisition. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the National Natural Science Foundation of China (31572500 to Z.L.). Acknowledgments: We would like to thank Pei Zhang at Wuhan Institute of Virology for TEM technical assistance
and Wanpo Zhang at the College of Veterinary Medicine, Huazhong Agricultural University for histopathology
technical assistance. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References Porcine Deltacoronavirus
Nucleocapsid Protein Suppressed IFN-beta Production by Interfering Porcine RIG-I dsRNA-Binding and
K63-Linked Polyubiquitination. Front. Immunol. 2019, 10, 1024. [CrossRef] 18. Xu, Z.; Zhong, H.; Huang, S.; Zhou, Q.; Du, Y.; Chen, L.; Xue, C.; Cao, Y. Porcine deltacoronavirus induces
TLR3, IL-12, IFN-alpha, IFN-beta and PKR mRNA expression in infected Peyer’s patches in vivo. Vet. Microbiol. 2019, 228, 226–233. 13 of 13 13 of 13 Viruses 2020, 12, 79 19. Guo, J.; Li, F.; Qian, S.; Bi, D.; He, Q.; Jin, H.; Luo, R.; Li, S.; Meng, X.; Li, Z. TGEV infection up-regulates
FcRn expression via activation of NF-kappaB signaling. Sci. Rep. 2016, 6, 32154. [CrossRef] 20. Hu, H.; Jung, K.; Vlasova, A.N.; Chepngeno, J.; Lu, Z.; Wang, Q.; Saif, L.J. Isolation and characterization of
porcine deltacoronavirus from pigs with diarrhea in the United States. J. Clin. Microbiol. 2015, 53, 1537–1548. [CrossRef] 21. Lorsirigool, A.; Saeng-Chuto, K.; Madapong, A.; Temeeyasen, G.; Tripipat, T.; Kaewprommal, P.;
Tantituvanont, A.; Piriyapongsa, J.; Nilubol, D. The genetic diversity and complete genome analysis
of two novel porcine deltacoronavirus isolates in Thailand in 2015. Virus Genes 2017, 53, 240–248. [CrossRef]
[PubMed] 22. Kumar, S.; Stecher, G.; Tamura, K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger
Datasets. Mol. Biol. Evol. 2016, 33, 1870–1874. [CrossRef] [PubMed] 23. Jung, K.; Wang, Q.; Scheuer, K.A.; Lu, Z.; Zhang, Y.; Saif, L.J. Pathology of US porcine epidemic diarrhea
irus strain PC21A in gnotobiotic pigs Emerg Infect Dis 2014 20 662 665 [CrossRef] [PubMed] 23. Jung, K.; Wang, Q.; Scheuer, K.A.; Lu, Z.; Zhang, Y.; Saif, L.J. Pathology of US porcine epidemic diarrhea
virus strain PC21A in gnotobiotic pigs. Emerg. Infect. Dis. 2014, 20, 662–665. [CrossRef] [PubMed] 24. Temeeyasen, G.; Sinha, A.; Gimenez-Lirola, L.G.; Zhang, J.Q.; Pineyro, P.E. Differential gene modulation of
pattern-recognition receptor TLR and RIG-I-like and downstream mediators on intestinal mucosa of pigs
infected with PEDV non S-INDEL and PEDV S-INDEL strains. Virology 2018, 517, 188–198. [CrossRef] 25. Song, D.; Zhou, X.; Peng, Q.; Chen, Y.; Zhang, F.; Huang, T.; Zhang, T.; Li, A.; Huang, D.; Wu, Q.; et al. Newly
Emerged Porcine Deltacoronavirus Associated With Diarrhoea in Swine in China: Identification, Prevalence
and Full-Length Genome Sequence Analysis. Transbound. Emerg. Dis. 2015, 62, 575–580. [CrossRef] 26. Chen, F.; Zhu, Y.; Wu, M.; Ku, X.; Yao, L.; He, Q. Full-Length Genome Characterization of Chinese Porcine
Deltacoronavirus Strain CH/SXD1/2015. Genome Announc. 2015, 3, e01284. [CrossRef] 27. References Liu, B.J.; Zuo, Y.Z.; Gu, W.Y.; Luo, S.X.; Shi, Q.K.; Hou, L.S.; Zhong, F.; Fan, J.H. Isolation and phylogenetic
analysis of porcine deltacoronavirus from pigs with diarrhoea in Hebei province, China. Transbound. Emerg. Dis. 2018, 65, 874–882. [CrossRef] 28. Madson, D.M.;
Magstadt, D.R.;
Arruda, P.H.;
Hoang, H.;
Sun, D.;
Bower, L.P.;
Bhandari, M.;
Burrough, E.R.; Gauger, P.C.; Pillatzki, A.E.; et al. Pathogenesis of porcine epidemic diarrhea virus
isolate (US/Iowa/18984/2013) in 3-week-old weaned pigs. Vet. Microbiol. 2014, 174, 60–68. [CrossRef] 29. Turula, H.; Wobus, C.E. The Role of the Polymeric Immunoglobulin Receptor and Secretory Immunog
during Mucosal Infection and Immunity. Viruses 2018, 10, 237. [CrossRef] 30. Liu, X.; Palaniyandi, S.; Zhu, I.; Tang, J.; Li, W.; Wu, X.; Ochsner, S.P.; Pauza, C.D.; Cohen, J.I.; Zhu, X. Human cytomegalovirus evades antibody-mediated immunity through endoplasmic reticulum-associated
degradation of the FcRn receptor. Nat. Commun. 2019, 10, 3020. [CrossRef] 31. Johansen, F.E.; Brandtzaeg, P. Transcriptional regulation of the mucosal IgA system. Trends Immunol. 2004,
25, 150–157. [CrossRef] [PubMed] 32. Pal, K.; Kaetzel, C.S.; Brundage, K.; Cunningham, C.A.; Cuff, C.F. Regulation of polymeric immunoglobulin
receptor expression by reovirus. J. Gen. Virol. 2005, 86, 2347–2357. [CrossRef] [PubMed] 33. Liu, X.; Ye, L.; Christianson, G.J.; Yang, J.Q.; Roopenian, D.C.; Zhu, X. NF-kappaB signaling regulates
functional expression of the MHC class I-related neonatal Fc receptor for IgG via intronic binding sequences. J. Immunol. 2007, 179, 2999–3011. [CrossRef] [PubMed] 34. Liu, X.; Ye, L.; Bai, Y.; Mojidi, H.; Simister, N.E.; Zhu, X. Activation of the JAK/STAT-1 signaling pathway by
IFN-gamma can down-regulate functional expression of the MHC class I-related neonatal Fc receptor for
IgG. J. Immunol. (Baltimore, Md. 1950) 2008, 181, 449–463. [CrossRef] [PubMed] 35. Wang, Y.; Yang, G.B. Alteration of Polymeric Immunoglobulin Receptor and Neonatal Fc Receptor Expression
in the Gut Mucosa of Immunodeficiency Virus-Infected Rhesus Macaques. Scand. J. Immunol. 2016, 83,
235–243. [CrossRef] 36. Li, D.; Wang, F.J.; Yu, L.; Yao, W.R.; Cui, Y.F.; Yang, G.B. Expression of pIgR in the tracheal mucosa of
SHIV/SIV-infected rhesus macaques. Zool. Res. 2017, 38, 44–48. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/). © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/). References © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Portraiture, Surveillance, and the Continuity Aesthetic of Blur
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Portraiture, Surveillance, and the Continuity Aesthetic of Blur Stefka Hristova Portraiture, Surveillance, and the Continuity
Aesthetic of Blur Stefka Hristova Frames Cinema Journal, Issue 18 (June 2021) Portraiture, Surveillance, and the Continuity Aesthetic of Blur
Stefka Hristova Portraiture, Surveillance, and the Continuity Aesthetic of Blur Introduction With the increasing transformation of photography away from a camera-based analogue
image-making process into a computerised set of procedures, the ontology of the photographic
image has been challenged. Portraits in particular have become reconfigured into what Mark B. Hansen has called “digital facial images” and Mitra Azar has subsequently reworked into
“algorithmic facial images.” 1 This transition has amplified the role of portraiture as a
representational device, as a node in a network of distribution, and as a process. Portraits now
function simultaneously as modes of self-expression, as networked data, and as the result of
algorithmic logics. This shift in the ways in which portraits circulate in culture speaks to what
Grace Kingston and Michael Goddard have described as the essence of the “networked image.” 2
They articulate the emergence of “dual beings with two habitations: one in a conventional organic
body, delimiting the space and time, a ‘here and now’; and the other taking the form of a data cloud
distributed across multiple networks and housed in who-knows-what and who-knows-where, in
server farms and databanks.”3 This transfiguration of the image from a visual to a data artefact is particularly evident in
the case of smartphone photography. The move from analogue camera-based portraits to mobile
device networked images has challenged the ontological status of the photograph. In its initial
ontology, photography was seen as a way to record the word visually and truthfully – to write with
light. As Daniel Rubenstein and Katrina Sluis write, 69 Frames Cinema Journal, Issue 18 (June 2021) photography has always been embedded in what Alan Sekula terms structures of representation
and repression, in the context of smartphone photography, these two trajectories have merged even
more profoundly.7 In this article, I investigate the ways in which smartphone images operate as
both self-portraits and as raw data harnessed in facial recognition and surveillance apparatuses. First, I outline a longer historical trajectory in which portraits have been used both as means of
representation as well as means for anthropometric research and surveillance in the context of
policing. Next, I highlight the use of smart phone portraits and selfies in AI-driven biometric
research that seeks to articulate biotypes about race, gender, sexual orientation, political
preference, etc. Further, I argue that the popularity of the selfie has led to the introduction of
pervasive surveillance technologies that uses front-facing cameras. These surveillance
technologies have become a staple of smart phone technology and now operate in a diverse set of
contexts: from border checkpoints to grocery store kiosks and autonomous vehicles driver assistant
technology. Last, I expose the mimicry of smartphone data images of people as portraits and selfies
through by highlighting the conventions that obscure their role as surveillance and biometric data. I argue that this masquerade is carried through the “aesthetics of continuity” of blur and bokeh,
which transposes the photographic portraiture convention of using shallow depth of field onto the
mobile image through the use of algorithms. photography has always been embedded in what Alan Sekula terms structures of representation
and repression, in the context of smartphone photography, these two trajectories have merged even
more profoundly.7 In this article, I investigate the ways in which smartphone images operate as
both self-portraits and as raw data harnessed in facial recognition and surveillance apparatuses. First, I outline a longer historical trajectory in which portraits have been used both as means of
representation as well as means for anthropometric research and surveillance in the context of
policing. Next, I highlight the use of smart phone portraits and selfies in AI-driven biometric
research that seeks to articulate biotypes about race, gender, sexual orientation, political
preference, etc. Further, I argue that the popularity of the selfie has led to the introduction of
pervasive surveillance technologies that uses front-facing cameras. Frames Cinema Journal, Issue 18 (June 2021) These surveillance
technologies have become a staple of smart phone technology and now operate in a diverse set of
contexts: from border checkpoints to grocery store kiosks and autonomous vehicles driver assistant
technology. Last, I expose the mimicry of smartphone data images of people as portraits and selfies
through by highlighting the conventions that obscure their role as surveillance and biometric data. I argue that this masquerade is carried through the “aesthetics of continuity” of blur and bokeh,
which transposes the photographic portraiture convention of using shallow depth of field onto the
mobile image through the use of algorithms. Frames Cinema Journal, Issue 18 (June 2021) An image on the screen of a smartphone or a laptop looks like a photograph not
because it has some ontological relationship to the object in the world, but because the
algorithmic interventions that ensure that what is registered on the camera’s
CCD/CMOS sensor is eventually output as something that a human would understand
as a photograph.4 The smartphone photograph was validated as an instance of “photography” by the continued use
of canonical visual devices. The image generated on our phones looks like a photograph or a
portrait, and thus we assume that it is one and can represent us in a fashion similar to that delivered
by traditional photography. This mimicry obscures the role of smartphone photographs as data-
sets used in both surveillance as well as algorithmic research about race, gender, age, sexual
orientation, political orientation, emotional state, etc. The transplanting of visual conventions
between different visual image-making processes is precisely what Lev Manovich refers to as an
“aesthetics of continuity.”5 The “aesthetics of continuity” patches over the disparate use of digital
images as data. Data that is relevant for machine vision and machine learning and which is relevant
to humans only in a secondary capacity. It is through the continuous use of conventions of portraiture that smartphone image-
making parades as photography-based portraiture, even though its main function as a “network
image” is to operate as “invisible” and further “operational” image rather than “visual” image.6 In
other words, while consumers believe that they are participating in a visual regime of photography-
based portraiture, the images that they generate are used in contemporary culture as raw data that
trains a wide range of algorithms. The image is created by and for a set of computer commands. It
is the “aesthetics of continuity” that obscures the important ways in which smartphone images,
posing as self-portraits, have come to fuel algorithmically-driven surveillance assemblages. While 70 Frames Cinema Journal, Issue 18 (June 2021) Frames Cinema Journal, Issue 18 (June 2021) Frames Cinema Journal, Issue 18 (June 2021) Facial Recognition and Surveillance From its inception, portraiture has acted both as a way of representing identity as well as a
way of articulating quantified selves.8 While this idea resonates with the contemporary use of AI
and facial recognition, I would like to highlight the ways in which scientists as well as
photographic critics of the time harnessed this idea. Joshua Lauer has detailed the ways in which 71 Frames Cinema Journal, Issue 18 (June 2021) Frames Cinema Journal, Issue 18 (June 2021) Frames Cinema Journal, Issue 18 (June 2021) as early as the 1880s, the portable camera was seen as a surveillance tool. Lauer writes that “the
respectable soft surveillance of family albums and honorific photography can be contrasted with
the camera’s repressive function as an instrument for detecting, classifying, and controlling social
deviance.”9 Alan Sekula has written extensively about the ways in which photography has been
coupled with both portraiture and police surveillance since its beginning. He argued that in the 19th
century, photographic portraiture came to “establish and delimit the terrain of the other, to define
both the generalized look – the typology – and the contingent instance of deviance and social
pathology.”10 These processes were made possible by the linkage of photography to a “truth-
apparatus” as the “camera is integrated into a larger ensemble: a bureaucratic clerical-statistic form
of ‘intelligence’.”11 In other words, photography became meaningful as a form of knowledge only
when accompanied by data. As Sekula demonstrates, Alphonse Bertillon’s system of policing as
well as Francis Galton’s anthropometric and racial human classification systems depended on both
photography and data – it is anthropometric data that anchored photography into an archive.12
Bertillon created the “first effective modern system of criminal identification” by coupling facial
measurements with photography.13 His system, however was rooted in racial hierarchies. Bertillon’s contribution to racial anthropology comes from his book Ethnographie moderne: Les
Racial Sauvages, in which he describes and measures the bodily structure of the “lower races”.14
In a passage on the cranial measurement of his subjects, he compares the Hottentot head to the
Parisian head (1250 vs 1500) in order to conclude that the typical Hottentot has the mental capacity
of an “idiot” in Paris.15 Galton similarly conducted extensive anthropometric studies that included
facial measurements and photographic documentation. He argued that by using composite
portraiture he would be able to identify a “biologically determined criminal type.”16 Galton coined
the phrase eugenics as a way to describe the science and idea of breeding “human stock” and was as early as the 1880s, the portable camera was seen as a surveillance tool. Lauer writes that “the
respectable soft surveillance of family albums and honorific photography can be contrasted with
the camera’s repressive function as an instrument for detecting, classifying, and controlling social
deviance.”9 Alan Sekula has written extensively about the ways in which photography has been
coupled with both portraiture and police surveillance since its beginning. Frames Cinema Journal, Issue 18 (June 2021) He argued that in the 19th
century, photographic portraiture came to “establish and delimit the terrain of the other, to define
both the generalized look – the typology – and the contingent instance of deviance and social
pathology.”10 These processes were made possible by the linkage of photography to a “truth-
apparatus” as the “camera is integrated into a larger ensemble: a bureaucratic clerical-statistic form
of ‘intelligence’.”11 In other words, photography became meaningful as a form of knowledge only
when accompanied by data. As Sekula demonstrates, Alphonse Bertillon’s system of policing as
well as Francis Galton’s anthropometric and racial human classification systems depended on both
photography and data – it is anthropometric data that anchored photography into an archive.12
Bertillon created the “first effective modern system of criminal identification” by coupling facial
measurements with photography.13 His system, however was rooted in racial hierarchies. Bertillon’s contribution to racial anthropology comes from his book Ethnographie moderne: Les
Racial Sauvages, in which he describes and measures the bodily structure of the “lower races”.14
In a passage on the cranial measurement of his subjects, he compares the Hottentot head to the
Parisian head (1250 vs 1500) in order to conclude that the typical Hottentot has the mental capacity
of an “idiot” in Paris.15 Galton similarly conducted extensive anthropometric studies that included
facial measurements and photographic documentation. He argued that by using composite
portraiture he would be able to identify a “biologically determined criminal type ”16 Galton coined 72 Frames Cinema Journal, Issue 18 (June 2021) Frames Cinema Journal, Issue 18 (June 2021) Frames Cinema Journal, Issue 18 (June 2021) the first to introduce statistical principles to the study of human intelligence. His work was also
rooted in deep-seeded racism. Galton travelled to South Africa in 1851 – a journey he
commemorated in his 1853 book Narrative of a Traveler to Tropical South Africa. In this book,
he describes the Hottentot people he encountered as having a face that is common among the
prisoners in England – a “felon face” as he put it.17 In both cases, the photograph acted as metadata
to the data of the catalogue card. In other words, the collection of data about subjects seen as
aberrant was conducted in the realm of the physical – the subject him/herself was subjected to
measurement. The photograph performed an important function of making data recognisable to
human agents of surveillance and thus legitimising the idea of biotypes. The idea of the face as a source of visual data was evoked not only by champions of
anthropometrics such as Bertillon and Galton, but also by photography critics writing about the
status of photography as art more broadly. The latter group is best represented by Lady Elizabeth
Eastlake who, in 1857, positioned portraiture as caught between representation and
quantification.18 In the contested case of portraiture, where photography replaced miniature
painting, she asks: What indeed are nine-tenths of those facial maps called photographic portraits, but
accurate landmarks and measurements for loving eyes and memories to deck with
beauty and animate with expression, in perfect certainty, that the ground-plan is
founded upon fact?19 These “facial maps” render visible one’s beauty, expression, as well as the “variable stages of
insanity.”20 Eastlake’s work echoes a number of contemporary studies that link photography to the 73 Frames Cinema Journal, Issue 18 (June 2021) Frames Cinema Journal, Issue 18 (June 2021) Frames Cinema Journal, Issue 18 (June 2021) Frames Cinema Journal, Issue 18 (June 2021) study of hysteria, and hence the surveillance of affect. 19th century neurologist Jean-Martin
Charcot studied hysteria by photographing the facial expressions of his medical subjects.21 The
facial maps deployed by Charcot attempted to taxonomize hysteria. The face, indeed, was to
become a truthful indicator of madness. Sander Gilman’s volume The Face of Madness is a primer
on the rise of psychiatric photography and the work of the English alienist Hugh W. Diamond in
particular.22 In another study from the 1850s, Guillaume-Benjamin-Armand Duchenne (de
Boulogne) used photographs to study the expression of emotions on human faces, “which he
provoked through electrical stimuli.”23 In opposition to these views, in the 1870s, Charles Darwin
conducted similar research, although he concluded that hysteria or insanity cannot be detected
from facial expressions, or indeed, from any photography at all. The portrait thus became
harnessed in anthropometric studies that attempted to justify the superiority of whiteness, in
systems and scientific discourses that claimed that both criminality and intelligence are
biologically defined by the size and shape of one’s head, and, last but not least, that hysteria and
human emotions more broadly can be determined accurately by one’s facial expression. These
discourses grounded photography in a knowledge domain driven by data and running counter to
the idea of photography as means of identity expression. The trend of using portraits to train surveillance and authentication systems because of
their ability to isolate faces and people permeates contemporary algorithmic culture as well. As
Joseph Ferenbok aptly points out, “[a]s faces, and the people behind them, are becoming more
readable by the surveillance authorities, the technologies and overall socio-technical assemblage
supporting the surveillance practices are becoming more sophisticated, complex, and opaque.”24
In algorithmic technology development, portraiture has been used in order to access one’s race,
gender, age, sexual orientation, emotional state, and political preference.25 74 74 Frames Cinema Journal, Issue 18 (June 2021) Frames Cinema Journal, Issue 18 (June 2021) Frames Cinema Journal, Issue 18 (June 2021) Smartphone photography has played an important role in the development of biometric
algorithms that aim to establish stable biotypes. Notable here is the Adience dataset, that has been
used extensively in training algorithms to detect gender and age based on selfies.26 Adience is a
large dataset that contains images taken with iPhone 5 or later smartphones.27 It contains 26,580
images, found “in the wild,” which means posted on the Internet. This database has been used by
the developers of the Face Image Project Gill Levi and Tal Hassner to conduct research on AI-
driven age and gender taxonomies.28 Smart phone images have also fuelled AI research on human emotion in particular. A
contemporary database that uses selfies and portraits in relation to affect technologies is the
infamous AffectNet: “a new database of facial expressions in the wild” which contains more than
one million facial images collected from the Internet.29 Numerous contemporary studies have
harnessed “loving eyes” as data points useful in recognizing human emotions. Affect recognition
technology has become even more pervasive and has thus revived 19th century conventions that
supported the problematic studies of hysteria.30 More specifically, it has renewed the belief that
hysteria, as well as emotions more broadly, can be read through a quantitative analysis of facial
features. While in the 19th century, photographic data of faces was disconnected from the portrait
studio, these two practices have become increasingly conflated in the contemporary algorithmic
landscape. Now, images taken by our mobile devices masquerade as photographs, as portraits, as
selfies; at the same time, they operate as data-points, as information, as the raw material for AI-
driven recognition. 75 Frames Cinema Journal, Issue 18 (June 2021) Frames Cinema Journal, Issue 18 (June 2021) Selfie to Self-Capture The doubling of photography as means of identity expression and as tool for visual data
gathering is evident in the case of selfie photography. Having outlined the ways in which portrait
photography from its beginning has been wedded to discourses of anthropometrics, I want to draw
attention to the significant role selfies have played in the emergence of contemporary algorithmic-
driven biometrics. Selfies first appeared in the early 2000s, initially as ways to document one’s own presence
through the use of mirrors, self-timers, and later, a forward-facing lens. Selfies are part of a longer
tradition of self-portraiture.31 In the context of mobile technologies, selfies became connected to
youth cultures and came to represent “self-performances where young people self-confidently
participate in representing their own narratives in playful ways.”32 Selfies were made possible by
the use of a front-facing camera on mobile devices. These cameras emerged in 2010 with the
introduction of Apple’s iPhone 4 and at first offered pixelated, low-quality visual images, since
the lens was of secondary quality compared to the rear one.33 Selfies entered the popular discourse
in 2013 when they were officially added to the Oxford English Dictionary, defined as: “a photo of
yourself that you take, typically with a smartphone or webcam, and usually put on social media.”34 These images were understood as taken by mobile phone or webcam and posted on social media
and became a visual signature for urban youth.35 As The Guardian wrote in 2013, selfies became
“the self-portrait of the digital age.”36 This mode of self-expression has been both condemned as
narcissistic and praised as an aspect of geek culture. Further, selfies have been connected to
political agency.37 As Mona Kasra has argued in relation to Aliaa Magda Elmahdy’s self-portraits,
selfies can also become “deliberate and personal acts of political expression” for youth that
“resituate political knowledge, power, and information distribution.”38 Clair Hampton’s analysis 76 Frames Cinema Journal, Issue 18 (June 2021) Frames Cinema Journal, Issue 18 (June 2021) Frames Cinema Journal, Issue 18 (June 2021) Frames Cinema Journal, Issue 18 (June 2021) Frames Cinema Journal, Issue 18 (June 2021) of the #nomakeupselfie provides yet another example of the ways in which the selfie has been
harnessed for the purposes of challenging hegemonic structures.39 This context is important
because the ubiquity of the selfie increased our comfort with front-facing cameras and articulated
a discourse in which images produced through such camera are seen as intrinsically linked to
questions of representation rather than surveillance. Selfies have also been harnessed as big data for algorithmic research. The Selfie Data Set
published by the University of Central Florida’s Center for Research in Computer Vision is a great
example.40 According to the website, … [the] Selfie dataset contains 46,836 selfie images annotated with 36 different
attributes divided into several categories as follows. Gender: is female. Age: baby,
child, teenager, youth, middle age, senior. Race: white, black, asian. Face shape: oval,
round, heart. Facial gestures: smiling, frowning, mouth open, tongue out, duck face. Hair color: black, blond, brown, red. Hair shape: curly, straight, braid. Accessories:
glasses, sunglasses, lipstick, hat, earphone. Misc.: showing cellphone, using mirror,
having braces, partial face. Lighting condition: harsh, dim.41 This selfie database is exemplary of the ways in which self-portraits have been harnessed for the
purposes of facial recognition. Here the selfies are transformed into data points and circulated in
big data structures. In another instance, selfie data sets were created by scraping Instagram
accounts for images tagged with the hashtag #selfie.42 As Kate Crawford and Trevor Paglen have
aptly noted, these definitions are 77 77 Frames Cinema Journal, Issue 18 (June 2021) Frames Cinema Journal, Issue 18 (June 2021) unsubstantiated and unstable epistemological and metaphysical assumptions about the
nature of images, labels, categorization, and representation [that] hark back to
historical approaches where people were visually assessed and classified as a tool of
oppression and race science43 In the context of algorithmic surveillance-based culture, selfie images have provided yet
another avenue for training facial recognition and surveillance systems and have undermined the
liberatory potential they were once endowed with. Recently, the term “selfie” itself has taken on a
definition that is more closely related to surveillance. On smartphone devices, facial recognition
authentication has become a standard feature. This harnessing of the selfie as a mode of facial
recognition is based on a new surveillance-based definition of what a selfie is. In a research article
from 2019 titled “DocFace+: ID Document to Selfie Matching,” Yichun Shi and Anil K. Jain argue
for the necessity to match accurately ID documents to “selfie” images. As part of this paper, the
authors offer a redefinition of what the term “selfie” means in the context of surveillance-oriented
algorithmic culture: “Technically, the word selfie refers to self-captured photos from mobile
phones. But here, we define “selfies” as any self-captured live face photos, including those from
mobile phones and kiosks.”44 What is new here is that “selfies” no longer require one to physically take the photograph
oneself. Selfies are images of “the self” captured by automated surveillance systems. The agency
behind consciously taking one’s own photograph is negated by the automation of the process. The
“selfie” is recognisable only through what Lev Manovich has coined as the “aesthetics of
continuity” in which one sees oneself as the image is being recorded. Here the “selfies” are taken
by surveillance systems such as Australia’s “SmartGate,” the e-Passport gates in the UK, 78 Frames Cinema Journal, Issue 18 (June 2021) Frames Cinema Journal, Issue 18 (June 2021) against a blurry background.48 Here, selfie biometrics is defined as “an authentication mechanism
where a user captures images of her biometric traits (such as the face or ocular region) by using
the imaging sensors available in the device itself.”49 The idea of the selfie here has again shifted
away from modes of representation and agency, towards an automated “capture” of biometric
traits. Indeed, the selfie functions no longer as a self-portrait, but rather as a data-gathering
mechanism – a “selfie capture.” Further, the authors distinguish between three types of selfie
biometrics: face, ocular biometrics (imaging and use of characteristic features extracted from the
eyes for personal recognition), and fingerphoto: “touchless fingerprint recognition technology,
where the back-facing smartphone cameras acquire high-resolution photographs of finger ridge
patterns.”50 These features have commonly been used in both anthropometrics and biometrics
and have been seen as staples of identification, policing, and surveillance. What is interesting in
this article is the articulation of the so-called “soft” biometrics. In this biometric profile,
ethnicity, gender, and age are assessed and recorded. Another chapter in this book explicitly
links the raise of selfie soft biometrics with the front-facing camera on mobile devices: “selfie
soft biometrics is gaining the most popularity due to the recent advancements in front-facing
cameras in smartphones.”51 It is worth noting that the same chapter details the ways in which
convolutional neural networks (CCN) networks are able to assess one’s age, gender, as well as
mood. Selfies, much like most smart phone portraiture, should thus been understood as an
extension of the 19th century projects of surveillance and the anthropometric articulation of
biotypes. Selfies today fuel algorithmically driven research similar to the work of Galton,
Bertillon, as well as Duchenne. Indeed, an increase in research on soft biometric data coincided with the release of against a blurry background.48 Here, selfie biometrics is defined as “an authentication mechanism
where a user captures images of her biometric traits (such as the face or ocular region) by using
the imaging sensors available in the device itself.”49 The idea of the selfie here has again shifted
away from modes of representation and agency, towards an automated “capture” of biometric
traits. Frames Cinema Journal, Issue 18 (June 2021) Automated Passport Control in the US, and the ID card gates in China. This is significant because
initially self-portraits and selfies were seen as ways of increasing the subject’s agency with regards
to representation. In a selfie, the subject indeed had great control over their representation as this
photographic genre required particular posing, and thus, a conscious construction of identity. The
Guardian playfully outlined the embodied conventions of the selfie: A doe-eyed stare and mussed-up hair denotes natural beauty, as if you've just woken
up and can't help looking like this. Sexiness is suggested by sucked-in cheeks,
pouting lips, a nonchalant cock of the head and a hint of bare flesh just below the
clavicle. Snap!45 When selfies are displaced into “self-captured live face photos” the agency is displaced away from
the self as the subject taking the photograph to the photograph emerging by itself. 46 Self here
refers to the autonomous process of photography – photography operating by itself. This significant shift in what the self means in regard to selfies and the self-captured face
photos has been addressed in a subfield of surveillance called “selfie biometrics.” A recent book
Selfie Biometrics: Advances and Challenges outlines the basic premises and techniques of this
burgeoning field.47 In the introduction of this edited volume, the editors Ajita Rattani, Reza
Derakhshani, and Arun Ross make an argument for the increasing viability of the selfie as a
valuable data-source for user authentication – in other words, for recognition and surveillance –
because of the advancements in image resolution and lens aperture size. The lens discussion is
important here, since proposed selfie lenses feature a wide aperture of f/1.4 – which, combined
with a longer focal length, mimics a portrait lens and allows for the articulation of a sharp face 79 Frames Cinema Journal, Issue 18 (June 2021) Frames Cinema Journal, Issue 18 (June 2021) collected with Apple’s front-facing and portrait mode cameras helped accelerate facial
recognition research on mobile devices. It ultimately resulted in the popularisation of the selfie
as an image of facial recognition and its mainstream acceptance as Apple’s new Face ID feature
on its iPhone X, introduced in 2017.53 This transition of the “selfie” from an instance of “self-portraiture” to “self-capture”
harnessed in biometrics speaks precisely of the ways in which smartphone photography has
helped to usher the distillation of the photograph from a visual form to a data entity. The
discourse of “capture” speaks precisely to the repressive function of photography. This time both
symbolic (actions are captured and used to determine once social and economic status) and at
times actualized imprisonment of the subject (captures are used to identify and convict criminals)
are enacted. The “here and now” indexicality that François Arago praised when announcing the
birth of photography is now parsed out into a set of distributed variables.54 No longer “here,” no
longer “now,” not even “us” for long, these facial maps speak to algorithmic logics and perform
for algorithmic visions that separate our images from ourselves in profound ways. This distinction
supports Kate Crawford’s claim that whereas anthropometrics and phrenology deployed
photography in analysing “human subjects,” AI driven assessments have further people into “data
subjects.”55 Frames Cinema Journal, Issue 18 (June 2021) Indeed, the selfie functions no longer as a self-portrait, but rather as a data-gathering
mechanism – a “selfie capture.” Further, the authors distinguish between three types of selfie
biometrics: face, ocular biometrics (imaging and use of characteristic features extracted from the
eyes for personal recognition), and fingerphoto: “touchless fingerprint recognition technology,
where the back-facing smartphone cameras acquire high-resolution photographs of finger ridge
patterns.”50 These features have commonly been used in both anthropometrics and biometrics
and have been seen as staples of identification, policing, and surveillance. What is interesting in
this article is the articulation of the so-called “soft” biometrics. In this biometric profile,
ethnicity, gender, and age are assessed and recorded. Another chapter in this book explicitly
links the raise of selfie soft biometrics with the front-facing camera on mobile devices: “selfie
soft biometrics is gaining the most popularity due to the recent advancements in front-facing
cameras in smartphones.”51 It is worth noting that the same chapter details the ways in which
convolutional neural networks (CCN) networks are able to assess one’s age, gender, as well as
mood. Selfies, much like most smart phone portraiture, should thus been understood as an
extension of the 19th century projects of surveillance and the anthropometric articulation of
biotypes. Selfies today fuel algorithmically driven research similar to the work of Galton,
Bertillon, as well as Duchenne. Indeed an increase in research on soft biometric data coincided with the release of Indeed, an increase in research on soft biometric data coincided with the release of
Apple’s front-facing camera in 2010 with the Iphone4 and portrait mode in 2016.52 The data 80 The Continuity Aesthetic of Blur From its beginning, photography was seen as a way to capture a slice of real life and more
specifically, to represent the people and places that make up everyday life. Prominent photo
historian Geoffrey Batchen called the photograph a “single vertical slice cut through the horizontal
passage of time and motion; a passage lived in the past.”56 In this slice of life capture, because of 81 Frames Cinema Journal, Issue 18 (June 2021) Frames Cinema Journal, Issue 18 (June 2021) Frames Cinema Journal, Issue 18 (June 2021) Image courtesy of the Library of
Congress, LC-USZ6-1957 83 Frames Cinema Journal, Issue 18 (June 2021) Frames Cinema Journal, Issue 18 (June 2021) Frames Cinema Journal, Issue 18 (June 2021) technical limitations, people were photographed in sharp focus while backgrounds receded into a
soft blur. This convention of using shallow depth of field in portraiture has remained a staple of
photographic portraiture up until today. In its early stages, the fixity of the image involved capture
of time with varying duration. Niepce’s first photograph took about 8 hours, while Daguerre
managed to reduce exposure time down to 3-15 minutes. As technology advanced, the subject was
captured not “in time” but “on time” – duration was reduced to the instant. In his essay “A Short
History of Photography,” Walter Benjamin laments for the earlier photographic portraits as the
subjects lived “into the instant not out of it” – they “grew as it were, into an image.”57 The long
exposures required subjects to sit still in front of the camera in order to emerge in sharper focus. Early portrait studios used blurry painted dioramas against which the subject appeared to be
sharper. This technique was necessitated by the long exposure times, where subjects were asked
to stay still in front of the camera for up to a minute and would often appear blurry against the
perfectly still – thus perfectly in-focus – background. The prolonged exposure in early photographic portraiture was necessitated by constraints
in photographic lenses. As Rudolph Kingslake notes in his extensive book A History of the
Photographic Lens, “the first lens to be used on a camera was the achromatic landscape lens of C. Chevalier (1804-1859).58 The aperture of this lens was only f/15.59 A portrait lens was introduced
in 1840, the following year, by J.M Petzval, but even that lens was “not good enough for practical
portraiture.”60 The Petzval lens had a “telephoto” mode in which the aperture was narrow at f/3.6. The Petzval portrait lens became a staple of the photographer’s toolkit and in the 1890s was
supplemented by the introduction of a telephoto lens.61 82 Frames Cinema Journal, Issue 18 (June 2021) Figure 1: “[Unidentified woman, three-quarter length portrait, facing front, seated before a painted
backdrop with column]” Daguerreotype portrait 1840-1860s. This image is exemplary of the 19th century Figure 1: “[Unidentified woman, three-quarter length portrait, facing front, seated before a painted
backdrop with column]” Daguerreotype portrait 1840-1860s. This image is exemplary of the 19th century
photographic convention of using blurred backgrounds in portraiture. Frames Cinema Journal, Issue 18 (June 2021) As photographic technology became more advanced, the photographic convention of
blurred background was achieved with macro and telephoto lenses that created a shallow depth of
field. In photographic terms, this means that portrait photographers use telephoto lenses spanning
over 70mm and then select a small aperture in the f/2.0-2.8 range. This convention is often taught
in photography books. For example, Erik Valind’s Portrait Photography: From Snapshots to
Great Shots, one among many photographic manual books, specifies that: [a] shallow depth of field is often desired because it draws attention to the subject’s
face while blurring out the less important features. This selective focus is a great way
to create strong portraits by directing the viewer straight to the subject’s eyes.62 This convention was carried forward and reintroduced as a dominant aesthetic with the rise of
digital photography in the late 1990s. Known as “bokeh,” a blurred orb-based background became
a visual trademark of the digital visual aesthetic. This effect requires a telephoto lens with wide
aperture in the range of f/1.4 to f/1.8.63 It produces a background effect in which the setting appears
to be a patchwork of fuzzy orbs. With the introduction of cell phone photography, the convention of blurring the
background when creating photographic portraits was delivered through algorithms that isolated
the human figure and scrambled the perceived “background.” This algorithmic mode of generating
blur mimics the physics of the dSLR camera. For example, the 2020 AIM challenge for rendering
a realistic blur used a Canon 7D dSLR camera as a base and attempted to create similar images
algorithmically with smartphone cameras.64 The process for articulating blur and bokeh was
introduced in 2014 and by 2016 was a common feature of most “portrait modes” of smartphone 84 Frames Cinema Journal, Issue 18 (June 2021) Frames Cinema Journal, Issue 18 (June 2021) Frames Cinema Journal, Issue 18 (June 2021) cameras. When Google’s Pixel phone introduced an algorithm for mimicking shallow depth of
field, they termed the effect “Lens Blur.” The camera lens on the smartphone camera is fairly basic
and operates at a level of sophistication similar to those in early photography: “standard cell phone
cameras cannot produce [blur] optically, as their short focal lengths and small apertures capture
nearly all-in-focus images”65 The software developers found a way to simulate telephoto lens
effects: “Lens Blur replaces the need for a large optical system with algorithms that simulate a
larger lens and aperture”.66 Figure 2: “Good mood lady with expansive smile enjoying started weekends and taking selfie on mobile
phone on blurred background” This image is exemplary of the simulation of blur or bokeh in mobile
photography. Shutterstock. Royalty-free stock photo ID: 1655727 Figure 2: “Good mood lady with expansive smile enjoying started weekends and taking selfie on mobile
phone on blurred background” This image is exemplary of the simulation of blur or bokeh in mobile
photography. Shutterstock. Royalty-free stock photo ID: 1655727 85 Frames Cinema Journal, Issue 18 (June 2021) Frames Cinema Journal, Issue 18 (June 2021) In 2016, Apple shifted the language around blur and bokeh to make it explicitly part of the
photographic portrait aesthetic with its “portrait mode.” As Sam Bayford writes, Apple makes use of this tech to drive its dual-camera phones’ portrait mode. The
iPhone’s image signal processor uses machine learning techniques to recognize people
with one camera, while the second camera creates a depth map to help isolate the
subject and blur the background. The ability to recognize people through machine
learning wasn’t new when this feature debuted in 2016, as it’s what photo organization
software was already doing. But to manage it in real time at the speed required for a
smartphone camera was a breakthrough.67 The articulation of blur and bokeh in relation to information processing has been a central
problem for AI developers. Researchers have focused a significant amount of work on attempting
to isolate subjects from backgrounds and introduce blurring effects that mimic the photographic
portraiture convention. This work has articulated both consumer practices – creating more realistic
blur for selfies – as well as surveillance structures – identifying subjects for the purposes of facial
recognition. A study on generating realistic bokeh notes specifically why selfies are a good
candidate for training the algorithm to recognize human/data subjects. As the argument goes,
“[s]uch images typically feature relatively large subject heads … further selfies are mostly
captured on a mobile phone, thus they have a large depth of field.”68 These features make them
the perfect candidates for creating an algorithmic effect that is physically impossible given the
limitation of the hardware itself. As is evident in convolutional neural networks (CNN) research, 86 Frames Cinema Journal, Issue 18 (June 2021) transformed from a visual element used to centre one’s attention on the foreground object or
subject to a data device made useful for information processing.71 In the context of Apple, their
facial recognition app “Recognizr” harnesses the ability to separate subject from background in
order to automate the recognition of subjects across collections of photographs taken on a mobile
device. This app renders the inner workings of surveillance systems as a “fun” consumer feature
and obscures the long history of portraiture-based surveillance.72 Much like its 19th century counterpart, contemporary AI-driven surveillance mechanisms
are laden with racial and gender bias. Joy Buolamwini and Timnit Gebru’s exceptional work on
algorithmic inequality is a prominent example of this adaptation. In their groundbreaking study
“Gender Shades,” Buolamwini and Gebru demonstrated that facial recognition software fails to
classify darker females accurately at a greater rate than it does with later males.73 Ruha Benjamin’s
book Race after Technology has further detailed the ways in which AI technology continues to
propagate anti-Blackness.74 Yet AI facial recognition technology is presented as convenient,
efficient, and fun. It is fueled by everyday consumer practices connected to cell phone portraiture
and self-portraiture. Frames Cinema Journal, Issue 18 (June 2021) Frames Cinema Journal, Issue 18 (June 2021) both bokeh and blur are being deployed as tools that allow for the isolation and recognition of the
most significant object of a picture. both bokeh and blur are being deployed as tools that allow for the isolation and recognition of the
most significant object of a picture. These features came again to the forefront when an image features multiple objects. As
Holly Chiang and colleagues write: These features came again to the forefront when an image features multiple objects. As
Holly Chiang and colleagues write: Another instance is if you have a photo of a target person of interest in front of a
famous landmark but there are too many tourists in the background, our detector will
be able to determine that the person and the landmark are the most significant objects
in the picture, and apply photography techniques to such as bokeh or blur to reduce
the background noise. Bokeh with focus on multiple objects, in particular, is very
difficult to achieve in the real world because cameras can only have one depth of view
for focusing. Therefore, if we can identify the important objects’ bounding boxes, we
can theoretically focus and blur multiple objects with a bokeh effect that is impossible
to do otherwise.69 As the authors of the multi-object recognition paper note, “[t]o simulate the bokeh effect we
applied a gaussian filter followed by randomly selecting pixels to enlarge into circles, followed by
another gaussian layer.”70 No longer a function of a camera lens, no longer aimed at now missing
human vision, the bokeh effect here is created by machine learning algorithms for machine vision. Algorithmically produced shallow depth of field (hence a blurry or bokeh background)
legitimises the status of the algorithmic image as a photograph and obscures the deployment of the
algorithmic image as a tool of surveillance. The algorithmic articulation of bokeh has provided
grounds for implementing depth maps that isolate subject from background for the purposes of
facial recognition and surveillance. Blur and bokeh, as aesthetics of continuity, have thus been 87 Frames Cinema Journal, Issue 18 (June 2021) Frames Cinema Journal, Issue 18 (June 2021) Frames Cinema Journal, Issue 18 (June 2021) to the straight and intent look required by Face ID authentication regimes. Portraits became portrait
modes in which algorithms were given an opportunity to train themselves at isolating human
subjects from perceived backgrounds. In reflecting on the ways in which photographic images
produced on our smartphone devices are increasingly created for machine seeing by machine
learning algorithms, it has become increasingly important to understand the history of photography
and its lasting conventions. These conventions are continuously used in order to legitimise data-
driven images as representative of our own image, as honourable. They appeal to the bourgeois
aesthetic of photographic portraiture, while at the same time articulating neoliberal surveillance
assemblages in which identities are constructed based on the intentionality of algorithms which
decide when an image is taken and how many data points are gathered rather than that of the
subject in front of the lens. Unpacking the photographic conventions, such as the “aesthetics of
continuity” of blur and bokeh, behind this new class of computational photography, produced with
ease on smartphone devices, is a crucial component of a newly emerging algorithmic literacy. It is
by grappling with the historical roots of photographic portraiture as both a mode of representation
and a mode of quantification that we are able to discern the new ways in which photography has
been summoned as a veil for our increasingly datafied selves. Understanding the historical
trajectory of the quantified self in relation to photography allows us to think critically about the
ways in which cell phone photography is used in contemporary surveillance and biometric
enterprises. Further, unpacking the visual conventions that conceal cell phone images as portraits
when in really they are raw data for algorithmic calculation helps foster a much needed critical
media literacy. to the straight and intent look required by Face ID authentication regimes. Portraits became portrait
modes in which algorithms were given an opportunity to train themselves at isolating human
subjects from perceived backgrounds. In reflecting on the ways in which photographic images
produced on our smartphone devices are increasingly created for machine seeing by machine
learning algorithms, it has become increasingly important to understand the history of photography
and its lasting conventions. These conventions are continuously used in order to legitimise data-
driven images as representative of our own image, as honourable. Conclusion Computational photography and digital imaging, harnessed in the service of biometrics and
facial recognition, have transformed loving eyes, pouting lips and sucked-in cheeks into data-
points. The processes of translating analogue photographic images into computer data have
transformed smartphone photography from a prominent device of self-expression to the ultimate
tool for surveillance. Initially articulated as self-portraits, “selfies” became “selfie captures” in the
context of selfie biometrics. We learned a new mode of posturing: away from making sassy faces 88 Frames Cinema Journal, Issue 18 (June 2021) Frames Cinema Journal, Issue 18 (June 2021) Frames Cinema Journal, Issue 18 (June 2021) Frames Cinema Journal, Issue 18 (June 2021) They appeal to the bourgeois
aesthetic of photographic portraiture, while at the same time articulating neoliberal surveillance
assemblages in which identities are constructed based on the intentionality of algorithms which
decide when an image is taken and how many data points are gathered rather than that of the
subject in front of the lens. Unpacking the photographic conventions, such as the “aesthetics of
continuity” of blur and bokeh, behind this new class of computational photography, produced with
ease on smartphone devices, is a crucial component of a newly emerging algorithmic literacy. It is
by grappling with the historical roots of photographic portraiture as both a mode of representation
and a mode of quantification that we are able to discern the new ways in which photography has
been summoned as a veil for our increasingly datafied selves. Understanding the historical
trajectory of the quantified self in relation to photography allows us to think critically about the
ways in which cell phone photography is used in contemporary surveillance and biometric
enterprises. Further, unpacking the visual conventions that conceal cell phone images as portraits
when in really they are raw data for algorithmic calculation helps foster a much needed critical
media literacy. 89 Frames Cinema Journal, Issue 18 (June 2021) Notes 1 Mark B.N. Hansen, “Affect as medium, or the ‘digital-facial-image’,” Journal of Visual
Culture, 2(2) (2003): 206-228. https://doi.org/10.1177%2F14704129030022004. Mitra Azar,
“Algorithmic Facial Image: Regimes of Truth and Datafication,” A Peer-Reviewed Journal
About APRJA, 7(1) (2018): 27-35. https://doi.org/10.7146/aprja.v7i1.115062. 2 2 Grace Kingston and Michael Goddard, “The Aesthetic Paradoxes of Visualizing the Networked
Image,” Contemporary Arts and Cultures (2017): 6. htt
//
t
t
it d /
b/
th ti
d 4 Daniel Rubinstein and Katrina Sluis, “The digital image in photographic culture: algorithmic
photography and the crisis in representation” In Martin Lister, ed. The photographic image in
digital culture (London and New York: Routledge, 2013): 22-40, 28. 5 Lev Manovich, The Language of New Media. (Cambridge, MA: MIT Press, 2002), 144. 6 See Trevor Paglen, “Invisible Images: Your Pictures Are Looking at You,” Architectural
Design, 89 (2019): 22-27, DOI: https://doi.org/10.1002/ad.2383 and Harun Faroki, “Phantom
Images,” Public 29 (2004). https://public.journals.yorku.ca/index.php/public/article/view/30354. And Trevor Paglen, “Operational Images,” e-flux 59 (November 2014) https://www.e-
flux.com/journal/59/61130/operational-images/. 4 Daniel Rubinstein and Katrina Sluis, “The digital image in photographic culture: algorithmic
photography and the crisis in representation” In Martin Lister, ed. The photographic image in
digital culture (London and New York: Routledge, 2013): 22-40, 28. 4 Daniel Rubinstein and Katrina Sluis, “The digital image in photographic culture: algorithmic
photography and the crisis in representation” In Martin Lister, ed. The photographic image in
digital culture (London and New York: Routledge, 2013): 22-40, 28. 5 Lev Manovich, The Language of New Media. (Cambridge, MA: MIT Press, 2002), 144. 6 4 Daniel Rubinstein and Katrina Sluis, “The digital image in photographic culture: algorithmic
photography and the crisis in representation” In Martin Lister, ed. The photographic image in
digital culture (London and New York: Routledge, 2013): 22-40, 28. 5 Lev Manovich, The Language of New Media. (Cambridge, MA: MIT Press, 2002), 144. 6 See Trevor Paglen, “Invisible Images: Your Pictures Are Looking at You,” Architectural
Design, 89 (2019): 22-27, DOI: https://doi.org/10.1002/ad.2383 and Harun Faroki, “Phantom
Images,” Public 29 (2004). https://public.journals.yorku.ca/index.php/public/article/view/30354. And Trevor Paglen, “Operational Images,” e-flux 59 (November 2014) https://www.e-
flux.com/journal/59/61130/operational-images/. digital cultu e ( o do a d New
o
:
out edge, 0 3):
0, 8. 5 Lev Manovich, The Language of New Media. (Cambridge, MA: MIT Press, 2002), 144. 6 See Trevor Paglen, “Invisible Images: Your Pictures Are Looking at You,” Architectural
Design, 89 (2019): 22-27, DOI: https://doi.org/10.1002/ad.2383 and Harun Faroki, “Phantom
Images,” Public 29 (2004). https://public.journals.yorku.ca/index.php/public/article/view/30354. Notes And Trevor Paglen, “Operational Images,” e-flux 59 (November 2014) https://www.e-
flux.com/journal/59/61130/operational-images/. j
p
g
7 Alan Sekula “The Body and the Archive” October 39 (Winter, 1986): 3-64, 6. 9 Josh Lauer, “Surveillance History and the History of New Media: An Evidential Paradigm.”
New Media & Society 14, no. 4 (June 2012): 566–82, 573. https://doi org/10 1177/1461444811420986 y
https://doi.org/10.1177/1461444811420986. 10 Sekula, 7. 11 Ibid., 16. 12 Ibid., 18. 13 Ibid., 18. ,
14 Alphonse Bertillon. Ethnographie moderne: les races sauvages (Paris: G. Masson, 1883). https://gallica.bnf.fr/ark:/12148/bpt6k104250m/texteBrut. 15 14 Alphonse Bertillon. Ethnographie moderne: les races sauvages (Paris: G. Masson, 1883). https://gallica.bnf.fr/ark:/12148/bpt6k104250m/texteBrut. 15 Ibid ,
17 Francis Galton. Narrative of a Traveler to Tropical South Africa (London: John Murray, 1853)
https://galton.org/books/south-west-africa/galton-1853-travels-in-south-africa-1up-linked.pdf. 18 Lady Elizabeth Eastlake. “Photography.” In Alan Trachtenberg, ed. Classic Essays on
Photography (New Haven, Conn: Leetes Island Books: 1981), 39-69. 19 Ibid., 65. 20 17 Francis Galton. Narrative of a Traveler to Tropical South Africa (London: John Murray, 1853)
https://galton.org/books/south-west-africa/galton-1853-travels-in-south-africa-1up-linked.pdf. Francis Galton. Narrative of a Traveler to Tropical South Africa (London: John Murray, 1853)
https://galton.org/books/south-west-africa/galton-1853-travels-in-south-africa-1up-linked.pdf. 18 Lady Elizabeth Eastlake. “Photography.” In Alan Trachtenberg, ed. Classic Essays on
Photography (New Haven, Conn: Leetes Island Books: 1981), 39-69. 19 Ibid., 65. 20 p
g
g
g
p
p
18 Lady Elizabeth Eastlake. “Photography.” In Alan Trachtenberg, ed. Classic Essays on
Photography (New Haven, Conn: Leetes Island Books: 1981), 39-69. 19 Ibid., 65. 21 Georges Didi-Huberman. Invention of Hysteria: Charcot and the Photographic Iconography
of the Salpêtrière (Cambridge, MA: MIT Press, 2004). 21 Georges Didi-Huberman. Invention of Hysteria: Charcot and the Photographic Iconography
of the Salpêtrière (Cambridge, MA: MIT Press, 2004). 23 Thy Phu and Linda M. Steer, “Introduction,” Photography and Culture 2, no. 3, (2019): 235-
239, 236, DOI: 10.2752/175145109X12532077132194 90 Frames Cinema Journal, Issue 18 (June 2021) 24 Joseph Ferenbok, “Configuring the Face as a Technology of Citizenship: Biometrics,
Surveillance and the Facialization of Institutional Identity.” In: Kalantzis-Cope P., Gherab-
Martín K. eds, Emerging Digital Spaces in Contemporary Society. (London: Palgrave
Macmillan, 2010), 126-127: 127. https://doi.org/10.1057/9780230299047_21. 25 For an example of race identification, see Alexander Todorov, Christopher Y. Olivola, and
others, “Social Attributions from Faces: Determinants, Consequences, Accuracy, and Functional
Significance.” Annual Review of Psychology, 66, (January, 2015): 519-545. https://doi.org/10.1146/annurev-psych-113011-143831. Notes For an example of gender identification,
see Rajeev Ranjan and Vishal Patel and others, “HyperFace: A Deep Multi-Task Learning
Framework for Face Detection, Landmark Localization, Pose Estimation, and Gender
Recognition,” IEEE Transactions on Pattern Analysis and Machine Intelligence, 41.1 (January
1, 2017): 121-135. DOI: 10.1109/TPAMI.2017.2781233. For an example of age identification,
see Angulu Raphael and Jules R. Tapamo and Adremi O. Adewumi, “Age estimation via face
images: a survey.” J Image Video Proc, 42 (2018). https://doi.org/10.1186/s13640-018-0278-6. For an example of sexual orientation identification, see Y. Wang and M. Kosinski, Deep neural
networks are more accurate than humans at detecting sexual orientation from facial
images. Journal of Personality and Social Psychology, 114.2 (2018), 246-257. https://doi.org/10.1146/annurev-psych-113011-143831. For an example of gender identification,
see Rajeev Ranjan and Vishal Patel and others, “HyperFace: A Deep Multi-Task Learning
Framework for Face Detection, Landmark Localization, Pose Estimation, and Gender
Recognition,” IEEE Transactions on Pattern Analysis and Machine Intelligence, 41.1 (January
1, 2017): 121-135. DOI: 10.1109/TPAMI.2017.2781233. For an example of age identification,
see Angulu Raphael and Jules R. Tapamo and Adremi O. Adewumi, “Age estimation via face
images: a survey.” J Image Video Proc, 42 (2018). https://doi.org/10.1186/s13640-018-0278-6. For an example of sexual orientation identification, see Y. Wang and M. Kosinski, Deep neural
networks are more accurate than humans at detecting sexual orientation from facial
images. Journal of Personality and Social Psychology, 114.2 (2018), 246-257. g
f
y
y
gy
https://doi.org/10.1037/pspa0000098. For an example of emotional state identification, see Avita
Saxena, Ashish Khanna, and Deepak Gupta, “Emotion Recognition and Detection Methods: A
Comprehensive Survey,” Journal of Artificial Intelligence and Systems, 2 (2020), 53-
79. https://doi.org/10.33969/AIS.2020.21005. For an example of political preference
identification, see Michal Kosinski, “Facial recognition technology can expose political
orientation from naturalistic facial images,” Scientific Reports 11.100 (2021). p
g
26 Tal Hassner, The OUI-Adience: Face Image Project, 26 Tal Hassner, The OUI-Adience: Face Image Project,
https://talhassner.github.io/home/projects/Adience/Adience-data.html
27 Ibid. g
j
https://talhassner.github.io/home/projects/Adience/Adience-data.html
27 Ibid 27 Ibid. 28 Gil Levi and Tal Hassner, Age and Gender Classification Using Convolutional Neural
Networks, IEEE Workshop on Analysis and Modeling of Faces and Gestures (AMFG), at the
IEEE Conf. on Computer Vision and Pattern Recognition (CVPR), Boston, June 2015. https://talhassner.github.io/home/projects/cnn_agegender/CVPR2015_CNN_AgeGenderEstimati
on.pdf. And Eran Eidinger, Roee Enbar, and Tal Hassner, Age and Gender Estimation of
Unfiltered Faces, Transactions on Information Forensics and Security (IEEE-TIFS), Special
Issue on Facial Biometrics in the Wild, Volume 9.12, (Dec, 2014): 2170 – 2179. https://talhassner.github.io/home/projects/Adience/Adience/EidingerEnbarHassner_tifs.pdf. Notes 51 Ajita Rattani and Mudit Agrawal. “Soft-Biometric Attributes from Selfie Images,” in
Rattani A., Derakhshani R., Ross A. eds, Selfie Biometrics. Advances in Computer Vision
and Pattern Recognition. (Springer, Cham. 2019). https://doi.org/10.1007/978-3-030-26972-
2_1. 52 See Attaullah Buriro, Zahid Akhtar, Bruno Crispo and Fillipo Del Frari, “Age, Gender and
Operating-Hand Estimation on Smart Mobile Devices,” 2016 International Conference of the
Biometrics Special Interest Group (BIOSIG) (Darmstadt, Germany, 2016): 1-5, DOI:
10.1109/BIOSIG.2016.7736910. 53 Russel Brandom, “The five biggest questions about Apple’s new facial recognition system.”
The Verge. (September 12, 2017). https://www.theverge.com/2017/9/12/16298156/apple-iphone-
x-face-id-security-privacy-police-unlock. 54 Dominique Francois Arago. “Report” In Alan Trachtenberg, ed. Classic Essays on
Photography (New Haven, Conn: Leetes Island Books: 1981): 15-26. 47 Ajita Rattani, Reza Derakhshani, Arun Ross, eds. Selfie Biometrics. Advances in Computer
Vision and Pattern Recognition (Springer, Cham, 2019). 48 47 Ajita Rattani, Reza Derakhshani, Arun Ross, eds. Selfie Biometrics. Advances in Computer
Vision and Pattern Recognition (Springer, Cham, 2019). 48 Ajita Rattani, Reza Derakhshani, Arun Ross, “Introduction to Selfie Biometrics.” in
Rattani A., Derakhshani R., Ross A. eds, Selfie Biometrics. Advances in Computer Vision
and Pattern Recognition. (Springer, Cham. 2019). https://doi.org/10.1007/978-3-030-26972-
2_1. 49 Ibid. 51 Ajita Rattani and Mudit Agrawal. “Soft-Biometric Attributes from Selfie Images,” in
Rattani A., Derakhshani R., Ross A. eds, Selfie Biometrics. Advances in Computer Vision
and Pattern Recognition. (Springer, Cham. 2019). https://doi.org/10.1007/978-3-030-26972-
2_1. 52 See Attaullah Buriro, Zahid Akhtar, Bruno Crispo and Fillipo Del Frari, “Age, Gender and
Operating-Hand Estimation on Smart Mobile Devices,” 2016 International Conference of the
Biometrics Special Interest Group (BIOSIG) (Darmstadt, Germany, 2016): 1-5, DOI:
10.1109/BIOSIG.2016.7736910. 53 Russel Brandom, “The five biggest questions about Apple’s new facial recognition system.”
The Verge. (September 12, 2017). https://www.theverge.com/2017/9/12/16298156/apple-iphone-
x-face-id-security-privacy-police-unlock. 54 Dominique Francois Arago. “Report” In Alan Trachtenberg, ed. Classic Essays on
Photography (New Haven, Conn: Leetes Island Books: 1981): 15-26. 51 Ajita Rattani and Mudit Agrawal. “Soft-Biometric Attributes from Selfie Images,” in
Rattani A., Derakhshani R., Ross A. eds, Selfie Biometrics. Advances in Computer Vision
and Pattern Recognition. (Springer, Cham. 2019). https://doi.org/10.1007/978-3-030-26972-
2_1. 35 Nicholas Mirzoeff, How to See the World: An Introduction to Images, from Self-Portraits to
Selfies, Maps to Movies, and More (New York: Basic Books, 2016).
36 Notes 36 35 Nicholas Mirzoeff, How to See the World: An Introduction to Images, from Self-Portraits to
Selfies, Maps to Movies, and More (New York: Basic Books, 2016). 36 Elizabeth Day, “How selfies became a global phenomenon,” The Guardian, (July 13, 2013). https://www.theguardian.com/technology/2013/jul/14/how-selfies-became-a-global-
phenomenon. p
37 Andre Gunhert, “The Consideration of the selfie: A cultural history” Julia Eckel, Jens Ruchatz,
and Sabine Wirth, eds., Exploring the Selfie: Historical, Theoretical, and Analytical Approaches
to Digital Self-Photography (Palgrave Macmillan, 2018). 37 Andre Gunhert, “The Consideration of the selfie: A cultural history” Julia Eckel, Jens Ruchatz,
and Sabine Wirth, eds., Exploring the Selfie: Historical, Theoretical, and Analytical Approaches
to Digital Self-Photography (Palgrave Macmillan, 2018). g
38 Mona Kasra, “Digital-networked images as personal acts of political expression: New
categories for meaning formation,” Media and Communication, 5(4) (2017): 51–64, 51, 53,
https://doi.org/10.17645/mac.v5i4.1065. 38 Mona Kasra, “Digital-networked images as personal acts of political expression: New
categories for meaning formation,” Media and Communication, 5(4) (2017): 51–64, 51, 53,
https://doi.org/10.17645/mac.v5i4.1065. 39 Claire Hampton, “#nomakeupselfies: The Face of Hashtag Slacktivism,” Networking
Knowledge: Journal of the MeCCSA Postgraduate Network 8(6) (2015). https://doi.org/10.31165/nk.2015.86.406 and Paul Frosh, The Poetics of Digital Media
(Cambridge, UK, and Medford, MA: Polity, 2019)
40 S lfi D t S t htt
//
f d /d t /S lfi / p
g
(Cambridge, UK, and Medford, MA: Polity, 2019) 40 Selfie Data Set, https://www.crcv.ucf.edu/data/Selfie/. 41 Ibid. 42 “Data Collection and Analysis,” Selfiecity, http://selfiecity.net/#dataset. 43 42 “Data Collection and Analysis,” Selfiecity, http://selfiecity.net/#dataset. 43 43 Kate Crawford and Trevor Paglen, Excavating AI: The politics of images in machine learning
training, https://www.excavating.ai. 44 43 Kate Crawford and Trevor Paglen, Excavating AI: The politics of images in machine learning
training, https://www.excavating.ai. 44 44 Yichun Shi and Anil K. Jain, “DocFace+: ID Document to Selfie Matching,” IEEE
Transactions on Biometrics, Behavior, and Identity Science 1.1 (January 2019): 56-67, 56. DOI:
10.1109/TBIOM.2019.2897807. 45 Ibid 44 Yichun Shi and Anil K. Jain, “DocFace+: ID Document to Selfie Matching,” IEEE
Transactions on Biometrics, Behavior, and Identity Science 1.1 (January 2019): 56-67, 56. DOI:
10.1109/TBIOM.2019.2897807. 45 Ibid. 46 Ibid. 47 Ajita Rattani, Reza Derakhshani, Arun Ross, eds. Selfie Biometrics. Advances in Computer
Vision and Pattern Recognition (Springer, Cham, 2019). 48 Ajita Rattani, Reza Derakhshani, Arun Ross, “Introduction to Selfie Biometrics.” in
Rattani A., Derakhshani R., Ross A. eds, Selfie Biometrics. Advances in Computer Vision
and Pattern Recognition. (Springer, Cham. 2019). https://doi.org/10.1007/978-3-030-26972-
2_1. 49 Ibid. 50 Ibid. Notes 29 AffectNet, http://mohammadmahoor.com/affectnet/. 30 Christoffer Heckman, “AI can now read emotions – should it? The Conversation. (January 8,
2020). https://theconversation.com/ai-can-now-read-emotions-should-it-128988
31 Marika Lüders, Lin Prøitz, Terje Rasmussen, “Emerging personal media genres,” New Media
& Society 12 (2010): 947–963, 959. 32 Ibid., 959. 33 Charles Arthur, “iPhone 4 unveiled by Apple,” The Guardian, (June 7, 2010)
https://www.theguardian.com/technology/2010/jun/07/iphone-4-apple-wwdc. 34 “Selfie,” Oxford Learner’s Dictionaries. https://www.oxfordlearnersdictionaries.com/us/definition/english/selfie. 28 Gil Levi and Tal Hassner, Age and Gender Classification Using Convolutio
Networks, IEEE Workshop on Analysis and Modeling of Faces and Gestures Networks, IEEE Workshop on Analysis and Modeling of Faces and Gestures (AMFG), at the
IEEE Conf. on Computer Vision and Pattern Recognition (CVPR), Boston, June 2015. https://talhassner.github.io/home/projects/cnn_agegender/CVPR2015_CNN_AgeGenderEstimati
on.pdf. And Eran Eidinger, Roee Enbar, and Tal Hassner, Age and Gender Estimation of
Unfiltered Faces, Transactions on Information Forensics and Security (IEEE-TIFS), Special
Issue on Facial Biometrics in the Wild, Volume 9.12, (Dec, 2014): 2170 – 2179. https://talhassner.github.io/home/projects/Adience/Adience/EidingerEnbarHassner_tifs.pdf. 29 p
g
p
j
g g
g
on.pdf. And Eran Eidinger, Roee Enbar, and Tal Hassner, Age and Gender Estimation of
Unfiltered Faces, Transactions on Information Forensics and Security (IEEE-TIFS), Special
Issue on Facial Biometrics in the Wild, Volume 9.12, (Dec, 2014): 2170 – 2179. https://talhassner.github.io/home/projects/Adience/Adience/EidingerEnbarHassner_tifs.pdf. 29 30 Christoffer Heckman, “AI can now read emotions – should it? The Conversation. (January 8,
2020). https://theconversation.com/ai-can-now-read-emotions-should-it-128988
31 Marika Lüders, Lin Prøitz, Terje Rasmussen, “Emerging personal media genres,” New Media
& Society 12 (2010): 947–963, 959. 32 Ibid 959 30 Christoffer Heckman, “AI can now read emotions – should it? The Conversation. (January 8,
2020). https://theconversation.com/ai-can-now-read-emotions-should-it-128988
31 Marika Lüders Lin Prøitz Terje Rasmussen “Emerging personal media genres ” New Media 2020). https://theconversation.com/ai can now read emotions should it 128988
31 Marika Lüders, Lin Prøitz, Terje Rasmussen, “Emerging personal media genres,” New Media
& Society 12 (2010): 947–963, 959. 32 Ibid., 959. 33 Charles Arthur, “iPhone 4 unveiled by Apple,” The Guardian, (June 7, 2010)
https://www.theguardian.com/technology/2010/jun/07/iphone-4-apple-wwdc. 34 “Selfie,” Oxford Learner’s Dictionaries. https://www.oxfordlearnersdictionaries.com/us/definition/english/selfie. https://www.theguardian.com/technology/2010/jun/07/iphone-4-apple-wwdc. 34 “Selfie,” Oxford Learner’s Dictionaries. https://www.oxfordlearnersdictionaries.com/us/definition/english/selfie. 91 Frames Cinema Journal, Issue 18 (June 2021) 35 Nicholas Mirzoeff, How to See the World: An Introduction to Images, from Self-Portraits to
Selfies, Maps to Movies, and More (New York: Basic Books, 2016). 36 35 Nicholas Mirzoeff, How to See the World: An Introduction to Images, from Self-Portraits to
Selfies, Maps to Movies, and More (New York: Basic Books, 2016). Notes 52 See Attaullah Buriro, Zahid Akhtar, Bruno Crispo and Fillipo Del Frari, “Age, Gender and
Operating-Hand Estimation on Smart Mobile Devices,” 2016 International Conference of the
Biometrics Special Interest Group (BIOSIG) (Darmstadt, Germany, 2016): 1-5, DOI:
10 1109/BIOSIG 2016 7736910 52 See Attaullah Buriro, Zahid Akhtar, Bruno Crispo and Fillipo Del Frari, “Age, Gender and
Operating-Hand Estimation on Smart Mobile Devices,” 2016 International Conference of the
Bi
t i
S
i l I t
t G
(BIOSIG) (D
t dt G
2016) 1 5 DOI p
g
f
f
Biometrics Special Interest Group (BIOSIG) (Darmstadt, Germany, 2016): 1-5, DOI:
10.1109/BIOSIG.2016.7736910. 53 Russel Brandom, “The five biggest questions about Apple’s new facial recognition system.”
The Verge. (September 12, 2017). https://www.theverge.com/2017/9/12/16298156/apple-iphone-
x-face-id-security-privacy-police-unlock. y p
y p
54 Dominique Francois Arago. “Report” In Alan Trachtenberg, ed. Classic Essays on
Photography (New Haven, Conn: Leetes Island Books: 1981): 15-26. 92 Frames Cinema Journal, Issue 18 (June 2021) 55 Jacob Metcalf and Kate Crawford, “Where are human subjects in Big Data research? The
emerging ethics divide,” Big Data & Society (January–June 2016), 1-14. https://doi.org/10.1177/2053951716650211
56 Geoffrey Batchen, Each Wild Idea: writing photography history (Cambridge, MA: MIT Press,
2002), 47. emerging ethics divide, Big Data & Society (January June 2016), 1 14. https://doi.org/10.1177/2053951716650211
56 Geoffrey Batchen, Each Wild Idea: writing photography history (Cambridge, MA: MIT Press,
2002), 47. )
57 Walter Benjamin, “Short History of Photography” (1931). Artforum. https://www.artforum.com/print/197702/walter-benjamin-s-short-history-of-photography-36010. 57 Walter Benjamin, “Short History of Photography” (1931). Artforum. https://www.artforum.com/print/197702/walter-benjamin-s-short-history-of-photography-36010. 58 Rudolph Kingslake A History of the Photographic Lens (Boston Academic Press 1989) j
,
y
g p y (
)
f
https://www.artforum.com/print/197702/walter-benjamin-s-short-history-of-photography-36010. 58 Rudolph Kingslake, A History of the Photographic Lens, (Boston, Academic Press, 1989). 59 Ibid., 7. p
p
j
y
p
g p y
58 Rudolph Kingslake, A History of the Photographic Lens, (Boston, Academic Press, 1989). 59 Ibid., 7. 61 Ibid., 8. ,
62 Erik Valind, Portrait Photography: From Snapshots to Great Shots (Peachpit Press, 2014), 25. 63 “7 Best Camera Lesnes for Bokeh Photography” Adorama.com, (May 21, 2020). htt
//
d
/ l /5 b
t
l
f
b k h
h t
h / 62 Erik Valind, Portrait Photography: From Snapshots to Great Shots (Peachpit Press, 2014), 25. 63 “7 Best Camera Lesnes for Bokeh Photography” Adorama.com, (May 21, 2020). https://www.adorama.com/alc/5-best-camera-lenses-for-bokeh-photography/. 63 “7 Best Camera Lesnes for Bokeh Photography” Adorama.com, (May 21, 2020). https://www.adorama.com/alc/5-best-camera-lenses-for-bokeh-photography/. 64 Andrey Ignatov, et al. 74 Ruha Benjamin, Race After Technology: Abolitionist Tools for the New Jim Code,
(Cambridge, UK and Malden, MA: Polity, 2019). 73 Joy Buolamwini and Timnit Gerbu. “Gender Shades: Intersectional Accuracy Disparities in
Commercial Gender Classification” Proceedings of Machine Learning Research. 81:1 (2018), 1-
15. Notes “AIM 2020 Challenge for Rendering Realistic Bokeh” ArXiv, (2020),
https://arxiv.org/abs/2011.04988. 65 64 Andrey Ignatov, et al. “AIM 2020 Challenge for Rendering Realistic Bokeh” ArXiv, (2020),
https://arxiv.org/abs/2011.04988. 65 Neal Wadhwa, et. al. “Synthetic depth-of-field with a single-camera mobile phone.” ACM
Transactions on Graphics, No 64 (July 2018). https://dl.acm.org/doi/10.1145/3197517.3201329. 66 Carlos Hernandez, “Lens Blur in the new Google Camera app” Google AI Blog (April 16,
2014). https://ai.googleblog.com/2014/04/lens-blur-in-new-google-camera-app.html. 67 67 Sam Bayford, “How AI is Changing Photography” The Verge (Jan 31, 2019). https://www.theverge.com/2019/1/31/18203363/ai-artificial-intelligence-photography-google-
photos-apple-huawei. 68 68 Friedrich, Nadine et al. “Faking it: Simulating background blur in portrait photography using a
coarse depth map estimation from a single image.” WSCG 2016: short communications
proceedings: The 24th International Conference in Central Europe on Computer Graphics,
Visualization and Computer Vision 2016 in co-operation with EUROGRAPHICS: University of coarse depth map estimation from a single image.” WSCG 2016: short communications
proceedings: The 24th International Conference in Central Europe on Computer Graphics,
Visualization and Computer Vision 2016 in co-operation with EUROGRAPHICS: University of
West Bohemia, Plzen, Czech Republic May 30 - June 3 2016, (2016): 17-23. https://dspace5.zcu.cz/bitstream/11025/29683/1/Friedrich.pdf. proceedings: The 24th International Conference in Central Europe on Computer Graphics,
Visualization and Computer Vision 2016 in co-operation with EUROGRAPHICS: University of
West Bohemia, Plzen, Czech Republic May 30 - June 3 2016, (2016): 17-23. https://dspace5.zcu.cz/bitstream/11025/29683/1/Friedrich.pdf. Visualization and Computer Vision 2016 in co-operation with EUROGRAPHICS: University of
West Bohemia, Plzen, Czech Republic May 30 - June 3 2016, (2016): 17-23. https://dspace5.zcu.cz/bitstream/11025/29683/1/Friedrich.pdf. 69 Holly Chiang, Yifan Ge, and Connie Wo, “Multiple Object Recognition with Focusing and
Blurring” http://cs231n.stanford.edu/reports/2016/pdfs/259_Report.pdf
70 Ibid 69 Holly Chiang, Yifan Ge, and Connie Wo, “Multiple Object Recognition with Focusing and
Blurring” http://cs231n.stanford.edu/reports/2016/pdfs/259_Report.pdf
70 Ibid 71 Antoly Nichvoloda “‘Hierarchical Bokeh’ Theory of Attention” in Dena Shottenkirk, Manuel
Curado, Steven S. Gouveia eds, Perception, Cognition, and Aesthetics (New York and London:
Routledge, 2019), 85-105. g
72 Joseph Ferenbok, “Configuring the Face as a Technology of Citizenship: Biometrics,
Surveillance and the Facialization of Institutional Identity.” In Kalantzis-Cope P., Gherab-
Martín K. eds, Emerging Digital Spaces in Contemporary Society (London: Palgrave
Macmillan, 2010), 126-127, 127. https://doi.org/10.1057/9780230299047_21. 72 Joseph Ferenbok, “Configuring the Face as a Technology of Citizenship: Biometrics,
Surveillance and the Facialization of Institutional Identity.” In Kalantzis-Cope P., Gherab-
Martín K eds Emerging Digital Spaces in Contemporary Society (London: Palgrave y
p
,
Martín K. eds, Emerging Digital Spaces in Contemporary Society (London: Palgrave
Macmillan, 2010), 126-127, 127. https://doi.org/10.1057/9780230299047_21. “7 Best Camera Lesnes for Bokeh Photography.” Adorama.com, (May 21, 2020).
https://www.adorama.com/alc/5-best-camera-lenses-for-bokeh-photography/ “7 Best Camera Lesnes for Bokeh Photography.” Adorama.com, (May 21, 2020). https://www.adorama.com/alc/5-best-camera-lenses-for-bokeh-photography/ Notes 73 Joy Buolamwini and Timnit Gerbu. “Gender Shades: Intersectional Accuracy Disparities in
Commercial Gender Classification” Proceedings of Machine Learning Research. 81:1 (2018), 1-
15. 93 Frames Cinema Journal, Issue 18 (June 2021) Buriro, Attaullah and Akhtar, Zahid and Crispo, Bruno and Del Frari, Fillipo. “Age, Gender and
Operating-Hand Estimation on Smart Mobile Devices.” 2016 International Conference of
the Biometrics Special Interest Group (BIOSIG) (Darmstadt, Germany, 2016): 1-5, DOI:
10.1109/BIOSIG.2016.7736910 AffectNet. http://mohammadmahoor.com/affectnet/ Arago, Dominique Francois. “Report.” In Alan Trachtenberg, ed. Classic Essays on
Photography. New Haven, Conn: Leetes Island Books: 1981: 15-26. Arthur, Charles. “iPhone 4 unveiled by Apple.” The Guardian, (June 7, 2010)
https://www.theguardian.com/technology/2010/jun/07/iphone-4-apple-wwdc Azar, Mitra. “Algorithmic Facial Image: Regimes of Truth and Datafication.” A Peer-Reviewed
Journal About APRJA, 7(1) (2018): 27-35. https://doi.org/10.7146/aprja.v7i1.115062 Batchen. Geoffrey. Each Wild Idea: writing photography history. Cambridge, MA: MIT Press,
2002. Bayford, Sam. “How AI is Changing Photography.” The Verge (Jan 31, 2019). https://www.theverge.com/2019/1/31/18203363/ai-artificial-intelligence-photography-
google-photos-apple-huawei Benjamin, Ruha. Race After Technology: Abolitionist Tools for the New Jim Code. Cambridge,
UK and Malden, MA: Polity, 2019. Benjamin, Walter. “Short History of Photography” (1931). Artforum. https://www.artforum.com/print/197702/walter-benjamin-s-short-history-of-photography-
36010 Bertillon. Alphonse. Ethnographie moderne: les races sauvages. Paris: G. Masson, 1883. https://gallica.bnf.fr/ark:/12148/bpt6k104250m/texteBrut Brandom, Russel. “The five biggest questions about Apple’s new facial recognition system.” The
Verge. (September 12, 2017). https://www.theverge.com/2017/9/12/16298156/apple-
iphone-x-face-id-security-privacy-police-unlock Buolamwini, Joy and Gerbu, Timnit. “Gender Shades: Intersectional Accuracy Disparities in
Commercial Gender Classification.” Proceedings of Machine Learning Research. 81(1)
(2018), 1-15. Buriro, Attaullah and Akhtar, Zahid and Crispo, Bruno and Del Frari, Fillipo. “Age, Gender and
Operating-Hand Estimation on Smart Mobile Devices.” 2016 International Conference of
the Biometrics Special Interest Group (BIOSIG) (Darmstadt, Germany, 2016): 1-5, DOI:
10.1109/BIOSIG.2016.7736910 94 Frames Cinema Journal, Issue 18 (June 2021) Chiang, Holly and Ge, Yifan and Wo, Connie. “Multiple Object Recognition with Focusing and
Blurring” http://cs231n.stanford.edu/reports/2016/pdfs/259_Report.pdf Chiang, Holly and Ge, Yifan and Wo, Connie. “Multiple Object Recognition with Focusing and
Blurring” http://cs231n.stanford.edu/reports/2016/pdfs/259_Report.pdf Crawford, Kate and Paglen, Trevor. Excavating AI: The politics of images in machine learning
training. https://www.excavating.ai “Data Collection and Analysis.” Selfiecity, http://selfiecity.net/#dataset Day, Elizabeth. “How selfies became a global phenomenon.” The Guardian, (July 13, 2013). https://www.theguardian.com/technology/2013/jul/14/how-selfies-became-a-global-
phenomenon Didi-Huberman, Georges. Invention of Hysteria: Charcot and the Photographic Iconography of
the Salpêtrière. Cambridge, MA: MIT Press, 2004. Eastlake, Lady Elizabeth. “Photography.” In Alan Trachtenberg, ed. Classic Essays on
Photography. New Haven, Conn: Leetes Island Books: 1981: 39-69. Eidinger, Eran and Enbar, Roee and Hassner, Tal. Age and Gender Estimation of Unfiltered
Faces. Transactions on Information Forensics and Security (IEEE-TIFS), Special Issue on
Facial Biometrics in the Wild, Volume 9.12, (Dec, 2014): 2170 – 2179. Faroki, Harun. “Phantom Images,” Public 29 (2004). https://public.journals.yorku.ca/index.php/public/article/view/30354 Ferenbok, Joseph. “Configuring the Face as a Technology of Citizenship: Biometrics,
Surveillance and the Facialization of Institutional Identity.” In: Kalantzis-Cope P.,
Gherab-Martín K. eds, Emerging Digital Spaces in Contemporary Society. London:
Palgrave Macmillan, 2010, 126-127. https://doi.org/10.1057/9780230299047_21 Galton, Francis. Hampton, Claire. “#nomakeupselfies: The Face of Hashtag Slacktivism.” Networking
Knowledge: Journal of the MeCCSA Postgraduate Network 8(6) (2015).
https://doi.org/10.31165/nk.2015.86.406 Rattani, Ajita and Agrawal, Mudit. “Soft-Biometric Attributes from Selfie Images.” in
Rattani A., Derakhshani R., Ross A. eds, Selfie Biometrics. Advances in Computer AffectNet. http://mohammadmahoor.com/affectnet/ Narrative of a Traveler to Tropical South Africa. London: John Murray, 1853. https://galton.org/books/south-west-africa/galton-1853-travels-in-south-africa-1up-
linked.pdf Gilman, Sander L. ed. The Face of Madness: Hugh Diamond and the Origin of Psychiatric
Photography. Brattleboro, VT: Echo Point Books and Media, 2014. Gunhert, Andre. “The Consideration of the selfie: A cultural history.” In Julia Eckel, Jens
Ruchatz, and Sabine Wirth, eds., Exploring the Selfie: Historical, Theoretical, and
Analytical Approaches to Digital Self-Photography. Palgrave Macmillan, 2018. Hampton, Claire. “#nomakeupselfies: The Face of Hashtag Slacktivism.” Networking
Knowledge: Journal of the MeCCSA Postgraduate Network 8(6) (2015). https://doi.org/10.31165/nk.2015.86.406 95 Frames Cinema Journal, Issue 18 (June 2021) Hansen, Mark B.N. “Affect as medium, or the ‘digital-facial-image’.” Journal of Visual Culture,
2(2) (2003): 206-228. https://doi.org/10.1177%2F14704129030022004 Hansen, Mark B.N. “Affect as medium, or the ‘digital-facial-image’.” Journal of Visual Culture,
2(2) (2003): 206-228. https://doi.org/10.1177%2F14704129030022004 Hassner, Tal. The OUI-Adience: Face Image Project. https://talhassner.github.io/home/projects/Adience/Adience-data.html Heckman, Christoffer. “AI can now read emotions – should it? The Conversation. (January 8,
2020). https://theconversation.com/ai-can-now-read-emotions-should-it-128988 Hernandez, Carlos. “Lens Blur in the new Google Camera app.” Google AI Blog (April 16,
2014). https://ai.googleblog.com/2014/04/lens-blur-in-new-google-camera-app.html Ignatov, Andrey et al. “AIM 2020 Challenge for Rendering Realistic Bokeh.” ArXiv, (2020),
https://arxiv.org/abs/2011.04988 Kasra, Mona. “Digital-networked images as personal acts of political expression: New
categories for meaning formation,” Media and Communication, 5.4 (2017): 51–64,
https://doi.org/10.17645/mac.v5i4.1065 Kingslake, Rudolph. A History of the Photographic Lens. Boston, Academic Press, 1989. Kingston, Grace and Goddard, Michael. “The Aesthetic Paradoxes of Visualizing the Networked
Image,” Contemporary Arts and Cultures (2017). https://contemporaryarts.mit.edu/pub/aestheticparadoxes Kosinski, Michal. “Facial recognition technology can expose political orientation from
naturalistic facial images.” Scientific Reports 11.100 (2021). https://doi.org/10.1038/s41598-020-79310-1 Lauer, Josh. “Surveillance History and the History of New Media: An Evidential Paradigm.”
New Media & Society 14(1) (June 2012): 566–82. https://doi.org/10.1177/1461444811420986 Levi, Gil and Hassner, Tal. Age and Gender Classification Using Convolutional Neural
Networks. IEEE Workshop on Analysis and Modeling of Faces and Gestures (AMFG), at
the IEEE Conf. on Computer Vision and Pattern Recognition (CVPR), Boston, June 2015. https://talhassner.github.io/home/projects/cnn_agegender/CVPR2015_CNN_AgeGenderEs
timation.pdf Lüders, Marika and Prøitz, Lin and Rasmussen, Terje. “Emerging personal media genres.” New
Media & Society 12 (2010): 947–963. Lupton, Deborah. The Quantified Self. Cambridge UK, and Medford, MA: Polity, 2016. Manovich, Lev. The Language of New Media. Cambridge, MA: MIT Press, 2002. 96 Frames Cinema Journal, Issue 18 (June 2021) Metcalf, Jacob and Crawford, Kate. “Where are human subjects in Big Data research? The
emerging ethics divide,” Big Data & Society (January–June 2016), 1-14. AffectNet. http://mohammadmahoor.com/affectnet/ https://doi.org/10.1177/2053951716650211 Metcalf, Jacob and Crawford, Kate. “Where are human subjects in Big Data research? The
emerging ethics divide,” Big Data & Society (January–June 2016), 1-14. https://doi.org/10.1177/2053951716650211 Mirzoeff, Nicholas. How to See the World: An Introduction to Images, from Self-Portraits to
Selfies, Maps to Movies, and More. New York: Basic Books, 2016. Nadine, Friedrich et al. “Faking it: Simulating background blur in portrait photography using a
coarse depth map estimation from a single image.” WSCG 2016: short communications
proceedings: The 24th International Conference in Central Europe on Computer Graphics,
Visualization and Computer Vision 2016 in co-operation with EUROGRAPHICS:
University of West Bohemia, Plzen, Czech Republic May 30 - June 3 2016, (2016): 17-23. https://dspace5.zcu.cz/bitstream/11025/29683/1/Friedrich.pdf Nichvoloda, Antoly. “‘Hierarchical Bokeh’ Theory of Attention.” in Dena Shottenkirk, Manuel
Curado, Steven S. Gouveia eds, Perception, Cognition, and Aesthetics. New York and
London: Routledge, 2019: 85-105. Paglen, Trevor. “Invisible Images: Your Pictures Are Looking at You.” Architectural Design 89
(2019): 22-27. https://doi.org/10.1002/ad.2383 ---. “Operational Images.” e-flux 59 (November 2014). https://www.e-
flux.com/journal/59/61130/operational-images/ Phu, Thy and Steer, Linda M. “Introduction.” Photography and Culture 2(3) (2019): 235-239,
DOI: 10.2752/175145109X12532077132194 Raphael, Angulu and Tapamo, Jules R. and Adewumi, Adremi O. “Age estimation via face
images: a survey.” J Image Video Proc, 42 (2018). https://doi.org/10.1186/s13640-018-
0278-6 Ranjan, Rajeev and Patel, Vishal and others, “HyperFace: A Deep Multi-Task Learning
Framework for Face Detection, Landmark Localization, Pose Estimation, and Gender
Recognition.” IEEE Transactions on Pattern Analysis and Machine Intelligence, 41.1
(January 1, 2017): 121-135. DOI: 10.1109/TPAMI.2017.2781233 Rattani, Ajita and Derakhshani, Reza and Ross, Arun eds. Selfie Biometrics. Advances in
Computer Vision and Pattern Recognition. Springer, Cham, 2019. --. “Introduction to Selfie Biometrics.” in Rattani A., Derakhshani R., Ross A. eds, Selfie
Biometrics. Advances in Computer Vision and Pattern Recognition. Springer, Cham. 2019. https://doi.org/10.1007/978-3-030-26972-2_1 Rattani, Ajita and Agrawal, Mudit. “Soft-Biometric Attributes from Selfie Images.” in
Rattani A., Derakhshani R., Ross A. eds, Selfie Biometrics. Advances in Computer 97 Frames Cinema Journal, Issue 18 (June 2021) Vision and Pattern Recognition. Springer, Cham. 2019. https://doi.org/10.1007/978-3-
030-26972-2_1 Vision and Pattern Recognition. Springer, Cham. 2019. https://doi.org/10.1007/978-3-
030-26972-2_1 Rubinstein, Daniel and Sluis, Katrina. “The digital image in photographic culture: algorithmic
photography and the crisis in representation.” In Martin Lister, ed. The photographic image
in digital culture. London and New York: Routledge, 2013: 22-40. Rubinstein, Daniel and Sluis, Katrina. “The digital image in photographic culture: algorithmic
photography and the crisis in representation.” In Martin Lister, ed. AffectNet. http://mohammadmahoor.com/affectnet/ The photographic image
in digital culture. London and New York: Routledge, 2013: 22-40. Saxena, Avita and Khanna, Ashish and Gupta, Deepak. “Emotion Recognition and Detection
Methods: A Comprehensive Survey.” Journal of Artificial Intelligence and Systems, 2
(2020), 53-79. https://doi.org/10.33969/AIS.2020.21005 Sekula, Alan. “The Body and the Archive.” October 39 (Winter, 1986): 3-64. “Selfie.” Oxford Learner’s Dictionaries. https://www.oxfordlearnersdictionaries.com/us/definition/english/selfie Selfie Data Set, https://www.crcv.ucf.edu/data/Selfie/ Shi, Yichun and Jain, Anil K. “DocFace+: ID Document to Selfie Matching’” IEEE
Transactions on Biometrics, Behavior, and Identity Science 1.1 (January 2019): 56-67, 56. DOI: 10.1109/TBIOM.2019.2897807 Todorov, Alexander and Olivola, Christopher Y. and others, “Social Attributions from Faces:
Determinants, Consequences, Accuracy, and Functional Significance.” Annual Review of
Psychology, 66, (January, 2015): 519-545. https://doi.org/10.1146/annurev-psych-113011-
143831 Valind, Erik. Portrait Photography: From Snapshots to Great Shots. Peachpit Press, 2014. Wadhwa, Neal et. al. “Synthetic depth-of-field with a single-camera mobile phone.” ACM
Transactions on Graphics, 64 (July 2018). https://dl.acm.org/doi/10.1145/3197517.3201329 Wang, Y. and M. Kosinski, “Deep neural networks are more accurate than humans at detecting
sexual orientation from facial images.” Journal of Personality and Social Psychology,
114.2 (2018), 246-257. https://doi.org/10.1037/pspa0000098 Author Biography Author Biography
Dr. Stefka D. Hristova is an Associate Professor of Digital Media at Michigan Technological
University. She holds a PhD in Visual Studies with emphasis on Critical Theory from the
University of California, Irvine. Her research analyses digital and algorithmic visual culture. Hristova’s work has been published in journals such as Transnational Subjects Journal, Visual
Anthropology, Radical History Review, TripleC, Surveillance and Security, Interstitial, Cultural
Studies, Transformations. She was a NEH Summer Scholar for “Material Maps In the Digital
Age” seminar in 2019. Hristova is the lead editor for Algorithmic Culture: How Big Data and
Artificial Intelligence are Transforming Everyday Life, Lexington Books, 2021. 98
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Sandra Požar, Inventar samostana Sv. Marije Magdalene u Portu na otoku Krku...
FLUMINENSIA, god. 30 (2018), br. 2, str. 29-43 Sandra Požar, Inventar samostana Sv. Marije Magdalene u Portu na otoku Krku... FLUMINENSIA, god. 30 (2018), br. 2, str. 29-43 29 This work is licensed under a Creative Commons Attribution 4.0 International License. Ovaj rad dostupan je za upotrebu pod međunarodnom licencom Creative Commons Attribution 4.0. https://doi.org/10.31820/f.30.2.4 Sandra Požar dr. sc. Sandra Požar, Staroslavenski institut, sudec@stin.hr, Zagreb izvorni znanstveni članak
UDK 003.349.1(497.5Krk)
2-523.6(497.5Krk)
811.163.42’282(497.5Krk)
rukopis primljen: 12. 10. 2018.; prihvaćen za tisak: 16. 11. 2018. izvorni znanstveni članak
UDK 003.349.1(497.5Krk)
2-523.6(497.5Krk)
811.163.42’282(497.5Krk)
rukopis primljen: 12. 10. 2018.; prihvaćen za tisak: 16. 11. 2018. U članku se donosi pregled grafijskih i glasovnih značajka Inventara
samostana sv. Marije Magdalene u Portu na otoku Krku (1734. – 1878.),
spomenika pragmatične glagoljske pismenosti franjevaca trećoredaca, čiji
sažeti upisi unatoč ustaljenosti i jezgrovitosti izraza nude vrijednu građu za
jezičnopovijesno istraživanje. Usporedba zatečena stanja s opisima krčkih
govora u literaturi (Lukežić, Turk 1998; Milčetić 1895) pokazala je uglavnom
podudaranje jezične slike Inventara s doseljeničkim krčkim govorima koji se
govore na prostoru nastanka rukopisa. No pojavljuju se i neke južnije
dijalektne inovacije koje se ne mogu pripisati pisarima jer povijesni izvori ne
potvrđuju prisutnost samostanskih članova s dotičnih područja. Ključne riječi: glagoljica; franjevci trećoredci glagoljaši; praktična glagoljska
pismenost; povijest hrvatskoga jezika; čakavsko narječje; govori otoka Krka INVENTAR SAMOSTANA SV. MARIJE
MAGDALENE U PORTU NA OTOKU KRKU
(1734. – 1878.) KAO JEZIČNI IZVOR:
GRAFIJA I FONOLOGIJA dr. sc. Sandra Požar, Staroslavenski institut, sudec@stin.hr, Zagreb Sandra Požar, Inventar samostana Sv. Marije Magdalene u Portu na otoku Krku... Sandra Požar, Inventar samostana Sv. Marije Magdalene u Portu na otoku Krku... 1 Rad je nastao na temelju pripreme za (neodržano) izlaganje na znanstvenom skupu
Trećoredska glagoljaška tradicija u europskom kontekstu, Zagreb, 2013. Istraživanje je
obuhvatilo i morfološku i sintaktičku te leksičku razinu, ali ovdje se zbog ograničenosti
prostora ti rezultati ne objavljuju. U zasebnom su radu također opisani rezultati jezičnoga
dodira s romanskim idiomima (Požar 2018). 2 Popisu iz te godine prethodi još jedan koji nije datiran.
3 Iako je autorica, sudjelujući u pripremi Badurinina izdanja, imala uvid u izvornik, u
vrijeme pisanja rada nije bila u mogućnosti ponovno provjeriti u njemu sumnjiva mjesta.
4 Uz primjere se navodi oznaka mjesta u izvornoj paginaciji. Zagrade unutar riječi sadrže
rekonstrukciju priređivača.
5 Priređivači su u transkripciji teksta dali grafičku obavijest o takvim znakovima
kurziviranjem. Ovdje su, budući da se primjeri navode u kurzivu, ta mjesta napisana obično. 1. Uvod 29-43 31 staviti da se u početku knjige piše na jeziku mjesta nastanka rukopisa
(ugledanjem na prethodne upise), a kasnije je iz nekoga razloga taj „standard”
počeo labaviti. U daljnjem se tekstu prikazuju pojave na grafijskoj i ortografskoj te
fonetsko-fonološkoj razini. Istraživanjem su obuhvaćeni svi glagoljicom
ispisani listovi: od 1r (prije 17342) do 80v b (1830). Latinični dio inventara
nije uzet u obzir zbog višesmislenosti grafije. Praktičnu pismenost, nasuprot svečanoj, bitno obilježava brzo pisanje
čiji su rezultat česte pisarske pogreške. Neke su od njih u članku proble
matizirane, dok su neke morale biti zanemarene: ako bi posrijedi mogao
biti pogrešan prijepis, previd ili pogrešna interpretacija priređivača (autora)
ili redaktora.3 1. Uvod Inventar samostana sv. Marije Magdalene u Portu na otoku Krku vođen je
kurzivnom glagoljicom i, manjim dijelom, latinicom od 1734. do 1878. godine Sandra Požar, Inventar samostana Sv. Marije Magdalene u Portu na otoku Krku... FLUMINENSIA, god. 30 (2018), br. 2, str. 29-43 30 u portanskom samostanu sv. Marije Magdalene franjevaca trećoredaca, a
objavio ga je 2013. godine A. Badurina, dok je prve podatke o njemu iznio V. Štefanić 1960. Inventar kao povijesni spomenik izvor je podataka o povijesti
redovničke zajednice franjevaca trećoredaca u Hrvatskoj, o lokalnoj povijesti
otoka Krka, te o crkvenoj, liturgijskoj, društvenoj i gospodarskoj povijesti. Hrvatski franjevci trećoredci, kojima pripada samostan sv. Marije Magdalene
u Portu, posebni su po tome što su najdulje u povijesti njegovali
crkvenoslavenski jezik u liturgiji i glagoljicu u pisanju (do početka primjene
odredaba Drugoga vatikanskoga koncila). Glagoljica se, kao što je poznato,
rabila do duboko u novi vijek i za svakodnevne praktične potrebe, dakako i
izvan trećoredskih krugova, a takvi su se tekstovi pisali čakavskim govornim
jezikom. Tomu korpusu pripada i ovdje obrađivani Inventar. Struktura popisa inventara određena je prostorima života i područjima
djelatnosti redovnika: crkva, sakristija, kuhinja, blagovaonica, konoba,
samostan, ljetina i blago. Inventar se popisivao svakih nekoliko godina,
kada bi bio izabran novi gvardijan, koji je najčešće i obavljao popisivanje. Upisi u Inventaru više su ili manje ustaljeni, a izraz im je u svojoj ograni
čenosti sveden uglavnom na imenovanje predmeta i navođenje njihove
vrste, kakvoće i količine. No unatoč oskudnosti izraza popisi pružaju
neočekivanu količinu podataka vrijednih za jezičnopovijesno istraživanje. Upravo je bogatstvo građe bilo poticaj nastanku ovoga članka.1 Narav je teksta praktična, te on stoga, doduše uz obojenost admini
strativnim stilom, bilježi govorni jezik. Osim toga, vrijedno je i to što je
zbog naravi teksta svaki izraz datiran. Međutim, u vezi s bilježenjem
govornoga jezika treba dodati važnu napomenu. U mlađem dijelu Inventara
pojavljuju se neke jugoistočnije čakavske značajke za koje bi se očekivalo da
upućuju na dolazak redovnika s dotičnih područja, a njima se mogu
pridružiti i morfološke značajke koje upućuju čak i na štokavski utjecaj. No
povijesni izvori, objavljeni u novije vrijeme, to ne potvrđuju. Naime, prema
tabličnom pregledu u Botica, Kovačić, Kuhar 2015: 29–45 redovnici iz
udaljenijih krajeva počeli su se u znatnijoj mjeri posinovljavati ovomu
samostanu tek nakon vremena pisanja Inventara. Stoga se može pretpo Sandra Požar, Inventar samostana Sv. Marije Magdalene u Portu na otoku Krku... NENSIA, god. 30 (2018), br. 2, str. 2 Popisu iz te godine prethodi još jedan koji nije datiran. 2. Grafija Grafiji Inventara svojstveno je da različiti pisari imaju različita grafijska
načela, ali na variranje se može naići i kod istoga pisara. Raznolikost je
osobito izražena u pisanju glasa /j/, koji se zbog svoje glasovne specifičnosti
i tradicionalno odlikuje različitim grafijskim ostvarajima. U glagoljaškoj je
tradiciji uobičajen postupak, naslijeđen iz staroslavenske pismenosti, da se
glas /j/ ne bilježi zasebnim znakom, nego se pretpostavlja prejotirani
izgovor /e/ (katkad i /i/) na početku riječi i iza samoglasnika. Tako nalazimo
tradicionalno pisanje elovine (74v a), esena (64v), ali i inovirane inačice:
jelovine (4v), jesena (5r).4 Tradicionalna mogućnost da se glas /j/ u pismu
obuhvati znakom za jat u Inventaru nije primijenjena jer je jat reflektiran,
pa znak za jat prenosi slijed j + a, što je učestalo iza samoglasnika (npr. Šakreštija, mobilija5 4r) ili iza mekih suglasnika /ĺ/ i /ń/ čija mekoća nije
drugačije obilježena, npr. stargulja (29v), Kuhinja, pinjate (40v). Slično
vrijedi za slovo ju, koje bilježi slijed j + u (npr. lušiju 29r). Druga je
tradicionalna mogućnost bilježenja glasa /j/ iskorištena: zasebnim grafemom
đerv, koji je izvorno bilježio refleks praslavenske skupine *dj. No posve je Sandra Požar, Inventar samostana Sv. Marije Magdalene u Portu na otoku Krku... FLUMINENSIA, god. 30 (2018), br. 2, str. 29-43 32 osobit način kojim neki pisari označavaju jotiranost odnosno bilježe glas /j/
pisanje znaka ‘ (apostrofa) iznad samoglasnika, npr. Rifitori’ (43r), što
predstavlja svojevrsnu inovaciju u glagoljskoj pismenosti. U kurzivnoj
glagoljici takav je znak doduše poznat, ali samo kao oznaka pojedinoga
samoglasnika u određenoj funkciji, npr. i kao veznik ili i na početku riječi,
slično kao što postoji inačica kurzivnoga slova i u tim položajima.6 Ovaj
znak ne treba poistovjetiti s „izvornim” apostrofom (jerokom) koji je – uz
manja ili veća odstupanja – kontinuanta znaka za stari poluglas. U Botica,
Kovačić, Kuhar 2015: 24 ova je pojava, na tragu paleografskih smjernica V. Štefanića (usp. Štefanić 1969: 20), prepoznata kao oponašanje postupaka u
priručnicima za opismenjavanje kojima su se trećoredci služili – glagoljskim
početnicama, tj. bukvarima R. Levakovića i M. Karamana.7 Raznolikost bilježenja glasa /j/ dobro ilustrira mjesto gdje je on u
prezentu glagola biti u tri uzastopna retka stranice 1v zabilježen na tri
načina: (...) kako e pismo / (...) je pismo / (...) pismo ’e. Slično je na stranici 50v
u dva susjedna retka: škrinja; škrin’e. 9 Jednako je stanje – i s obzirom na raznovrsnost rješenja i s obzirom na nedosljednosti
– u pragmatičnom glagoljskom spomeniku s istoga prostora i iz podudarna vremena:
dubašljanskom Trećem notarskom protokolu Jura Sormilića iz 18. stoljeća (Bošnjak Botica,
Botica, Galović 2016: 26). Upravo praktična namjena i njome uvjetovana brzina pisanja
(koja povećava mogućnost pogreške) pokazuju se kao ključna potvrda Štefanićeve tvrdnje o
17. i 18. stoljeću kao o vremenu „nazadovanja” glagoljske pismenosti (Štefanić 1969: 17;
nazadovanje se odnosi na ljepotu rukopisa, pa se shvaća uvjetno). 6 Osim toga, u 17. i 18. stoljeću može se u knjiškom kurzivu pojaviti točka iznad slova i,
koja je bez funkcije, a tumači se kao rezultat latiničnoga utjecaja (usp. Žagar 2013.a: 362). 6 Osim toga, u 17. i 18. stoljeću može se u knjiškom kurzivu pojaviti točka iznad slova i,
koja je bez funkcije, a tumači se kao rezultat latiničnoga utjecaja (usp. Žagar 2013.a: 362).
7 Ovo nije jedina potvrda takva načina pisanja. Ono je pronađeno u još jednom krčkom
rukopisu iz podudarna vremena Zbirci legendi popa Duma Grege iz Omišlja (1773) 8 Usp. Mihaljević 2014: 64–65. Ovo nije jedina potvrda takva načina pisanja. Ono je pronađeno u još jednom krčkom
isu iz podudarna vremena, Zbirci legendi popa Duma Grege iz Omišlja (1773). 10 Kada ne bi bilo stalnoga variranja i da nije poznato današnje stanje, moglo bi se
pretpostaviti da grafemi bez oznake mekoće bilježe depalatalizirani glas. 2. Grafija Glas /j/ može se, nadalje, pisati i grafemom i, katkad čak i ispred
grafema i kojim se bilježi glas /i/: kolonica na koii stoii kašetica (16r). Konačno, glas /j/ može, doduše rijetko, biti zabilježen i grafemom šta, pri
čemu je šta zamjena za đerv, obrnuto od starije glagoljske pismenosti u
kojoj je postojala zamjena slova šta slovom đerv.8 Primjerice, oblik majkača
(‘vrsta motike’) ponegdje se ostvaruje kao maĉkača (22v), a da je riječ o
glasu /j/, pokazuje pisanje sa i (maikače, 64v). Navedeno stanje pokazuje da
su pisari poznavali stariju glagoljsku pismenost, ali su, s obzirom na
praktičnu namjenu teksta, uzimali slobodu za iznalaženje novih rješenja.9 Sandra Požar, Inventar samostana Sv. Marije Magdalene u Portu na otoku Krku... FLUMINENSIA, god. 30 (2018), br. 2, str. 29-43 3. Ortografija Vrijedi spomenuti i uvođenje ortografije bliske suvremenomu latinič
nom pravopisu, što se može pratiti i u pravnim tekstovima.12 To se odnosi
na pisanje velikoga početnoga slova u imenima, npr. Jivu Žgaliću (2r), te u
pridjevu svet: Svetim ulem (16v).13 Druga je ortografska značajka to što je punktuacija uglavnom prešla u
interpunkciju (usp. Žagar 2013: 287). Upotrebljavaju se ovi interpunkcijski
znakovi: točka, zarez i točka-zarez. Točka se kao interpunkcijski znak
pojavljuje na kraju uvodne rečenice svakoga popisa, gdje se navodi tko
komu i kada izručuje inventar. Punktuacijsku ulogu točka ima još jedino iza
brojeva.14 U oba je slučaja točka još uvijek na sredini retka. Zarez se može
upotrijebiti za odvajanje rečenica, npr. krave ke su na pašu, jesu na drugi libar
(20r); štrite od mostira pol, a pol S(veto)ga Mikule (48r); te za nabrajanje: Slidi
intrada, žito, vino i ule (68v b). Slično vrijedi i za točku sa zarezom: pod
nastojanje; aliti prigledanje p(oštovanomu) ocu (31v). Sandra Požar, Inventar samostana Sv. Marije Magdalene u Portu na otoku Krku...
FLUMINENSIA, god. 30 (2018), br. 2, str. 29-43 Na nekim mjestima nije jasno jesu li pojedina slova
namjerno ispuštena, radi kraćenja – budući da je riječ o poznatim pojmo Sandra Požar, Inventar samostana Sv. Marije Magdalene u Portu na otoku Krku... FLUMINENSIA, god. 30 (2018), br. 2, str. 29-43 34 vima koji se ponavljaju – ili je riječ o pisarovoj pogrešci, ili pak o bilježenju
glasovne pojave, npr. bola(n)čun (61v), ku(hi)ni (68r a).11 Sandra Požar, Inventar samostana Sv. Marije Magdalene u Portu na otoku Krku...
FLUMINENSIA, god. 30 (2018), br. 2, str. 29-43 33 Označavanje mekoće odnosno jotiranosti osobito dolazi do izražaja
pri bilježenju suglasnika /ĺ/ i /ń/ odnosno sljedova l + j i n + j te njihovu
grafijskom razlikovanju od suglasnika /l/ i /n/ koji se bilježe istim grafemima. Treba naglasiti da to označavanje nije svojstveno glagoljskoj tradiciji. Ipak,
mekoću ne bilježe svi pisari.10 Usp. ule (10v) ~ ul’e (22v); ula (11r) ~ ul’a
(24r), ulja (31v); stergula (40v) ~ stargulja (29v); šuplača (17v), šupla (25r) ~
šupljača (7v); zemle (45v) ~ zemlje (72v a); badni (60r) ~ bad’ni (26r); kon
(68v b) ~ kon’ (55v); kostana (10r) ~ kostan’a (30r), kostanja (46r); ražni (5r)
~ ražn’i (29v); škrina (44r) ~ škrin’a (14v), škrinja (41v); noi (32r) ~ n’oi (28v);
sridni (36r) ~ sridn’i (29r); manih (14r) ~ man’ih (71r a). Nadalje, smjene su,
kako je naprijed spomenuto, moguće i kod istoga pisara, u dva susjedna
retka, npr. zeml’e ~ zemle (70v b), manih ~ man’ih (72v a), ili u razmaku od
nekoliko redaka, npr. kostana ~ kostanja (60r). Na neujednačenost u pisanju
apostrofa autori Botica, Kovačić, Kuhar 2015: 24 upozoravaju kao na opću
grafijsku značajku trećoredskih spisa. Znak sličan apostrofu (’), osim u navedenoj službi označavanja
mekoće, rabi se mjestimično i kao tradicionalan grafijski ostatak nekadaš
njega znaka za poluglas, npr. z’ manigi (5r), z’ naviželu (12r). No to je slučaj
samo u starijem dijelu Inventara: posljednja je potvrda z’ Mikom na stranici
26v iz 1752. godine. Ponegdje se tim znakom razbijaju konsonantske
skupine: z’del, z’dola (24r), što je također poznato iz starije pismene tradicije
kao mehanički unos znaka (usp. Mihaljević 2014: 68). Kao grafijska osobitost može se spomenuti i upotreba grafema ju za
glas /u/, npr. ljušiju 17r ~ lušiju, dakako, ako se isključi da je riječ o bilježenju
glasovne pojave. Takva upotreba grafema ju nije nepoznata ni starijoj
glagoljaškoj tradiciji. Na grafijskoj su razini zanimljiva i kraćenja riječi. Skraćene riječi mogu
biti označene titlom, kao što je bilo uobičajeno u crkvenoslavenskoj
glagoljskoj tradiciji, npr. sg̃a = s(veto)ga, st̃a = s(ve)ta. Neka kraćenja nisu
označena titlom, ali su, zbog česta pojavljivanja ili na temelju tradicije, obična
i očekivana, npr. otc za otac. No ima i kraćenja koja nisu česta i sustavna,
nego su provedena ad hoc, pa se ne mogu smatrati nastavljanjem tradicije,
npr. Sakramet̃ (24r). 11 Više usp. u odjeljku 4.3.
12 Usp. primjerice upotrebu zareza već u prijepisu Istarskoga razvoda iz 1546. (usp. Bratulić
1989).
13 Veliko se početno slovo piše katkad i na početku cjeline, tj. u naslovima: Po crikvi roba
(67v a), što je bilo poznato i u crkvenoslavenskoj ortografiji.
14 Pitanje je je li u potvrdi klišća . popečak . kana (78r b) točka u funkciji zareza ili je bilo
predviđeno da stoji uz broj koji je ipak izostavljen kada je zatečen po jedan primjerak
svakoga navedenog predmeta. 11 Više usp. u odjeljku 4.3. 12 Usp. primjerice upotrebu zareza već u prijepisu Istarskoga razvoda iz 1546. (usp
1989).
13 (67v a), što je bilo poznato i u crkvenoslavenskoj ortografiji.
14 Pitanje je je li u potvrdi klišća . popečak . kana (78r b) točka u funkciji zareza ili je bilo
predviđeno da stoji uz broj koji je ipak izostavljen kada je zatečen po jedan primjerak
svakoga navedenog predmeta. 15 Autor je upisa gvardijan fra Mikel Brzac, kojemu podrijetlo nije pobliže poznato, nego
se navodi samo „otok Krk” (usp. Botica, Kovačić, Kuhar 2015: 30). Sandra Požar, Inventar samostana Sv. Marije Magdalene u Portu na otoku Krku...
FLUMINENSIA, god. 30 (2018), br. 2, str. 29-43 Sandra Požar, Inventar samostana Sv. Marije Magdalene u Portu na otoku Krku... FLUMINENSIA, god. 30 (2018), br. 2, str. 29-43 35 17 Brojke označuju broj mjesta (stranica), a treba napomenuti da se na nekim stranicama
dotični oblik pojavljuje i više puta (dva ili tri). 16 Usp. odjeljak 4.2.6. 4. Fonetika i fonologija Na fonetsko-fonološkom planu mogu se identificirati tipične srednjo
čakavske glasovne pojave. One u osnovnim crtama odgovaraju današnjemu
stanju konzervativnijega modela doseljeničkoga mikrosustava krčkih
govora, tj. govorima Dubašnice, kako su ih opisale Iva Lukežić i Marija Turk
(Lukežić, Turk 1998: 74–83). Međutim, u vezi s refleksima nekih praje
zičnih glasova ili skupina slika koju pruža Inventar nije konzistentna. U tim
je slučajevima provedeno dodatno istraživanje na obilno potvrđenim
oblicima, na što se upozorava na dotičnim mjestima. 18 Konačno, polazni nominativni oblik se i smatra jedinim relevantnim (usp. Lukežić,
Turk 1998: 24–25). Ima pojavnica koje se samo mjestimično ne slažu s pravilom, ali je o
njima teško raspravljati jer je izvornik mjestimično dosta teško čitljiv, pa je pri njegovu
prijepisu moglo doći do zamjene slova i i e, koji su u kurzivu slični. Naime, slovo e mjestimice
je vrlo slično desnomu dijelu slova i, a početni potez lijevoga dijela slova i – koje je u
kurzivnoj glagoljici polegnuto – vučen odozgo prema dolje, može biti slabije vidljiv. 4.1. Samoglasnici 4.1.1. Refleks reduciranoga samoglasnika /ə/ u pravilu je uvijek /a/, npr. bačav (5r), ovac (20r), ražan (72v a). Jedina je iznimka oblik sežen’ (15r, 1742.).15 Jaka vokalnost uočena je, osim u zamjenici ča, u vezniku z (od sə) (→
za): sedlo (...) za s(vo)imi foramenti (20r), iako je u takvoj okolini češće veznik
stegnut s početnim suglasnikom sljedeće riječi.16 4.1.2. Jat je reflektiran kao /e/ i /i/ u skladu s Jakubinski-Meyerovim
pravilom, npr. leto (19v), leta (21r, 24r, 28r, 31v itd.), testo (22v, 26r, 30r);
prid (2r), drivenih (5r), mriže, mihi, sviće, sikir (19v), misit (22v), nemirena,
vrić (20v), crikva (24r), oriha, vrić (30v), sviće, mihi, mriže, svitila, potriba,
sikir, slidi (31r), sridn’i (33r), pinica (48v), cipat (64v), Slidi (68v b). Ikavskim
refleksima može se dodati i ostvarivanje na kraju riječi (brojeva i priloga):
dvi (1v); ovdi (20r), drugdi (31v), kadi (44v). Budući da su uočena i odstupanja od pravila, stanje je provjereno na
tri obilno potvrđene osnove: *zděl-, *běl- i *cěl-. Potvrđeni oblici razvrstani
su s obzirom na usklađenost s pravilom, odstupanje od pravila zbog
ujednačenosti paradigme (u korist ekavskih oblika) ili ujednačenosti s
osnovom i nemotivirano odstupanje od pravila (u korist ikavskih oblika). Provjera je dala rezultate koji su prikazani u tablici 1. Tablica 1. Refleks jata u riječima s osnovama *zděl- *běl- i *cěl-17
REFLEKS
OSNOVA
PRAVILNOST
(Jakubinski – Meyer)
ODSTUPANJE
E
I
Analogijsko
Nemotivirano
*zděl-
4
7
11
5
*běl-
15
25
53
3
*cěl-
7
6
12
14
Ukupno
26
38
76
22
Ukupno
Prav./ Odstup. 64
98 Tablica 1. Refleks jata u riječima s osnovama *zděl- *běl- i *cěl-17 Sandra Požar, Inventar samostana Sv. Marije Magdalene u Portu na otoku Krku... FLUMINENSIA, god. 30 (2018), br. 2, str. 29-43 36 Iz tablice je vidljivo da ima znatno više slučajeva koji odstupaju od
pravila nego onih koji su s njim u skladu, ali da se velika većina njih ipak
može tumačiti ujednačenjem s polaznim oblikom.18 Ipak, količina nemotivi
ranih odstupanja u korist ikavskih oblika upućuje na prisutnost ikavskoga
elementa, odnosno labavljenje lokalne norme. 4.1.3. Uz slogotvorno /r/ pojavljuju se kao popratni samoglasnici /e/ i
/a/, a katkad je on zabilježen bez toga samoglasnika, npr. 4.1. Samoglasnici černimi (6r), derva
(16r), černe, mertvih (17r), čerlenih (24v), stergula (40v), kerčice (50r), serpi
(78v a) ~ svardli (14r), darva (24r), svardal (26v), karčica (33v), stargulja
(36r), naiparvo (73v a), srebarni (74r a) ~ drva (4r), strgula (13r), svrdal,
svrdli (14v), črnu (21v), crni (56v). Ni kod ove pojave nema ujednačenosti,
nego se inačice uočavaju i kod istoga pisara, i to u neposrednoj blizini. Tako
primjerice na stranici 24v najprije nalazimo z’ muštre crne, dva retka niže: z’
muštru čarnu, potom četiri retka niže, na 25r: od beden černih bilih i čerlenih,
i opet dva retka niže: paramenat S(veto)ga Mikule crni stari. Usp. i u dva
susjedna retka: svrdal za glavine 1 / sverdli drugi (18v), te u istom retku:
verči od već varsti (80r a). Načelan razlog tomu variranju može se tražiti u
artikulacijskom položaju popratnoga samoglasnika, koji se u dubašljanskim
govorima (danas) nalazi između kratkoga /a/ i /ə/ (Lukežić, Turk 1998: 77),
što je kod pisara moglo izazvati nesigurnost u način njegova bilježenja. Drugi bi razlog mogao biti nedovoljno posvećivanje pozornosti jeziku s
obzirom na to da dokument ima samo praktičnu namjenu. Istraživanje
dobro
potvrđenih
osnova
pokazuje
da
osjetno
prevladavaju potvrde sa er: derv- (93 potvrde) ~ darv- (20) ~ drv- (19); cern-
(45) ~ čern- (24) ~ crn- (9) ~ carn- (7) ~ čarn- (2) ~ črn- (2); verč- (28) ~ vrč-
(13) ~ varč- (1); cerlen- (22) ~ čerlen- (16) ~ crlen- (8) ~ črlen- (2) ~ čarlen- (2)
~ carlen- (1); mertv- (16) ~ martv- (11) ~ mrtv- (8). Međutim, kod nekih je
osnova stanje drugačije. Može prevladavati slijed ar, kao u slučaju leksema
karpatur- (28) ~ kerpatur- (9) ~ krpatur- (8) i stargul- (6) ~ stergul- (2) ~
strgul- (2); može prevladavati inačica bez popratnoga samoglasnika, kao u
slučaju svrd- (9) ~ svard- (3) ~ sverd- (1); ili broj potvrda er i ar može biti Sandra Požar, Inventar samostana Sv. Marije Magdalene u Portu na otoku Krku... FLUMINENSIA, god. 30 (2018), br. 2, str. 29-43 37 izjednačen, kao u karč- (10) ~ kerč- (10) ~ krč- (3). Teško je iz ovoga nešto
zaključiti, s obzirom na spomenuto variranje unutar istih inventarnih popisa,
osim to da je popratni samoglasnik češći nego slogotvorno /r/ bez poprat
noga samoglasnika. izjednačen, kao u karč- (10) ~ kerč- (10) ~ krč- (3). 20 Takvo je stanje i u potvrdama koje navodi Milčetić 1895: 110, a isto naglašava I. Miloš
za suvremeno stanje (Miloš 2015: 173). 4.1. Samoglasnici Teško je iz ovoga nešto
zaključiti, s obzirom na spomenuto variranje unutar istih inventarnih popisa,
osim to da je popratni samoglasnik češći nego slogotvorno /r/ bez poprat
noga samoglasnika. 4.1.4. Sekundarno slogotvorno /r/ običnije je u stranim riječima:
tabrnakul (9r, 24r), frmentuna (11r), Petrlin (21r, 21v). U čakavskom
glasovnom materijalu o njemu bi mogao posredno svjedočiti oblik prenašli
u potvrdi zač smo ih prenašli (19r), gdje se oblik sa sekundarnim slogo
tvornim /r/ može pretpostaviti kao prijelazni oblik od polaznoga pronašli,
koji zatim dobiva popratni samoglasnik /e/, a koji pak može biti metate
ziran (pronašli → *prnašli → *pernašli → prenašli).19 4.1.5. Slogotvorno /l/ nije sačuvano, usp. vuna. To jasno upućuje na
doseljeničke krčke govore (usp. Lukežić, Turk 1998: 77, 91). 4.1.6. Protetsko /j/ ispred /i/ očekivano je s obzirom na stanje opisano
u literaturi (Lukežić, Turk 1998: 81; Miloš 2015: 170; usp. i Milčetić 1895:
113), npr. Jivu (2r). Nasuprot tomu susreće se obrnuta pojava da se /j/ se
ispred /i/ na početku riječi na nekim mjestima ne bilježi: idro (1r) ~ jidro
(usp. glagol jadrit). 19 Usp. takav slučaj kod posuđenice frementun (Požar 2018).
20 19 Usp. takav slučaj kod posuđenice frementun (Požar 2018). 4.2. Suglasnici 4.2.1. U suglasničkom sustavu uglavnom se zatječu glasovne pojave
koje upućuju na čakavsku težnju artikulacijskomu rasterećenju, koja se
ostvaruje kao pojednostavnjenje (usp. Lukežić, Turk 1998: 30). To se odnosi
i na pojedine suglasnike i na suglasničke skupine. Tako se afrikate mogu
zamjenjivati frikativima, npr. č, ć → š (bnetaških 6r, razlišni 10v, težaške 15v,
mertvaški 75v; pokušne 9r). Međutim, treba napomenuti da su neki od tih
oblika, kao razlišni i pokušne, iznimni u odnosu na suparnički oblik s afrika
tom.20 4.2.2. Na djelu je i zamjena sonanata m → n: rinski (17r), ozinca (20r),
ozinčene (20r). Treba dodati da prijelaz m → n na kraju riječi u pravilu
izostaje, barem koliko se može utvrditi. Naime, rijetki su oblici u kojima bi
ta pojava mogla biti posvjedočena, usp. s’ patenom (21r), svoim kavadunom Sandra Požar, Inventar samostana Sv. Marije Magdalene u Portu na otoku Krku... FLUMINENSIA, god. 30 (2018), br. 2, str. 29-43 38 (53v), svoim jarmižem (77r a); dim (15r), a brojevi su češće pisani brojkama. Osamljene su potvrde prijelaza m → n kod imenice s Anton Dubašlančićen
(75r)21 i kod zamjenice na istin (69r a, 77v a). (53v), svoim jarmižem (77r a); dim (15r), a brojevi su češće pisani brojkama. Osamljene su potvrde prijelaza m → n kod imenice s Anton Dubašlančićen
(75r)21 i kod zamjenice na istin (69r a, 77v a). 4.2.3. Pojednostavnjuju se suglasničke skupine, početne: pš → š (šenica,
posvuda),22 str → sr (srić 34v),23 štr → šr (šrapuntini 68v a), škr → šr (šrinja
41r); i nepočetne: ts → s (hrvaske 15r), kt → ht (lahti 65v). Težnji pojednostavnjenju odupire se suglasnik /ĺ/. Naime, promjena ĺ →
j nije provedena: ule (5v), postilu (6r), deblega (14r), žmuli (19v),24 stargulja
(29v), bolih (48v), konople (51v), zemle (74r b), osim u dvjema stranim
riječima (luja 47v, Veju 68v a).25 To donekle odudara od današnjega stanja, u
kojem postoje obje mogućnosti, čak i kod istih govornika (usp. Lukežić, Turk
1998: 80).26 O odupiranju navedenoj težnji može se vjerojatno govoriti i u
slučaju čuvanja završnoga /l/: maštel (30v), kotal (48v), karatel (58r); bil (79v
a).27 U suprotnosti je s težnjom pojednostavnjivanju prijelaz ps → pc u
obliku pca (veruge ke stoe na pca, 50r, 55r), koji je zabilježio i I. Milčetić
(1895: 110). Pojava bi se mogla objasniti preventivnim razlogom, odnosno
nastojanjem da se izbjegne pojednostavnjivanje ps → s koje bi dovelo do
manjka obavijesti zbog oskudna glasovnoga sastava. 27 Isto je i danas (Lukežić, Turk 1998: 81–82). 26 U Miloš 2015: 173 navodi se samo /j/. 23 Budući da autorica nije mogla provjeriti stanje u izvorniku i utvrditi je li posrijedi
možda ligatura slova t i r (pa je mjesto pogrešno preneseno), taj primjer treba uzeti sa
zadrškom. 21 U riječi Anton nije provedena ta glasovna promjena, nego se ime, očito zbog pogreške,
pojavljuje u nesklonjenu obliku. 25 Samo ĺ i u Milčetić 1895: 108. 22 Bilježi i Milčetić 1895: 110. 24 Budući da je današnjemu dubašljanskom govoru svojstven ostvaraj žmuj (Spicijarić
2009: 19), može se zaključiti da se navedeni oblik ostvarivao sa /ĺ/, a ne sa /l/. 4.2. Suglasnici 4.2.4. Zabilježeno je daljinsko jednačenje: kupnena (75r) ~ kuplena
(usp. Miloš 2015: 174). No u slučaju većine potvrda (šežen’ 15r, žežan
‘sežanj’ 11r, zelezna 25v, zelezni 26v, 30r, ožimčene 31v, zelezo 76r, želeža
70v b) možda se može pomišljati na utjecaj cakavizma drugoga tipa koji je
svojstven govorima na jugu otoka (Baška, Batomalj i Jurandvor; Lukežić,
Turk 1998: 62–63), pri čemu bi pisar bio nesiguran u to kako u pismu
fiksirati /ź/, iako treba reći da povijesni izvori (Botica, Kovačić, Kuhar Sandra Požar, Inventar samostana Sv. Marije Magdalene u Portu na otoku Krku... FLUMINENSIA, god. 30 (2018), br. 2, str. 29-43 39 2015) pokazuju samo iznimnu zastupljenost samostanskih posinovljenika
s toga područja u vrijeme nastajanja Inventara.28 4.2.5. Potvrđeno je dodirno razjednačivanje, npr. slavnice (8v) ~
slamnice, kao i razjednačivanje na daljinu: bervijali (74r a) ~ bervijari,
škandalet (1r) ~ škaldalet, multar (57v) ~ mortar, rankurić (19v) ~ rankunić.29 4.2.5. Potvrđeno je dodirno razjednačivanje, npr. slavnice (8v) ~
slamnice, kao i razjednačivanje na daljinu: bervijali (74r a) ~ bervijari,
škandalet (1r) ~ škaldalet, multar (57v) ~ mortar, rankurić (19v) ~ rankunić.29 4.2.5. Potvrđeno je dodirno razjednačivanje, npr. slavnice (8v) ~
slamnice, kao i razjednačivanje na daljinu: bervijali (74r a) ~ bervijari,
škandalet (1r) ~ škaldalet, multar (57v) ~ mortar, rankurić (19v) ~ rankunić.29 4.2.6. Udvojeni se suglasnici na granici riječi u pisanju stežu: ss → s (važi
Svetim ulem 16v, krave domaće svoimi telci 77r a), kaleži od srebra svoimi
patenami 79v b); dd → d (propelce o drva 6v); tt → t (za misi testo 26r); (ćš →)
šš → š30 (vrči od vešurte 10r, ← već šurte). 4.2.6. Udvojeni se suglasnici na granici riječi u pisanju stežu: ss → s (važi
Svetim ulem 16v, krave domaće svoimi telci 77r a), kaleži od srebra svoimi
patenami 79v b); dd → d (propelce o drva 6v); tt → t (za misi testo 26r); (ćš →)
šš → š30 (vrči od vešurte 10r, ← već šurte). 4.2.6. Udvojeni se suglasnici na granici riječi u pisanju stežu: ss → s (važi
Svetim ulem 16v, krave domaće svoimi telci 77r a), kaleži od srebra svoimi
patenami 79v b); dd → d (propelce o drva 6v); tt → t (za misi testo 26r); (ćš →)
šš → š30 (vrči od vešurte 10r, ← već šurte). 4.2.7. 29 Zanimljivo je da je ista promjena (s drugim rezultatima) već ranije bila provedena u toj
riječi. Naime, posuđenica rankun postala je daljinskim jednačenjem l → n prema talijanskom
roncolo, odnosno mletačkom roncola (usp. Turk, Spicijarić Paškvan 2015: 208). 30 S prethodnim jednačenjem po mjestu tvorbe. 28 Samo fra Ivan Čeperić iz Baške, posinovljen 1740. godine (usp. Botica, Kovačić, Kuhar
2015: 31). 31 Usp. odjeljak 4.1.3. 31 Usp. odjeljak 4.1.3. 28 Samo fra Ivan Čeperić iz Baške, posinovljen 1740. godine (usp. Botica, Kovačić, Kuhar
2015: 31).
29 Zanimljivo je da je ista promjena (s drugim rezultatima) već ranije bila provedena u toj
riječi. Naime, posuđenica rankun postala je daljinskim jednačenjem l → n prema talijanskom
roncolo, odnosno mletačkom roncola (usp. Turk, Spicijarić Paškvan 2015: 208). 32 Usp. kako Milčetić govori o nosnom izgovoru: „Dobašļanin će reći: k5mo, t5mo, a na
Cresu kažu: ka̯n, ta̯n, i to nekako na nos.” (Milčetić 1895: 111). 4.2.13. Početna odnosno veznička skupina iz prelazi u z: z Rudine (14r). 4.2.13. Početna odnosno veznička skupina iz prelazi u z: z Rudine (14r). 4.2. Suglasnici Potvrđena je druga palatalizacija u nominativu množine
muškoga roda, koju Milčetić u Dubašnici bilježi samo u slučaju g → z (vrazi
~ grihi, orihi; Milčetić 1895: 110). Zbog neujednačenosti oblika pojava je
provjerena na češćim riječima, što je potvrdilo dvojnost: banci (30 potvrda)
~ banki (14), naglavnici (8) ~ naglavniki (3). Pritom treba naglasiti da su
palatalizirani oblici prisutni u cijelom Inventaru, dok se oni nepalatalizirani
susreću u pravilu u njegovu mlađem dijelu (od 1773. godine). 4.2.8. Sekundarni nosni sonant više je puta zabilježen u pridjevu
dunbok (npr. dunboki 49r). 4.2.8. Sekundarni nosni sonant više je puta zabilježen u pridjevu
dunbok (npr. dunboki 49r). 4.2.9. Unatoč čuvanju suglasničke skupine čr, češća je skupina cr, usp. crlen (8 puta) ~ črlen (2 puta); crn (9) ~ črn (2). No takvih je potvrda ukupno
manje nego onih gdje je skupina razbijena samoglasnikom, npr. cernih,
cerlenih (16v); čerlen (42v).31 Takva neujednačenost nije strana ni
srednjočakavskomu ni južnočakavskomu dijalektu, a često je, osobito na
jugu, uvjetovana cakavizmom (usp. Lisac 2009: 102; 144). 4.2.10. Skupina və u prijedlogu reflektirana je kao u, i to ne samo u
vrlo čestu spoju usve odnosno use nego i kad je prijedlog slobodan: u noi
(9v), u crikvi (32r), u noii, u komu, u pišidu (42r), u kuhini mobilija (66v a). To
je očekivano s obzirom na stanje u dubašljanskim govorima (Lukežić, Turk
1998: 79). Sandra Požar, Inventar samostana Sv. Marije Magdalene u Portu na otoku Krku... FLUMINENSIA, god. 30 (2018), br. 2, str. 29-43 40 4.2.11. Skupina sə u vezniku prelazi u z ispred samoglasnika ili
sonanta: z uzdu (20r), z’ voli (15v), z’ Mikom (8r), z’ merli (22r), z manigi
(57v), z nihovimi (12r), z negovimi (74v). Takav ostvaraj potpuno prevladava
nad onim izjednačenim po mjestu tvorbe, koji je potvrđen samo jedanput:
š’ nihovimi (72r b). 4.2.12. Početna skupina vəs- ostvaruje se na dva načina, zastupljena u
podjednakoj mjeri: s redukcijom početnoga /v/, kao s-, i s metatezom, a ta
dvojnost odgovara doseljeničkim govorima (redukcija u konzervativnijim, a
metateza u inovativnijim; usp. Lukežić, Turk 1998: 211; 252). Refleksi
skupine najčešće su potvrđeni u oblicima use i usve. Unatoč mjestimičnu
smjenjivanju tih dvaju oblika u starijem dijelu Inventara, može se utvrditi
da oblik s metatezom preteže u njegovu mlađem dijelu, počevši s godinom
1784. (str. 62v). No budući da je navedeni oblik okamenjen, kao važniji su
promotreni oblici slobodne zamjenice i njezinih izvedenica, koji su potvrdili
dvojnost, npr. 4.2. Suglasnici se (5 potvrda); sega (4); sih (2); sagdani (5); sakidani (5) ~ sve
(5); svega (2), svaka (8). 4.3. Mogućnost tumačenja pojava koje se doimaju kao pogreške U uvodu je napomenuto da su u Inventaru zbog praktične naravi teksta
i nastajanja njegovih popisa u brzini česte pisarske pogreške. Ipak, pri
proglašavanju nekoga oblika riječi pogreškom valja biti na oprezu jer u
nekim slučajevima može biti riječ o govornoj osobitosti. Tako se primjerice
za potvrdu tavalol (18r) ~ tavajol može, umjesto pogreške, pretpostaviti
izgovor [tavaljol] i stoga se posuđenica može odrediti kao talijanizam, a ne
venecijanizam. U potvrdi pak dupriri (63v) ~ dupliri prvo r, ako nije po
greška uzrokovana anticipacijom sljedećega sloga, moglo bi biti rezultat
jednačenja na daljinu, iako je težnja razjednačivanju osjetnija nego težnja
jednačenju. U obliku lancu(n) (18v) u prijepisu je rekonstruirano završno n jer mu
je tu svakako mjesto. No možda oblik bez n u izvorniku ne treba smatrati
pisarskom pogreškom, nego je riječ o nazalnom ostvaraju fonema /u/, na
koji je prenesena nazalnost završnoga /n/ koji je ispao. Takav je ostvaraj
značajka nekih srednjočakavskih govora (usp. Lisac 2009: 100).32 Da posri Sandra Požar, Inventar samostana Sv. Marije Magdalene u Portu na otoku Krku... NENSIA, god. 30 (2018), br. 2, str. 29-43 41 jedi nije pogreška, mogla bi govoriti činjenica da se oblik lancu(n) pojavljuje
dvaput u istom retku (18v), dok u obliku lancuni u sljedećem retku n nije
izostalo zbog heterosilabičkoga položaja slijeda un. Slične su potvrde Sakramet̃ (24r) i bola(n)čun (61v) za koje nije sigurno
je li riječ o nazalizaciji vokala ili su posrijedi prigodne (ad hoc) skraćenice,
kakve su uobičajene i drugdje u Inventaru, ili ipak pisarske pogreške nastale
u brzini. Oblik pet (64r) umjesto peć mogao bi upućivati na reduciranje čakav
skoga donjoalveolarnoga /ć/ [t’], odnosno pokazivati utjecaj izgovora na
pisanje. 5. Zaključak U članku se donosi dio rezultata istraživanja jezika Inventara samo
stana sv. Marije Magdalene u Portu na otoku Krku (1734. – 1878.): grafijske,
ortografske i glasovne pojave. Nekim podatcima treba pristupati s oprezom
jer su kod pojedinih fonoloških pojava uočene neujednačenosti i velike
varijacije. To se odnosi na reflekse jata, popratne vokale uz slogotvorno r i
skupinu čr/cr. Ipak, opća jezična slika Inventara, osobito njegova starijega
dijela, upućuje na doseljeničke krčke govore kako su opisani u literaturi,
suvremenoj (Lukežić, Turk 1998) i onoj vremenski bližoj istraživanomu
izvoru (Milčetić 1895), uz blagu obilježenost administrativnim stilom. Utvrđena variranja – s navedenim iznimkama – više su odraz grafijske
neujednačenosti, labavosti norme i nedoumica nego stvarnih jezičnih
razlika. U grafiji ima nekih sasvim osobitih rješenja, koja su jedva poznata u
ostatku glagoljske pismenosti (bilježenje mekoće i glasa /j/ znakom sličnim
apostrofu iznad dotičnoga odnosno prethodnoga grafema), a treba naglasiti
i naznake modernoga pravopisa: sustavno pisanje velikoga početnoga slova
i interpunkcijske znakove. Badurina, Anđelko (2013) Inventar samostana sv. Marije Magdalene u Portu
na otoku Krku (1734. – 1878.), Glosa Rijeka i Provincijalat franjevaca
trećoredaca glagoljaša Zagreb. Izvor Badurina, Anđelko (2013) Inventar samostana sv. Marije Magdalene u Portu
na otoku Krku (1734. – 1878.), Glosa Rijeka i Provincijalat franjevaca
trećoredaca glagoljaša Zagreb. Sandra Požar, Inventar samostana Sv. Marije Magdalene u Portu na otoku Krku... FLUMINENSIA, god. 30 (2018), br. 2, str. 29-43 42 Literatura Botica, Ivan, Vinko Kovačić, Kristijan Kuhar (2015) Knjige posinovljenja,
novicijata i zavjetovanja franjevaca trećoredaca glagoljaša otoka Krka
(1717. – 1914.), Provincija franjevaca trećoredaca glagoljaša i Staro
slavenski institut, Zagreb. Tomislava Bošnjak Botica, Ivan Botica, Tomislav Galović, prir. (2016)
Hrvatskoglagoljski notarijat otoka Krka : Notari Dubašnice, sv. 1. Treći
notarski protokol Jura Sormilića (1726. – 1734.), Hrvatski državni arhiv,
Staroslavenski institut, Filozofski fakultet Sveučilišta u Zagrebu,
Povijesno društvo otoka Krka, Zagreb. Bratulić, Josip, prir. (1989) Istarski razvod, Istarska književna kolonija
Grozd, Pula. Lisac, Josip (2009) Hrvatska dijalektologija 2: Čakavsko narječje, Golden
marketing i Tehnička knjiga, Zagreb. Lukežić, Iva, Marija Turk (1986) „Mjesni govor Milohnića”, Otok Krk: Zapadni
dio. Krčki zbornik, 16, 231–253. Lukežić, Iva, Marija Turk (1998) Govori otoka Krka, Libellus, Crikvenica Mihaljević, Milan (2014) „Pismo i fonemski sustav”, Hrvatski crkvenosla
venski jezik, priredio M. Mihaljević, Hrvatska sveučilišna naklada i
Staroslavenski institut, Zagreb, 49–90. Milčetić, Ivan (1895) „Čakavština Kvarnerskih otoka”, Rad JAZU, 121, 92–
131. Miloš, Irena (2015) „Fonološki opis govora Dubašnice”, Hrvatski dijalekto
loški zbornik, 19, 169–177. Požar, Sandra (2018) Čakavsko-romanski jezični dodir u Inventaru
samostana sv. Marije Magdalene u Portu na otoku Krku (1734. – 1878.),
Od fonologije do leksikologije: Zbornik u čast Mariji Turk, ur. Diana
Stolac, Filozofski fakultet, Rijeka, 291–311. Štefanić, Vjekoslav (1960) Glagoljski rukopisi otoka Krka, Jugoslavenska
akademija znanosti i umjetnosti, Zagreb. Žagar, Mateo (2013) Uvod u glagoljsku paleografiju 1 (X. i XI. stoljeće), Institut
za hrvatski jezik i jezikoslovlje, Zagreb. Žagar, Mateo (2013a) Hrvatska pisma i pravopisi u 17. i 18. stoljeću,
Povijest hrvatskoga jezika, 3. knjiga: 17. i 18. stoljeće, ur. Radoslav
Katičić i Josip Lisac, Croatica, Zagreb, 343–369. 43 Sandra Požar, Inventar samostana Sv. Marije Magdalene u Portu na otoku Krku...
FLUMINENSIA, god. 30 (2018), br. 2, str. 29-43 SUMMARY
Sandra Požar
INVENTORY OF THE MONASTERY OF ST. MARY MAGDALENE IN
PORAT ON THE ISLAND OF KRK (1734 – 1878) AS A LINGUISTIC
SOURCE: WRITING AND PHONOLOGY The paper presents a review of the writing system and the orthographic and
phonological features of the Inventory of St. Mary Magdalene in Porat on the island of
Krk (1734–1878), a source representative of pragmatic Glagolitic literacy of the
Franciscan Third Order Regular. The intention was to show that a text that seems to be
scarce and monotonous can be a valuable source of dialectological material. The
recorded phenomena were compared with the extant descriptions of the Krk dialects
in literature: those of present-day dialects (Lukežić, Turk 1998) and of those that are
almost contemporary to the respective source (Milčetić 1895). The general linguistic
picture of the Inventory, especially its older part, shares traits with the immigrant
dialects present on the island of Krk, slightly marked by the administrative style. On
the phonological level the picture that the Inventory provides is, however, not
consistent. This refers to the reflexes of the phoneme yat, to the vowels accompanying
the syllabic r, and to the group cr / čr. Although we would expect that these innovations
were introduced by the speakers of dialects which contain these traits, recently
published historical sources containing information on the origin of monastic
members do not support this assumption. The identified variations in writing are more of a reflection of the lack of uniformity in
writing, of looser norm and doubts, than of actual language differences. Some writing
solutions which are barely known in other Glagolitic texts (marking of palatality and
the phoneme j with a mark similar to the apostrophe above the respective or preceding
grapheme) and indications of modern orthography (capital letters and punctuation
marks) must also be emphasized. Keywords: Glagolitic script; Glagolitic friars of the Franciscan Third Order
Regular; pragmatic Glagolitic literacy; history of the Croatian
language; Chakavian dialect; dialects of the island of Krk
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Consultant medical trainers, modernising medical careers (MMC) and the European time directive (EWTD): tensions and challenges in a changing medical education context
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BMC medical education
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BioMed Central BioMed Central BioMed Central Open Acc
Research article
Consultant medical trainers, modernising medical careers (MMC)
and the European time directive (EWTD): tensions and challenges
in a changing medical education context
Maria Tsouroufli*1 and Heather Payne2 Open Access Address: 1School of Medicine, Health Policy and Practice, University of East Anglia, Norwich, NR4 7TJ, UK and 2Wales College of Medicine, School
of Postgraduate Medical and Dental Education, Cardiff University, UK Email: Maria Tsouroufli* - M.Tsouroufli@uea.ac.uk; Heather Payne - payneeh@cf.ac.uk
* Corresponding author Published: 20 May 2008
BMC Medical Education 2008, 8:31
doi:10.1186/1472-6920-8-31
Received: 6 August 2007
Accepted: 20 May 2008
This article is available from: http://www.biomedcentral.com/1472-6920/8/31
© 2008 Tsouroufli and Payne; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Received: 6 August 2007
Accepted: 20 May 2008 y
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Page 1 of 7
(page number not for citation purposes) Recruitment Research participants were recruited from attendees at a
short training course organised by the Postgraduate Dean-
ery at Cardiff University (n = 13). Snowballing was also
used. 7 research participants were recruited through inter-
viewees' personal contacts and had not attended the train-
ing course. The course which looked at teaching,
assessment and educational supervision under the new
training system (MMC) was delivered at many sites across
Wales and was attended by a total of 60 consultants. The
Principal Investigator was a non-participant observer at
the course delivered at Cardiff. Invitations to participate
in the study were sent to all attendees. Those who returned
their consent forms (20) were followed up by email or
phone. However, 'Next Steps' [4] stated that the apprenticeship
model should not be abandoned but rather managed
appropriately within the new training system and the
requirements of the EWTD [11]. Unfortunately, when
MMC was implemented Trainers and Trainees were not
equipped with a clear plan for managing and maintaining
the apprenticeship model in a context of reduced working
hours. Consultant Trainers were also expected to under-
take new responsibilities (assessment and educational
supervision) under the new training system and to be sup-
ported in their new role [2]. Although, training in assess-
ment, supervision and teaching was offered by
Postgraduate Deaneries, most Consultant Trainers did not
have a suitable job plan with an appropriate workload
and time to develop trainees, nor were supported by an
education team when MMC was implemented. Abstract Background: We analysed the learning and professional development narratives of Hospital
Consultants training junior staff ('Consultant Trainers') in order to identify impediments to
successful postgraduate medical training in the UK, in the context of Modernising Medical Careers
(MMC) and the European Working Time Directive (EWTD). Methods: Qualitative study. Learning and continuing professional development (CPD), were
discussed in the context of Consultant Trainers' personal biographies, organisational culture and
medical education practices. We conducted life story interviews with 20 Hospital Consultants in
six NHS Trusts in Wales in 2005. Results: Consultant Trainers felt that new working patterns resulting from the EWTD and MMC
have changed the nature of medical education. Loss of continuity of care, reduced clinical exposure
of medical trainees and loss of the popular apprenticeship model were seen as detrimental for the
quality of medical training and patient care. Consultant Trainers' perceptions of medical education
were embedded in a traditional medical education culture, which expected long hours' availability,
personal sacrifices and learning without formal educational support and supervision. Over-reliance
on apprenticeship in combination with lack of organisational support for Consultant Trainers' new
responsibilities, resulting from the introduction of MMC, and lack of interest in pursuing training in
teaching, supervision and assessment represent potentially significant barriers to progress. Conclusion: This study identifies issues with significant implications for the implementation of
MMC within the context of EWTD. Postgraduate Deaneries, NHS Trusts and the new body; NHS:
Medical Education England should deal with the deficiencies of MMC and challenges of ETWD and
aspire to excellence. Further research is needed to investigate the views and educational practices
of Consultant Medical Trainers and medical trainees. long learning are key features of Modernising Medical
Careers (MMC) [1]. This new scheme replaced the current
medical training grades with a 2-year Foundation Pro- Background
Structured training, clearly defined competencies, trans-
parent assessment, and emphasis on self-directed and life- Page 1 of 7
(page number not for citation purposes) Page 1 of 7
(page number not for citation purposes) BMC Medical Education 2008, 8:31 http://www.biomedcentral.com/1472-6920/8/31 http://www.biomedcentral.com/1472-6920/8/31 Supervisors, giving attention to Trainers' competency and
support in their role [12]. Supervisors, giving attention to Trainers' competency and
support in their role [12]. gramme followed by a 3–7 year specialty training pro-
gramme [2]. Methods
h
l This qualitative, interview based study was conducted in
2005 on 20 practising NHS Hospital Consultants with
responsibilities for supervising trainee Doctors, from six
NHS Trusts in Wales. The study was funded by Cardiff
University. This research study was conducted in compli-
ance with the Helsinki Declaration [13]. It was reviewed
and approved by MREC Wales (Ref. no. 05/MRE09/53). R&D approval was also granted by six NHS Trusts in
Wales. Well before the publication of the Tooke report (2008)
concerns regarding MMC had been expressed by both
trainers and trainees [6], particularly whether the Founda-
tion schemes will adequately prepare trainees for special-
ist programmes. There was also concern about the impact
of MMC and EWTD on the quality of medical training,
surgical training [7] in particular and the quality of patient
care [8]. Fears had been expressed that the 'apprenticeship
model' will be destroyed [9] and that shorter training will
undermine the authority and status of the medical profes-
sion [10]. Abstract Consultant Trainers were also expected to un
take new responsibilities (assessment and educatio
supervision) under the new training system and to be s
ported in their new role [2]. Although, training in ass
ment, supervision and teaching was offered
Postgraduate Deaneries, most Consultant Trainers did
have a suitable job plan with an appropriate workl
and time to develop trainees, nor were supported by
education team when MMC was implemented. Recently, Postgraduate Medical Education and Train
Board (PMETB), a further challenge for Consultant Tr
ers, have set high standards for Clinical and Educatio However, Consultant Trainers' views about postgraduate
training, their new roles and their everyday experiences
under the new training system and the constraints of
EWTD have received less attention in research, and subse-
quently Continuing Professional Development (CPD)
objectives. However, the recently published Tooke report (2008) rec-
ommended changes to MMC and in particular the return
to a 1-year house officer training followed by a three-year
broader-based training prior to higher specialist training. It also identified issues that hampered MMC. Lack of pol-
icy objectives; no guiding principles shared by all stake-
holders; lack of clarity about the role of doctors; erosion
of health-education partnership; lack of involvement of
the medical profession in medical management, leader-
ship and policy. The Tooke report also addressed the effect
of the current interpretation of EWTD by UK legislation
and the implications on the acquisition of clinical experi-
ence, confidence and the ability to shoulder responsibility
[5]. In this paper we explore Consultant Trainers' views on
postgraduate medical education and the implications of
cultural changes, resulting from MMC and EWTD, aiming
to identify impediments in the successful implementation
of MMC, within a context of reduced working hours. In
view of the potential confusion about the exact roles of
the 'Educational' and 'Clinical' Supervisor we will use the
generic term 'Consultant Trainer' throughout this paper. Page 2 of 7
(page number not for citation purposes) Abstract It aimed to provide trainees with generic
skills and experience in a variety of settings and speciali-
ties [3] leading to the award of a Certificate of Completed
Training (CCT), described in the document 'The Next
Steps' [4]. gramme followed by a 3–7 year specialty training pro-
gramme [2]. It aimed to provide trainees with generic
skills and experience in a variety of settings and speciali-
ties [3] leading to the award of a Certificate of Completed
Training (CCT), described in the document 'The Next
Steps' [4]. skills and experience in a variety of settings and spec
ties [3] leading to the award of a Certificate of Comple
Training (CCT), described in the document 'The N
Steps' [4]. However, the recently published Tooke report (2008)
ommended changes to MMC and in particular the ret
to a 1-year house officer training followed by a three-y
broader-based training prior to higher specialist train
It also identified issues that hampered MMC. Lack of p
icy objectives; no guiding principles shared by all sta
holders; lack of clarity about the role of doctors; eros
of health-education partnership; lack of involvemen
the medical profession in medical management, lea
ship and policy. The Tooke report also addressed the ef
of the current interpretation of EWTD by UK legislat
and the implications on the acquisition of clinical exp
ence, confidence and the ability to shoulder responsib
[5]. Well before the publication of the Tooke report (20
concerns regarding MMC had been expressed by b
trainers and trainees [6], particularly whether the Foun
tion schemes will adequately prepare trainees for spec
ist programmes. There was also concern about the imp
of MMC and EWTD on the quality of medical train
surgical training [7] in particular and the quality of pat
care [8]. Fears had been expressed that the 'apprentices
model' will be destroyed [9] and that shorter training
undermine the authority and status of the medical pro
sion [10]. However, 'Next Steps' [4] stated that the apprentices
model should not be abandoned but rather mana
appropriately within the new training system and
requirements of the EWTD [11]. Unfortunately, w
MMC was implemented Trainers and Trainees were
equipped with a clear plan for managing and maintain
the apprenticeship model in a context of reduced work
hours. http://www.biomedcentral.com/1472-6920/8/31 http://www.biomedcentral.com/1472-6920/8/31 BMC Medical Education 2008, 8:31 (qualitative social scientist-clinician/medical educator)
enriched the analytical process. who throughout their careers had been involved in formal
or informal teaching, clinical and educational supervision
and staff appraisal. The interviews were coded by the PI under the broad
descriptive categories (table 1) of the interview schedule
with the purpose of easy retrieval of data in Nvivo 2.0. Authors worked independently and met regularly to dis-
cuss their thoughts on literal and interpretive reading of
the data [18] Ideas emerged from these two analytical
stages which were developed further during the last stage
of the analysis; the reflective reading [19]. Authors were
able to access, read and discuss each other's reflective
notes in Nvivo 2.0. Through the process of discussion and
comparison of data [20] and reading of relevant literature,
the change of working practices resulting from EWTD and
MMC; and Consultant Medical Teachers' traditional dis-
courses of medical education emerged as key issues in our
analysis. • What are Consultant Medical Teachers' perceptions of
medical education? • What are Consultant Medical Teachers' perceptions of
medical education? • What are the links between organisational and educa-
tional changes? • What are the links between organisational and educa-
tional changes? Procedure Interviews were conducted by the Principal Investigator –
a female social scientist aged 35 – in Consultants' offices,
a seminar room in the hospital or University. Consultants'
unfamiliarity with qualitative research and their busy
schedule were important challenges for the interviewer
and the interviewees who seemed to hesitate to express
opinions or feelings. Interviews lasted around an hour or
more in some cases and were audio recorded, transcribed,
anonymised and imported into the qualitative analysis
package Nvivo 2.0. Field-notes were also written up after
each interview, recording reflections on the interviews and
initial analytical comments. • What is the relationship between personal experience
of training (as a junior doctor) and current views? • What is the relationship between personal experience
of training (as a junior doctor) and current views? Life-story approach and the interview schedule A life story approach was employed because of its orienta-
tion towards exploring the importance of biography in
interviewees' current views and practices [14]. The narra-
tive approach gave interviewees the opportunity to discuss
their own knowledge and experience of adult learning and
professional development as complex processes, emerg-
ing from dynamic relationships between learners' per-
sonal biographies, organisational and professional
culture and educational practices. Interviews contained
discussion of MMC and the doctors' own medical teach-
ing role. They also contained discussion of the interview-
ees' lifetime experiences of general and medical
education. The themes discussed in interview were based
upon literature in education [15] medical education
[16,17] and the authors' clinical, educational and research
experience. The interview schedule (table 1) was devel-
oped collaboratively and piloted with volunteers
employed by the Postgraduate Deanery at Cardiff Univer-
sity. The analysis presented in this paper was guided by three
research questions: Results In what follows we explore Consultant Trainers' views
about the cultural changes that have occurred as a result of
MMC and EWTD. We also discuss Consultant Trainers'
views of the effect of these changes on trainees' compe-
tency, Trainers' professional-educational role and Contin-
uing Professional Development (CPD). Sample The sample size was determined by saturation of data. This sample of consultants gave diversity of age (35 – 55),
sex (11 female, 9 male), ethnicity (1 Asian, 2 Arab, 1
Greek, 3 English, 2 Irish, 1 Scot and 10 Welsh), and clini-
cal specialty (general medicine, surgery, radiology, cardi-
ology, obstetrics and gynaecology, ENT, paediatrics,
emergency care, clinical pathology, anaesthetics, psychia-
try). All research participants were committed educators Recently, Postgraduate Medical Education and Training
Board (PMETB), a further challenge for Consultant Train-
ers, have set high standards for Clinical and Educational Page 2 of 7
(page number not for citation purposes) Page 2 of 7
(page number not for citation purposes) Analysis
d Continuity is very difficult, and if
you are going to give a hot report on somebody's performance
when you are not there when they are doing it, and then you
don't see them until two weeks afterwards, you can't do that
and I think that makes the learning more difficult. If it is not
hot taking on the message, 'well what did you think about that,
or that might have been another way to do' it doesn't mean very
much if it is two weeks later. (Doctor 12) Well they (trainees) go through a very structured process, you
know the amount of time they spend, particularly when they are
qualified, actually doing their job it's substantially theirs,, so
when they have to, which they're not suppose to, but when they
have to work, out of hours or work very intensively for a short
period of time, I don't think they're really got that sort of expe-
rience that they can necessarily cope and some people, obviously
do, but, I think a lot of them do find it quite stressful when they
have to say look after three very sick patients all at the same
time, you know, well I've heard in silence that they just can't
cope. Whereas, in the old days it would be part and parcel of
you training really and you would cope. (Doctor 7) The implications of shorter training, in particular the defi-
ciencies of current trainees, dominated the interviews
with Consultant Trainers, whereas reflection and evalua-
tion of Consultant Trainers' teaching and supervision
practices was a non-issue, irrespective of interviewees' age,
gender, ethnicity and clinical speciality. The doctor in the
following excerpt was the only interviewee who expressed
concerns about her teaching practices and a need for
adjustment to the new reality of reduced working hours
and shorter medical training. The limited availability of trainees has also resulted to
reduced clinical exposure and experience. In the following
excerpt a surgeon expresses concerns about the quality of
surgical training, as reduced working hours have inadvert-
ently resulted to reduced surgical experience. Hands on training, right ok I want to do the hands on training,
I said I have 3 theatre sessions a week and so I want my FP2 to
be with me in three theatre sessions so that I can give him the
hands on training, that is my commitment in the bid. Analysis
d Old 'apprenticeship' and the new EWTD
Either explicitly or implicitly, Consultant Trainers
expressed positive attitudes towards the apprenticeship
model of learning or some aspects of it such as A record of analysis as well as detailed information about
the research process and the participants is available in
Nvivo 2.0. We adopted collaborative analysis and writing
to maximise the confirmability and credibility of our find-
ings [18]. The authors' different academic backgrounds 'stability' (Doctor 3). They focused on familiarity with many clinical procedures
and fast learning which occurred Table 1: Interview schedule topics
Employing organisation
Interviewee's life and career
Experiences of learning
Experiences of teaching
Experiences of supervision
Continuing Professional Development Table 1: Interview schedule topics
Employing organisation
Interviewee's life and career
Experiences of learning
Experiences of teaching
Experiences of supervision
Continuing Professional Development Table 1: Interview schedule topics 'simply by being there, asking questions and learning from mis-
takes' (Doctor 11). Sharing information and discussing decision-making
approaches informally with senior staff throughout the
disease trajectory was seen as conducive to learning. How- Page 3 of 7
(page number not for citation purposes) Page 3 of 7
(page number not for citation purposes) Page 3 of 7
(page number not for citation purposes) http://www.biomedcentral.com/1472-6920/8/31 BMC Medical Education 2008, 8:31 in real life situations, due to their limited clinical expo-
sure. ever, similar opportunities have disappeared since the
introduction of EWTD and the disintegration of the firm. The introduction of shift work has resulted in reduced
availability of trainees, reduced contact between trainers
and trainees and limited opportunities for communica-
tion and learning as the following excerpt illustrates: Consultant Trainers also felt that structured training
(MMC) and reduced working hours is creating a genera-
tion of doctors unable to deal with the pressures and chal-
lenges of the medical profession as the following excerpt
illustrates: Now, if I feel someone could have improved the care they gave
somebody and would like to talk about it, I'd have to try and
make a formal appointment with them which makes it seem
very big, and formal, or I'll say I'll just mention it in passing
when I see them, and then I don't see them weeks could go by
so time for that sort of pointed follow-up of an individual case
care is very, very difficult. ... Analysis
d But, they
can't be with me because they are on call for that day, they don't
come to the theatre with me. Because they have been taking his-
tory examinations to all this emergency cases and then the next
day they are off, they are not allowed to join me, even if they,
er I mean voluntary, because they have been asked to go home. So, when my trainee is around I don't have the theatre. How
am I going to give him the hands on training? (Doctor 6) So I think it's been pointed out that the change in working
hours means a need for change in the way of teaching the trou-
ble is from the experience I've had of teaching, is of that appren-
ticeship method, so I find it difficult to think about well how
else do I go about getting this message across, when I can't do
it the way that worked for me and it was a good method for me. I know it's not a good method for some people but it was a good
method for me. (Doctor 12) New working patterns resulting from the EWTD have
altered the nature of medical communities and had impli-
cations for the quality of medical training. MMC-new roles and responsibilities for Consultant
Trainers Complaints about time constraints and increased work-
load, resulting from Consultant Trainers' involvement in
MMC were common in the interviewees. The majority of
interviewees perceived their new responsibilities, includ-
ing educational supervision and assessment as conducive
to trainees' learning and important for the effectiveness of
MMC. However, all Interviewees expressed concerns
about lack of time for performing teaching, supervision
and assessment of trainees, due to clinical commitments
and pressures from NHS employers. The doctor in the fol-
lowing excerpt expresses concerns about the implications Page 4 of 7
(page number not for citation purposes) Consultant Trainers' educational expertise and perceived
training needs Consultant Trainers' preference for apprenticeship and
resistance to training, pertinent to Consultants' new role,
might be associated with their personal experience of
medical training. Long hours' availability, personal sacri-
fices, emotional and physical robustness and strength,
self-reliance and independence, were subtle expectations
of medicine when Consultants were junior doctors. Con-
sultant Trainers felt that current trainees should make sim-
ilar sacrifices and accept that medicine is not a 9-5 job as
the following excerpts illustrate: Consultants Trainers felt that changes in working patterns
and medical training had implications for trainees as well
as their employing organisation. However, further train-
ing in teaching, supervision and assessment was not iden-
tified as an appropriate response to the changes brought
about by MMC and the EWTD. There was a general feeling
that competency in these areas was acquired through
experience and informal learning. Interestingly Consult-
ants Trainers, who had formal training; a formal role in
undergraduate of postgraduate Medical Education; and a
strong role model as a teacher/mentor/supervisor; were
more enthusiastic about addressing professional chal-
lenges resulting from MMC and EWTD. 'to come out of their (trainees) way to learn... rather than
thinking as a nine to five clerical assistant' (doctor 13). You have to be organised, say for instance today I am not going
to do anything I will keep this time to myself for studying or
after five by boss is having an emergency gastrectomy and I will
go and see that. If you go and see the elective emergency gast-
rectomy starting at six you have to sacrifice whatever activities
you have in the evening. (Doctor 6) The doctor in the following excerpt came from a family
who valued good teaching and mentoring. He used to
consult his mother, a teacher, on educational and profes-
sional development issues. After reflection and careful
needs assessment, he decided to do a Bachelor of Educa-
tion to address challenges, resulting from his involvement
in the Foundation Program and specialist training. As junior doctors, Consultant Trainers had ample oppor-
tunity for informal learning due to different working pat-
terns. However, in the old days, relationships were not
always easy and some Consultant Trainers commented on
difficulties in raising concerns and getting constructive
criticism from their seniors as the following excerpt
shows: The FP2s have an appraisal every two months and certainly
every 4 months. of her supervision commitments arising from MMC and
the lack of organisational support. Consultant Trainers' accounts show limited perceived
benefit of teaching and supervision for their educational
and professional development and variation in percep-
tions of their supervisory role. All interviewees described
themselves as facilitators of the trainees' learning but most
thought that their main task as MMC educational supervi-
sors was to organise learning events, discuss aims and
objectives with the trainee and inform them of the learn-
ing opportunities in their department. Providing regular
feedback on performance and progress through formal or
informal learning events was felt to be desirable by fewer
interviewees (and not always achievable, due to organisa-
tional constraints). Supporting trainees in identifying
learning needs, providing pastoral care and career advice
were the least frequently mentioned activities mentioned
by interviewees. Yeah, well the FP1's I've, I've put an application to get a FP1,
um, I think it's, it's definitely the way forward but I don't think
it's been particularly well thought through. I mean, I just super-
vise 1 FP2 at the moment and it takes an awful lot of time and
to go through all the materials and, and to do it properly, I don't
know how we're coping because if you're in speciality with
maybe one or two you can but from next August I'll have 5 and
it takes me about an hour a week to, if I'm doing it properly, to
supervise that particular FP2 and go through all their materials
and assessments with them and I don't know how it's going to
be possible, we've got sort of, 15 of them to do. The level of sup-
port will drop because we just won't have the time to do it. We're not being given any extra consultant time to, to perform
that role. (Doctor 8) Consultant Trainers' perceptions of MMC trainees Consultant Trainers felt that cultural changes, resulting
from EWTD and MMC, impinged on trainees' compe-
tency. All interviewees described current trainees as less
confident and less able to work independently in compar-
ison to themselves, however sound their theoretical
knowledge, and thought that trainees required a lot more
support and direction than themselves when they were
being trained. Consultant Trainers felt that trainees were
struggling with the application of theoretical knowledge Page 4 of 7
(page number not for citation purposes) Page 4 of 7
(page number not for citation purposes) http://www.biomedcentral.com/1472-6920/8/31 http://www.biomedcentral.com/1472-6920/8/31 BMC Medical Education 2008, 8:31 of her supervision commitments arising from MMC and
the lack of organisational support. of her supervision commitments arising from MMC and
the lack of organisational support. Page 5 of 7
(page number not for citation purposes) Competing interests Maria Tsouroufli has no competing interests. Heather Payne is employed by a Postgraduate Medical
Deanery. The implementation of MMC within the context of EWTD
has created new responsibilities and challenges for Con-
sultant Trainers. Our study findings show that lack of
organisational support for Consultant Trainers and man-
agement strategies that would balance clinical and admin-
istrative impinged on the quality of medical training and
Consultants' educational role. http://www.biomedcentral.com/1472-6920/8/31 BMC Medical Education 2008, 8:31 Consultant Trainers acknowledged the problems arising
from unstructured postgraduate training with no pro-
tected time for learning, no formal educational supervi-
sion and lack of constructive criticism, impinging on their
learning. However, all interviewees felt that their difficult
experiences enabled them to develop into independent
learners and hardworking consultants as the following
excerpt illustrates: embedded in a 'sink or swim' medical education culture
[22]. Having received limited supervision and support as
juniors and little training in teaching and supervision [23]
as Consultants, interviewees seemed to be heavily reliant
on the 'old apprenticeship', focusing mainly on the defi-
ciencies of their trainees and the constraining environ-
ments in which they work and teach. In our study,
interviewees did not often appear to use more advanced
teaching skills such as seeking to diversify educational
techniques in the face of insuperable obstructions (time
and synchronicity constraints), engagement in shared and
reciprocal learning practices [24], reflection and evalua-
tion of their own teaching and supervisory practices [25]. This is a matter for concern in a sample explicitly commit-
ted to training. It is also a key issue that needs to be
addressed as Consultant Trainers are now expected to be
involved in creating a learning culture, and provide a level
of supervision appropriate to the competence and experi-
ence of the trainee [12]. My 1st job was in the A&E department as a pre-registration
house officer with four of us together who graduated together
from medical school and not a great deal of supervision from
anyone more senior, we could go to people if we did have prob-
lems but we generally tried to resolve the problems as far as we
could amongst ourselves. Lots of experience, it probably
moulded me for the future that every job I did involve hard work
and quite a lot of independent problem solving and taking
responsibility for your own education and learning. (Doctor 4) Abbreviations NHS: National Health Service, MMC: Modernising Medi-
cal Careers, EWTD: European Working Time Directive,
MREC: Multi-site Research Ethics Committee, R&D:
Research and Development and CPD: Continuing Profes-
sional Development. Authors' contributions Maria Tsouroufli was principal investigator for the study. Heather Payne was co-applicant on the study research
proposal and contributed to each stage of the study devel-
opment, process, analysis and reporting. Lack of clarity about Consultants' educational role in
combination with their resistance to further training
might have also been a barrier to the successful imple-
mentation of MMC, within a context of reduced working
hours. This qualitative study has identified Consultant
Trainers' predominant assumptions about learning, Conclusion Analysis of educational narratives of 20 Consultant Train-
ers indicates that the EWTD has led to working practices
which have altered the Trainee-Consultant Trainer rela-
tionship. The introduction of shift duties was perceived by
our interviewees, to have fragmented medical communi-
ties, and significantly reduced contact between trainers
and trainees. Similar findings were reported in McKee's
ethnographic study of senior house officers [21]. Establishing appropriate mechanisms for the selection,
organisational and educational support of Trainers [12]
and fostering strong links between health and theeduca-
tion sector are potentially ways forward [5]. Also promot-
ing effective involvement of the medical profession in
training policy-making could lead to clear, shared princi-
ples about the implementation and management of post-
graduate training [5]. Further qualitative research is also
needed to investigate the quality of postgraduate training
as perceived and experienced by Trainers and Trainees. New working practices resulting from the EWTD have
implications for medical training. Our qualitative data
shows that limited clinical exposure of trainees, lack of
continuity, and limited Consultant Trainer-Trainee con-
tact at the workplace, has inadvertently led to the loss of
'apprenticeship' long used and still favoured by both
trainers and trainees. This causes concern to the medical
community and is seen as a threat to the profession. Fur-
ther research is needed to explore shared understanding of
'apprenticeship', but there was common reference to 'see
one, do one, teach one'. Consultant Trainers' educational expertise and perceived
training needs The GP trainee will need assessment every six
months before they move on to their next placement, a specialist
registrar will need an appraisal every twelve months before they
move onto their next placement. So I had a need to produce var-
ious educational material and a lot of my personal education
has been around improving my teaching technique so that I can
deliver that better. So it hasn't been about learning old age psy-
chiatry, it is learning how to teach about old age psychiatry in
a different setting or a better setting or more effective. (Doctor I would have like to feel able to ask questions and if I wasn't
sure or wanted something explained I thought I would get sup-
port but it was more about you don't do it like that, you should
have done this and then there's like you know, you want to
know why but that was never given, it was just everything you
did was wrong. (Doctor 8) 1) Page 5 of 7
(page number not for citation purposes) Page 5 of 7
(page number not for citation purposes) http://www.biomedcentral.com/1472-6920/8/31 References 1. Modernising Medical Careers [http://www.mmc.nhs.uk] 1. Modernising Medical Careers [http://www.mmc.nhs.uk] 2. Modernising Medical Careers [http://www.mmc.nhs.uk/ 2. Modernising Medical Careers [http://www.mmc.nhs.uk/
download_files/What%20is%20changing%20at%20MMC.pdf] 2. Modernising Medical Careers [http://www.mmc.nhs.uk
download_files/What%20is%20changing%20at%20MMC.pdf] g
[
p
download_files/What%20is%20changing%20at%20MMC.pdf] 3. The Foundation Programme Committee of the Academy of Medical
Royal Colleges, in co-operation with Modernising Medical Careers in
the Departments of Health: Curriculum for the Foundation Years in Post-
graduate Education and Training Department of Health, London; 2005. g
g
p
4. UK Health Departments: The Next Steps: The Future Shape of the Foun-
dation, Specialist and General Practice Training Programmes Department
of Health, London; 2004. ,
;
5. MMC Inquiry 2008 [http://www.inquiry.org.uk?_Jan_08_MMC-
all.pdf] 6. Kang SN, Sanghrajka A, Amin A, Lee J, Briggs T: Modernising Med-
ical Careers: orthopaedic trainees' perspectives. Annals of
Royal College of Surgeons in England 2005, 873(Suppl):310-312. y
g
f
g
g
(
pp )
7. Payne SR, Shaw MKB: What impact will shortened training
have on urological service delivery? Annals of Royal College of Sur-
geons in England 2005, 87(Suppl):373-378. g
g
(
pp )
8. Devey L: Will modernised medical careers produce a better
surgeon? BMJ 2005, 331:1346. g
J
9. Anwar M: EWTD has negative impact on training for sur-
geons. BMJ 2005, 331:1476. g
J
10. Clough CG: New development in medical speciality training. Clinical Medicine 2005, 5:349-353. 11. Department
of
Health
European
Time
Directive
[http:library.nhs.uk/healthmanagement/ViewRe
source.aspx?reID=51961&tabID-290&CATID=4031] p
]
12. PMETB Standards for Trainers [http://www.pmetb/org/uk/file
admin/user/QA/Assessment/
PMETB_Standards_FORTRAINERS_JAN_2008.pdf] 12. PMETB Standards for Trainers [http://www.pmetb/org/uk/file
admin/user/QA/Assessment/ 13. The Helsinki Declaration [http://www.wma.net/e/policy/b3.htm
14
C
i
k B N
i
i S
i l S i
R
h L
d
S
2004 13. The Helsinki Declaration [http://www.wma.net/e/policy/b3.htm]
14. Czarniawska B: Narrative in Social Science Research London: Sage; 2004. 13. The Helsinki Declaration [http://www.wma.net/e/policy/b3.htm]
14. Czarniawska B: Narrative in Social Science Research London: Sage; 2004. 15. Osler A: Teachers' biographies and educational develop-
ment: A Kenyan case study. International Journal of Educational
Development 1997, 17:361-371. p
16. Allery L, Owen PA, Robbing M: Why general practitioners and
consultants change their clinical practice: a critical incident
study. BMJ 1997, 314:870. y
J
17. Bennett N, Davis AP, Easterling WE, Friedman P, Green JS, Koeppen
BM, Mazmanian P, Waxman HS: Continuing Medical Education. Academic Medicine 2000, 75:1167-1172. 18. Miles MB, Huberman MA: Qualitative analysis: an expanded sourcebook
Thousand Oaks, CA: Sage Publications; 1994. g
19. Mason J: Qualitative Researching London: Sage; 1990. 20. Acknowledgements We thank the participants for their time, referees Dr Simon Murphy and
Professor Gareth Rees, advisers Dr Alan Dowler and Dr Chris Taylor. We
also thank Professor Derek Gallen, Dr Sally Davies and Dr Michael Shepard
for their comments. Last but not least we are indebted to Professor Sam
Leinster for commenting on drafts. Page 6 of 7
(page number not for citation purposes) (page number not for citation purposes) http://www.biomedcentral.com/1472-6920/8/31 http://www.biomedcentral.com/1472-6920/8/31 http://www.biomedcentral.com/1472-6920/8/31 BMC Medical Education 2008, 8:31 References Weber R: Basic Content Analysis London: Sage; 1990. y
g
21. Mc Kee A: Working and Learning in hospitals: Junior doctors
adrift in fragmented communities. Learning in Health and Social
Care 2002, 3:158-169. 22. Doherty C: Introducing mentoring to doctors: Challenging
the sink or swim culture. Development and Learning in Organisations
2004, 1:6-8. 23. Rogstad K, Talbot M: A preliminary study comparing attitudes
of trainee doctors and their mentors, to compulsory educa-
tional supervision in postgraduate medicine. Mentoring and
Tutoring 2001, 1:77-83. g
24. Carrington G: Supervision as a Reciprocal Learning Process. Educational Psychology in Practice 2004, 1:31-42. 25. Dewar BJ, Walker E: Experiential learning: Issues for supervi-
sion. Journal of Advanced Nursing 1999, 6:1459-1467. Publish with BioMed Central and every
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English
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Significant Changes in Metabolic Profiles after Intervention with Selenium and Coenzyme Q10 in an Elderly Population
|
Biomolecules
| 2,019
|
cc-by
| 11,663
|
Urban Alehagen 1,*, Peter Johansson 2,3,4, Jan Aaseth 5
, Jan Alexander 6
, Izabella Surowiec 7,
Katrin Lundstedt-Enkel 7
and Torbjörn Lundstedt 7 1
Division of Cardiovascular Medicine, Department of Medical and Health Sciences, Linköping University,
SE-581 85 Linköping, Sweden
2
Department of Social and Welfare Studies, Linköping University, SE-581 83 Linköping, Sweden;
peter.b.johansson@liu.se
3
Department of Internal Medicine, Linköping University, SE-581 83 Linköping, Sweden
4
Department of Medical and Health Sciences, Linköping University, SE-581 83 Linköping, Sweden
5
Research Department, Innlandet Hospital Trust, N-2380 Brumunddal, Norway; jaol-aas@online.no
6
Norwegian Institute of Public Health, N-0403 Oslo, Norway; Jan.Alexander@fhi.no
7
AcureOmics AB, Tvistevägen 48, SE-907 36 Umeå, Sweden; izabella.surowiec@acureomics.com (I.S.);
katrin.lundstedt-enkel@telia.com (K.L.-E.); torbjorn.Lundstedt@acureomics.com (T.L.)
*
Correspondence: urban.alehagen@liu.se *
Correspondence: urban.alehagen@liu.se biomolecules biomolecules Significant Changes in Metabolic Profiles after
Intervention with Selenium and Coenzyme Q10 in
an Elderly Population Urban Alehagen 1,*, Peter Johansson 2,3,4, Jan Aaseth 5
, Jan Alexander 6
, Izabella Surowiec 7,
Katrin Lundstedt-Enkel 7
and Torbjörn Lundstedt 7 1. Introduction Selenium (Se) is a trace element that is essential for all living cells within the human body. There are 25 genes encoding for selenoproteins in humans [1]. Several of the selenoproteins have
important roles in anti-oxidative stress defence, and in immune surveillance [2,3]. Some of the most
important selenoproteins are the glutathione peroxidases, thioredoxin reductase 1, selenoprotein P, and
iodothyronine deiodinases [4]. There is a relation between the Se content in soil, food grown on these
soils and dietary Se intake, where both high and low intakes have been reported [5,6]. In Europe there is
in general a low concentration of Se in the soil [7–11] and, hence, a low intake, estimated at about 40 µg
Se/day [1]. The daily intake of selenium needed for the optimal expression of selenoprotein P in plasma
has been debated. Xia et al. demonstrated that an intake of at least 75 µg/day, to achieve a plasma
concentration of Se above 90 µg/L was required in normal weight Caucasians [12], whereas Hurst et al. reported that an intake of at least 105 µg/day was required [13]. In Sweden, reports have shown an
intake of selenium below the recommended levels [14,15], which appears to increase the cardiovascular
risk [16]. The soils in North America have a significantly higher content of selenium than in Europe,
with reported serum selenium levels generally above 120 µg/L in the US population [17,18]. In a
population of elderly healthy Swedish persons with a mean serum selenium concentration of 67 µg/L,
we showed that those with low selenium concentration had a higher cardiovascular risk compared to
those with higher selenium concentration, thus confirming previous reports in the literature [16,19]. There is an important relationship between Se and ubiquinone (coenzyme Q10, hereafter
abbreviated Q10) in living cells. In order to synthesise selenocysteine-containing enzymes a functional
mevalonate pathway is required, and Q10 is a product in this pathway [20]. Additionally, the
selenoenzyme TrxR may reduce Q10 to its active form, ubiquinol; hence, the presence of an adequate
concentration of selenium is required in the cell for optimal Q10 function. In addition to being a strong antioxidant and essential for all living cells, Q10 is of particular
importance for the crucial ATP-generating steps within the mitochondrial respiratory chain and in
oxidative phosphorylation mediated through the pentose phosphate pathway [21]. Received: 27 August 2019; Accepted: 26 September 2019; Published: 30 September 2019 Abstract: Selenium and coenzyme Q10 (SeQ10) are important for normal cellular function. Low
selenium intake leads to increased cardiovascular mortality. Intervention with these substances with
healthy elderly persons over a period of four years in a double-blind, randomised placebo-controlled
prospective study showed reduced cardiovascular mortality, increased cardiac function, and a lower
level of NT-proBNP. Therefore, we wanted to evaluate changes in biochemical pathways as a result
of the intervention with SeQ10 using metabolic profiling. From a population of 443 healthy elderly
individuals that were given 200 µg selenium and 200 mg coenzyme Q10, or placebo daily for four
years, we selected nine males on active intervention and nine males on placebo for metabolic profiling
in the main study. To confirm the results, two validation studies (study 1 n = 60 males, study 2 n =
37 males) were conducted. Principal component analyses were used on clinical and demographic
data to select representative sets of samples for analysis and to divide the samples into batches for
analysis. Gas chromatography time-of-flight mass spectrometry-based metabolomics was applied. The metabolite data were evaluated using univariate and multivariate approaches, mainly T-tests and
orthogonal projections to latent structures (OPLS) analyses. Out of 95 identified metabolites, 19 were
significantly decreased due to the intervention after 18 months of intervention. Significant changes
could be seen in the pentose phosphate, the mevalonate, the beta-oxidation and the xanthine oxidase
pathways. The intervention also resulted in changes in the urea cycle, and increases in the levels of
the precursors to neurotransmitters of the brain. This adds information to previous published results
reporting decreased oxidative stress and inflammation. This is the first-time metabolic profiling
has been applied to elucidate the mechanisms behind an intervention with SeQ10. The study is
small and should be regarded as hypothesis-generating; however, the results are interesting and,
therefore, further research in the area is needed. This study was registered at Clinicaltrials.gov, with
the identifier NCT01443780. Keywords: selenium; coenzyme Q10; elderly; metabolic profiling Biomolecules 2019, 9, 553; doi:10.3390/biom9100553 www.mdpi.com/journal/biomolecules Biomolecules 2019, 9, 553 2 of 20 2. Results The baseline characteristics of the total male study population are presented in Table 1. The baseline characteristics of the total male study population are presented in Table 1. The baseline characteristics of the total male study population are presented in Table 1. Table 1. Baseline characteristics of the male study population receiving an intervention of dietary
supplementation of selenium and coenzyme Q10 combined over four years. Active
Placebo
p Value
N
55
44
Age (years)
76.1 (2.9)
76.3 (2.9)
History
Smokers (present) n (%)
5 (7.3)
6 (13.6)
0.47
Diabetes n (%)
11 (20.0)
8 (18.2)
0.82
Hypertension n (%)
34 (61.8)
31 (70.5)
0.91
IHD n (%)
12 (21.8)
10 (22.7)
0.91
NYHA class I n (%)
37 (67.3)
22 (50.0)
0.08
NYHA class II n (%)
12 (21.8)
18 (40.9)
0.04
NYHA class III n (%)
6 (10.9)
4 (9.1)
0.77
NYHA class IV n (%)
0
0
Unclassified NYHA n (%)
0
0
Medications
ACEI or ARBn (%)
11 (20.0)
12 (27.3)
0.39
Beta-blockers n (%)
20 (36.4)
17 (38.6)
0.82
Digitalis n (%)
3 (5.5)
0 (0)
n/a
Diuretics n (%)
13 (23.6)
13 (29.5)
0.51
Statins n (%)
13 (23.6)
7 (15.9)
0.34
Examinations
EF < 40% n (%)
6 (10.9)
2 (4.5)
0.25
Note: ACEI: ACE- inhibitors; ARB; Angiotension receptor blockers; EF: Ejection fraction; IHD; Ischaemic heart
disease; NYHA: New York Heart Association functional class; p values indicate the Chi-square test was used for
discrete variables. Table 1. Baseline characteristics of the male study population receiving an intervention of dietary
supplementation of selenium and coenzyme Q10 combined over four years. Note: ACEI: ACE- inhibitors; ARB; Angiotension receptor blockers; EF: Ejection fraction; IHD; Ischaemic heart
disease; NYHA: New York Heart Association functional class; p values indicate the Chi-square test was used for
discrete variables. As can be seen, all variables were balanced between the two groups included in this evaluation,
with the exception that among those receiving a placebo there were more participants in the NYHA
functional group II and fewer in group I. 1. Introduction The endogenous
production of Q10 decreases with increasing age, and in the myocard, by the age of 80 years it is
reduced to half the level that exists at 20 years [22]. Furthermore, the need for Q10 is even greater
during diseases [23,24]. In 2013, we presented the results of four years of intervention using Se and Q10 combined
(SeQ10) as a dietary supplement in an elderly healthy population in a randomised prospective
double-blind, placebo-controlled study [25]. The main result of the intervention was a significant
reduction of cardiovascular mortality by more than 50%, better cardiac function as demonstrated on
echocardiography, and a lower plasma concentration of the natriuretic peptide N-terminal fragment
of proBNP (NT-proBNP) indicating less myocardial wall tension [25]. Due to the striking positive
clinical results, in an effort to shed light on possible mechanisms behind the clinical effects, we used a
metabolic profiling approach to pinpoint changes in metabolites and metabolic pathways following
SeQ10 administration. Studies of metabolic patterns, by analysing body fluids or sampled tissue, have evolved into an
important routine in several fields of the clinical and pharmacological discipline [26]. Non-targeted
metabolite profiling—known as metabolomics—provides a functional molecular picture of the
biochemistry connecting both genomics and proteomics to a physiological state, and it also incorporates
environmental interactions. Metabolomics is, thus, a mapping of the actual state of the phenotype. The metabolomic approach in characterising biological systems is frequently applied in several
situations; (1) In the diagnostic process, i.e., where the metabolic profiles of healthy subjects, and
subjects with a disease are used for classification of new cases. (2) In the identification of perturbed
metabolic pathways, i.e., the detection of biomarkers and connecting them to, e.g., disease pathogenesis,
and (3) as a tool in disease follow-up and treatment, i.e., following changes of metabolites after changes
in lifestyle and/or food and/or pharmacological intervention [27–30]. 3 of 20 Biomolecules 2019, 9, 553 Biomolecules 2019, 9, 553 In the present work, we used metabolomics to identify the impacts of SeQ10 intervention on the
plasma metabolome. As previous results from this intervention study indicated that males and females
differed strongly in cardiovascular mortality, and as we could not exclude a hormonal influence, we
decided to include only males in this study in order to have a more homogenous group to analyse. 2.1. Main Study OPLS-DA score plot based on each individual’s metabolic profile. (R2X
g,
model with one predictive and one orthogonal component including 17 Figure 1. OPLS-DA score plot based on each individual’s metabolic profile. (R2X = 0.19, R2Y = 0.57,
Q2Y = 0.18). Note: Round circles indicate the men that received the placebo (Pl, green; n = 9), and the
squares are men treated with SeQ10 (Ki, blue; n = 9). Note: The metabolite pattern was determined in
plasma at the time point of 18 months (T18). Note: One of the individuals receiving placebo took SeQ10
by self-administration. Figure 1. OPLS-DA score plot based on each individual’s metabolic profile. (R2X = 0.19, R2Y = 0.57,
Q2Y = 0.18). Note: Round circles indicate the men that received the placebo (Pl, green; n = 9), and the
squares are men treated with SeQ10 (Ki, blue; n = 9). Note: The metabolite pattern was determined in
plasma at the time point of 18 months (T18). Note: One of the individuals receiving placebo took SeQ10
by self-administration. model with one predictive and one orthogonal component including 17 individuals (R2X =
R2(cum) = 0.91, Q2(cum) = 0.41). The difference in the whole metabolome between the two stu
groups was evident when comparing predicted cross-validated Y values for samples from
groups (p < 0.01) (Figure 2). One of the samples from the placebo group had a position in the plot that indicated scores th
eviated from those of the rest of the placebo group and, thus, negatively influenced the power
he OPLS-DA model used. After checking with this individual, it was revealed that despite being
articipant of the placebo group, the person with sample no. 186 supplemented himself wit
elenium on his own initiative, which the model including each individual’s metabolite pattern
lasma, could detect and demonstrate (Figure 1). This individual was thus excluded from all further modelling, resulting in the final OPLS-D
model with one predictive and one orthogonal component including 17 individuals (R2X = 0.1
2(cum) = 0.91, Q2(cum) = 0.41). The difference in the whole metabolome between the two studie
roups was evident when comparing predicted cross-validated Y values for samples from bot
roups (p < 0.01) (Figure 2). Figure 2. Predicted cross-validated Y values (from the OPLS-DA model). 2.1. Main Study The active treatment group was evaluated against the placebo group using the OPLS-DA model
assessment at 18 months (T18) where 95 metabolites analysed in plasma were included. This evaluation
demonstrated significant differences between the two groups by using seven-fold cross-validation (the
OPLS-DA model with one significant component that explained 57% of the variance in Y, R2X = 0.19,
R2(cum) = 0.57 and Q2(cum) = 0.18) (Figure 1). One of the samples from the placebo group had a position in the plot that indicated scores that
deviated from those of the rest of the placebo group and, thus, negatively influenced the power of
the OPLS-DA model used. After checking with this individual, it was revealed that despite being a
participant of the placebo group, the person with sample no. 186 supplemented himself with selenium
on his own initiative, which the model including each individual’s metabolite pattern in plasma, could
detect and demonstrate (Figure 1). This individual was thus excluded from all further modelling, resulting in the final OPLS-DA
model with one predictive and one orthogonal component including 17 individuals (R2X = 0.13,
R2(cum) = 0.91, Q2(cum) = 0.41). The difference in the whole metabolome between the two studied 4 of 20 Biomolecules 2019, 9, 553 groups was evident when comparing predicted cross-validated Y values for samples from both groups
(p < 0.01) (Figure 2). Biomolecules 2019, 9, 553
4 of 20 Figure 1. OPLS-DA score plot based on each individual’s metabolic profile. (R2X = 0.19, R2Y = 0.57,
Q2Y = 0.18). Note: Round circles indicate the men that received the placebo (Pl, green; n = 9), and the
squares are men treated with SeQ10 (Ki, blue; n = 9). Note: The metabolite pattern was determined in
plasma at the time point of 18 months (T18). Note: One of the individuals receiving placebo took SeQ10
by self-administration. One of the samples from the placebo group had a position in the plot that indicated scores
deviated from those of the rest of the placebo group and, thus, negatively influenced the powe
the OPLS-DA model used. After checking with this individual, it was revealed that despite bei
participant of the placebo group, the person with sample no. 186 supplemented himself w
selenium on his own initiative, which the model including each individual’s metabolite patter
plasma, could detect and demonstrate (Figure 1). 2.1. Main Study This individual was thus excluded from all further modelling resulting in the final OPLS Figure 1. OPLS-DA score plot based on each individual’s metabolic profile. (R2X = 0.19, R2Y = 0.57,
Q2Y = 0.18). Note: Round circles indicate the men that received the placebo (Pl, green; n = 9), and the
squares are men treated with SeQ10 (Ki, blue; n = 9). Note: The metabolite pattern was determined in
plasma at the time point of 18 months (T18). Note: One of the individuals receiving placebo took SeQ10
by self-administration. Figure 1. OPLS-DA score plot based on each individual’s metabolic profile. (R2X = 0.19, R2Y = 0.57,
Q2Y = 0.18). Note: Round circles indicate the men that received the placebo (Pl, green; n = 9), and the
squares are men treated with SeQ10 (Ki, blue; n = 9). Note: The metabolite pattern was determined in
plasma at the time point of 18 months (T18). Note: One of the individuals receiving placebo took SeQ10
by self-administration. Figure 1. OPLS-DA score plot based on each individual’s metabolic profile. (R2X = 0.19, R2Y = 0.57,
Q2Y = 0.18). Note: Round circles indicate the men that received the placebo (Pl, green; n = 9), and the
squares are men treated with SeQ10 (Ki, blue; n = 9). Note: The metabolite pattern was determined in
plasma at the time point of 18 months (T18). Note: One of the individuals receiving placebo took SeQ10
by self-administration. One of the samples from the placebo group had a position in the plot that indicated scores
deviated from those of the rest of the placebo group and, thus, negatively influenced the pow
the OPLS-DA model used. After checking with this individual, it was revealed that despite bei
participant of the placebo group, the person with sample no. 186 supplemented himself
selenium on his own initiative, which the model including each individual’s metabolite patter
plasma, could detect and demonstrate (Figure 1). This individual was thus excluded from all further modelling, resulting in the final OPLS
model with one predictive and one orthogonal component including 17 individuals (R2X =
R2(cum) = 0.91, Q2(cum) = 0.41). The difference in the whole metabolome between the two stu
groups was evident when comparing predicted cross-validated Y values for samples from
groups (p < 0.01) (Figure 2). Figure 1. OPLS-DA score plot based on each individual’s metabolic profile. (R2X
Figure 1. 2.1. Main Study Note: Actual values for
controls are denoted 0 and for SeQ10-treated are denoted 1, showing that the two groups’ metabolic
patterns are significantly different (p = 0.006). Figure 2. Predicted cross-validated Y values (from the OPLS-DA model). Note: Actual values for
controls are denoted 0 and for SeQ10-treated are denoted 1, showing that the two groups’ metabolic
patterns are significantly different (p = 0.006). Figure 2. Predicted cross-validated Y values (from the OPLS-DA model). Note: Actual values fo
controls are denoted 0 and for SeQ10-treated are denoted 1, showing that the two groups’ metabol
patterns are significantly different (p = 0.006). Figure 2. Predicted cross-validated Y values (from the OPLS-DA model). Note: Actual values for
controls are denoted 0 and for SeQ10-treated are denoted 1, showing that the two groups’ metabolic
patterns are significantly different (p = 0.006). Using the jack-knifing method in the multivariate analyses and applying a 95% confidence
interval, the results showed that 19 metabolites out of the 95 identified and modelled metabolites were
significantly decreased in the SeQ10-treated men. A further examination applying a T-test analysis,
showed that 13 of these 19 metabolites were significantly lower in the SeQ10 -treated men when using
univariate statistics (Table 2). Fi
2 P
di t d
lid t d Y
l
(f
th
OPLS DA
d l) N t
A t
l
l
f
No metabolites were significantly higher in the SeQ10 men as compared with the controls. The p(corr) loading plot from the OPLS-DA model indicated that the difference could largely be
attributed to changes in amino acids, which were at lower levels in the SeQ10-treated group in
comparison with the placebo group (Figure 3). 5 of 20
6
f 20 Biomolecules 2019, 9, 553 r) loading vector from the OPLS-DA model with the 95 metabolites ide
receiving a daily addition of SeQ10 for 18 months as compared with
n = 8). Note: Bars pointing to the left from mean (0) indicate metab
treated men Amino acids are marked in light blue
r) loading vector from the OPLS-DA model with the 95 metabolites id
eceiving a daily addition of SeQ10 for 18 months as compared with pla
). Note: Bars pointing to the left from mean (0) indicate metabolites w
A
i
id
k d i
li ht bl Figure 3. 2.1. Main Study Note: No metabolite showed significantly higher levels in SeQ10 men; Note: VIP: Variables’ Importance to the
Projection. Note: T-tests were used in the statistical evaluation. Note: NS: Not significant. However, a decrease could also be seen in the polyunsaturated fatty acids arachidonic acid,
eicosapenatenoic and docosahexaenoic acid, and an increase in the saturated fatty acids (hexadecanoic
acid, myristic and stearic acid) and monounsaturated fatty acids hexadecenoic acid and oleic acid. 2.2. Projection of Deceased Participants Two individuals that participated in the project for at least 18 months, but later died from
cardiovascular disease, delivered samples at T0 and T18. The metabolic profiles from these individuals
were projected into the principal component analysis (PCA) models to investigate whether the T18
blood samples from these individuals deviated from the other T18 samples. In the score plots (not
shown) it could be seen that the metabolic patterns of the samples from the deceased participants did
not deviate from the surviving participants. It was, therefore, not possible to use the existing models to
predict the fate of individual participants after T18. 2.1. Main Study P(corr) loading vector from the OPLS-DA model with the 95 metabolites identified in plasma
of men (n = 9) receiving a daily addition of SeQ10 for 18 months as compared with placebo-treated
men (controls; n = 8). Note: Bars pointing to the left from mean (0) indicate metabolites with lower
levels in SeQ10-treated men. Amino acids are marked in light blue. Figure 3. P(corr) loading vector from the OPLS-DA model with the 95 metabolites identified in plasma
of men (n = 9) receiving a daily addition of SeQ10 for 18 months as compared with placebo-treated men
controls; n = 8). Note: Bars pointing to the left from mean (0) indicate metabolites with lower levels in
SeQ10-treated men. Amino acids are marked in light blue. Figure 3. P(corr) loading vector from the OPLS-DA model with the 95 metabolites identified in plasma
of men (n = 9) receiving a daily addition of SeQ10 for 18 months as compared with placebo-treated
men (controls; n = 8). Note: Bars pointing to the left from mean (0) indicate metabolites with lower
levels in SeQ10-treated men. Amino acids are marked in light blue. Figure 3. P(corr) loading vector from the OPLS-DA model with the 95 metabolites identified in plasma
of men (n = 9) receiving a daily addition of SeQ10 for 18 months as compared with placebo-treated men
(controls; n = 8). Note: Bars pointing to the left from mean (0) indicate metabolites with lower levels in
SeQ10-treated men. Amino acids are marked in light blue. 6 of 20 Biomolecules 2019, 9, 553 Table 2. Metabolites that showed significantly lower levels in plasma of men receiving a daily addition
of SeQ10 for 18 months as compared with the controls, the placebo-treated men. Metabolite
p-Value
VIP (OPLS-DA)
Decreased in SeQ10 group
Alanine
0.001
1.79
Alanine-3-cyano
0.016
1.62
Arachidonic acid
NS
1.30
Aspartic acid
0.017
1.65
Butyric acid, 2-amino
NS
1.16
Cysteine
0.048
1.05
Fucose
NS
1.21
Glutamic acid
0.022
1.60
Glycine
0.010
1.50
Isoleucine
0.031
1.49
Leucine
NS
1.45
Methionine
0.030
1.51
Myo-inositol
0.022
1.42
Ornithine-1,5-lactam
0.013
1.29
Phenylalanine
NS
1.25
Proline-4-hydroxy
0.026
1.39
Salicylic acid
NS
1.34
Serine
0.032
1.44
Threonine
0.049
1.36
Note: No metabolite showed significantly higher levels in SeQ10 men; Note: VIP: Variables’ Importance to the
Projection. Note: T-tests were used in the statistical evaluation. Note: NS: Not significant. 2.3. Validation Studies Both univariate analyses and multivariate modelling were carried out in each batch separately. The results are summarised in Tables 3 and 4 where the T-tests with corresponding variables’ importance
to the projection (VIP) values from the OPLS-DA models are presented, and the values of the combined
loading vectors obtained are presented in Table 5. In that table, 62 out of 107 (57.9%) evaluated
metabolites showed congruence in change, or no change between the main study and at least one of
the validation studies. 7 of 20 Biomolecules 2019, 9, 553 Table 3. Compounds that were found significant according to T-test in at least one batch when
SeQ10 and controls at 18 months were compared, with the corresponding p-value and VIP value from
this batch. Compound
p Value
(95% Confidence Level)
VIP
Decreased in the SeQ10
group
Alanine
0.026
1.94
Glycine
0.025
1.95
Isoleucine
0.016
1.93
Leucine
0.031
1.60
Lysine
0.025
1.37
Tryptophan
0.025
0.95
Increased
1-monohexadecanoylglycerol
0.004
1.79
Aspartic acid
0.007
2.19
Hexadecanoic acid
0.013
1.71
Hexadecenoic acid
0.039
1.63
Lauric acid
0.008
1.51
Myristic acid
0.026
1.44
Oleic acid
0.033
1.72
Note: p-values obtained from t-tests. Note: VIP values were obtained from the OPLS-DA model. Table 4. Compounds that were found significant according to t-test in at least one batch when SeQ10
and controls for men at 18 months were compared, with the corresponding p-value and VIP value from
this batch. Compound
p Value
(95% Confidence Level)
VIP
Decreased in the SeQ10 group
Arabinose
0.013
1.83
Ribose
0.014
1.76
Sucrose
0.034
1.83
Xylitol
0.025
1.41
Increased
Fructose
0.022
1.96
1,5-anhydroglucitol
0.016
1.93
Note: p values obtained from t-tests; VIP values were obtained from the OPLS-DA model. Table 5. Metabolites identified in the main study and validation studies 1 and 2 together with the
combined loading vectors from these studies. Table 5. Metabolites identified in the main study and validation studies 1 and 2 together with the
combined loading vectors from these studies. Compound
Main Study
Validation 1
Validation 2
1,5-anhydro-d-glucitol
N
↑
↑
1-dodecanoyl-sn-glycero-3-phosphocholine
N
N
↑
1-Monohexadecanoylglycerol
N
↑
↑
1-Palmitoyl-sn-glycero-3-phosphocholine
N
↓
N 8 of 20 Biomolecules 2019, 9, 553 Table 5. Cont. Table 5. Cont. 2.3. Validation Studies Compound
Main Study
Validation 1
Validation 2
2-aminobutyric acid
N
↓
↓
3-hydroxybutyric acid
N
↑
↑
Alanine
↓
↓
↓
Alanine, 3-cyano
↓
N
N
Allothreonine
N
↓
N
Alpha-ketoglutarate
↓
N
N
Aminomalonic acid
N
N
↓
Arabinose
↓
↓
↓
Arabitol/ribitol
↓
N
N
Arachidonic acid
↓
N
N
Arginine
↓
↓
↓
Asparagine, DL-
↓
↓
↓
Aspartic acid, DL
↓
↓
↑
Benzyl alcohol
↓
N
N
Beta-Alanine
↓
N
N
Butanoic acid, 2,4-dihydroxy-
↑
N
N
Butyric acid, 2-amino
↓
N
N
Campesterol
↑
↑
↑
Cholesterol
↑
N
N
Citric acid
↓
↑
↑
Creatinine
↓
↓
↑
Cysteine
↓
↓
↓
Cystine
↓
↑
↓
Docosanoic acid
↑
↑
N
Docosahexaenoic acid,
4,7,10,13,16,19-(Z,Z,Z,Z,Z,Z)
↓
N
↓
Eicosanoic acid, n-
↓
N
N
Eicosapentaenoic acid
↓
N
N
Ethanolamine
↓
↑
↑
Fructose
↑
↑
↑
Fucose
↓
N
N
Glucose
↑
N
N
Glucose, 1,6-anhydro, beta
↓
↓
N
Glutamic acid
↓
↓
↓
Glutamine
↓
↓
↑
Glyceric acid
↓
↓
↓
Glycerol
↑
N
N
Glycerol-2-phosphate
↓
N
N
Glycerol-3-phosphate
↑
↓
↓
Glycine
↓
↓
↑
Glycolic acid
N
N
↑
Heptadecanoic acid, n-
↑
↓
N Main Study
Validation 1
Validation 2 9 of 20 Biomolecules 2019, 9, 553 Table 5. Cont. Table 5. Cont. 2.4. Validation Studies 1 and 2 Whereas no metabolites by univariate analysis were significantly changed in any of the studies,
comparison of metabolic profiles in relation to the SeQ10 administration supports the results from the
main study at T18. There is also consistency between the two validation studies, where 62% of the
metabolites detected in at least two studies showed the same changes (higher or lower metabolite
levels) in SeQ10-treated individuals as compared with those receiving a placebo. The differences
between the groups were largely attributed to amino acids that were at lower levels in the actively
treated group compared with the placebo group (Table 3). 2.3. Validation Studies “N” stands
for no change as a result of the intervention. 2.3. Validation Studies Compound
Main Study
Validation 1
Validation 2
Heptanoic acid
↑
N
N
Hexadecanoic acid
↑
↑
↑
Hexadecenoic acid
↑
↑
↑
Hippuric acid
↓
N
N
Histidine
↓
↓
↓
Indole-3-acetic acid
↓
N
N
Inosine
↑
N
N
Inositol, myo
↓
↑
↓
Inositol-1-phosphate
↑
↓
↑
Isoleucine
↓
↓
↓
Itaconic acid
↓
N
N
Lactic acid,
↓
↓
↑
Lauric acid (dodedecanoic acid)
↑
↑
↑
Leucine
↓
↓
↓
Linoleic acid
↑
↑
↑
Lysine
↓
↓
↓
Malic acid
↓
↓
↑
Malonic acid, 2-amino
↑
N
N
Maltose
↓
↓
↑
Mannitol
N
N
↓
Mannose
↑
N
N
Methionine
↓
N
N
Monomethylphosphate
↑
↑
↑
Myristic acid
↑
↑
↑
Nonanoic acid,n
↓
↑
↓
Oleic acid
↑
↑
↑
Ornithine
↓
↓
↓
Ornithine-1,5-lactam
↓
N
N
Oxalic acid
N
N
↓
Phenylalanine
↓
↓
↓
Phosphoric acid
↑
N
N
Pipecolic acid
↓
N
N
Proline
↓
↓
↓
Proline, 4-hydroxy
↓
N
N
Protocatechuic acid
↓
N
N
Pseudouridine
N
↓
↑
Putrescine
↓
N
N
Pyridine, 3-hydroxy
↓
N
N
Pyroglutamic acid
↓
↓
↑
Pyruvic acid
↑
↓
↓
Ribose
↓
↓
↓
Salicylic acid
↓
↓
↓ Main Study
Validation 1
Validation 2 10 of 20 Biomolecules 2019, 9, 553 Table 5. Cont. Compound
Main Study
Validation 1
Validation 2
Serine
↓
↓
↓
Sitosterol
↑
↓
N
Sorbitol
↑
N
N
Stearic acid
↑
↑
↑
Succinic acid
↑
N
↑
Sucrose
↑
↑
↑
Taurine
↓
↓
↓
Threitol
↑
N
N
Threonic acid
↓
↑
↓
Threonine
↓
↓
↓
Tocopherol, alpha-
↑
↑
↓
Tocopherol, gamma-
↓
↓
↑
Tryptophan
↓
↓
↓
Tyrosine
↓
↓
↓
Urea
↓
N
↓
Uric acid
↓
↓
↑
Valine
↓
↓
↓
Xylitol
↑
↓
↓
Xylose
↓
↓
↑
Xylulose
↓
N
↑
Note: Arrows pointing down are metabolites with lower levels in the SeQ10-treated men compared with the placebo
group, and arrows pointing up are those metabolites that showed higher levels in the SeQ10 treated men. “N” stands
for no change as a result of the intervention. Note: Arrows pointing down are metabolites with lower levels in the SeQ10-treated men compared with the placebo
group, and arrows pointing up are those metabolites that showed higher levels in the SeQ10 treated men. 3. Discussion Our group has recently presented clinical data showing reduced cardiovascular mortality, increased
cardiac function according to echocardiographic evaluation, and decreased concentration of the cardiac
biomarker NT-proBNP, as a result of intervention with SeQ10 as a dietary supplement given to an elderly
healthy population over four years. To acquire knowledge of the possible biochemical mechanisms
behind these clinical results, we have applied metabolic profiling to investigate possible metabolic
changes due to the intervention. We have shown an effect on the metabolome in the SeQ10-treated men after 18 months when
compared with those receiving the placebo (Figures 1–3, Tables 2 and 3). For a majority of the changes observed in the main study (57.9%), the same changes could be
seen in one or both validation studies, supporting the validity of the results from the main study. 11 of 20 Biomolecules 2019, 9, 553 Together, the changes in the metabolite patterns after SeQ10 treatment could be attributed to biochemical
pathways discussed further in the following paragraphs. An interesting example of the effect of the intervention could be seen in the level of 1,5-anhydro-
D-glucitol, a monosaccharide that is found in many foods. This substance is reabsorbed by the proximal
renal tubule in competition with glucose. A high blood concentration of 1,5-anhydro-D-glucito is
indicative of a low level of glucose in primary urine, and is therefore regarded as positive [31]. It has also
been reported in the literature that low levels of 15-anhydro-D-glycitol are associated with increased
cardiovascular mortality or increased coronary artery disease risk [32], even in cases without signs
of diabetes [33]. In our study we reported a higher concentration of the substance in those receiving
SeQ10, compared with those on placebo, indicating a change in the metabolomic profile that could be a
clinically important effect of the intervention. 3.1. Biochemical Pathways Altered by the Intervention with SeQ10 (A) The pentose phosphate pathway, which is an alternative metabolic pathway to that of glycolysis
with generation of, e.g., NADPH and 5-carbon sugars (pentoses), was enhanced. In those receiving
SeQ10, a decrease in 1,6-anhydro-β-D-glucose, as well as an increase in glycerol-3-phosphate, fructose
and inositol-1-phosphate could be seen. This strongly indicates that the equilibria are shifted towards
the generation of reducing equivalents in the form of NADPH, i.e., further providing selenoproteins, e.g.,
thioredoxin reductase, glutathione peroxidases and selenop, that may scavenge reactive oxygen species
(ROS) within the cell. Previously, increased oxidative stress with ROS formation has been discussed
as one of the main mechanisms in the development of heart disease [34]; therefore, intervention to
decrease oxidative stress is of great interest [35]. Our group have previously reported signs of less
oxidative stress as a result of the SeQ10 intervention [36]. (B) The mevalonate pathway, in which squalene is an important precursor in the biosynthesis
of cholesterol, heme, vitamin K, coenzyme Q and all steroid hormones [37], was influenced by the
intervention with selenium and coenzyme Q10. We observed that several phytosterols e.g., campesterol,
and sitosterol showed higher levels in the SeQ10-treated participants than in those receiving the placebo. Usually, phytosterols are exogenous compounds produced by plants and found in the diet. Some
phytosterols may be added to food (usually margarine), and they compete with cholesterol as regards
intestinal absorption and can thereby lower LDL cholesterol [38]. It has been reported that phytosterols
reduce LDL levels and increase HDL in the blood and, thus, have a potential place in the treatment of
hyperlipidaemia [39–41]. A potential source of the phytosterols in the present study could be the yeast
in the tablets containing yeast selenium. One might also speculate that the intervention modified the
gastro-intestinal flora, resulting in increased intestinal release of phytosterols from the food matrix or
uptake of such compounds [42]. (B) The mevalonate pathway, in which squalene is an important precursor in the biosynthesis
of cholesterol, heme, vitamin K, coenzyme Q and all steroid hormones [37], was influenced by the
intervention with selenium and coenzyme Q10. We observed that several phytosterols e.g., campesterol,
and sitosterol showed higher levels in the SeQ10-treated participants than in those receiving the placebo. Usually, phytosterols are exogenous compounds produced by plants and found in the diet. 3.1. Biochemical Pathways Altered by the Intervention with SeQ10 Some
phytosterols may be added to food (usually margarine), and they compete with cholesterol as regards
intestinal absorption and can thereby lower LDL cholesterol [38]. It has been reported that phytosterols
reduce LDL levels and increase HDL in the blood and, thus, have a potential place in the treatment of
hyperlipidaemia [39–41]. A potential source of the phytosterols in the present study could be the yeast
in the tablets containing yeast selenium. One might also speculate that the intervention modified the
gastro-intestinal flora, resulting in increased intestinal release of phytosterols from the food matrix or
uptake of such compounds [42]. (C) The beta-oxidation pathway is increased by the treatment with SeQ10. This is supported by
the decrease of unsaturated fatty acids with an even number of double bonds and the increased levels
of monounsaturated fatty acids (Figure 3). Thus, fatty acids with multiple double bonds decrease,
whereas less unsaturated fatty acids increase as a result of the intervention. In combination with the
cardio-protective steroids produced in the mevalonate pathway that could be demonstrated in the
analyses in the present study, the altered fatty acid composition indicates that SeQ10 should have a
positive effect on individuals with metabolic syndrome and/or type 2 diabetes [43]. (D) The xanthine oxidase pathway, which is a metabolic pathway for uric acid formation, is
down-regulated as the levels of uric acid in plasma are decreased in SeQ10 -treated men. Uric acid
is a major antioxidant in a hydrophilic environment. Reduced synthesis might indicate a reduced
need for antioxidants because ROS are taken care of by selenoproteins and/or the reduced form of
Q10 in the supplemented group. This supports our previous results that SeQ10 appeared to have an
anti-inflammatory effect [44]. From a clinical point of view, reports indicating an important association
between inflammation and vascular disease can be found in the literature. Therefore, this aspect is
important [45,46]. An anti-oxidative effect caused by the intervention with SeQ10 and shown by the 12 of 20 Biomolecules 2019, 9, 553 decrease in uric acid levels is also supported by previously presented findings in the same study
population on the concentrations of copeptin and the midregional segment of proadrenomedullin
(MR-proADM) as markers for anti-oxidative activity [36]. 3.1. Biochemical Pathways Altered by the Intervention with SeQ10 (E) An interesting observation is that an influence on the urea cycle could be seen, with
decreased levels of non-essential amino acids as well as creatinine, but without increased levels
of the corresponding ketoacids. It could be inferred that the non-essential amino acids were part of
the production of proteins. This suggests that the treatment with SeQ10 favours an anabolic process
instead of an oxidative process with catabolism. Decreased levels of the essential amino acids could also be seen in those who were supplemented
with SeQ10, compared with those on placebo. As it is reported in the literature that a high concentration
of some essential, branched-chain amino acids (leucine, isoleucine and valine) is associated with
diabetes mellitus [47,48], and also with oxidative stress and inflammation [49], decreased levels could
be interpreted as positive. 3.2. General Effects of SeQ10 Treatment In addition to the changes discussed above we also observed that the levels of the precursors
to neurotransmitters of the brain, i.e., the aromatic amino acids, were decreased in those receiving
SeQ10. This might explain the observed reduction in sense of fatigue in the intervention group reported
previously [50] The same has also been reported for Q10 given in monotherapy [51,52]. An interesting observation is that the participants treated with SeQ10 had changes in their
metabolome that were opposite to changes in patients suffering from severe systemic lupus
erythematosus (SLE) [53]. Therefore, patients with severe SLE might also benefit from intervention
with SeQ10, as this could normalise their pathological metabolic profile. At T18 months we also observed that the SeQ10-treated men had lower levels of salicylic acid in
their plasma, which could imply less consumption of salicylic acid-containing painkillers than the
placebo group. However, we do not have any anamnestic data to support this interesting observation,
even though there are reports in the literature indicating such an effect by coenzyme Q10 [54–56]. Another observation was decreased levels of the essential amino acid methionine in the
SeQ10-treated group. Methionine is the main precursor for cysteine and glutathione and a central
molecule in one-carbon metabolism as a precursor for S-adenosylmethionine (SAM), the principal
donor of methyl groups. In mice, selenium modulates one-carbon metabolism and increases hepatic
methylation capacity, resulting in increased global DNA methylation and affecting lipogenesis [43,57]. The DNA methylation regulating gene expression is also related to ageing [58]. In a previous publication
our group presented data indicating less fibrosis in seven out of eight evaluated biomarkers for fibrosis
in the population receiving SeQ10 compared with those on placebo. This could indicate a possible
anti-ageing effect on the cardiovascular system, as the ageing process is highly associated with increased
fibrosis of the cardiovascular system [59]. Increased expression and synthesis of selenoprotein P
following selenium supplementation requires SAM-dependent protein methylation [57]. Taken together, the presented changes in metabolic profiles as seen in the present evaluation could
provide important information to support the positive clinical results that have been demonstrated in
the previous publications. Therefore, for those with insufficient selenium and coenzyme Q10 intake,
the metabolic profiling results could strengthen the argument for supplementation. 4. Material 4.1. Aim 4.1. Aim The aim of the present work was to investigate changes in biochemical pathways after intervention
with SeQ10 to a healthy elderly male community-living population by metabolic profiling of
plasma metabolites. 13 of 20 Biomolecules 2019, 9, 553 13 of 20 5.2. Blood Samples Blood samples were collected while the participants were resting in a supine position. Pre-chilled,
EDTA vials (Terumo EDTA K-3) were used. The vials were placed on ice and centrifuged for 10 min at
3000× g, +4 ◦C, and the plasma was immediately transferred to storage at −70 ◦C until further analysis. No sample was thawed more than once. 5.1. Echocardiography Doppler-echocardiography examinations were performed with the participants in the left lateral
position. The ejection fraction (EF) readings were categorised into four classes with interclass limits
placed at 30%, 40% and 50% [61,62]. Normal systolic function was defined as EF ≥50%, while severely
impaired systolic function was defined as EF < 30%. 5.3. Sample Selection The available biobank contained samples from 443 individuals, including both males and females,
and the male population was chosen. From those, the individuals who delivered blood samples at one
(T0), two (T0 + T18 months), or three time points (T0 + T18 months + T48 months) were chosen after
taking into account clinical and demographic variables in order to get balanced groups. The samples
were divided into a main study as well as two validation studies. In each of the validation studies the
samples were divided into analytical batches run on separate days. Each batch contained a balanced
mix of treatment and placebo samples, with all time point samples from an individual included in the
same batch. As explained below, samples selected for analysis were representative of the whole study
and covered the whole expected intervention period, the same was true for batch separation where
each batch itself contained representative information found in the whole cohort [63,64]. Therefore,
the individuals included in the study were selected to meet those criteria. 4.2. Design A double-blind randomised placebo-controlled study where intervention with a dietary
supplement of Se and Q10 combined (SeQ10) in an elderly healthy rural population of 443 individuals
in the age range of 70–88 years has been previously reported [25,60]. The first participant was included
in January 2003, and the last participant concluded the study in February 2010. The participants
received the intervention for 48 months during which time they were re-examined every six months,
and throughout the study, all mortality was registered. During the study 221 individuals received
active supplementation of 200 µg/day organic Se (SelenoPrecise®, PharmaNord, Denmark), plus
200 mg/day of Q10 (Bio-Quinon®, PharmaNord, Denmark), and 222 individuals received a placebo. At inclusion, all participants underwent clinical examination, a new patient record was obtained, the
New York Heart Association (NYHA) functional class was assessed (this classification grades how
a patient with heart disease experiences symptoms of tiredness, breathlessness or chest pain; it is
graded from I–IV, where IV is symptoms already at rest), an ECG and a Doppler-echocardiography
were performed. Informed consent was obtained from each patient. The study was approved by the
Regional Ethical Committee in Linköping (Forskningsetikkommmitten, Hälsouniversitetet, SE-581 85
Linköping, Sweden; no. D03-176), and it conforms to the ethical guidelines of the 1975 Declaration of
Helsinki. The Medical Product Agency declined to review the study protocol since the study was not
considered a trial of a medication for a certain disease, but rather one of food supplement commodities
that are commercially available. The study was registered at clinicaltrials.gov. (NCT01443780). 5.5. Validation Studies To confirm the results from the main study, it was decided to perform two validation studies;
“Validation study 1” and “Validation study 2”, each made up of several batches. Each batch contained
a balanced mix of SeQ10 and placebo samples, selected according to the MVD approach (as described
for the main study). 5.4. Main Study In the participant database, clinical and demographic data were used for the selection of
participants. From the original dataset, a selection was made by use of multivariate design (MVD), 14 of 20 14 of 20 Biomolecules 2019, 9, 553 which, in turn, was based on a combination of an experimental design and multivariate data analysis
(MVDA) [63,64]. By using MVD we ensured that the selected subjects together spanned as large an
area of the available properties domain as possible, thus including as large a natural variation as
possible, and at the same time this ensured that the properties’ domain in the placebo group covered
the same domain as the group receiving SeQ10. Principal component analysis of the clinical and
demographic information that was collected on the inclusion date of each participant was applied. A total of 182 variables representing all available demographic and clinical data were used in the
PCA model. Based on two separate overview models of the SeQ10 and the placebo-treated men, a
representative selection for an initial metabolomic study was obtained. From the PCA score plot
(which plotted the subjects in the first two principal components) 10 individuals were selected from
each group (two from each of the four quadrants plus two from the centre, thus spanning the whole
properties domain). In addition to these 20 participants, seven others (two SeQ10 and five placebo)
were selected representing the participants that had participated up until T18 but had later died of
cardiovascular disease (CVD). Those who died a cardiovascular death (n = 2) during the intervention
time were not included in the main analyses but were evaluated separately. When retrieving the samples from the freezer it was noticed that one of the placebo samples was
contaminated with hepatitis and this was therefore excluded from analysis. During sample preparation,
one of the SeQ10 samples did not derivatise so no metabolite analysis could be performed. All derivatised samples were analysed by GC-MS, and 315 metabolites were detected. Using
metabolite databases, 95 of these were identified and were used in the modelling of metabolite profiles. All derivatised samples were analysed by GC-MS, and 315 metabolites were detected. Using
metabolite databases, 95 of these were identified and were used in the modelling of metabolite profiles. 5.4. Main Study Ultimately, we included nine male SeQ10 and nine placebo participants (T18) in the main study, Ultimately, we included nine male SeQ10 and nine placebo participants (T18) in the main study,
and we treated the samples from the individuals that died from CVD as a separate group. 5.5.1. Validation Study 1 Metabolic profiles from male individuals were investigated. A total of 60 male individuals
(31 SeQ10, 29 placebo) were included, all having three samples each (T0, T18 and T48). One individual
(SeQ10) from the first study with two time points was included as well. All samples belonging to
Validation study 1 were divided into five analytical batches (subsets). 5.5.2. Validation Study 2 In validation study 2, samples from 37 men (23 SeQ10, 14 placebo) were included. 5.6. Metabolomic Profiling 5.6. Metabolomic Profiling 5.6.1. Metabolite Analysis (Extraction, Quantification and Identification) 5.6.1. Metabolite Analysis (Extraction, Quantification and Identification) Plasma samples were extracted, derivatised, and analysed using GC–TOF–MS as previously
described [65]. Masses were acquired in the mass range 50–800 mass/charge numbers of ions (m/z) at a
rate of 30 spectra/s. The acceleration voltage was turned on after a solvent delay of 150 s. An alkane
series (C10–C32) was run together with all samples. Non-processed MS files from GC/TOFMS analysis
were exported in NetCDF format to MATLAB software 7.3 (Mathworks, Natick, MA, USA), where all
data pre-treatment procedures, such as baseline correction, chromatogram alignment, time-window
setting and multivariate curve resolution were performed using custom scripts [66]. For the identification of metabolites, The National Institute of Standards and Technology (NIST)
MS Search software (version 2.0, Gaithersburg, MD, USA) was used. With this we compared the mass 15 of 20 Biomolecules 2019, 9, 553 spectra of all detected compounds with spectra in the NIST library, the in-house mass spectra library
established by Umeå Plant Science Centre and Swedish Metabolomics Centre and the mass spectra
library maintained by the Max Planck Institute, Golm, Germany (http://csbdb.mpimp-golm.mpg.de/
csbdb/gmd/gmd.html). A retention index comparison was performed, with a retention index deviation
< ±10 (in addition to a high spectral match, above 800). 5.6.2. Statistical Analysis 5.6.2. Statistical Analysis OPLS-DA Models The orthogonal partial least-squares discriminant analysis (OPLS-DA) models were calculated
between the two intervention groups (SeQ10 and placebo) to identify the metabolites that showed
different levels in these groups. Multivariate Data Analysis Metabolite levels reflect biochemical processes, and as few processes in an organism are truly
independent, metabolite levels are also highly correlated. The MVDA approaches are best suited
to analyses of highly correlated data and allow the identification of the following: small changes in
the variable patterns between different groups; internal relations/correlations between samples and
between metabolites; and how samples and metabolites are connected to each other. These differences
can be missed by using a univariate approach alone [67–69]. Univariate Statistics Univariate analysis was performed on each metabolite separately, with p values calculated by
performing two-sample, unequal variance, two-tailed t-tests. Data Normalisation Data were normalised using 11 internal standards (eluting over the whole chromatographic time
range). To obtain accurate peak areas for the internal standards, two unique masses for each compound
were specified and the samples were reprocessed using an in-house MATLAB-based script. Principal
component analyses were performed on the peak areas of internal standards with Unit Variance
Normalisation and the first component (t1) score value for each sample was used to normalise the
resolved data. This was done by dividing the peak areas in each sample by the corresponding score
value [70]. All multivariate modelling was performed using the software SIMCA version 14 (Umetrics, Umeå,
Sweden). The PCA approach was used for a metabolomics data overview [71], and the OPLS-DA
approach was used to elucidate the differences concerning metabolomics between the two groups of
subjects (SeQ10 and placebo) [72,73]. Unit Variance scaling was used for both types of models [74]. The significant differences between the SeQ10 group and the placebo-treated individuals were identified
by using confidence intervals calculation by use of jack-knifing [75]. A potential model significance
was found by means of cross-validation [75]. Furthermore, cross-validated predicted Y values from
the cross-validation procedure were used to visualise the ability of the OPLS-DA model to separate
individuals receiving SeQ10 from those receiving a placebo. The VIP values were calculated (SIMCA v.14) as VIP scores. This was done in order to estimate
the importance of each variable in the projection used in the OPLS-DA model. A variable with a VIP
score of about 0.8 or greater was considered significant in the model. In order to obtain an overview model of the different batches, the p(corr)-loadings for each of the
SeQ10 treated vs. placebo OPLS-DA models were combined into an X-matrix which was used to create
a PCA model where no scaling was applied. The p [1] loading profile from this PCA model was used
as a combined metabolic profile representing the metabolic effects of the SeQ10 intake in validation
studies 1 and 2, and compared with the p(corr) vector from the main study. Biomolecules 2019, 9, 553 16 of 20 Small differences in the number of significant compounds when detected by univariate and
multivariate approaches were expected, since both approaches are based on different testing principles;
the former (t-tests) is a statistical hypothesis test, whereas the multivariate testing, using jack-knifing,
is a resampling technique applied to the multivariate space. Data Normalisation These differences were, thus, included in
the evaluation of the results. 6. Conclusions There was a clear difference in the metabolic profile in plasma from men receiving SeQ10 as
compared with men receiving placebo. The major differences can be seen in the pentose phosphate
pathway, in the mevalonate pathway, and in the beta-oxidation pathway. An interesting observation
was downregulation of plasma methionine, indicating increased methylating activity, as also previously
observed experimentally. It can also be reported that xanthine oxidase was down-regulated. Moreover,
there were effects on the urea cycle where the non-essential amino acids were down-regulated. All these
effects, together with signs of less inflammation and less oxidative stress observed in the previous
studies of this group, indicate a positive health effect by the supplementation with selenium and
coenzyme Q10 compared with those on placebo. However, as this is the first intervention with the SeQ10 combination in an elderly population low
in Se, the results should be regarded as hypothesis-generating, and more research is needed to confirm
the positive health impact of the SeQ10 combination in elderly persons. Author Contributions: U.A. and P.J. conceived and designed the research project. U.A. and P.J. conducted the
research. T.L. and I.S. provided the essential reagents, and performed the analyses. U.A., T.L. and K.L.-E. analysed
data and performed the statistical analyses. U.A., P.J., J.A. (Jan Aaseth), J.A. (Jan Alexander), T.L., I.S. and K.L.-E. wrote the paper. U.A. had the final responsibility for the final content. All the authors have read and approved the
final manuscript. Funding: The analysis costs were partially supported by grants from Pharma Nord Aps, Denmark, the County
Council of Östergötland, Linköping University. The funding organisations had no role in the design, management,
analysis, or interpretation of the data, nor in the preparation, review or approval of the manuscript. No economic
compensation was distributed. Conflicts of Interest: The authors declare no conflicts of interest. References 1. Rayman, M.P. Selenium and human health. Lancet 2012, 379, 1256–1268. [CrossRef] 1. Rayman, M.P. Selenium and human health. Lancet 2012, 379, 1256–1268. [CrossRef] 2. Selenius, M.; Rundlöf, A.K.; Olm, E.; Fernandes, A.P.; Björnstedt, M. Selenium and the Selenoprotein
Thioredoxin Reductase in the Prevention, Treatment and Diagnostics of Cancer. Antioxid. Redox Signal. 2010,
12, 867–880. [CrossRef] [PubMed] 2. Selenius, M.; Rundlöf, A.K.; Olm, E.; Fernandes, A.P.; Björnstedt, M. Selenium and the Selenoprotein
Thioredoxin Reductase in the Prevention, Treatment and Diagnostics of Cancer. Antioxid. Redox Signal. 2010,
12, 867–880. [CrossRef] [PubMed] 2. Selenius, M.; Rundlöf, A.K.; Olm, E.; Fernandes, A.P.; Björnstedt, M. Selenium and the Selenoprotein
Thioredoxin Reductase in the Prevention, Treatment and Diagnostics of Cancer. Antioxid. Redox Signal. 2010,
12, 867–880. [CrossRef] [PubMed] 3. Huang, Z.; Rose, A.H.; Hoffmann, P.R. The Role of Selenium in Inflammation and Immunity: From Molecular
Mechanisms to Therapeutic Opportunities. Antioxid. Redox Signal. 2012, 16, 705–743. [CrossRef] [PubMed] 3. Huang, Z.; Rose, A.H.; Hoffmann, P.R. The Role of Selenium in Inflammation and Immunity: From Molecular
Mechanisms to Therapeutic Opportunities. Antioxid. Redox Signal. 2012, 16, 705–743. [CrossRef] [PubMed]
4. Fairweather-Tait, S.J.; Bao, Y.; Broadley, M.R.; Collings, R.; Ford, D.; Hesketh, J.E.; Hurst, R. Selenium in
Human Health and Disease. Antioxid. Redox Signal. 2011, 14, 1337–1383. [CrossRef] g,
;
,
;
,
y
Mechanisms to Therapeutic Opportunities. Antioxid. Redox Signal. 2012, 16, 705–743. [CrossRef] [PubMed]
4. Fairweather-Tait, S.J.; Bao, Y.; Broadley, M.R.; Collings, R.; Ford, D.; Hesketh, J.E.; Hurst, R. Selenium in
Human Health and Disease. Antioxid. Redox Signal. 2011, 14, 1337–1383. [CrossRef] p
pp
g
4. Fairweather-Tait, S.J.; Bao, Y.; Broadley, M.R.; Collings, R.; Ford, D.; Hesketh, J.E.; Hurst, R. Selenium in
Human Health and Disease. Antioxid. Redox Signal. 2011, 14, 1337–1383. [CrossRef] 5. Yang, G.; Zhou, R. Further observations on the human maximum safe dietary selenium intake in a seleniferous
area of China. J. Trace Elem. Electrolytes Health Dis. 1994, 8, 159–165. 5. Yang, G.; Zhou, R. Further observations on the human maximum safe dietary selenium intake in a seleniferous
area of China. J. Trace Elem. Electrolytes Health Dis. 1994, 8, 159–165. heng, Y.Y.; Qian, P.C. The effect of selenium-fortified table salt in the prevention of Keshan disease on a
pulation of 1.05 million. Biomed. Environ. Sci. 1990, 3, 422–428. 7. Akbaraly, N.T.; Arnaud, J.; Hininger-Favier, I.; Gourlet, V.; Roussel, A.-M.; Berr, C. Selenium and Morta
the Elderly: Results from the EVA Study. Clin. Chem. References Relatively high mortality risk in
elderly Swedish subjects with low selenium status. Eur. J. Clin. Nutr. 2016, 70, 91–96. [CrossRef] [PubMed] 17. Kafai, M.R.; Ganji, V. Sex, age, geographical location, smoking, and alcohol consumption influence serum
selenium concentrations in the USA: Third national health and nutrition examination survey, 1988–1994. J. Trace Elements Med. Boil. 2003, 17, 13–18. [CrossRef] 18. Bleys, J.; Navas-Acien, A.; Laclaustra, M.; Pastor-Barriuso, R.; Menke, A.; Ordovas, J.; Stranges, S.;
Guallar, E. Serum Selenium and Peripheral Arterial Disease: Results From the National Health and Nutrition
Examination Survey, 2003–2004. Am. J. Epidemiol. 2009, 169, 996–1003. [CrossRef] [PubMed] 19. Alehagen, U.; Alexander, J.; Aaseth, J. Supplementation with Selenium and Coenzyme Q10 Reduces
Cardiovascular Mortality in Elderly with Low Selenium Status. A Secondary Analysis of a Randomised
Clinical Trial. PLoS ONE 2016, 11, e0157541. [CrossRef] 20. Xia, L.; Nordman, T.; Olsson, J.M.; Damdimopoulos, A.; Björkhem-Bergman, L.; Nalvarte, I.; Eriksson, L.C.;
Arnér, E.S.; Spyrou, G.; Björnstedt, M. The mammalian cytosolic selenoenzyme thioredoxin reductase reduces
ubiquinone. A novel mechanism for defense against oxidative stress. J. Biol. Chem. 2003, 278, 2141–2146. [CrossRef] 21. Acosta, M.J.; Fonseca, L.V.; Desbats, M.A.; Cerqua, C.; Zordan, R.; Trevisson, E.; Salviati, L. Coenzyme Q
biosynthesis in health and disease. Biochim. Biophys. Acta 2016, 1857, 1079–1085. [CrossRef] [PubMed] biosynthesis in health and disease. Biochim. Biophys. Acta 2016, 1857, 1079–1085. [CrossRef] [PubMed]
22. Kalén, A.; Appelkvist, E.-L.; Dallner, G. Age-related changes in the lipid compositions of rat and human
tissues. Lipids 1989, 24, 579–584. [CrossRef] [PubMed] 22. Kalén, A.; Appelkvist, E.-L.; Dallner, G. Age-related changes in the lipid compositions of rat and human
tissues. Lipids 1989, 24, 579–584. [CrossRef] [PubMed] 23. Folkers, K.; Moesgaard, S.; Morita, M. A one year bioavailability study of coenzyme Q10 with 3 months
withdrawal period. Mol. Asp. Med. 1994, 15, s281–s285. [CrossRef] 24. Molyneux, S.L.; Florkowski, C.M.; George, P.M.; Pilbrow, A.P.; Frampton, C.M.; Lever, M.; Richards, A.M. Coenzyme Q10: An independent predictor of mortality in chronic heart failure. J. Am. Coll. Cardiol. 2008, 52,
1435–1441. [CrossRef] 25. Alehagen, U.; Johansson, P.; Björnstedt, M.; Rosén, A.; Dahlström, U. Cardiovascular mortality and
N-terminal-proBNP reduced after combined selenium and coenzyme Q10 supplementation: A 5-year
prospective randomized double-blind placebo-controlled trial among elderly Swedish citizens. Int. J. Cardiol. 2013, 167, 1860–1866. [CrossRef] 26. Madsen, R.K.; Lundstedt, T.; Gabrielsson, J.; Sennbro, C.-J.; Alenius, G.-M.; Moritz, T.; Rantapää-Dahlqvist, S.;
Trygg, J. Diagnostic properties of metabolic perturbations in rheumatoid arthritis. Arthritis Res. Ther. References 2005, 51, 2117–2123. [CrossRef] 8. Adame, E.M.; Florea, D.; Pérez, L.S.; López, J.M.; López-González, B.; De La Cruz, A.P.; Del Pozo, E.P. Deficient selenium status of a healthy adult Spanish population. Nutrición Hospitalaria 2012, 27, 524–528. 9. Letsiou, S.; Nomikos, T.; Panagiotakos, D.; Pergantis, S.A.; Fragopoulou, E.; Antonopoulou, S.; Pitsavos, C.;
Stefanadis, C. Serum total selenium status in Greek adults and its relation to age. The ATTICA study cohort. Biol. Trace Elem. Res. 2009, 128, 8–17. [CrossRef] 9
ets ou, S ; No
kos,
;
a ag otakos,
;
e ga t s, S
;
agopou ou,
;
to opou ou, S ;
tsavos, C ;
Stefanadis, C. Serum total selenium status in Greek adults and its relation to age. The ATTICA study cohort. Biol. Trace Elem. Res. 2009, 128, 8–17. [CrossRef] 17 of 20 17 of 20 Biomolecules 2019, 9, 553 10. Benes, B.; Spevácková, V.; Smíd, J.; Batáriová, A.; Cejchanová, M.; Zítková, L. Effects of age, BMI, smoking
and contraception on levels of Cu, Se and Zn in the blood of the population in the Czech Republic. Central
Eur. J. Public Health 2005, 13, 202–207. 11. Reszka, E.; Wieczorek, E.; Jablonska, E.; Janasik, B.; Fendler, W.; Wasowicz, W. Association between plasma
selenium level and NRF2 target genes expression in humans. J. Trace Elem. Med. Biol. 2015, 30, 102–106. [CrossRef] [PubMed] 12. Xia, Y.;
Hill, K.E.;
Li, P.;
Xu, J.;
Zhou, D.;
Motley, A.K.;
Wang, L.;
Byrne, D.W.;
Burk, R.F. Optimization of selenoprotein P and other plasma selenium biomarkers for the assessment of the selenium
nutritional requirement: A placebo-controlled, double-blind study of selenomethionine supplementation in
selenium-deficient Chinese subjects. Am. J. Clin. Nutr. 2010, 92, 525–531. [CrossRef] [PubMed] 13. Hurst, R.; Armah, C.N.; Dainty, J.R.; Hart, D.J.; Teucher, B.; Goldson, A.J.; Broadley, M.R.; Motley, A.K.;
Fairweather-Tait, S.J. Establishing optimal selenium status:
Results of a randomized, double-blind,
placebo-controlled trial. Am. J. Clin. Nutr. 2010, 91, 923–931. [CrossRef] [PubMed] 14. Gao, H.; Hägg, S.; Sjögren, P.; Lambert, P.C.; Ingelsson, E.; Van Dam, R.M. Serum selenium in relation to
measures of glucose metabolism and incidence of Type 2 diabetes in an older Swedish population. Diabet. Med. 2014, 31, 787–793. [CrossRef] [PubMed] 5. Harris, H.R.; Bergkvist, L.; Wolk, A. Selenium intake and breast cancer mortality in a cohort of Swed
women. Breast Cancer Res. Treat. 2012, 134, 1269–1277. [CrossRef] [PubMed] 16. Alehagen, U.; Johansson, P.; Björnstedt, M.; Rosén, A.; Post, C.; Aaseth, J. References Alehagen, U.; Aaseth, J.; Johansson, P. Less increase of copeptin and MR-proADM due to intervention
with selenium and coenzyme Q10 combined: Results from a 4-year prospective randomized double-blind
placebo-controlled trial among elderly Swedish citizens. BioFactors 2015, 41, 443–452. [CrossRef] 37. Holstein, S.A.; Hohl, R.J. Isoprenoids: Remarkable diversity of form and function. Lipids 2004, 39, 293–309. [CrossRef] 38. Shaghaghi, M.A.; AbuMweis, S.S.; Jones, P.J. Cholesterol-Lowering Efficacy of Plant Sterols/Stanols Provided
in Capsule and Tablet Formats: Results of a Systematic Review and Meta-Analysis. J. Acad. Nutr. Diet. 2013,
113, 1494–1503. [CrossRef] 9. Marangoni, F.; Poli, A. Phytosterols and cardiovascular health. Pharmacol. Res. 2010, 61, 193–199. [Cross 39. Marangoni, F.; Poli, A. Phytosterols and cardiovascular health. Pharmacol. Res. 2010, 61, 193–199. [CrossRef]
40. Dumolt, J.H.; Rideout, T.C. The Lipid-lowering Effects and Associated Mechanisms of Dietary Phytosterol
Supplementation. Curr. Pharm. Des. 2017, 23, 5077–5085. [CrossRef] 40. Dumolt, J.H.; Rideout, T.C. The Lipid-lowering Effects and Associated Mechanisms of Dietary Phytosterol
Supplementation. Curr. Pharm. Des. 2017, 23, 5077–5085. [CrossRef] 41. Cabral, C.E.; Klein, M.R.S.T. Phytosterols in the Treatment of Hypercholesterolemia and Prevention of
Cardiovascular Diseases. Arq. Bras. Cardiol. 2017, 109, 475–482. [CrossRef] [PubMed] 42. Shandilya, U.K.; Sharma, A. Functional Foods and Their Benefints: An Overview. J. Nutr. Health Food Eng. 2017, 7, 247. 43. Speckmann, B.; Schulz, S.; Hiller, F.; Hesse, D.; Schumacher, F.; Kleuser, B.; Geisel, J.; Obeid, R.; Grune, T.;
Kipp, A.P. Selenium increases hepatic DNA methylation and modulates one-carbon metabolism in the liver
of mice. J. Nutr. Biochem. 2017, 48, 112–119. [CrossRef] [PubMed] 44. Alehagen, U.; Lindahl, T.L.; Aaseth, J.; Svensson, E.; Johansson, P. Levels of sP-selectin and hs-CRP Decrease
with Dietary Intervention with Selenium and Coenzyme Q10 Combined: A Secondary Analysis of a
Randomized Clinical Trial. PLoS ONE 2015, 10, e0137680. [CrossRef] [PubMed] 45. Walker, K.A.; Power, M.C.; Hoogeveen, R.C.; Folsom, A.R.; Ballantyne, C.M.; Knopman, D.S.; Windham, B.G.;
Selvin, E.; Jack, C.R.; Gottesman, R.F. Midlife Systemic Inflammation, Late-Life White Matter Integrity, and
Cerebral Small Vessel Disease: The Atherosclerosis Risk in Communities Study. Stroke 2017, 48, 3196–3202. [CrossRef] [PubMed] 46. Harrington, R.A. Targeting Inflammation in Coronary Artery Disease. N. Engl. J. Med. 2017, 377, 1197–1198. [CrossRef] [PubMed] 47. Würtz, P.; Soininen, P.; Kangas, A.J.; Rönnemaa, T.; Lehtimäki, T.; Kähönen, M.; Viikari, J.S.; Raitakari, O.T.;
Ala-Korpela, M. Branched-Chain and Aromatic Amino Acids Are Predictors of Insulin Resistance in Young
Adults. Diabetes Care 2013, 36, 648–655. [CrossRef] [PubMed] 48. References 2011,
13, R19. [CrossRef] 27. Madsen, R.; Lundstedt, T.; Trygg, J. Chemometrics in metabolomics—A review in human disease diagnosis. Anal. Chim. Acta 2010, 659, 23–33. [CrossRef] 28. Nordstrom, A.; Lewensohn, R. Metabolomics: Moving to the clinic. J. Neuroimmune Pharmacol. 2010, 5, 4–17. [CrossRef] 18 of 20 18 of 20 Biomolecules 2019, 9, 553 29. Vinayavekhin, N.; Homan, E.A.; Saghatelian, A. Exploring disease through metabolomics. ACS Chem. Biol. 2010, 5, 91–103. [CrossRef] 30. Scholz, S.; Fischer, S.; Gündel, U.; Küster, E.; Luckenbach, T.; Voelker, D. The zebrafish embryo model in
environmental risk assessment—applications beyond acute toxicity testing. Environ. Sci. Pollut. Res. 2008,
15, 394–404. [CrossRef] 31. Yamanouchi, T.; Minoda, S.; Yabuuchi, M.; Akanuma, Y.; Akanuma, H.; Miyashita, H.; Akaoka, I. Plasma
1,5-anhydro-D-glucitol as new clinical marker of glycemic control in NIDDM patients. Diabetes 1989, 38,
723–729. [CrossRef] [PubMed] 32. Fujiwara, T.; Yoshida, M.; Yamada, H.; Tsukui, T.; Nakamura, T.; Sakakura, K.; Wada, H.; Arao, K.;
Katayama, T.; Funayama, H.; et al. Lower 1,5-anhydroglucitol is associated with denovo coronary artery
disease in patients at high cardiovascular risk. Heart Vessels 2015, 30, 469–476. [CrossRef] 33. Ouchi, S.; Shimada, K.; Miyazaki, T.; Takahashi, S.; Sugita, Y.; Shimizu, M.; Murata, A.; Kadoguchi, T.;
Kato, T.; Aikawa, T.; et al. Low 1,5-anhydroglucitol levels are associated with long-term cardiac mortality in
acute coronary syndrome patients with hemoglobin A1c levels less than 7.0. Cardiovasc. Diabetol. 2017, 16,
151. [CrossRef] [PubMed] 34. Münzel, T.; Camici, G.G.; Maack, C.; Bonetti, N.R.; Fuster, V.; Kovacic, J.C. Impact of Oxidative Stress on the
Heart and Vasculature: Part 2 of a 3-Part Series. J. Am. Coll. Cardiol. 2017, 70, 212–229. [CrossRef] [PubMed]
35. Siti, H.N.; Kamisah, Y.; Kamsiah, J. The role of oxidative stress, antioxidants and vascular inflammation in 34. Münzel, T.; Camici, G.G.; Maack, C.; Bonetti, N.R.; Fuster, V.; Kovacic, J.C. Impact of Oxidative Stress on the
Heart and Vasculature: Part 2 of a 3-Part Series. J. Am. Coll. Cardiol. 2017, 70, 212–229. [CrossRef] [PubMed] Heart and Vasculature: Part 2 of a 3-Part Series. J. Am. Coll. Cardiol. 2017, 70, 212–229. [CrossRef] [PubMed]
35. Siti, H.N.; Kamisah, Y.; Kamsiah, J. The role of oxidative stress, antioxidants and vascular inflammation in
cardiovascular disease (a review). Vasc. Pharmacol. 2015, 71, 40–56. [CrossRef] [PubMed] 35. Siti, H.N.; Kamisah, Y.; Kamsiah, J. The role of oxidative stress, antioxidants and vascular inflammation in
cardiovascular disease (a review). Vasc. Pharmacol. 2015, 71, 40–56. [CrossRef] [PubMed] 36. References Tillin, T.; Hughes, A.D.; Wang, Q.; Würtz, P.; Ala-Korpela, M.; Sattar, N.; Forouhi, N.G.; Godsland, I.F.;
Eastwood, S.V.; McKeigue, P.M.; et al. Diabetes risk and amino acid profiles: Cross-sectional and prospective
analyses of ethnicity, amino acids and diabetes in a South Asian and European cohort from the SABRE
(Southall And Brent REvisited) Study. Diabetologia 2015, 58, 968–979. [CrossRef] 19 of 20 19 of 20 Biomolecules 2019, 9, 553 49. Zhenyukh, O.; Civantos, E.; Ruiz-Ortega, M.; Sánchez, M.S.; Vázquez, C.; Peiró, C.; Egido, J.; Mas, S. High
concentration of branched-chain amino acids promotes oxidative stress, inflammation and migration of
human peripheral blood mononuclear cells via mTORC1 activation. Free Radic. Boil. Med. 2017, 104, 165–177. [CrossRef] [PubMed] 50. Johansson, P.; Dahlstrom, O.; Dahlstrom, U.; Alehagen, U. Improved health-related quality of life, and more
days out of hospital with supplementation with selenium and coenzyme Q10 combined. Results from a
double blind, placebo-controlled prospective study. J. Nutr. Health Aging 2015, 19, 870–877. [CrossRef]
[PubMed] 51. Iwase, S.; Kawaguchi, T.; Yotsumoto, D.; Doi, T.; Miyara, K.; Odagiri, H.; Kitamura, K.; Ariyoshi, K.; Miyaji, T.;
Ishiki, H.; et al. Efficacy and safety of an amino acid jelly containing coenzyme Q10 and L-carnitine in
controlling fatigue in breast cancer patients receiving chemotherapy: A multi-institutional, randomized,
exploratory trial (JORTC-CAM01). Support. Care Cancer 2016, 24, 637–646. [CrossRef] [PubMed] 52. Sanoobar, M.; Dehghan, P.; Khalili, M.; Azimi, A.; Seifar, F. Coenzyme Q10 as a treatment for fatigue and
depression in multiple sclerosis patients: A double blind randomized clinical trial. Nutr. Neurosci. 2016, 19,
138–143. [CrossRef] [PubMed] 53. Bengtsson, A.A.; Trygg, J.; Wuttge, D.M.; Sturfelt, G.; Theander, E.; Donten, M.; Moritz, T.; Sennbro, C.-J.;
Torell, F.; Lood, C.; et al. Metabolic Profiling of Systemic Lupus Erythematosus and Comparison with
Primary Sjögren’s Syndrome and Systemic Sclerosis. PLoS ONE 2016, 11, 0159384. [CrossRef] [PubMed] 54. Di Pierro, F.; Rossi, A.; Consensi, A.; Giacomelli, C.; Bazzichi, L. Role for a water-soluble form of CoQ10 in
female subjects affected by fibromyalgia. A preliminary study. Clin. Exp. Rheumatol. 2017, 35 (Suppl. 105),
20–27. 55. Zhang, Y.P.; Eber, A.; Yuan, Y.; Yang, Z.; Rodriguez, Y.; Levitt, R.C.; Takacs, P.; Candiotti, K.A. Prophylactic
and Antinociceptive Effects of Coenzyme Q10 on Diabetic Neuropathic Pain in a Mouse Model of Type 1
Diabetes. Anesthesiology 2013, 118, 945–954. [CrossRef] 56. Zhang, Y.P.; Mei, S.; Yang, J.; Rodriguez, Y.; Candiotti, K.A. Acute Hypoglycemia Induces Painful Neuropathy
and the Treatment of Coenzyme Q10. References J. Diabetes Res. 2016, 2016, 1–7. [CrossRef] [PubMed] 57. Jackson, M.I.; Cao, J.; Zeng, H.; Uthus, E.; Combs, G.F. S-Adenosylmethionine-dependent Protein Methylation
Is Required for Expression of Selenoprotein P and Gluconeogenic Enzymes in HepG2 Human Hepatocytes. J. Boil. Chem. 2012, 287, 36455–36464. [CrossRef] 58. Jung, M.; Pfeifer, G.P. Aging and DNA methylation. BMC Biol. 2015, 13, 7. [CrossRef] 59. Alehagen, U.; Aaseth, J.; Alexander, J.; Svensson, E.; Johansson, P.; Larsson, A. Less fibrosis in elderly subjects
supplemented with selenium and coenzyme Q10-A mechanism behind reduced cardiovascular mortality? BioFactors 2017, 44, 137–147. [CrossRef] 60. Johansson, P.; Dahlström, Ö; Dahlström, U.; Alehagen, U. Effect of selenium and Q10 on the cardiac biomarker
NT-proBNP. Scand. Cardiovasc. J. 2013, 47, 281–288. [CrossRef] 61. Jensen-Urstad, K.; Bouvier, F.; Höjer, J.; Ruiz, H.; Hulting, J.; Samad, B.; Thorstrand, C.; Jensen-Urstad, M. Comparison of Different Echocardiographic Methods With Radionuclide Imaging for Measuring Left
Ventricular Ejection Fraction During Acute Myocardial Infarction Treated by Thrombolytic Therapy. Am. J. Cardiol. 1998, 81, 538–544. [CrossRef] 62. van Royen, N.; Jaffe, C.C.; Krumholz, H.M.; Johnson, K.M.; Lynch, P.J.; Natale, D.; Atkinson, P.; Deman, P.;
Wackers, F.J. Comparison and reproducibility of visual echocardiographic and quantitative radionuclide left
ventricular ejection fractions. Am. J. Cardiol. 1996, 77, 843–850. [CrossRef] 63. Surowiec, I.; Johansson, E.; Torell, F.; Idborg, H.; Gunnarsson, I.; Svenungsson, E.; Jakobsson, P.-J.;
Trygg, J. Multivariate strategy for the sample selection and integration of multi-batch data in metabolomics. Metabolomics 2017, 13, 114. [CrossRef] [PubMed] 64. Surowiec, I.; Vikström, L.; Hector, G.; Johansson, E.; Vikström, C.; Trygg, J. Generalized Subset Designs in
Analytical Chemistry. Anal. Chem. 2017, 89, 6491–6497. [CrossRef] [PubMed] 65. A, J.; Trygg, J.; Gullberg, J.; Johansson, A.I.; Jonsson, P.; Antti, H.; Marklund, S.L.; Moritz, T. Extraction and
GC/MS analysis of the human blood plasma metabolome. Anal. Chem. 2005, 77, 8086–8094. [CrossRef]
[PubMed] 66. Jonsson, P.; Johansson, A.I.; Gullberg, J.; Trygg, J.; A, J.; Grung, B.; Marklund, S.; Sjöström, M.; Antti, H.;
Moritz, T. High-Throughput Data Analysis for Detecting and Identifying Differences between Samples in
GC/MS-Based Metabolomic Analyses. Anal. Chem. 2005, 77, 5635–5642. [CrossRef] [PubMed] 20 of 20 20 of 20 Biomolecules 2019, 9, 553 67. Trygg, J.; Wold, S. PLS regressionon waveletcompressed NIR spectra. Chemom. Intell. Lab. Syst. 1998, 42,
209–220. [CrossRef] 68. Wold, S.; Trygg, J.; Berglund, A.; Antti, H. Some recent developments in PLS modeling. Chemom. Intell. Lab. Syst. 2001, 58, 131–150. [CrossRef] 69. Wold, S. © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/). References Pattern-Recognition by Means of Disjoint Principal Component Analysis. Pattern Recognit. 1976, 8,
127–139. [CrossRef] 70. Redestig, H.; Fukushima, A.; Stenlund, H.; Moritz, T.; Arita, M.; Saito, K.; Kusano, M. Compensation for
Systematic Cross-Contribution Improves Normalization of Mass Spectrometry Based Metabolomics Data. Anal. Chem. 2009, 81, 7974–7980. [CrossRef] 71. Wold, S.; Esbensen, K.; Geladi, P. Principal Component Analysis. Chemom. Intell. Lab. Syst. 1987, 2, 37–52. [CrossRef] 72. Trygg, J.; Wold, S. Orthogonal projections to latent structures (O-PLS). J. Chemom. 2002, 16, 119–128. [CrossRef] 73. Bylesjo, M.; Rantalainen, M.; Cloarec, O.; Nicholson, J.K.; Holmes, E.; Trygg, J. OPLS discriminant analysis:
Combiningthe strengths of PLS-DA and SIMCA classification. J. Chemom. 2006, 20, 341–351. [CrossRef] 74. Wiklund, S.; Johansson, E.; Sjöström, L.; Mellerowicz, E.J.; Edlund, U.; Shockcor, J.P.; Gottfries, J.; Moritz, T.;
Trygg, J. Visualization of GC/TOF-MS-Based Metabolomics Data for Identification of Biochemically Interesting
Compounds Using OPLS Class Models. Anal. Chem. 2008, 80, 115–122. [CrossRef] [PubMed] 75. Efron, B.; Gong, G. A Leisurely Look at the Bootstrap, the Jackknife, and Cross-Validation. Am. Stat. 1983,
37, 36–48. © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Novel Photocatalytic Reactor Development for Removal of Hydrocarbons from Water
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International journal of photoenergy
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Novel photocatalytic reactor development for removal of hydrocarbons
from water Adams, M., Campbell, I., & Robertson, P. K. J. (2008). Novel photocatalytic reactor development for removal of
hydrocarbons from water. International Journal of Photoenergy, 2008, Article 674537. https://doi.org/10.1155/2008/674537 Queen's University Belfast - Research Portal:
Link to publication record in Queen's University Belfast Research Portal Queen s University Belfast - Research Portal:
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estricted use, distribution, and reproduction in any medium, provided the original work is properly cited. py g
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INTRODUCTION bit, but also prevents blowouts. On the platform, the cuttings
are separated from the fluid with the fluid being reinjected. Most of the cuttings will also, at some point, come into
contact with hydrocarbons which are difficult to remove in
an environmentally friendly manner. In the past, almost all of
the drill cuttings from the North Sea were dumped overboard
onto the seabed. Hydrocarbons can contaminate the aqueous environment
through several routes, for example, as by-products of the
oil and gas industry such as drill cuttings, or as surface run
offfrom petrol stations and garages. Pollution resulting from
hydrocarbons [1, 2] contaminating the marine environment
must be addressed due to the potential toxic effects associated
with these compounds can cause considerable harm to a
range of targets within the environment [3–5]. Produced
water presents a significant environmental problem to the oil
industry internationally. In one year over 8500 tonnes of oil
was discharged from oil and gas installations to the North
Sea from produced water discharges [6]. This is a particular
problem now as the offshore industry is moving towards zero
discharges from platforms. Since the detrimental environmental impacts [8] of
these cuttings were established, the government legislation
has reduced the amount of drill cuttings permitted to be
discharged into the sea and is moving towards banning the
practice altogether [9, 10]. Consequently, the oil and gas
industries have investigated alternative methods for dispos-
ing of drill cuttings. Processes that have been investigated
include reinjecting the cuttings back into the well (well
injection) [7] or shipping the cuttings to shore for treatment. The on-shore treatment and disposal options which include
techniques such as thermal desorption, thermal distillation,
solvent extraction, solidification, incineration/combustion,
and landfill [11]. Drill cuttings are one of the by-products of oil explo-
ration and recovery, with around 8000 m3 of diesel and low
toxicity oil contaminated drill cuttings deposited around the
base of platforms in the North Sea [7]. Drill cuttings consist
of small pieces of rock which are generated when drilling
a well, which vary in size from gravel to fine silt. These
cuttings are carried from down hole to the oil platform by
drilling fluid which not only lubricates and cools the drill With thermal desorption [12], the drill cuttings are
treated by heating the materials, which results in the vapor-
isation of water and hydrocarbons. Open Access
Thi
h Open Access
This research has been made openly available by Queen's academics and its Open Research team. We would love t
this research benefits you. – Share your feedback with us: http://go.qub.ac.uk/oa-feedback Download date:24. Oct. 2024 Hindawi Publishing Corporation
International Journal of Photoenergy
Volume 2008, Article ID 674537, 7 pages
doi:10.1155/2008/674537 Hindawi Publishing Corporation
International Journal of Photoenergy
Volume 2008, Article ID 674537, 7 pages
doi:10.1155/2008/674537 Centre for Research in Energy and the Environment, School of Engineering, The Robert Gordon University, Aberdeen AB10 1FR, UK Centre for Research in Energy and the Environment, School of Engineering, The Robert Gordon University, Aberdeen AB10 1FR, UK Correspondence should be addressed to Peter K. J. Robertson, peter.robertson@rgu.ac.uk Received 22 July 2007; Accepted 19 August 2008 Recommended by Russell Howe Hydrocarbons contamination of the marine environment generated by the offshore oil and gas industry is generated from a
number of sources including oil contaminated drill cuttings and produced waters. The removal of hydrocarbons from both
these sources is one of the most significant challenges facing this sector as it moves towards zero emissions. The application of
a number of techniques which have been used to successfully destroy hydrocarbons in produced water and waste water effluents
has previously been reported. This paper reports the application of semiconductor photocatalysis as a final polishing step for the
removal of hydrocarbons from two waste effluent sources. Two reactor concepts were considered: a simple flat plate immobilised
film unit, and a new rotating drum photocatalytic reactor. Both units proved to be effective in removing residual hydrocarbons
from the effluent with the drum reactor reducing the hydrocarbon content by 90% under 10 minutes. Copyright © 2008 Morgan Adams et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited. 2.1.
Multiplate thin film reactor design There are several important parameters in the reactor
design; one of the most significant of which is the active
photocatalyst coating and the underlying substrate material. In addition, the coating preparation and the surface area
of the catalyst available to the pollutant molecules are also
crucial considerations. Two substrate materials were studied
in this investigation: polymethylmethacrylate (PMMA) and
titanium metal. Hydrocarbon contamination of water can also occur at
the distribution stage as well as extraction. A particular
example of such contamination results from “surface runoff”
from Garage Forecourts. This effluent frequently contains
petrol, diesel, oils, brake fluids, and also dust from brake
parts and exhaust particles. The effluent is collected from
drainage tanks for specialised treatment and disposal. Typ-
ically, the waste water effluent is passed through filtration
systems but cannot fully remove volatile organic compounds
(VOCs), and hence secondary treatment is often required. It is well known that certain forms of PMMA are
transparent to ultraviolet radiation making the material
ideally suited for an optical type chemical reactor where
ultraviolet light is used to activate the photocatalyst. The
other material under investigation is titanium. Although
expensive, there is an important property in that when the
titanium is oxidised, titanium dioxide is produced which is
the photocatalyst being used in the reactor. There may also
be advantages in that; there is likely to be good adhesion
between the titanium and titanium dioxide. Semiconductor photocatalysis is a rapidly developing
process which may have a significant impact on the reduction
and removal of these harmful and toxic compounds from
produced water and waste water effluents and has even been
used for potable water treatment [13–17]. This technology
should be therefore a highly feasible process for the treatment
of both produced waters and waste water runofffrom garage
forecourts. Although the use of the technology for removal of
a vast range of compounds from water has been previously
reported, one of the main challenges to date has been the
up-scaling of the process to a size where it can practically
treat large volumes of water. Many processes reported are
usually treating litres per hour or even tens of litres per
hour. Most practical effluent treatment processes require at
least 5–10 m3hr−1, and some offshore oil and gas platforms
generate up to 105 m3hr−1. A review by Alfano et al. 1.
INTRODUCTION This vapour is separated
and subsequently recondensed giving an oil/water liquid and International Journal of Photoenergy 2 TiO2 coated
PMMA plate
Effluent
recirculation
reservoir
500 W UV
lamp
Effluent
delivery
reservoir
Effluent
pump
Figure 1: Prototype flat plate reactor treatment system under a UV source. TiO2 coated
PMMA plate Effluent
recirculation
reservoir Figure 1: Prototype flat plate reactor treatment system under a UV source. 2.
MATERIALS AND METHODS clean drill cuttings. The drill cuttings are then bagged and
used for landfill whilst oil and water are separated. The
recovered oil is burned in oil-fired power stations whilst
the water is filtered to remove any residual hydrocarbons
before being discharged into the sea. This water may still have
residual hydrocarbon content. 2.2.
Drum reactor design The drum reactor was designed to be a single pass continuous
flow system for produced water/effluents. If after one pass the
water was still above the discharge level for hydrocarbons, the
water was allowed to run into a lower reservoir. Typically,
the residence time in each drum was just over 3 minutes,
with a total treatment time after passing through three drum
modules being around 10 minutes. If at this stage the sample
was still contaminated, it was then recirculated. Normalised fluorescence The addition of hydrogen peroxide to the photocatalytic
system has been previously reported to enhance photocat-
alytic degradation rates through the generation of additional
OH radicals via the conductance band reaction with the
peroxide molecule [20, 21]. This was also found to be the
case for the degradation of hydrocarbons in both our systems
so was introduced into the final reactor setup. The hydrogen
peroxide concentration was 0.5% v/v total concentration in
the effluent. This recirculation process was continued until
the hydrocarbons had been removed. Figure 3: Demonstration of the decrease in both absorbance and
fluorescence activity of produced water with reaction time. The TiO2 utilised in the reactor was a Hombikat C mate-
rial supplied by Sachtleben Chemie, Duisburg, Germany. The
reactor drums were irradiated using 36 W Philips PL-L sun-
lamp UV tubes supplied by RS Components Ltd, Northants,
UK. Figures 4(a) and 4(b) show the patented photocatalytic
drum reactor configuration [22]. Sampling was achieved via
the open air vents at the fluid inlet side of the reactor drum. from a thermal desorption plant used to treat drill cuttings. The water sample typically contained 100–200 ppm hydro-
carbons. This is significantly higher than the permissible
discharge consent level for the UK controlled waters which
is currently 30 mg/L [19]. The waste water sample was taken from an interceptor
waste water collecting effluent from a Garage Forecourt. This
sample contained a mixture of hydrocarbons at a total COD
level of between 3500 and 4000 ppm. We have previously reported the use of fluorescence
spectroscopy for in situ monitoring of hydrocarbons in the
marine environment. As part of this study, the applicability
of absorption spectroscopy as an alternative technique to
fluorescence spectroscopy was assessed. Figure 3 shows the
results obtained for the analysis of a produced water sample
using absorption and fluorescence spectroscopy, when the
sample was treated using a flat plate reactor. 2.2.
Drum reactor design As can be seen
from the figure for this study, a good correlation between
the two techniques was obtained and hence absorption
spectroscopy was used for monitoring the hydrocarbon
reduction for this investigation. The analysis was performed
using a Novaspec II absorption spectrometer monitoring the
decrease of the broadband peaking at 335 nm. The destruction of the hydrocarbons was monitored by
both measuring the chemical oxygen demand of the sample
and also by gas chromatography/mass spectrometry (GCMS)
using a Hewlett Packard model 5890 series II GC connected
to a Hewlett Packard model 5971A mass selective detector. 2.1.
Multiplate thin film reactor design (a) (b) (b) (a) Figure 2: Multiple thin film plate reactor stack (a) lab-based unit, (b) concept design for scaled-up unit. 135
120
105
90
75
60
45
30
15
0
Reaction time (mins)
0
0.2
0.4
0.6
0.8
1
1.2
Normalised fluorescence
Absorption
Normalised
fluorescence
0
0.05
0.1
0.15
0.2
0.25
Absorbance
Figure 3: Demonstration of the decrease in both absorbance and
fluorescence activity of produced water with reaction time. 135
120
105
90
75
60
45
30
15
0
Reaction time (mins)
0
0.2
0.4
0.6
0.8
1
1.2
Normalised fluorescence
Absorption
Normalised
fluorescence
0
0.05
0.1
0.15
0.2
0.25
Absorbance
Figure 3: Demonstration of the decrease in both absorbance and
fl
i i
f
d
d
i h
i
i 2.1.
Multiplate thin film reactor design [18]
provides an excellent overview of many of the practical
processes that have been developed for photocatalytic water
treatment, particularly those utilising solar energy. In this
paper, we report the development of both flat bed and drum
reactor designs for the treatment of two real contaminated
water samples, that is, a pretreated produced water and
a waste water sample from a garage forecourt. The basic
concept of these processes has been assessed and described
herein. The initial system developed for treating contaminated
water was based on a thin film photocatalytic reactor. Figure 1 shows the prototype design of the photocatalytic
reactor where the UV source was mounted on a support
frame above the coated plate. The plate was placed on a water
tight channel which had an effluent delivery tank at the head
and an effluent collection reservoir at the base. The plate was
mounted at an angle inducing effluent flow when introduced
at the top of the unit. The thin film plates of either PMMA or titanium
were coated with TiO2 in a 50 mL methanol suspension,
containing between 200 and 250 mg of photocatalyst. This
was achieved by stirring the solution for 10 minutes to obtain
an evenly distributed mixture; this was then applied to the
PMMA or titanium plates. The plate to be coated was placed
in a shallow vessel with the TiO2/methanol solution applied
centrally, and the vessel gently tipped from side to side to
produce an even coating. In this type of unit, the plates and plate reservoirs ulti-
mately could be jointed creating a “concertina” multiple plate
reactor module for large-scale water treatment (see Figure 2). The contaminated water sample used to assess the
efficiency of this reactor was a sample of effluent taken In this type of unit, the plates and plate reservoirs ulti-
mately could be jointed creating a “concertina” multiple plate
reactor module for large-scale water treatment (see Figure 2). In this paper, we describe a method of substituting the
water filtration system, as an addition to the current system,
to achieve very low levels of hydrocarbons in water. The contaminated water sample used to assess the
efficiency of this reactor was a sample of effluent taken 3 3 Morgan Adams et al. (a)
(b)
Figure 2: Multiple thin film plate reactor stack (a) lab-based unit, (b) concept design for scaled-up unit. 3.1.
Flat plate reactor 16
14
12
10
8
6
4
2
0
Treatment time (mins)
0
0.2
0.4
0.6
0.8
1
Normalised absorbance
Titanium sub 37 deg
Titanium sub 26 deg
Titanium sub 15 deg
PMMA Sub 37 deg
PMMA Sub 26 deg
PMMA Sub 15 deg
Figure 6: Comparison of substrate material with reactor plate angle
at the optimum TiO2 loading. g
To determine increase in efficiency of the reaction by
the addition of an alternative electron acceptor to oxygen
(air), hydrogen peroxide solution was added at an optimum
initial concentration of 0.5% to the produced water sample. Figure 7 shows the results of bubbling air only through the
produced water, using a combination of air and hydrogen
peroxide and hydrogen peroxide only. It can be seen that a
significant enhancement was obtained with the addition of
the peroxide alone. In addition bubbling air through this
system did not lead to any additional enhancement of the
destruction of the hydrocarbons in the produced water. This
supports similar observations previously reported by our
own group and others [26–31] and indicates that the rate of
aeration of the solution by ambient air is faster than the rate
of oxygen consumption associated with the photocatalytic
destruction of the hydrocarbons. Figure 6: Comparison of substrate material with reactor plate angle
at the optimum TiO2 loading. 3.1.
Flat plate reactor Initial experiments focused on the optimisation of the TiO2
coating on the plate substrate were performed by preparing International Journal of Photoenergy 4 (a)
Plexiglas supports
containing effluent
transfer chambers
Plexiglas XT
tubes with
Hombicat
36 W UV
tubes
(b)
Figure 4: (a) Photocatalytic reactor drum setup with patented paddle design [20] and (b) drum reactor configuration in UV box. Plexiglas supports
containing effluent
transfer chambers Plexiglas supports
containing effluent
transfer chambers
Plexiglas XT
tubes with
Hombicat
36 W UV
tubes
(b) (a) Plexiglas XT
tubes with
Hombicat (b) (a) Figure 4: (a) Photocatalytic reactor drum setup with patented paddle design [20] and (b) drum reactor configuration in UV box. 140
120
100
80
60
40
20
0
Reaction time (mins)
0
0.2
0.4
0.6
0.8
1
1.2
Normalised absorbance
Room temp drying-100 mg/50 ml
High temp drying-100 mg/50 ml
Room temp drying-200 mg/50 ml
Room temp drying-250 mg/50 ml
Figure 5: Comparison of TiO2 loading and heat treatment. 140
120
100
80
60
40
20
0
Reaction time (mins)
0
0.2
0.4
0.6
0.8
1
1.2
Normalised absorbance different coatings using the slurry method described above. It can be seen from Figure 5 that the most effective coatings
were achieved using suspensions of between 200 and 250 mg
of TiO2 (Degussa P25) in 50 mL of pure methanol followed
by air drying at room temperature. Experiments were also
carried out using elevated temperatures for evaporating off
the methanol. As can be seen from Figure 5, the effect of
catalyst loading on the system was only marginal, which
would be expected in this type of unit where mass transfer
kinetics would be expected to predominate [23–25]. After establishing loading parameters, the reaction rate
was studied as a function of plate angle and substrate mate-
rial. It can be seen from the plot that the PMMA substrate
plate consistently outperformed the titanium substrate when
both are coated with the optimal TiO2 loading at a sub 15◦
angle. The primary reason that the shallower plate angle had
greater destructive efficiency is most likely due to a greater
contact time of the effluent on the catalyst plate due to
the slower flow rates and hence longer residence time (see
Figure 6). Figure 5: Comparison of TiO2 loading and heat treatment. 3.2.
Drum reactor 135
120
105
90
75
60
45
30
15
0
Reaction time (mins)
0
0.2
0.4
0.6
0.8
1
1.2
Normalised absorbance 27
26
25
24
23
22
21
20
19
18
17
16
TIC: MOG1PW0.D
0
4
8
12
16
20
24
×104
Abundance
(a)
27
26
25
24
23
22
21
20
19
18
17
16
TIC: MOG1PW1.D
0
4
8
12
16
20
24
×104
Abundance
(b) Reaction time (mins) Figure 7: Effect of bubbling air and adding hydrogen peroxide on
the destruction rate of produced water with the plate reactor. Table 1: Mean COD values for pretreatment, 1st pass and 2nd pass
through the drum reactor. Sample pass
Mean COD mg/L
0 pass pretreatment
3618
1st pass through drum reactor
2166
2nd pass through drum reactor
868 Table 1: Mean COD values for pretreatment, 1st pass and 2nd pass
through the drum reactor. (b)
27
26
25
24
23
22
21
20
19
18
17
16
TIC: MOG1PW2.D
0
4
8
12
16
20
24
×104
Abundance
(c) TIC: MOG1PW2.D 27
26
25
24
23
22
21
20
19
18
17
16
TIC: MOG1PW2.D
0
4
8
12
16
20
24
×104
Abundance
(c)
27
26
25
24
23
22
21
20
19
18
17
16
TIC: MOG1PW3.D
0
4
8
12
16
20
24
×104
Abundance
(d) could affect the photocatalytic reaction by providing a higher
surface area of catalyst within the reactor drum. This would
also cause secondary problems for the setup of the reactor
as the excess particulates could block the effluent transfer
pathways. TIC: MOG1PW3.D Figure 8 shows the GC/MS results which clearly show a
90% overall destruction over 10 minutes of VOCs present
in the waste water effluent treated through a total of 600 g
of the TiO2 catalyst. This was achieved by passing the waste
water effluent through three consecutive reactor drums each
containing 200 g loads of photocatalyst. It can be seen that
with 10 minutes and after passing through the third drum,
the level of hydrocarbons in the water sample had virtually
disappeared. pp
The chemical oxygen demand (COD) of the water
samples was also measured as an indicator of the total
hydrocarbon level in the untreated and treated samples. Table 1 shows the mean COD values obtained from 5
experimental runs with each point being an average of 3
samples. 3.2.
Drum reactor It can be seen that the COD value decreases very
quickly during the time it takes to process through the 3
consecutive reaction drums (10-minute reaction time). As
these experiments were performed on different days and
the waste water effluent was decanted from a large storage
drum, it is possible that the effluent content was not 100%
consistent. (d) Figure 8: GC/MS chromatogram of waste water effluent, (a) pure
sample with no treatment, (b) 1st pass through drum reactor, (c)
2nd pass through drum reactor, and (d) 3rd pass through drum
reactor (accumulative total) catalyst treatments over a 10-minute
irradiation. Figure 8: GC/MS chromatogram of waste water effluent, (a) pure
sample with no treatment, (b) 1st pass through drum reactor, (c)
2nd pass through drum reactor, and (d) 3rd pass through drum
reactor (accumulative total) catalyst treatments over a 10-minute
irradiation. 3.2.
Drum reactor was initially washed with distilled water to remove excess
particulates TiO2 from the surface of the pellets which was initially washed with distilled water to remove excess
particulates TiO2 from the surface of the pellets which Initial experiments using the drum reactor were configured
for continuous flow effluent treatment. The photocatalyst 5 Morgan Adams et al. 5
27
26
25
24
23
22
21
20
19
18
17
16
TIC: MOG1PW0.D
0
4
8
12
16
20
24
×104
Abundance
(a)
27
26
25
24
23
22
21
20
19
18
17
16
TIC: MOG1PW1.D
0
4
8
12
16
20
24
×104
Abundance
(b)
27
26
25
24
23
22
21
20
19
18
17
16
TIC: MOG1PW2.D
0
4
8
12
16
20
24
×104
Abundance
(c)
27
26
25
24
23
22
21
20
19
18
17
16
TIC: MOG1PW3.D
0
4
8
12
16
20
24
×104
Abundance
(d)
Figure 8: GC/MS chromatogram of waste water effluent, (a) pure
sample with no treatment, (b) 1st pass through drum reactor, (c)
2nd pass through drum reactor, and (d) 3rd pass through drum
reactor (accumulative total) catalyst treatments over a 10-minute
irradiation. 4. CONCLUSION
The results of this study have demonstrated that both reactor 27
26
25
24
23
22
21
20
19
18
17
16
TIC: MOG1PW0.D
0
4
8
12
16
20
24
×104
Abundance
(a)
27
26
25
24
23
22
21
20
19
18
17
16
TIC: MOG1PW1.D
0
4
8
12
16
20
24
×104
Abundance
(b)
27
26
25
24
23
22
21
20
19
18
17
16
TIC: MOG1PW2.D
0
4
8
12
16
20
24
×104
Abundance
(c)
TIC: MOG1PW3.D
×104 135
120
105
90
75
60
45
30
15
0
Reaction time (mins)
0
0.2
0.4
0.6
0.8
1
1.2
Normalised absorbance
PMMA + air
PMMA + H2O2
PMMA + air + H2O2
Figure 7: Effect of bubbling air and adding hydrogen peroxide on
the destruction rate of produced water with the plate reactor. 135
120
105
90
75
60
45
30
15
0
Reaction time (mins)
0
0.2
0.4
0.6
0.8
1
1.2
Normalised absorbance
PMMA + air
PMMA + H2O2
PMMA + air + H2O2
Figure 7: Effect of bubbling air and adding hydrogen peroxide on
the destruction rate of produced water with the plate reactor. 4.
CONCLUSION The results of this study have demonstrated that both reactor
designs proved effective for the removal of hydrocarbon International Journal of Photoenergy 6 contamination from waster water effluents. With the thin
film plate reactor, the assessment of PMMA and titanium
metal plates as substrate materials was investigated under
different conditions. By varying the mounting angle of the
plates, the PMMA plate mounted at 15 degrees produced
60% destruction after 15 minutes. This demonstrated that
the lower plate angle increases the retention time of the
pollutant and therefore the chance of a successful catalyst–
pollutant interface. An investigation into the effect of adding
air and H2O2 to the system showed that the addition of air
alone to the reactor produced 40% degradation, compared
to the 80% degradation of H2O2, over 135 minutes. [2] K. Hylland, K.-E. Tollefsen, A. Ruus, et al., “Water column
monitoring near oil installations in the North Sea 2001–2004,”
Marine Pollution Bulletin, vol. 56, no. 3, pp. 414–429, 2008. pp
[3] J. Zha, Z. Wang, and D. Schlenk, “Effects of pentachlorophenol
on the reproduction of Japanese medaka (Oryzias latipes),”
Chemico-Biological Interactions, vol. 161, no. 1, pp. 26–36,
2006. [4] A. Brouwer, U. G. Ahlborg, M. Van den Berg, et al., “Func-
tional aspects of developmental toxicity of polyhalogenated
aromatic hydrocarbons in experimental animals and human
infants,” European Journal of Pharmacology, vol. 293, no. 1, pp. 1–40, 1995. [5] C. L. Waldner, C. S. Ribble, E. D. Janzen, and J. R. Campbell,
“Associations between oil- and gas-well sites, processing facili-
ties, flaring, and beef cattle reproduction and calf mortality in
western Canada,” Preventive Veterinary Medicine, vol. 50, no. 1-2, pp. 1–17, 2001. With the development of the pelletised TiO2, it was
possible to develop an alternative reactor configuration with
a smaller foot print. Conventional powder catalyst systems
have traditionally posed removal problems with filtration,
and settling is required to remove powder from the effluent. This limits the type of reactor design to batch, as it is
impractical to provide online filtration for a continuous flow
reactor system. [6] OSPAR, “Discharges, Waste Handling and Air Emissions from
Offshore Installations for 1998-1999,” OSPAR Commission
Report , 2001. [7] OSPAR, “Environmental aspects of on and off-site injection
of drill cuttings and produced water,” OSPAR Commission
Report, 2001. 4.
CONCLUSION y
The drum reactor reported in this study was configured
for continuous flow through 3 reactor tubes (see Figure 4(b))
each containing the same quantity of catalyst; GC/MS results
showed the effective 90% removal of VOCs over 5 minutes. The mechanical mixing action of the paddle array within
the reactor tubes greatly increases the pollutant-catalyst
interface, enhances mass transport, and also removes the
need for additional air to be added to the system. The
patented paddle array also maintains an even spread of
the catalyst pellet within the drum which would normally
suffer from “corkscrew” effect of the turning drum. Using a
standard indicator for organic compounds in water, chemical
oxygen demand, the drum reactor showed an 85% reduction
of organic content. [8] M. Laake, T. Bakke, and Schaanning, “Joint Study of Environ-
mental Impact of Aquamul Cuttings and Test on Degradation
and Environmental Effects of Thermal Treated Oily Cuttings
under Natural Conditions,” Report 2744, Norwegian Institute
for Water Research, Oslo, Norway, 1992. [9] U. Government, “Offshore Petroleum Activities (Oil Pollution
Prevention and Control) Regulations,” 2005. [10] U. Government, “The Offshore Chemicals Regulations,”
Statutory Instrument (1355), 2002. y
[11] “Technical Report—Offshore Drilling Waste Management
Review,” Canadian Association of Petroleum Producers 2001-
0007, 2001. [12] I. A. o. O. a. G. Producers, “E&P Forum Joint Study, Summary
Report,” Report 2.61/202, International Association of Oil and
Gas Producers, London, UK, 1996. It should finally be noted that for both reactors assessed
in this paper, the processes had been developed as “polishing”
units and a complementary technology to existing tech-
niques. The technique would not be viable for more heavily
contaminated water samples as the kinetics of the process
would require very significant reaction times and the photo-
catalytic process cannot compete with existing technologies,
where semiconductor photocatalysis has demonstrated a
particular effectiveness in such a final polishing step for
removal of more resilient compounds that traditional waste
water technologies are not capable of removing. [13] A. Rachel, B. Lav´edrine, J.-P. Aguer, and P. Boule, “Photo-
chemical study of 4,4′-dinitrostilbene-2,2′-disulfonate (DSD)
degradation in water,” Journal of Photochemistry and Photobi-
ology A, vol. 151, no. 1–3, pp. 137–143, 2002. gy
pp
[14] S. Malato, “New large solar photocatalytic plant: set-up and
preliminary results,” Chemosphere, vol. 47, no. 3, pp. 235–240,
2002. [15] R. Dillert, A. E. Cassano, R. Goslich, and D. Bahnemann,
“Large scale studies in solar catalytic wastewater treatment,”
Catalysis Today, vol. 4.
CONCLUSION 54, no. 2-3, pp. 267–282, 1999. [16] M. Abdullah, G. K. C. Low, and R. W. Matthews, “Effects of
common inorganic anions on rates of photocatalytic oxidation
of organic carbon over illuminated titanium dioxide,” Journal
of Physical Chemistry, vol. 94, no. 17, pp. 6820–6825, 1990. ACKNOWLEDGMENT [17] J. M. C. Robertson, P. K. J. Robertson, and L. A. Lawton, “A
comparison of the effectiveness of TiO2 photocatalysis and
UVA photolysis for the destruction of three pathogenic micro-
organisms,” Journal of Photochemistry and Photobiology A, vol. 175, no. 1, pp. 51–56, 2005. This project was funded under the Scottish Enterprise Proof
of Concept Programme. This project was funded under the Scottish Enterprise Proof
of Concept Programme. REFERENCES [18] O. M. Alfano, D. Bahnemann, A. E. Cassano, R. Dillert, and R. Goslich, “Photocatalysis in water environments using artificial
and solar light,” Catalysis Today, vol. 58, no. 2-3, pp. 199–230,
2000. [1] M. Russell, L. Webster, P. Walsham, et al., “The effects
of oil exploration and production in the Fladen Ground:
composition and concentration of hydrocarbons in sediment
samples collected during 2001 and their comparison with
sediment samples collected in 1989,” Marine Pollution Bulletin,
vol. 50, no. 6, pp. 638–651, 2005. [1] M. Russell, L. Webster, P. Walsham, et al., “The effects
of oil exploration and production in the Fladen Ground:
composition and concentration of hydrocarbons in sediment
samples collected during 2001 and their comparison with
sediment samples collected in 1989,” Marine Pollution Bulletin,
vol. 50, no. 6, pp. 638–651, 2005. [19] https://www.og.berr.gov.uk/environment/opaoppcr.htm. [20] S. Malato, J. Blanco, C. Richter, B. Braun, and M. I. Mal-
donado, “Enhancement of the rate of solar photocatalytic 7 Morgan Adams et al. mineralization of organic pollutants by inorganic oxidizing
species,” Applied Catalysis B, vol. 17, no. 4, pp. 347–356, 1998. [21] T. Hirakawa, K. Yawata, and Y. Nosaka, “Photocatalytic
reactivity for O−
2 . and OH. radical formation in anatase and
rutile TiO2 suspension as the effect of H2O2 addition,” Applied
Catalysis A, vol. 325, no. 1, pp. 105–111, 2007. [22] P. K. J. Robertson, I. Campbell, and D. Russell, “Apparatus
and method for treating a fluid by means of a transparent
container,” World Patent (WO2005033016), 2006. [23] M. d. l. M. Ballari, R. Brandi, O. Alfano, and A. Cassano, “Mass
transfer limitations in photocatalytic reactors employing
titanium dioxide suspensions. I. Concentration profiles in the
bulk,” Chemical Engineering Journal, vol. 136, no. 1, pp. 50–65,
2008. [24] M. d. l. M. Ballari, R. Brandi, O. Alfano, and A. Cassano, “Mass
transfer limitations in photocatalytic reactors employing
titanium dioxide suspensions. II. External and internal particle
constrains for the reaction,” Chemical Engineering Journal, vol. 136, no. 2-3, pp. 242–255, 2008. [25] D. A. Hickman, M. Weidenbach, and D. P. Friedhoff, “A
comparison of a batch recycle reactor and an integral reactor
with fines for scale-up of an industrial trickle bed reactor from
laboratory data,” Chemical Engineering Science, vol. 59, no. 22-
23, pp. 5425–5430, 2004. [26] D. F. Ollis, E. Pelizzetti, and N. Serpone, “Destruction of water
contaminants,” Environmental Science and Technology, vol. 25,
no. 9, pp. 1523–1529, 1991. pp
[27] R. W. REFERENCES Matthews, “Photooxidative degradation of coloured
organics in water using supported catalysts. TiO2 on sand,”
Water Research, vol. 25, no. 10, pp. 1169–1176, 1991. [28] T. Hisanaga, K. Harada, and K. Tanaka, “Photocatalytic
degradation of organochlorine compounds in suspended
TiO2,” Journal of Photochemistry and Photobiology A, vol. 54,
no. 1, pp. 113–118, 1990. [29] V. Auguliaro, E. Davi, L. Palmisano, M. Schiavello, and A. Sclafani, “Influence of hydrogen peroxide on the kinetics
of phenol photodegradation in aqueous titanium dioxide
dispersion,” Applied Catalysis, vol. 65, no. 1, pp. 101–116,
1990. [30] B. J. P. A. Cornish, L. A. Lawton, and P. K. J. Robertson,
“Hydrogen peroxide enhanced photocatalytic oxidation of
microcystin-lR using titanium dioxide,” Applied Catalysis B,
vol. 25, no. 1, pp. 59–67, 2000. [31] I. Liu, L. A. Lawton, B. Cornish, and P. K. J. Robertson, “Mech-
anistic and toxicity studies of the photocatalytic oxidation of
microcystin-LR,” Journal of Photochemistry and Photobiology
A, vol. 148, no. 1–3, pp. 349–354, 2002. REFERENCES Submit your manuscripts at
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Mammalian Epidermis: A Compendium of Lipid Functionality
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Citation for published version (APA):
Vietri Rudan, M., & Watt, F. M. (2022). Mammalian Epidermis: A Compendium of Lipid Functionality. Frontiers in
Physiology, 12, Article 804824. https://doi.org/10.3389/fphys.2021.804824 Citation for published version (APA):
Vietri Rudan, M., & Watt, F. M. (2022). Mammalian Epidermis: A Compendium of Lipid Functionality. Frontiers in
Physiology, 12, Article 804824. https://doi.org/10.3389/fphys.2021.804824 Citing this paper
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again advised to check the publisher's website for any subsequent corrections. Citation for published version (APA):
Vietri Rudan, M., & Watt, F. M. (2022). Mammalian Epidermis: A Compendium of Lipid Functionality. Frontiers in
Physiology, 12, Article 804824. https://doi.org/10.3389/fphys.2021.804824 Mammalian Epidermis: A
Compendium of Lipid Functionality Matteo Vietri Rudan and Fiona M. Watt* Mammalian epidermis is a striking example of the role of lipids in tissue biology. In
this stratified epithelium, highly specialized structures are formed that leverage the
hydrophobic properties of lipids to form an impermeable barrier and protect the humid
internal environment of the body from the dry outside. This is achieved through tightly
regulated lipid synthesis that generates the molecular species unique to the tissue. Beyond their fundamental structural role, lipids are involved in the active protection of
the body from external insults. Lipid species present on the surface of the body possess
antimicrobial activity and directly contribute to shaping the commensal microbiota. Lipids belonging to a variety of classes are also involved in the signaling events that
modulate the immune responses to environmental stress as well as differentiation of the
epidermal keratinocytes themselves. Recently, high-resolution methods are beginning
to provide evidence for the involvement of newly identified specific lipid molecules in
the regulation of epidermal homeostasis. In this review we give an overview of the wide
range of biological functions of mammalian epidermal lipids. Edited by: Edited by:
Pasquale Simeone,
University of Studies G. d’Annunzio
Chieti and Pescara, Italy Reviewed by:
Christoph Reinhardt,
Johannes Gutenberg University
Mainz, Germany
Sandra Iden,
Saarland University, Germany General rights General rights
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the work immediately and investigate your claim. Download date: 24. Oct. 2024 REVIEW
published: 12 January 2022
doi: 10.3389/fphys.2021.804824 REVIEW
published: 12 January 2022
doi: 10.3389/fphys.2021.804824 INTRODUCTION The evolution of an impermeable barrier that could preserve the internal aqueous environment of
the body away from water in ancestral reptiles was one of the most important events that allowed
the colonization of dry land by vertebrates. That barrier, the “cornified” or “horny” epidermis,
leveraged the physical properties of hydrophobic lipid and protein molecules to achieve the
separation between the “wet” inside and the “dry” outside. Since then, over the course of evolution,
the epidermis has acquired numerous additional structures and adaptations. However, the core
physical mechanisms through which it exerts its most fundamental functions as well as its basic
architecture have remained similar in all terrestrial vertebrates (reptiles, birds and mammals). *Correspondence:
Fiona M. Watt
fiona.watt@kcl.ac.uk *Correspondence:
Fiona M. Watt
fiona.watt@kcl.ac.uk Specialty section:
This article was submitted to
Lipid and Fatty Acid Research,
a section of the journal
Frontiers in Physiology
Received: 29 October 2021
Accepted: 13 December 2021
Published: 12 January 2022
Citation:
Vietri Rudan M and Watt FM
(2022) Mammalian Epidermis:
A Compendium of Lipid Functionality. Front. Physiol. 12:804824. Specialty section:
This article was submitted to
Lipid and Fatty Acid Research,
a section of the journal
Frontiers in Physiology In mammals, the epidermis is the outermost part of the skin, overlying the connective tissue,
the dermis. Mammalian epidermis is a stratified epithelium comprising a series of layers of
progressively more differentiated cells, called keratinocytes. At the dermal interface, resting on
a basement membrane, the basal keratinocyte layer contains cycling cells, responsible for the
renewal of the tissue. Basal cells undergo a phase of commitment and then start the process of
differentiation, exiting the cell cycle and beginning to migrate upward toward the body surface. As the cells progress through differentiation, they move through the spinous layer and the
granular layer, all the while accumulating specific lipids and proteins and building functional
ultrastructures. The keratinocytes ultimately lose their nucleus and become flattened “bricks” of
insoluble protein called corneocytes, surrounded by lipid “mortar” to hold them together in the Received: 29 October 2021
Accepted: 13 December 2021
Published: 12 January 2022 Keywords: lipids, epidermis, keratinocytes, ceramides, signaling, fatty acids, lipidomics Granular Layer As keratinocytes progress through differentiation and become
more specialized, their lipid and protein makeup change. Starting
from the upper spinous layer and in the granular layer the
keratinocytes begin forming cross-linked bundles of keratin
fibers and filaggrin in their cytoplasm. Their organelles begin
degenerating and they assemble lamellar bodies (LB), also known
as membrane-coating granules, lamellar granules, or Odland
bodies (Selby, 1957; Odland, 1960; Proksch et al., 2008; Wertz,
2018). LB have been traditionally described as membrane-bound
organelles of ∼200 nm in diameter containing a series of bilayer
membranes 6–7 nm thick that are closely stacked together
(Matoltsy and Parakkal, 1965; Figure 1), although more recent
evidence suggests that they form a tubuloreticular network
derived from the trans-Golgi apparatus (Matoltsy and Parakkal,
1965; Elias et al., 1998; Norlén et al., 2003). Lipids are essential for the fundamental function of the
epidermis since they are a key element in the water-insulating
properties of the cornified layer. The relative abundance of
different lipid species changes across the various layers of the
epidermis, underscoring how the correct establishment of a
functional barrier requires tightly controlled regulation of lipid
production (Lampe et al., 1983). Indeed, dysregulation of the
lipid balance can cause a number of pathological conditions,
notably ichthyoses (Akiyama, 2017; Vahlquist and Törmä,
2020). Beyond their structural role in the formation of the physical
barrier of the epidermis, several studies have unraveled the
participation of lipid molecules in the active protection of
the body from external harmful agents, such as pathogenic
microbes and damaging UV-radiation. Moreover, several studies
have shown how specific lipids may be directly involved
in the regulation of keratinocyte differentiation itself, and
new techniques are now allowing the exploration of this
field of research. Early characterization of the contents of LBs revealed that
they are mostly made up of lipids and are specifically rich
in glycolipids (mostly glucosylceramides), phospholipids and
cholesterol as well as smaller amounts of sterol esters, ceramides,
and fatty acids (Wertz et al., 1984; Grayson et al., 1985). An
especially remarkable lipid species found uniquely in LB is
an acylglucosylceramide comprising a linoleic acid (C18:2ω6)
molecule esterified to the ω-hydroxy group of a very long chained
(30–34 carbons) fatty acid moiety of the glucosylceramide (Wertz
et al., 1984; Bowser et al., 1985). Granular Layer A specific role has been proposed
for this particular lipid in the correct structural assembly of LBs,
whereby its very long ω-hydroxyacid chain is able to span an
entire lipid bilayer while the linoleate tail can insert itself in a
neighboring bilayer, thus acting as a “molecular rivet” holding
together the tightly packed series of membranes found in LBs
(Wertz and Downing, 1982; Wertz, 2018). Importantly, besides
its potential role in structuring the LB’s contents, the linoleic
acid-containing acylglucosylceramide is an essential component
of the LB’s bounding membrane, where the majority of it is found
(Wertz, 2000, 2018). This review covers the multi-faceted roles, from structural
to regulatory, that lipids play in epidermal homeostasis and
protection from the external environment. The epidermis
is a model of the versatility of this class of molecules,
highlighting their important, often understated, and still
potentially uncharacterized activities. We first describe the
central role of lipids in the formation of the epidermal water
permeability barrier. Then we detail the antimicrobial and
immunomodulatory activity of certain epidermal lipids. Finally,
we analyze how epidermal lipids of many different classes can
influence epidermal cell signaling. This review will not, however,
cover lipid vitamins. For details of the extremely important part
vitamin A, vitamin D and their derivatives play in the biology of
the epidermis, the reader is referred to recent reviews (Piotrowska
et al., 2016; Szyma´nski et al., 2020). Besides lipid molecules, LBs contain several hydrolytic
enzymes such as carboxypepdidase, cathepsin B, acid hydrolase,
as
well
as
acid
lipase,
β-glucosidases,
phospholipase
A,
sphingomyelinase,
ceramidases,
and
steroid
sulfatase
that
play an important role during the subsequent differentiation
steps (Freinkel and Traczyk, 1985; Grayson et al., 1985). Cornified Layer (Stratum corneum) Cornified Layer (Stratum corneum)
The boundary between the granular and cornified layers
represents the water-permeability barrier of the body, as
demonstrated by dermal injection of water-soluble tracers (Elias
and Friend, 1975). As the cells move toward and through this
boundary, they become corneocytes, the “bricks” of the stratum
corneum: they form an extremely resistant protein shell, the
cornified cell envelope, just beneath their plasma membrane,
made of crosslinked involucrin, loricrin, small proline-rich
proteins and other proteins. The nucleus starts to degenerate
and ultimately disappears, as the cytoplasm becomes filled
with keratin bundles and the cell becomes flattened. The LBs
approach the apical plasma membrane of the cell, their bounding Long, 1970). Citation: Vietri Rudan M and Watt FM
(2022) Mammalian Epidermis:
A Compendium of Lipid Functionality. Front. Physiol. 12:804824. doi: 10.3389/fphys.2021.804824 January 2022 | Volume 12 | Article 804824 1 Frontiers in Physiology | www.frontiersin.org Lipids Roles in Mammalian Epidermis Vietri Rudan and Watt outermost impermeable cornified layer or stratum corneum
(Nemes and Steinert, 1999; Watt, 2014; Figure 1). THE BODY’S OUTER WALL – THE
FUNDAMENTAL STRUCTURAL ROLE OF
EPIDERMAL LIPIDS The lipid composition varies dramatically along the thickness of
mammalian epidermis (Figure 1). Early dissections of the lipid
makeup of the epidermis of different mammals revealed striking
differences between the basal and spinous layers, the granular
layer and the cornified layer (stratum corneum) (Kooyman, 1932;
Long, 1970). Basal and Spinous Layers In the basal and lower spinous layers, phospholipids are
most common, with high levels of phosphatidylcholines,
phosphatidylethanolamines,
phosphatidylserines
and
sphingomyelins
(Gray
et
al.,
1980). This
phospholipid
predominance likely underlies the main roles that lipids
have
in
these
cells:
membrane
maintenance,
energy
production and signaling. January 2022 | Volume 12 | Article 804824 Frontiers in Physiology | www.frontiersin.org 2 Lipids Roles in Mammalian Epidermis Vietri Rudan and Watt FIGURE 1 | Role of lipids in the formation of the epidermal water permeability barrier. Illustration of the structure of the skin with the main lipid components of the
various layers indicated on the right. The left inset shows the microstructure at the boundary between the granular layer and the stratum corneum. The right inset
displays the possible molecular arrangement of the main stratum corneum lipids in the intercellular lamellae according to the “sandwich model.” Alternative models
have also been proposed. FIGURE 1 | Role of lipids in the formation of the epidermal water permeability barrier. Illustration of the structure of the skin with the main lipid components of the
various layers indicated on the right. The left inset shows the microstructure at the boundary between the granular layer and the stratum corneum. The right inset FIGURE 1 | Role of lipids in the formation of the epidermal water permeability barrier. Illustration of the structure of the skin with the main lipid components of the
various layers indicated on the right. The left inset shows the microstructure at the boundary between the granular layer and the stratum corneum. The right inset
displays the possible molecular arrangement of the main stratum corneum lipids in the intercellular lamellae according to the “sandwich model.” Alternative models
have also been proposed. membrane finally fusing with it, releasing the LB contents
in the extracellular space (Wertz, 1996; Proksch et al., 2008;
Figure 1). Upon extrusion, the lipids contained in the LBs undergo
dramatic changes, likely becoming substrates for the lipid
hydrolases that convert them into ceramides (∼50%), cholesterol
(∼25%), and fatty acids (∼10%), the main constituents of the
intercellular lipid “mortar” of the cornified layer (Figure 2),
which also comprises smaller amounts of cholesterol sulfate and
cholesterol esters (Nicolaides, 1974; Wertz and van den Bergh,
1998). Basal and Spinous Layers Notably, the release of fatty acids from phospholipids
by phospholipase A2 contributes to the creation of an acidic
environment in the stratum corneum, which in turn regulates
the activity of other enzymes, such as β-glucocerebrosidase, acid
sphingomyelinase as well as serine proteases. This is necessary The
linoleate-containing
acylglucosylceramide
in
the
bounding
membrane
is
processed
to
become
ω-hydroxyceramide, which is covalently bound to the cornified
cell envelope’s proteins, notably involucrin, forming an additional
layer around the cell, known as the corneocyte lipid envelope
(Swartzendruber et al., 1987; Wertz and Downing, 1987; Nemes
et al., 1999; Grond et al., 2017). Together, the cornified cell
envelope and the lipid envelope replace the plasma membrane
of the corneocyte. January 2022 | Volume 12 | Article 804824 Frontiers in Physiology | www.frontiersin.org 3 Lipids Roles in Mammalian Epidermis Vietri Rudan and Watt FIGURE 2 | Main lipids of the stratum corneum. Ceramide classes are labeled both according to the classical nomenclature (Cer1-9) when applicable and by the
letter code indicating the sphingoid base-fatty acyl moiety combination. In the example structures of the ceramides, all sphingoid bases and fatty acid moieties are
shown as C18, but can be substituted by other chain lengths according to what is indicated at the top of the figure or described in the main text. Cer[EO] structures
are presented as C26 fatty acids esterified to linoleic acid (as that is what is most commonly found in the epidermis). Both ceramide fatty acyl groups and stratum
corneum free fatty acids are generally saturated or monounsaturated. FIGURE 2 | Main lipids of the stratum corneum. Ceramide classes are labeled both according to the classical nomenclature (Cer1-9) when applicable and by the
letter code indicating the sphingoid base-fatty acyl moiety combination. In the example structures of the ceramides, all sphingoid bases and fatty acid moieties are
shown as C18, but can be substituted by other chain lengths according to what is indicated at the top of the figure or described in the main text. Cer[EO] structures
are presented as C26 fatty acids esterified to linoleic acid (as that is what is most commonly found in the epidermis). Both ceramide fatty acyl groups and stratum
corneum free fatty acids are generally saturated or monounsaturated. to ensure the correct formation of the epidermal barrier and its
homeostatic regulation (Hachem et al., 2003). chains of the acylceramides acting as a zipper (Swartzendruber
et al., 1989). Frontiers in Physiology | www.frontiersin.org Basal and Spinous Layers X-ray diffraction studies of the human cornified
layer’s intercellular lipid matrix instead describe two lamellar
phases, referred to as the long-periodicity phase (LPP, ∼13 nm)
and the short-periodicity phase (SPP, ∼5 nm), with the former
likely to be relevant for the barrier function, having been
identified in multiple animal species (Bouwstra et al., 1991,
1992). The LPP has in fact been equated to one Landmann unit,
in which the ceramides, cholesterol and fatty acids in the outer
bilayers arrange themselves as a dense crystalline orthorhombic
lattice, responsible for hindering the movement of compounds
through the outer epidermis, and form the impermeable barrier
that lines the body, while the intervening monolayer made up
of the ω-esterified unsaturated fatty acid (mostly linoleate) and The
chemical
changes
in
lipids
are
accompanied
by
morphological ones, as the disk-like bilayers seen in the
LBs fuse to become a series of broad lamellar sheets (Elias
et al., 1977; Landmann, 1986; Madison et al., 1987; Figure 1). The corneocyte lipid envelope plays a fundamental role in
the assembly of these lamellae, acting as a template for their
orientation with respect to the corneocytes (Behne et al., 2000). More specifically, lipids arrange themselves into repeating units
that appear as a series of broad-narrow-broad electron-lucent
bands in electron micrographs. Each of these units (sometimes
referred to as Landmann units) are thought to be juxtaposed
lipid bilayers with an intervening monolayer formed by the long January 2022 | Volume 12 | Article 804824 Frontiers in Physiology | www.frontiersin.org Lipids Roles in Mammalian Epidermis Vietri Rudan and Watt cholesterol forms a liquid phase that could provide flexibility and
resistance to shear stress. This is referred to as the “sandwich
model” (Bouwstra et al., 2000, 2002; Figure 1). the epidermis also actively participates in the protection of the
organism against potential threats from opportunistic pathogenic
micro-organisms (Natsuga et al., 2016; Fischer, 2020). Given its
position as the outer boundary of the organism, the epidermal
surface is host to a diverse set of commensal microbes, whose
composition varies at different body sites, depending on certain
physiological characteristics such as degree of moisture or
abundance of sebaceous secretions (Table 1; Grice et al., 2009;
Grice and Segre, 2011). Free Sphingoid Bases One source of antimicrobial lipids comes from the stratum
corneum lipids themselves. Among the enzymes contained in
LBs are ceramidases, which cleave stratum corneum ceramides
into a sphingosine base and a fatty acid. The fatty acids
generated from these ceramides and more generally all stratum
corneum fatty acids synthesized by keratinocytes have very
long, mostly saturated carbon chains and do not possess any
antiseptic activity (Rothman et al., 1946; Kabara et al., 1972,
1977; Zheng et al., 2005; Brogden et al., 2011). However,
multiple studies have demonstrated the antimicrobial potency
of sphingosine, dihydrosphingosine, and 6-hydroxysphingosine, The functional importance of such lipid complexity is
underscored by several studies that attempt to replicate the
ultrastructure of cornified layer lipid lamellae in chemically
defined model membranes (Bouwstra et al., 2002; de Jager et al.,
2003; Opálka et al., 2016; Školová et al., 2017; Schmitt et al.,
2019; Beddoes et al., 2021). It is further revealed by a variety of
ichthyotic skin conditions in which different lipid biosynthetic
pathways are perturbed (Akiyama, 2017; Vahlquist and Törmä,
2020). TABLE 1 | Main members of epidermal surface bacterial communities at different
body sites grouped based on their microenvironmental characteristics. Moist (e.g., armpit)
Sebaceous (e.g., glabella)
Dry (e.g., forearm)
Corynebacterium spp. (28%)
Cutibacterium spp. (46%)
Betaproteobacteria (32%)
Betaproteobacteria (22%)
Staphylococcus spp. (16%)
Corynebacterium spp. (15%)
Staphylococcus spp. (21%)
Corynebacterium spp. (10%)
Flavobacteriales (14%)
Flavobacteriales (9%)
Betaproteobacteria (9%)
Cutibacterium spp. (13%)
Cutibacterium spp. (7%)
Flavobacteriales (3%)
Gammaproteobacteria (7%)
Gammaproteobacteria
Lactobacillales (3%)
Staphylococcus spp. Lactobacillales
Clostridiales
Lactobacillales
Bacteria are in order of abundance, with approximate average percentages in each
microenvironment indicated. Data adapted from Grice et al. (2009). TABLE 1 | Main members of epidermal surface bacterial communities at different
body sites grouped based on their microenvironmental characteristics. Moist (e.g., armpit)
Sebaceous (e.g., glabella)
Dry (e.g., forearm)
Corynebacterium spp. (28%)
Cutibacterium spp. (46%)
Betaproteobacteria (32%)
Betaproteobacteria (22%)
Staphylococcus spp. (16%)
Corynebacterium spp. (15%)
Staphylococcus spp. (21%)
Corynebacterium spp. (10%)
Flavobacteriales (14%)
Flavobacteriales (9%)
Betaproteobacteria (9%)
Cutibacterium spp. (13%)
Cutibacterium spp. (7%)
Flavobacteriales (3%)
Gammaproteobacteria (7%)
Gammaproteobacteria
Lactobacillales (3%)
Staphylococcus spp. Lactobacillales
Clostridiales
Lactobacillales
Bacteria are in order of abundance, with approximate average percentages in each
microenvironment indicated. Data adapted from Grice et al. (2009). Basal and Spinous Layers Lipids produced in the epidermis
can directly target surface-dwelling microbes to shape the
commensal microbiota and quell the growth of potentially
pathogenic species as well as participate in regulation of the
innate and adaptive immune responses that follow after invasion
of foreign pathogens. Once the corneocytes reach the surface of the body, they are
detached from the epidermis by desquamation. This process is
mediated by the action of certain serine proteases that attack
the desmosomes that connect corneocytes together (Suzuki et al.,
1993). Cholesterol sulfate present in the stratum corneum acts as
an inhibitor of these proteases. The concentration of cholesterol
sulfate is highest in the granular layer, and from there it gradually
decreases along the thickness of the cornified layer due to the
action of steroid sulfatase. The lower concentration of cholesterol
sulfate near the surface thus allows the proteases to become active
and enables the eventual desquamation of the corneocytes (Elias
et al., 1984; Sato et al., 1998). Heterogeneity of Stratum corneum
Lipids The surface of the epidermis is a generally inhospitable
environment for microbes owing to its mild acidity (pH 4-5.5),
scarcity of water and poor availability of essential metabolites
like phosphate (Aly et al., 1972, 1975; Forslind et al., 1995). Moreover, during differentiation, keratinocytes produce and
secrete peptides and proteins that possess antimicrobial activity,
such as defensins, cathelicidins (LL-37 in humans) and RNAse 7. Production of these peptides is stimulated upon damage to the
epidermis (Fulton et al., 1997; Dorschner et al., 2001; Falconer
et al., 2001). While the acylceramides, uniquely found in the epidermis, are
crucial for the establishment of the water permeability barrier,
there is considerable heterogeneity in the pool of stratum
corneum ceramides and fatty acids, which is likely to contribute
to the proper architecture of the lipid matrix (Figure 2). Ceramides show a remarkably wide range of diversity due
to heterogeneity in each of their sub-components. One of four
different sphingoid bases – sphingosine, dihydrosphingosine,
phytosphingosine, 6-hydroxysphingosine, usually C18 or C20
in length – can be paired with fatty acids that vary in carbon
chain size (C15 to C34, though most are between C20 to C30),
hydroxylation profile (nonhydroxy, α-hydroxy- or ω-hydroxy-),
and presence of an ω-esterified fatty acid moiety (almost always
linoleic acid) (Lampe et al., 1983; Masukawa et al., 2009; van
Smeden et al., 2011). Based on the different combinations of
these components, epidermal ceramides have been categorized
into several classes (Figure 2). Free fatty acids of the cornified
layer are nearly all saturated but their carbon chain length can
vary between C18 and C28, with the most abundant species being
C22 and C24 (Nicolaides, 1974; Wertz et al., 1987; Wertz and van
den Bergh, 1998). In addition to peptides, one of the main antimicrobial
components of the epidermis resides in its lipid fraction
(Burtenshaw, 1942). Indeed, two major components of lipid-
mediated immunity have been identified: free sphingoid bases in
the stratum corneum and free fatty acids in the sebum (Figure 3). BEYOND THE MORTAR – LIPIDS AS
ACTIVE PROTECTORS OF EPIDERMAL
INTEGRITY Besides its role as an impermeable physical barrier between
the internal environment of the body and the outside world, January 2022 | Volume 12 | Article 804824 Frontiers in Physiology | www.frontiersin.org 5 Lipids Roles in Mammalian Epidermis Vietri Rudan and Watt FIGURE 3 | Main antimicrobial lipids of the epidermis. Rough spectra of activity are indicated on the right with a few examples. The lists of target micro-organisms
are not exhaustive and efficacy can vary among the different stratum corneum sphingoid bases or the various sebaceous fatty acids. FIGURE 3 | Main antimicrobial lipids of the epidermis. Rough spectra of activity are indicated on the right with a few examples. The lists of target micro-organisms
are not exhaustive and efficacy can vary among the different stratum corneum sphingoid bases or the various sebaceous fatty acids. FIGURE 3 | Main antimicrobial lipids of the epidermis. Rough spectra of activity are indicated on the right with a few examples. The lists of target micro-organisms
are not exhaustive and efficacy can vary among the different stratum corneum sphingoid bases or the various sebaceous fatty acids. which are found in the outer epidermis (Figure 3; Bibel
et al., 1992). Phytospingosine also displays similar properties
(Pavicic et al., 2007), but it is not found in free form in
human stratum corneum (Wertz and Downing, 1990). The
spectrum of antimicrobial activity of sphingosines is quite broad,
including activity against numerous Gram-positive bacteria such
as Cutibacterium acnes, Staphylococcus aureus, and Streptococcus
pyogenes; some Gram-negative bacteria are also affected, such as
Escherichia coli and Porphyromonas gingivalis; finally, fungi such
as Candida albicans can also be targeted by these compounds
(Figure 3; Bibel et al., 1992, 1993; Fischer et al., 2012b). The
varying degrees of efficacy exhibited by sphingoid bases against
different micro-organisms point to a certain level of specificity in
their mode of action. In addition to their direct effect on bacterial
growth and survival, sphingosines have proven very effective at
interfering with bacterial biofilm formation (Seitz et al., 2019;
Beck et al., 2020). in which the cell wall is not synthesized, are more resistant
to sphingoid base treatment. Interestingly, the cell wall of the
Gram-negative E. Coli is not affected, implying that the effect
on cell wall biosynthesis is likely a secondary consequence of the
treatment. BEYOND THE MORTAR – LIPIDS AS
ACTIVE PROTECTORS OF EPIDERMAL
INTEGRITY Given that the sphingoid bases become incorporated
into the bacteria, one possibility is that they may insert themselves
into the bacterial envelope/plasma membranes and render these
structures non-functional. Alternatively, they may enter the
cytoplasm and accumulate intracellularly where they might
interfere with cellular metabolism (Bibel et al., 1993; Fischer
et al., 2012b). This latter possibility is supported by the fact that
sphingosines can participate in cellular signaling by inhibiting
protein kinase C (PKC) in mammalian cells (Hannun et al., 1986). PKC is an important hub of cellular signaling; the different PKC
isoforms vary in their regulation and can play numerous roles in
influencing cellular behavior. Some PKC isoforms can be directly
regulated by lipids such as diacylglycerol, as described below. The mechanisms through which sphingoid bases enact their
antimicrobial activity have not been fully elucidated. Treatment
of S. aureus and E. Coli with sphingosine or dihydrosphingosine
causes shrinking and distortion of the cells with alterations
of cell membranes and emergence of inclusion bodies. In the
Gram-positive S. aureus, the cell wall is lost and L-form strains, Frontiers in Physiology | www.frontiersin.org Sebaceous Free Fatty Acids been implicated in helping to maintain the integrity of the
epidermal barrier, in thermoregulation, photoprotection, and
helping to deliver vitamin E to the skin (Zouboulis, 2004). Alterations in sebaceous secretions can lead to imbalances in
the skin microbiome and are linked to pathological conditions. More specifically, the mutual influence between sebum and the
commensal microbiota is exemplified by the fact that sebum-
rich areas of the body host a specific subset of microbial species
(Table 1), by the direct influence of bacterial lipases on sebum
composition, and by the changes in sebum composition observed
in acne patients (Scheimann et al., 1960; Lovászi et al., 2017;
Dréno et al., 2020; Oulès et al., 2020). Sebum composition is species-specific. The main components
in humans are triglycerides (∼45%), wax esters (∼25%), squalene
(∼12%), and fatty acids (∼10%). In contrast, mouse sebum is
mostly made up of wax esters (∼70%), with a much smaller
fraction of triglycerides (∼6%) and no fatty acids or squalene
(Wilkinson and Karasek, 1966; Nicolaides et al., 1968; Nikkari,
1974; Pappas, 2009). Among human sebum components, fatty
acids are the ones responsible for the antiseptic action of sebum. As sebum flows through the pilosebaceous duct, fatty acids are
released from sebaceous triglycerides by the action of bacterial
and possibly host lipases (Nicolaides and Wells, 1957; Scheimann
et al., 1960; Marples et al., 1971; Götz et al., 1998; Zouboulis et al.,
1999; Drake et al., 2008). When compared to those found in the
stratum corneum, sebum fatty acids have shorter, in some cases
odd-numbered, carbon chains, a higher degree of unsaturation,
and display potent antimicrobial activity (Weitkamp et al., 1947). Among these, enanthic (C7:0), pelargonic (C9:0), undecylic acid
(C11:0), and tridecylic acids (C13:0) extracted from human hair
fat have antifungal activity that is implicated in protection against
ringworm of the scalp (Rothman et al., 1946). Undecylenic acid
(C11:1) is found in sweat and has widespread use as an antifungal
treatment (Landau, 1983). Oleic (C18:1) and linoleic (C18:2)
acid are abundant in sebum and have efficacy as antibacterials
(Weitkamp et al., 1947; Kabara et al., 1972). Some of the most
potent sebaceous fatty acids include lauric acid (C12:0), present
in relatively minor amounts, and sapienic acid (C16:1ω10), thus
named because it is the most abundant fatty acid found in Homo
sapiens sebum (Figure 3; Weitkamp et al., 1947; Downing and
Strauss, 1974; Stewart and Downing, 1991). Sebaceous Free Fatty Acids Another important source of antimicrobial lipids is sebum,
a liquid mixture of neutral lipids assembled and secreted by
sebaceous glands that coats and lubricates the outer epidermis. The function of sebum is to a degree still debated, but it has January 2022 | Volume 12 | Article 804824 6 Lipids Roles in Mammalian Epidermis Vietri Rudan and Watt The
molecular
basis
of
the
antimicrobial
activity
of
sebaceous fatty acids is not understood completely (Desbois
and Smith, 2010). The amphipathic nature of fatty acids
is necessary for their antibacterial action, as replacing the
-OH group at one end of the molecule with a methyl
group abolishes any effect (Kodicek and Worden, 1945;
Kabara et al., 1972; Zheng et al., 2005). The shape of the
fatty acid molecule appears to affect its activity, with key
determinants being carbon chain length as well as presence,
number, position, and orientation of unsaturations. More
specifically, for saturated species there is a tendency for
antimicrobial potency to be highest at C10–12 and decrease
with either longer or shorter carbon chains (Kabara et al.,
1972; Bergsson et al., 2001; Wille and Kydonieus, 2003). The presence and number of unsaturations tend to increase
efficacy at a given carbon length, with the cis- orientation
(which is most often found in endogenous compounds)
most effective in boosting activity (Kabara et al., 1972;
Galbraith and Miller, 1973; Saito et al., 1984; Knapp and
Melly, 1986). For example, despite its relatively long carbon
chain, the double bond in sapienic acid is an unusual
cis C-6 unsaturation that allows the molecule to adopt
a
conformation
resembling
a
shorter-chained
fatty
acid
(Fischer, 2020). been implicated in helping to maintain the integrity of the
epidermal barrier, in thermoregulation, photoprotection, and
helping to deliver vitamin E to the skin (Zouboulis, 2004). Alterations in sebaceous secretions can lead to imbalances in
the skin microbiome and are linked to pathological conditions. More specifically, the mutual influence between sebum and the
commensal microbiota is exemplified by the fact that sebum-
rich areas of the body host a specific subset of microbial species
(Table 1), by the direct influence of bacterial lipases on sebum
composition, and by the changes in sebum composition observed
in acne patients (Scheimann et al., 1960; Lovászi et al., 2017;
Dréno et al., 2020; Oulès et al., 2020). Sebaceous Free Fatty Acids The principal target of sebum fatty acids seems to be
the bacterial cytoplasmic membrane, where they are thought
to interfere with oxidative phosphorylation-mediated energy
production by way of disruption of the electron transport
chain or dissipation of the membrane potential necessary
for ATP synthesis (Sheu and Freese, 1972; Galbraith and
Miller, 1973; Greenway and Dyke, 1979). One hypothesis
is that the shorter-chained or cis- unsaturated fatty acids
cause membrane fluidification and destabilization of electron
transport proteins (Greenway and Dyke, 1979; Chamberlain
et al., 1991). Other proposals are that they increase membrane
permeability to protons (Borst et al., 1962; Greenway and
Dyke, 1979; Gutknecht, 1988), or inhibit components of
the ATP synthase machinery (Wojtczak and Załuska, 1967). Besides their action at the cell membrane, unsaturated fatty
acids can inhibit cellular enzymes such as those responsible
for nutrient uptake (Galbraith and Miller, 1973), and can
inhibit fatty acid biosynthesis (Zheng et al., 2005; Sado-
Kamdem et al., 2009). They can increase oxidative stress
after undergoing peroxidation (Knapp and Melly, 1986) or
undergo auto-oxidation yielding other antibacterial compounds
(Gutteridge et al., 1974). Finally, fatty acids can cause leakage
of intracellular components and cell lysis (Galbraith and
Miller, 1973; Greenway and Dyke, 1979; Carson and Daneo-
Moore, 1980). Understanding which of these mechanisms
is responsible for the antimicrobial action of fatty acids is
challenging, because some are connected (e.g., the decrease
in energy production and the suppression of nutrient uptake,
or the effect on membrane fluidity and the inhibition of
fatty acid biosynthesis) and because different mechanisms
may involve different fatty acid-microbe pairings and different
environmental conditions. Sebum fatty acids tend to be more active toward Gram-
positive bacteria (e.g., C. acnes, S. aureus) and lack efficacy
against Gram-negative bacteria (e.g., E. coli, P. aeruginosa). This
has been associated with the fact that the lipopolysaccharide-
coated outer membrane of Gram-negative bacteria represents an
effective barrier against penetration of hydrophobic compounds
(Nikaido, 1976; Greenway and Dyke, 1979). Sebaceous fatty acid
species can also be effective against certain viruses (Figure 3). Interestingly, the antimicrobial spectra of lauric and sapienic
acids do not completely overlap (Kabara et al., 1972, 1977;
Bergsson et al., 2001; Wille and Kydonieus, 2003; Thormar and
Hilmarsson, 2007; Nakatsuji et al., 2009; Fischer et al., 2012a;
Huang et al., 2014). As is the case with sphingosines, the variation
in effectiveness of different compound-bacterium pairings may
indicate some specificity in their action. Eicosanoids and Related Lipids Eicosanoids are a heterogeneous group of lipids derived
from the metabolism of C20 poly-unsaturated fatty acids
(PUFAs)
such
as
arachidonic
acid
(C20:4ω6),
dihomo-
γ-linolenic
acid
(C20:3ω6),
and
eicosapentaenoic
acid
(C20:5ω3). These
molecules,
together
with
some
related
PUFAs, can be found in the epidermis and they can become FIGURE 4 | Impact of epidermal bioactive lipids on the immune response. 9-HODE, 9-hydroxyoctadecadienoic acid; 12-HETE,
12 h d
i
t t
i
id 15 HET E 15 h d
i
t i
i
id In addition to their direct antimicrobial activity, fatty acids
contribute to the acidification of the surface of the skin – the so-
called “acid mantle” (Schade and Marchionini, 1928) – which in
turn influences which micro-organisms can successfully colonize
the skin and contributes to epidermal integrity (Puhvel et al.,
1975; Fluhr et al., 2001; Hachem et al., 2003). A fascinating example of the crosstalk between the immune
system and the epidermal “shield” is the ability of epidermis-
resident innate lymphoid cells to limit the growth of sebocytes
and influence the presence of antimicrobial fatty acid in the
sebum. To be retained in the tissue, innate lymphoid cells require
secretion of chemokines and cytokines by the epithelium. If
the lymphoid cells are lost, the epidermis reacts by producing
more sebum and increasing sebaceous antimicrobial lipid
content, in turn regulating the composition of the commensal
microbiota (Kobayashi et al., 2019). In another instance that
underscores the close relationship between the regulation of
lipid production and immune response, overexpression of the
transcription factor GATA6 in cultured sebocytes leads to
alterations in the expression of lipid-modifying enzymes and
reduced accumulation of lipids after stimulation with the PPARγ
agonist troglitazone, while at the same time increasing the
levels of anti-inflammatory mediators such as PD-L1 and IL10
(Oulès et al., 2020). Some
antimicrobial
lipids
have
anti-inflammatory
activity. Sapienic
acid
inhibits
gene
expression
resulting
from lipopolysaccharide stimulation in murine macrophages
(Astudillo et al., 2018). Though a relatively high concentration
of the fatty acid was necessary to see the effect (25 µM), it
is still consistent with an anti-inflammatory action in skin,
given the abundance of sapienic acid in sebum (>100 µM). Phytosphingosine
demonstrates
effective
inhibition
of
interleukin-1α (IL-1α) secretion and PKC activation in skin
explants and artificial human epidermis (Pavicic et al., 2007). Similar effects have been reported on a broader range of cytokines
and chemokines (Klee et al., 2007; Brogden et al., 2012). Sebaceous Free Fatty Acids January 2022 | Volume 12 | Article 804824 Frontiers in Physiology | www.frontiersin.org 7 Lipids Roles in Mammalian Epidermis Vietri Rudan and Watt Lipids as Part of Epidermal Innate and
Adaptive Immunity the physiological equilibrium of the skin or to intervene in the
regulation of epidermal cell behavior (Figure 4). The active role of epidermal lipids in the protection of the
organism should be considered in the wider context of the innate
immune response. The combination of environmental (acidity,
humidity) and biochemical – either protein or lipid – factors of
both host and microbial origin shapes the composition of the skin
microbiota (Grice et al., 2009; Grice and Segre, 2011). All these
components can directly interact with one another; for example,
lipid and proteinaceous antimicrobial factors can have synergistic
action, as in the case of sphingosine and LL-37 or sebaceous fatty
acids and the antimicrobial histone H4 (Lee et al., 2009; Brogden
et al., 2012). Frontiers in Physiology | www.frontiersin.org Eicosanoids and Related Lipids In atopic
dermatitis, a skin condition with an inflammatory component,
levels of both sapienic acid and sphingosines are found to be
downregulated (Arikawa et al., 2002; Takigawa et al., 2005). FIGURE 4 | Impact of epidermal bioactive lipids on the immune response. 9-HODE, 9-hydroxyoctadecadienoic acid; 12-HETE,
12-hydroxyeicosatetraenoic acid; 15-HETrE, 15-hydroxyeicosatrienoic acid;
cysLT, cysteinyl-leukotrienes; PGD2, prostaglandin D2; PGE2, prostaglandin
E2; S1P, sphingosine-1-phosphate; SPC, sphingosylphosphorylcholine. FIGURE 4 | Impact of epidermal bioactive lipids on the immune response. 9-HODE, 9-hydroxyoctadecadienoic acid; 12-HETE,
12-hydroxyeicosatetraenoic acid; 15-HETrE, 15-hydroxyeicosatrienoic acid;
cysLT, cysteinyl-leukotrienes; PGD2, prostaglandin D2; PGE2, prostaglandin
E2; S1P, sphingosine-1-phosphate; SPC, sphingosylphosphorylcholine. Lipids from numerous different classes and cellular origins
can work as paracrine or autocrine mediators through cell-
surface receptors to help determine the type of adaptive immune
response that is triggered after an external stimulus perturbs January 2022 | Volume 12 | Article 804824 8 Lipids Roles in Mammalian Epidermis Vietri Rudan and Watt proposed to lead to cytoskeletal rearrangements and inhibition
of cellular movement (Angeli et al., 2001, 2004). substrates of a set of different enzymes. Cyclooxygenases
can generate prostanoids (prostaglandins, prostacyclins, and
thromboxanes). Lipoxygenases can give rise among others
to leukotrienes, a variety of hydroxy-fatty acids [namely
hydroxyeicosatrienoic acids (HETrEs), hydroxyeicosatetraenoic
acids (HETEs), and hydroxyoctadecadienoic acids (HODEs)],
and resolvins. In addition, cytochrome P450 enzymes lead to
the production of such molecules as dihydro-eicosatetraenoic
acids (DHETs) and epoxyeicosatetraenoic acid (EET). All
these lipid species are known autocrine or paracrine biological
mediators, with some of them being active in the epidermis
(Kendall and Nicolaou, 2013). While there is some evidence that resident epidermal cells
express 5-lipoxygenase (5-LOX) – one of the key enzymes for
the generation of leukotrienes – the major source of leukotriene
production in the epidermis is thought to be the infiltrating
immune cells (Janssen-Timmen et al., 1995; Breton et al., 1996). Leukotrienes, particularly cysteinyl-leukotrienes such as LTC4,
have been implicated in the migration of Langerhans cells
toward the lymph nodes, too, as mice lacking 5-LOX display
a strongly reduced Langerhans cell movement (Robbiani et al.,
2000; Doepping et al., 2007). Among prostanoids, prostaglandin E2 is one of the main
mediators of inflammation in skin. PGE2 exerts its action by
binding to four different G-protein-coupled receptors (GDPR),
termed EP1−4 (Tober et al., 2007). In the context of normal
epidermis, PGE2 has been mostly implicated as a vasodilation-
promoting agent in the first phase of the sunburn response
to acute UVB radiation. Eicosanoids and Related Lipids In human epidermis, levels of PGE2
increase between 6 and 24 h after UVB irradiation, leading to the
development of erythema within 48 h (Black et al., 1978; Rhodes
et al., 2001, 2009). In mice, it has been shown that PGE2 produced
in the epidermis exerts its vasodilatory action by binding to EP2
and EP4 receptors on blood vessels (Kabashima et al., 2007). In
addition to its effects following UVB irradiation, PGE2 possesses
activity in Langerhans cells (epidermal dendritic cells). In a
murine model, ablation of EP4 receptor impaired Langerhans cell
migration toward the lymph nodes following antigen stimulation
and the ability of Langerhans cells to stimulate T lymphocytes,
suggesting a positive modulation of these processes by PGE2
(Kabashima et al., 2003). Epidermal cells can also express 12-LOX and 15-LOX. While
12-HETE is able to attract neutrophils and monocytes to the
epidermis (Dowd et al., 1985), 15-LOX downstream products
appear to have anti-inflammatory activity. 15-HETE and 15-
HETrE can inhibit the release of inflammatory mediators such
as leukotriene B4 (LTB4) from immune cells (Ziboh et al., 2000). Mice deficient for 15-LOX exhibit dramatic inflammatory skin
phenotypes, including extensive immune infiltrates in the skin,
epidermal hyperproliferation and compromised barrier function. These effects are at least partially due to a deficiency in resolvin
D2 (Kim et al., 2018). Oxidative stresses such as UVB radiation can cause oxidation
of linoleic acid in the stratum corneum to the eicosanoid-related
species 9-HODE. This mediator can bind the G2A receptor on
keratinocytes, leading to the release of inflammatory cytokines
and inhibition of proliferation through cell cycle arrest and
DNA synthesis suppression. G2A is also induced by oxidative
stress, making for a co-ordinated ligand/receptor response to
an external insult (Hattori et al., 2008). This mechanism is
particularly interesting, as it shows that an essential component
of the epidermal barrier structure also functions as a sensor for
damage to the barrier itself, highlighting how lipids can have
multi-faceted functions within the epidermis. PGD2 can be produced in the epidermis by Langerhans cells
or by sub-epidermal mast cells (Lewis et al., 1982; Maciejewski-
Lenoir et al., 2006). PGD2 can skew the profile of the T-cell
response induced by antigen presentation toward Th2 cell
activation by inhibiting the secretion of IL-12 from dendritic
cells (Theiner et al., 2006). Accordingly, PGD2 promotes
the recruitment and activation of mast cells and eosinophils
characteristic of a Th2-type immune response. Eicosanoids and Related Lipids This effect is
mediated by PGD2 binding to prostaglandin receptor DP2, also
known as CRTH2, a GPCR that triggers inhibition of adenylate
cyclase and lowers cAMP levels (Gαi signaling), ultimately
leading to cellular mobilization (Hirai et al., 2001; Spik et al.,
2005). PGD2 is able to induce expression of β-defensin-3 (hBD-
3) in keratinocytes by engaging a src/MEK/ERK/c-Fos signaling
axis, through DP2, and can thus bolster the antimicrobial defense
of the epidermis. Moreover, since hBD-3 can trigger mast
cell activation and further release of PGD2, this leads to the
establishment of a positive feedback loop of communication
between mast cells and keratinocytes (Kanda et al., 2010). Endocannabinoids Consistent with this inflammatory phenotype, SPC can also
induce keratinocytes to express intercellular adhesion molecule-1
(ICAM-1), a surface protein necessary for leukocyte recruitment
and retention, as well as the secretion of cytokines TNFα
and IL-6. These phenotypes are in line with the remarkable
increase in SPC levels seen in atopic dermatitis patients
(Imokawa et al., 1999). Endocannabinoids Endocannabinoids are endogenous lipids that can bind and
activate GPC cannabinoid receptors (CB1 and CB2). The most
prominent members of this class of compounds are arachidonic
acid metabolites N-arachidonoyl-ethanolamine (anandamide)
and 2-arachdonoylglycerol (2-AG). Anandamide can work as a
partial agonist for CB1 and CB2 receptors, while 2-AG functions
as a full ligand for both receptors (Sugiura et al., 2006; Smita et al.,
2007). Biosynthesis of these compounds happens “on demand”
in cell membranes through the action of N-acyltransferase
and N-acyl-phosphatidylethanolamine phospholipase D in the
case of anandamide, or the activity of phospholipase C
and diacylglycerol lipase in the case of 2-AG (Wang and
Ueda, 2009). The production of these two endocannabinoids
occurs in epidermal keratinocytes and melanocytes, which
also express both CB1 and CB2 as well as transient receptor
potential vanilloid-1 (TRPV-1) which can also be activated by
endocannabinoids (Maccarrone et al., 2003; Tóth et al., 2011;
Pucci et al., 2012). PGD2
can also be released by the helminth parasite
Schistosoma mansoni to inhibit the migration of Langerhans cells
to the lymph nodes following tumor necrosis factor-α (TNF-
α) stimulation. In this case, the release of Langerhans cells is
impaired by PGD2 binding to prostaglandin receptor DP1 and
is dependent on the subsequent adenylate cyclase activation and
rise in cAMP levels (Gαs signaling). Increased Gαs signaling is Generally,
endocannabinoids
possess
anti-inflammatory
activity, being able to suppress T cell receptor signaling Frontiers in Physiology | www.frontiersin.org January 2022 | Volume 12 | Article 804824 9 Lipids Roles in Mammalian Epidermis Vietri Rudan and Watt and
inhibit
dendritic
cell-mediated
immune
stimulation
(Wacnik et al., 2008; Börner et al., 2009). In skin, the
endocannabinoid system has been implicated in the attenuation
of the inflammatory response during contact hypersensitivity
reactions. When challenged with an obligate contact allergen,
mice lacking cannabinoid receptors or treated with receptor
antagonists show increased signs of inflammation and immune
infiltration. Conversely, mice that accumulate anandamide due
to the lack of its catabolic enzyme fatty acid amide hydrolase
(FAAH) have reduced inflammatory responses after allergen
treatment. Interestingly, stimulation by the contact allergen
produces an increase in the levels of anandamide and 2-AG. Monocyte chemotactic protein 2 (MCP2) is one of the genes
most upregulated in the absence of cannabinoid receptors
(Karsak et al., 2007). Consistent with this, anandamide can lower
the levels of IL-6 and MCP1 released by keratinocytes following
TNF-α stimulation (Leonti et al., 2010). Sphingosine-1-Phosphate The hydrolysis of ceramides by ceramidases can release
sphingosine bases that become substrates for sphingosine kinases,
yielding sphingosine-1-phosphate (S1P). Due to its relatively low
hydrophobicity and presence of a polar headgroup, S1P can
exit the membrane space and move into solution but is not
able to readily flip-flop across membrane leaflets. S1P is mostly
found in serum and interstitial fluid, is strongly released during
platelet degranulation, and exerts its actions in a paracrine or
autocrine manner through GPCRs (termed S1P1−5) as well as
intracellularly (Hannun and Obeid, 2008; van Meer et al., 2008;
Gomez-Larrauri et al., 2020). Lipid Modulation of Epidermal Cell
Behavior Sphingosine-1-phosphate generally behaves as a mitogen
and a migration-stimulatory agent. However, some of its
effects in skin go against this trend. S1P interferes with the
migration of Langerhans cells toward the lymph node (Reines
et al., 2009). Additionally, it affects how these dendritic cells
stimulate T lymphocytes by promoting Th2 cell activation
(Müller et al., 2005). The journey of a keratinocyte from basal layer stem cell to
stratum corneum corneocyte is influenced by several cellular
signaling pathways that control the balance between proliferation
and differentiation, exit from the stem cell compartment
and progression through the epidermal layers. Some of the
prominent signaling molecules involved in these processes
include β-catenin, extracellular-regulated kinase (ERK) and p38
mitogen-activated protein kinases (MAPK), phosphoinositide-3-
kinase (PI3K)/RACα serine/threonine-protein kinase (Akt) and
PKC. More specifically, accumulation of β-catenin in the nucleus
promotes expansion of the stem cell compartment (Zhu and
Watt, 1999; Watt and Collins, 2008) and high ERK activity
is associated with more proliferative/stem-like keratinocytes
(Hiratsuka et al., 2020). Conversely, p38 MAPK inhibition blocks
the induction of differentiation of cultured human keratinocytes
(Connelly et al., 2011). Activation of the PI3K/Akt pathway has
been linked with the stimulation of keratinocyte differentiation
(Janes et al., 2004, 2009). PKC is required for keratinocyte
differentiation in response to a range of stimuli; for example
blocking PKC can prevent cells from undergoing suspension-
induced commitment and differentiation (Adhikary et al., 2010;
Mishra et al., 2017). Sphingosine-1-phosphate is also involved in the response to
bacterial invasion. Secretions from P. aeruginosa and S. aureus
contain ceramidase or sphingomyelinase, respectively, that can
stimulate the production of S1P. Subsequent binding to S1P1 and
S1P2 receptors on the surface of keratinocytes can trigger the
expression and release of inflammatory cytokines such as TNFα,
IL-8, and IL-36γ, making S1P function as an “alarm signal” for
the immune response (Oizumi et al., 2014; Igawa et al., 2019). UNDER-RECOGNIZED ACTORS – THE
ROLE OF LIPID SIGNALING IN
EPIDERMAL HOMEOSTASIS For nearly a century of research, lipids have played a
center-stage role in studies of the structural integrity and
protection of the skin barrier. However, recent evidence
is beginning to reveal an even more dynamic role for
lipid mediators in signaling events that are key to the
maintenance of epidermal homeostasis. Lipid molecules can
act in an autocrine, paracrine or intracellular fashion to
modulate several aspects of epidermal cell behavior, particularly
keratinocyte differentiation, and the extent of their action
has likely been underappreciated so far. In this respect, it
is important to understand the enormous diversity of lipid
species present in the epidermis and how this impacts the
biology of the skin. Sphingosylphosphorylcholine Another sphingolipid, sphingosylphosphorylcholine (SPC, also
known as lysosphingomyelin), arising from the action of
sphingomyelin deacylase on sphingomyelin, has biological
activity in epidermal cells. SPC is sufficiently polar to be able
to move between different membranes and enter solution; it is
thought to exert its effects through low-affinity binding to S1P
receptors as well as intracellularly (Nixon et al., 2008; van Meer
et al., 2008). SPC has pro-inflammatory effects on keratinocytes,
increasing production of reactive oxygen species, activating
COX-2 and increasing PGE2 production (Choi et al., 2010). Complementing our understanding of these well characterized
signaling proteins, various different classes of lipids play
a role in the regulation and maintenance of epidermal
homeostasis (Figure 5). January 2022 | Volume 12 | Article 804824 Frontiers in Physiology | www.frontiersin.org 10 Lipids Roles in Mammalian Epidermis Vietri Rudan and Watt n bioactive lipid species active in the regulation of keratinocyte differentiation. Use of the indeterminate article in front of compound name denotes if
es for a certain compound are possible; in these cases, all “variable” fatty acid moieties are shown as C18:0 but can be substituted by alternative
ng in chain length and degree of unsaturation. Parentheses denote abbreviations, while brackets indicate the specific name of the structure
applicable. FIGURE 5 | Main bioactive lipid species active in the regulation of keratinocyte differentiation. Use of the indeterminate article in front of compound name denotes if
multiple structures for a certain compound are possible; in these cases, all “variable” fatty acid moieties are shown as C18:0 but can be substituted by alternative
fatty acids differing in chain length and degree of unsaturation. Parentheses denote abbreviations, while brackets indicate the specific name of the structure
displayed, when applicable. Eicosanoids and Related Lipids in this context does not affect adenylate cyclase activity but
results in an increase in the levels of intracellular diacylglycerol
and ceramide, both of which can promote keratinocyte
differentiation. This suggests the tantalizing possibility of a
lipid-mediated coupling mechanism between proliferation and
differentiation in basal keratinocytes (Konger et al., 2005b;
Figure 6). Besides their role in the modulation of the immune response,
eicosanoids and related lipids also intervene in the regulation of
keratinocyte differentiation. Epidermal keratinocytes express all
four prostaglandin EP receptors bound by PGE2. The different
receptors possess varying affinity for PGE2, with EP1 and
EP2 having affinities in the nanomolar range, while EP3 and
EP4 bind to PGE2 in the sub-nanomolar range. The lower-
affinity EP2 receptor, expressed both basally and suprabasally,
is responsible for stimulating keratinocyte proliferation, possibly
in response to increased PGE2 levels during inflammatory
events. This mitogenic effect is mediated by Gαs signaling
and activation of adenylate cyclase (Konger et al., 1998,
2005a; Tober et al., 2007). In contrast, binding of PGE2
to the high affinity, basally expressed, EP3 receptor leads
to keratinocyte growth inhibition, indicating a potential role
for PGE2 in the limitation of basal cell proliferation in
homeostatic conditions. Interestingly, EP3 receptor activation The
linoleic
acid
derivative
13-HODE
can
promote
keratinocyte
differentiation
through
activation
of
NF-κB
(Ogawa et al., 2011). While the NF-kB pathway is mainly
associated with inhibition of proliferation and protection form
apoptosis in keratinocytes (Seitz et al., 2000), it was also shown to
promote expression of differentiation-associated proteins such
as Keratin 10 and Keratin 1. Treatment of keratinocytes with 13-
HODE activates IκB kinase (IKK), which in turn phosphorylates
and inactivates Inhibitor of κB (IκB) and stimulates NF-κB
activity, leading to an increase in the expression of Keratin 1
(Ogawa et al., 2011). January 2022 | Volume 12 | Article 804824 Frontiers in Physiology | www.frontiersin.org 11 Lipids Roles in Mammalian Epidermis Vietri Rudan and Watt FIGURE 6 | Receptor-mediated lipid signaling in keratinocytes. Yellow halos around proteins denote activation, while gray halos denote inhibition. Arrows next to
metabolites/proteins denote increase or decrease in levels/expression. cAMP, cyclic adenosine monophosphate; LPA, lysophosphatidic acid; PGE2, prostaglandin
E2; S1P, sphingosine-1-phosphate. FIGURE 6 | Receptor-mediated lipid signaling in keratinocytes. Yellow halos around proteins denote activation, while gray halos denote inhibition. Arrows next to
metabolites/proteins denote increase or decrease in levels/expression. cAMP, cyclic adenosine monophosphate; LPA, lysophosphatidic acid; PGE2, prostaglandin
E2; S1P, sphingosine-1-phosphate. Diacylglycerols and the Protein Kinase C Pathway Diacylglycerols and the Protein Kinase C Pathway
As mentioned above, the PKC pathway can be directly
regulated by lipids. Depending on the isoform, PKC activity is
modulated by both intracellular calcium levels and presence of
diacylglycerol/lipids (-α -β -γ), by diacylglycerol/lipids alone (-
δ, -ε, -η, -θ), or by allosteric interaction with other proteins (-ζ
-ι/λ) (Nishizuka, 1992; Rosse et al., 2010). The two PKC isoforms
that are most relevant to keratinocyte differentiation – PKCδ and
PKCη – are insensitive to calcium but activated by the presence
of phosphatidylserine and diacylglycerol (Adhikary et al., 2010). Moreover, lysophosphatidylcholines and unsaturated fatty acids
can further enhance the activation of PKC (Bronfman et al.,
1988; Shinomura et al., 1991; Nishizuka, 1992). Presence and
activation of PKCδ and η, and the subsequent triggering of
specific MAPK enzymes (MEKK, MEK-6, MEK-3, and p38δ),
are necessary for the induction of keratinocyte differentiation in
response to a number of stimuli (Figure 7; Adhikary et al., 2010;
Mishra et al., 2017). Eicosanoids and Related Lipids endocannabinoid system potentially represents a direct link
between exposure to damaging sunlight and melanin production
by melanocytes (Pucci et al., 2012). CYP2B19, expressed in the granular layer of mouse epidermis,
produces 14,15-EET, which enhances the local activity of
transglutaminases in the formation of the cornified cell envelope. The mechanisms through which this is achieved remain unclear,
but no receptors for 14,15-EET have been robustly identified,
suggesting that this mediator may be acting intracellularly on
the complex regulation of transglutaminase activity (Figure 7;
Gibson et al., 1996; Kalinin et al., 2002; Ladd et al., 2003). Frontiers in Physiology | www.frontiersin.org Endocannabinoids Anandamide
inhibits
keratinocyte
differentiation
through
interaction with CB1, suppression of PKC and subsequent
suppression of activator protein 1 (AP-1) transcription factor
action (Figure 6). Keratinocytes treated with anandamide
show decreased expression of cornified cell envelope proteins
(such as involucrin and loricrin) and reduced transglutaminase
activity. Keratinocyte anandamide levels are regulated during
differentiation by increasing their degradation through FAAH
(Maccarrone et al., 2003). In melanocytes, endocannabinoids (likely from neighboring
keratinocytes) can enhance melanin production by increasing
tyrosinase expression following p38/ERK1/ERK2 and CREB
activation downstream of CB1. Since keratinocytes can increase
their anandamide synthesis in response to UVB radiation, the The specific acyl residues contained in diacylglycerol, as well
as its hydrophilicity/hydrophobicity profile, can influence the January 2022 | Volume 12 | Article 804824 Frontiers in Physiology | www.frontiersin.org 12 Lipids Roles in Mammalian Epidermis Vietri Rudan and Watt FIGURE 7 | Intracellular lipid signaling in keratinocytes. Arrows denote activation, while “T” symbols denote inhibition. Arrows crossing the DNA symbol indicate
transcription, while dotted lines represent translation. Arrows going through PPARs are depicted with different shadings for visual clarity. Protein colors indicate
approximate function: transcription factors in dark blue, signaling mediators in green, and structural/differentiation-associated proteins in dark red. 13-HODE,
13-hydroxyoctadecadienoic acid; 14,15-EET, 14,15-epoxyeicosatrienoic acid; 15d-PGJ2, 15-deoxy-prostaglanding J2; 15-HETrE, 15-hydroxyeicosatrienoic acid;
C1P, ceramide-1-phopshate; DAG, diacylglycerol; S1P, sphingosine-1-phosphate; SPC, sphingosylphosphorylcholine. FIGURE 7 | Intracellular lipid signaling in keratinocytes. Arrows denote activation, while “T” symbols denote inhibition. Arrows crossing the DNA symbol indicate
transcription, while dotted lines represent translation. Arrows going through PPARs are depicted with different shadings for visual clarity. Protein colors indicate
approximate function: transcription factors in dark blue, signaling mediators in green, and structural/differentiation-associated proteins in dark red. 13-HODE,
13-hydroxyoctadecadienoic acid; 14,15-EET, 14,15-epoxyeicosatrienoic acid; 15d-PGJ2, 15-deoxy-prostaglanding J2; 15-HETrE, 15-hydroxyeicosatrienoic acid;
C1P, ceramide-1-phopshate; DAG, diacylglycerol; S1P, sphingosine-1-phosphate; SPC, sphingosylphosphorylcholine. Similar findings were also reported for diacylglycerol containing
the
anti-inflammatory
eicosanoid-related
lipid
15-HETrE
(Cho and Ziboh, 1997). degree to which PKC becomes regulated (Mori et al., 1982;
Molleyres and Rando, 1988). In support of this, the eicosanoid-
related
molecule
13-HODE,
a
hydroxylated
linoleic
acid
metabolite produced in the epidermis, becomes incorporated
into membrane phosphatidylcholines and phosphatidylinositols,
which in turn can be converted to 13-HODE-containing
diacylglycerol
(Cho
and
Ziboh,
1994b). This
particular
diacylglycerol is able to downmodulate the activity and
expression of PKCβ (Figure 7). Endocannabinoids Interestingly, inhibition of
this PKC isoform by 13-HODE has been correlated with
the rescue of a keratinocyte hyperproliferation phenotype in
guinea pigs (Cho and Ziboh, 1994a, 1995; Ziboh et al., 2000). Frontiers in Physiology | www.frontiersin.org Sphingolipids Sphingolipids are one of the most functionally multifaceted
lipid classes in the epidermis. They play a fundamental role in
the formation and the structure of the epidermal barrier and
also participate in the protection of the epidermis. Additionally,
several sphingolipid species take part in epidermal signaling
(Hannun and Obeid, 2008; Gomez-Larrauri et al., 2020). These
include ceramides, glycosylceramides, ceramides-1-phosphate, January 2022 | Volume 12 | Article 804824 Frontiers in Physiology | www.frontiersin.org 13 Lipids Roles in Mammalian Epidermis Vietri Rudan and Watt produces an expansion of the hair follicle stem cell compartment
(Peters et al., 2015). The different effects of alkaline ceramidase
and CerS4 deletion can be reconciled by the fact that while the
absence of alkaline ceramidase produces a generalized increase
of all ceramide species, lack of CerS4 produces a decrease of
some ceramide species, but an increase in others, indicating that
only specific ceramide subspecies might influence the behavior
of murine epidermal stem cells. In addition to its effect on the
stem cell compartment, epidermal deletion of CerS4 impairs
maintenance of the epidermal barrier in adult mice, leading
to increased trans-epidermal water loss, hyperkeratosis and
accumulation of immune cells. This effect is notable because
CerS4 catalyzes the synthesis of shorter-chained ceramide species
that are not directly involved in the structure of the barrier. These
species or other lipids indirectly affected by CerS4 knockdown
may thus be involved in signaling events that are necessary for
epidermal homeostasis (Peters et al., 2020). sphingosine-1-phosphate,
and
sphingosylphosphorylcholine. The metabolism of sphingolipids is remarkably interconnected,
with interconversion between different species occurring readily
depending on the relevant enzymes’ presence and abundance
(Hannun, 1994; Hannun et al., 2001; Hannun and Obeid, 2008). Ceramides can be formed de novo by the condensation of
palmitate and serine catalyzed by serine palmitoyl transferase
and the subsequent activity of ceramide synthase. Alternatively,
they can be derived from either sphingomyelin through the
action of sphingomyelinases or glucosylceramides through the
action of glucocerebrosidase. They can be mainly found in the
ER membranes (where their de novo synthesis occurs) or at
the plasma membrane. Due to their physico-chemical properties
they are unable to transfer freely between different cellular
compartments, needing either vesicular transport or protein
mediators to move across membranes. They can, however,
flip-flop between membrane leaflets, though this may too be
subject to regulation (Hannun and Obeid, 2008; van Meer
et al., 2008). Sphingolipids Ceramides act as signaling molecules in a variety
of settings and are mostly known for inducing apoptosis and
responding to cellular stress (Uchida, 2014; Uchida and Park,
2021). In vitro, treatment of keratinocytes with exogenous short-
chained (C2–C8) ceramides can inhibit their proliferation and
induce differentiation (Wakita et al., 1994). In contrast to ceramides, glucosylceramide has mitogenic
effects
on
epidermal
keratinocytes
when
administered
subcutaneously in mice (Figure 7; Marsh et al., 1995). Conversely, certain glycosylceramides such as the ganglioside
GM3, which are present on the outer leaflet of keratinocyte
plasma membranes, have an antiproliferative effect when
supplemented
in
the
medium
of
cultured
keratinocytes
(Paller et al., 1993). Gangliosides (ceramides conjugated to
oligosaccharides containing sialic acid) have been implicated
in some important membrane dynamics in the early phases
of
keratinocyte
differentiation. When
basal
keratinocytes
commit to differentiation, the adherens junctions connecting
the basal cells to one another must relax to allow the cells to
delaminate to the suprabasal layers. In order for this process to
occur, desmosomes containing the differentiation-dependent
cadherin desmoglein 1 (Dsg1) must be localized within insoluble
membrane compartments enriched in gangliosides. Interfering
with the compartmentalization of Dsg1 in these lipid domains
impairs the release of tension at the adherens junctions and
leads to reduced keratinocyte delamination from monolayers
(Figure 7; Nekrasova et al., 2018). Both exogenous short-chained (C2 and C6) ceramides and
endogenous ceramides can directly regulate the expression
of certain differentiation-related genes in keratinocytes. One
example is glucosylceramide transporter ABCA12, involved in
the translocation of glucosylceramides into lamellar bodies,
whose transcription is enhanced by ceramide through increased
PPARβ/δ expression (Jiang et al., 2009). Another case is the
expression of caspase-14 – a protein present in differentiated
keratinocytes and responsible for the processing of profilaggrin
into filaggrin, important for the appropriate hydration of
the stratum corneum. Caspase-14 expression can be directly
stimulated by ceramides, although the mechanism does not
involve PPARβ/δ (Jiang et al., 2013). C2 ceramide administered
in vitro has been shown to strongly reduce Akt activity in
melanocytes, to inhibit their growth, and to decrease tyrosinase
activity and consequent melanin production (Kim et al., 2001). Ceramides can also intervene in the response of epidermal
keratinocytes to UVB radiation. Exposing keratinocytes to UVB
increases the de novo synthesis of ceramides, which in turn
participate in the subsequent induction of apoptosis by a caspase-
independent mechanism (Uchida et al., 2003; Figure 7). Frontiers in Physiology | www.frontiersin.org Lysophosphatidic Acid Besides its activity through GPCRs, S1P can act intracellularly
to influence the behavior of keratinocytes. Activation of
sphingosine kinase 1, one of the two enzymes catalyzing S1P
biosynthesis, mobilizes calcium from intracellular stores, leading
to inhibition of keratinocyte proliferation and stimulation of
differentiation (Hong et al., 2008). Similarly, inhibition of
S1P lyase, the main enzyme responsible for S1P catabolism,
increases intracellular S1P levels and causes cell cycle arrest
and expression of differentiation markers (Jeon et al., 2020). In response to ER-stress, which can be caused for example
by perturbation of the epidermal barrier, S1P levels rise and
promote NF-κB activation via assembly of an intracellular
signaling complex, causing the stimulation of C/EBPα-dependent
transcription, and ultimately leading to increased expression
of the cathelicidin LL-37 and enhanced epidermal protection
(Park et al., 2013, 2016; Figure 7). The effect of ER-stress-
induced S1P on cathelicidin LL-37 mimics that of ER-stress-
induced C1P on β-defensins (Kim et al., 2014), resulting
in a synergistic response to external injury via two related
sphingolipid metabolites. Lysophosphatidic acids (LPAs) are the simplest phospholipid,
formed by a glycerol backbone attached to a phosphate group,
a fatty acid, and a hydroxyl moiety. LPA can be synthesized
intracellularly by glycerophosphate acyltransferase, acylglycerol
kinase or phospholipase A. Alternatively, it can be generated
extracellularly by the secreted lysophospholipase D autotaxin. Degradation of LPA is mainly carried out by lipid phosphate
phosphatases. The single aliphatic chain of LPA makes it
sufficiently soluble to leave membranes (van Meer et al., 2008; Lei
et al., 2018). In addition to being a precursor in phospholipid biosynthesis,
LPA is a ligand of a series of GPCRs – LPA1−6 – through which
it exerts its biological activity. Some of these receptors belong
to the same family as S1P receptors, and S1P and LPA share
some structural similarity (Geraldo et al., 2021). A dependence
of the biological activity of LPA on its acyl-chain length has been
reported (van Corven et al., 1992). During wound repair, LPA is delivered to the injury site mainly
through platelet degranulation and its effects are akin to S1P:
keratinocyte migration is enhanced, with increases in expression
of laminin-332, TGF-α and TGF-β. Unlike S1P, however, LPA
appears to stimulate keratinocyte proliferation, although this may
be an indirect consequence of the induction of TGF-α expression
rather than direct LPA-mediated signaling (Figure 6; Piazza et al.,
1995; Demoyer et al., 2000; Amano et al., 2004). Sphingolipids Ceramides-1-phosphate (C1Ps) are sphingolipid mediators
that generally exert opposing effects to those of ceramides
(e.g., increased cell survival, increased proliferation), and
phosphorylation by ceramide kinase is usually considered a
way in which ceramides can be inactivated. C1Ps can be
found in the Golgi apparatus and the plasma membrane. They
are limited in their mobility between membranes, and owing
to their polar head group are unlikely to flip-flop between
membrane leaflets (Hannun and Obeid, 2008; Uchida, 2014;
Gomez-Larrauri et al., 2020). In keratinocytes, a role for C1P
has been described during cellular stress. Serum starvation of
mouse keratinocytes induces apoptosis which is mitigated by
the activity of PPARβ/δ. Among its targets is ceramide kinase,
whose activity is necessary to reduce the levels of intracellular
ceramides and prevent excessive apoptosis (Tsuji et al., 2008). C1P produced in response to endoplasmic reticulum stress leads
to an increase in the expression of β-defensin 2 and 3 (hBD-
2/3). This suggests a model whereby C1P activates cellular
phospholipase A2, causing the release of arachidonic acid from Interfering with sphingolipid metabolism in vivo not only,
as predicted, leads to disruption of epidermal homeostasis but
has also a surprising impact on keratinocyte differentiation. Eliminating alkaline ceramidase, an enzyme responsible for
ceramide degradation, in mouse skin leads to an abnormal
accumulation of ceramides, which in turn compromise the
barrier function of the epidermis and cause alterations in
the final stages of keratinocyte differentiation and an increase
in the epidermal stem cell population of the hair follicle
(Liakath-Ali et al., 2016). In apparent contrast, knockout of ceramide synthase 4
(CerS4), an enzyme responsible for ceramide synthesis, in mice January 2022 | Volume 12 | Article 804824 Frontiers in Physiology | www.frontiersin.org 14 Lipids Roles in Mammalian Epidermis Vietri Rudan and Watt urokinase-type plasminogen activator (uPA) and its receptor
(uPA-R) (Figure 7; Wakita et al., 1998). phospholipids and an increase of 15-deoxy-PGJ2; this leads to
activation of PPARα or -β/δ, which promotes expression of Src
and stimulates STAT1/3-mediated expression of the β-defensin
genes (Figure 7; Kim et al., 2014). phospholipids and an increase of 15-deoxy-PGJ2; this leads to
activation of PPARα or -β/δ, which promotes expression of Src
and stimulates STAT1/3-mediated expression of the β-defensin
genes (Figure 7; Kim et al., 2014). Sphingomyelins (SMs) are synthesized in the Golgi apparatus
and found at the plasma membrane. Sphingolipids Their double aliphatic chain
makes them insoluble in water and their polar phosphocholine
group
impairs
flip-flopping
between
membrane
leaflets
(Hannun and Obeid, 2008; van Meer et al., 2008). Unlike
other sphingolipids, SMs are a major component of cellular
membranes, which makes them less likely to be able to act as
bioactive mediators. In fact, SMs are often considered a reservoir
for the generation of bioactive sphingolipids (Hannun, 1994;
Hannun et al., 2001; Wanner et al., 2004). However, variation
in SM levels can influence the biophysical characteristics of
the membrane such as, for example, fluidity, stiffness and
mechanosensory properties, which in turn can drive epidermal
cellular responses (Mobasseri et al., 2019). Moreover, by virtue
of the mostly saturated nature of its acyl chains, SMs can form
lipid aggregates with cholesterol within membranes which can
influence protein interaction dynamics. While the existence
of such lipid “rafts” in biological membranes in vivo has been
controversial, they have been observed in artificial bilayers
mimicking the lipid composition of the plasma membrane
(Dietrich et al., 2001; Samsonov et al., 2001; Simons and Sampaio,
2011; Levental et al., 2020). Early studies showed that epidermal
desmosome preparations are enriched in cholesterol, SM and
gangliosides (Drochmans et al., 1978); more recently, rafts have
been implicated in desmosome assembly and dismantling in
keratinocytes (Stahley et al., 2014). Along with its function in immunity, spiingosine-1-phosphate
(S1P) also inhibits proliferation and promotes differentiation
of keratinocytes (Vogler et al., 2003). Binding of S1P to S1P2
antagonizes the proliferative signals coming from the insulin
receptors by activating PKCδ, causing dephosphorylation of Akt
and leading to inhibition of cyclin D2 and upregulation of cyclin-
dependent kinase inhibitors p21 and p27Kip1 (Kim et al., 2004;
Schüppel et al., 2008). Moreover, signals mediated through S1P3
are reported to increase the intracellular calcium concentration
in keratinocytes, which has been linked to differentiation (Watt
et al., 1984; Lichte et al., 2008; Figure 6). In addition to proliferation inhibition and in sharp contrast
to its role in Langerhans cells, S1P stimulates keratinocyte
migration. This effect is enacted by S1P receptors through
transforming growth factor-β (TGF-β)-receptor I-mediated
phosphorylation and activation of Smad3 (Figure 6). In fact, S1P
and TGF-β treatment cause similar phenotypes in keratinocytes,
both in terms of general cellular behavior and in terms of
specific gene expression changes; for example, both ligands
can increase laminin 5 production (Amano et al., 2004; Sauer
et al., 2004). Sphingolipids The overlap between S1P and TGF-β signaling is
likely important during the complex orchestration of the wound
healing process, when both these ligands as well as multiple
other mediators are abundantly produced (Vogler et al., 2003;
Rognoni and Watt, 2018). Frontiers in Physiology | www.frontiersin.org An Unbiased Screen for Lipid Regulators
of Differentiation While we have presented evidence that separate lipid classes
play a role in signaling to impact keratinocyte proliferation,
differentiation and other processes, this is inevitably an
oversimplification. The lipid species produced in the cell and the
enzymes that modify them form an extremely intricate network
and it is difficult to predict the multiple perturbations that ensue
from a specific intervention, such as depletion of a single lipid
modifying enzyme (Muro et al., 2014). Lipid subspecies can
influence cellular biology in ways that go beyond their direct
interaction with proteins as ligands or allosteric modulators, by
for example working as ensembles that can facilitate (or impede)
the activity of groups of molecular partners (Muro et al., 2014). Activation of all PPAR isoforms or LXRs pushes keratinocytes
to differentiate both in vitro in human keratinocytes and in vivo
in mouse epidermis (Hanley et al., 1998, 2000b,a; Mao-Qiang
et al., 2004; Schmuth et al., 2004). More specifically, while all
receptors stimulate expression of differentiation markers, PPARα
and LXR also cause inhibition of keratinocyte proliferation
(Hanley et al., 1998, 2000b). PPARs or LXR activation induce
accumulation of lipids as well as expression of lipid metabolism
enzymes, including ones involved in lamellar body biogenesis,
and stimulate lamellar body secretion when activated topically
in mouse epidermis (Rivier et al., 2000; Man et al., 2006; Jiang
et al., 2008, 2010; Lu et al., 2010). While the expression of
lipid-modifying enzymes appears to be directly regulated by the
receptors’ transcriptional activity, the kinetics of induction of
differentiation markers suggests an indirect regulation (Hanley
et al., 1998; Schmuth et al., 2004). Despite these challenges, newly developed techniques or
refinement of established methods are beginning to shed more
light on the potential contribution of endogenous cellular
lipid subspecies to a variety of cellular processes. Prompted
by the finding that the alteration of the ceramide profile in
mouse epidermis could lead to an effect on the stem cell
compartment (Peters et al., 2015; Liakath-Ali et al., 2016),
our lab recently performed lipidomics in a well characterized
in vitro keratinocyte differentiation system combined with a
lipid-modifying enzyme siRNA screen to evaluate the changes in
cellular lipid composition occurring during differentiation and to
try to understand which changes might be important for the early
phases of this process. Our study identified several ceramide and glucosylceramide
subspecies that could induce keratinocyte differentiation when
administered exogenously in vitro (Vietri Rudan et al., 2020). Peroxisome Proliferator-Activated Receptors
Signaling g
g
Other lipid-sensitive proteins that have been shown to be
involved in the regulation of keratinocyte differentiation are
peroxisome proliferator-activated receptors (PPARs) and liver
X receptors (LXRs). When activated by a lipid ligand, PPARs
or LXR heterodimerize with retinoid X receptor (RXR) and
regulate gene expression. Three PPAR isoforms (α-, β/δ-, γ-)
exist, all of which are expressed in keratinocytes: while PPARβ/δ
expression is constitutive, PPARα and PPARγ levels increase with
differentiation (Rivier et al., 1998). Both LXR isoforms – α and
β – are constitutively expressed in human keratinocytes (Russell
et al., 2007). PPAR endogenous ligands are fatty acids and their
metabolites, including eicosanoids, while LXRs are activated by
the cholesterol metabolite oxysterol (Bocos et al., 1995; Janowski
et al., 1996). Cholesterol Sulfate Cholesterol metabolites can also participate in the modulation
of keratinocyte differentiation. An example of this is cholesterol
sulfate, whose levels progressively increase over the course
of differentiation, peaking in the granular layer. Treatment
of
human
keratinocytes
with
cholesterol
sulfate
causes
induction of the expression of the retinoic acid receptor-
related orphan receptor α (RORα), which in turn is able to
elicit transcription of the profilaggrin gene (Hanyu et al., 2012;
Figure 7). This mechanism provides another link between the
constantly changing protein and lipid landscape of differentiating
keratinocytes, further underscoring the functional connection of
these changes in the creation of the epidermal barrier. Lysophosphatidic Acid An interesting
study examining skin blisters showed that LPA production is
increased in blister fluid by autotaxin activity and expression of Sphingosylphosphorylcholine (SPC) appears to promote an
abnormal route of differentiation, with increased intracellular
calcium levels and activation of transglutaminase 1, at the same
time as inhibiting the expression of cornified cell envelope
proteins such as profilaggrin and loricrin (Higuchi et al.,
2001; Choi et al., 2010). SPC also promotes re-epithelialization
during wound healing by acting as a keratinocyte mitogen and
increasing migration through stimulation of the expression of January 2022 | Volume 12 | Article 804824 Frontiers in Physiology | www.frontiersin.org 15 Lipids Roles in Mammalian Epidermis Vietri Rudan and Watt LPA1 is also enhanced, pointing to the relevance of this signaling
in vivo (Mazereeuw-Hautier et al., 2005). transcription of differentiation proteins has been proposed as
a coupling mechanism to coordinate the changes in lipids and
proteins occurring during epidermal maturation (Figure 7; Rivier
et al., 2000; Man et al., 2006; Jiang et al., 2008, 2010; Lu et al.,
2010). When treating keratinocytes with LPA, an induction of
differentiation can be observed, potentially through multiple
pathways. LPA induces an increase of intracellular calcium
concentration, likely through binding to LPA2 (Lichte et al.,
2008). Binding to LPA1 or LPA5 instead leads to an increase in
the differentiation marker pro-filaggrin and alteration of cellular
morphology, signaling via Gα12/13 to RhoA-ROCK and activating
Serum Response Factor (SRF) (Figure 6). The SRF pathway is
involved in keratinocyte differentiation induced by restricted
spreading on micropatterned substrates (Connelly et al., 2010;
Sumitomo et al., 2019). The increase in filaggrin triggered
by LPA treatment provides a likely molecular explanation
for the beneficial effects of LPA on skin moisturization
(Yahagi et al., 2011). Frontiers in Physiology | www.frontiersin.org An Unbiased Screen for Lipid Regulators
of Differentiation Using such a combined approach it was possible to leverage
the high specificity of protein-targeting techniques while keeping
the focus on the lipid changes. This is key because of the
cascading effects of lipid enzyme perturbation and the potential
presence of compensatory mechanisms. In line with this, while
the enzymes that emerged from our siRNA screen (ELOVL1 and In the case of PPARα and LXL, differentiation proceeds
through the activation of AP-1 transcription factors (Hanley
et al., 2000a,b). While there is overlap between the consequences
of activating different PPAR isoforms, the different isoforms
are able to exert their effect independently of one another,
which suggests a certain degree of redundancy. The fact that
PPARs and LXRs are sensitive to endogenous lipid mediators
generated during keratinocyte differentiation and can affect the January 2022 | Volume 12 | Article 804824 Frontiers in Physiology | www.frontiersin.org 16 Lipids Roles in Mammalian Epidermis Vietri Rudan and Watt FATP1) were directly associated with generation of specific chain-
length saturated and mono-unsaturated fatty acids and fatty acyl
CoAs, the bioactive lipids identified belonged to various different
phospholipid and sphingolipid classes. Conversely, the relative abundance of sebaceous secretions can
determine the abundance of different bacterial species at specific
body sites (Table 1; Grice et al., 2009; Grice and Segre, 2011). Some parasites can secrete lipids to interfere with the host’s
immune response, as is the case for PGD2 secreted by S. Mansoni
(Angeli et al., 2001). The full range of lipid mediators of
microbial origin that participate in their interaction with the host
remains to be explored. While our approach demonstrates the power of an unbiased
lipidomic screen, it is important to note that our lipidomic panel
did not include a number of potentially relevant lipid classes. For example, we did not include fatty acids and fatty acyl-
CoAs, which may regulate keratinocyte differentiation through,
for example, PPAR or PKC signaling (Bronfman et al., 1988;
Bocos et al., 1995). In addition, the size of the effect of bioactive
lipids we observed experimentally will depend as how efficiently
they reach the appropriate subcellular destination. Furthermore,
lipids operate by affecting the functionality of whole assemblies
of molecular partners, including proteins and carbohydrates,
which makes it harder to evaluate their role using the linear
approaches that are typically employed when studying protein
signaling pathways. DISCUSSION With lipids playing such an important functional role
in the epidermis, it is not surprising that they intervene
in the regulation of keratinocyte differentiation and in the
maintenance of epidermal homeostasis. In this review, we
have highlighted numerous examples of how bioactive lipid
mediators from several different classes can affect in these
processes, whether by influencing membrane characteristics or
participating in cellular signaling. The importance of membrane
properties in keratinocytes is highlighted for example by the
interaction between key proteins involved in their differentiation,
desmosomal cadherins, and membrane lipid domains to ensure
the appropriate assembly and disassembly of these functional
ultrastructures and in turn guarantee proper delamination from
the basal layer (Stahley et al., 2014; Nekrasova et al., 2018). We have described how in the epidermis the physical, chemical
and biological characteristics of lipids are all functionally
leveraged to build, protect, and maintain the outer barrier
of the organism. The hydrophobic and fluidity properties of
ceramides, cholesterol and fatty acids are exploited to create
an impermeable, resistant, yet flexible, dynamic structure in
the stratum corneum. Nevertheless, despite nearly one hundred
years of research into the physiology of the body’s outer
boundary, questions still remain as to how exactly different
ceramide species participate in the generation of the exquisite
biophysical properties of the cornified layer. This is not
surprising, considering that the composition of the stratum
corneum can vary quite extensively in different species (e.g., in
birds), while maintaining its fundamental microstructure and
functional characteristics (Champagne et al., 2012). Similarly,
mammals living in cold environments, such as arctic foxes
or reindeer, have dramatically lower melting points for the
lipid mixtures isolated from their extremities as compared
to the ones obtained from the body core, reflecting the
fact that their peripheral tissues can be up to 40◦C colder
than their central tissues (Irving et al., 1957). How this
adaptation can influence the properties, microstructure, and
composition of the epidermis of these animals is yet to be
fully elucidated. More generally, the apico-basal polarity of the plasma
membrane is a fundamental feature of epithelial cells, with
keratinocytes being no exception; the relevance of lipids in the
establishment of such oriented cell membranes remains poorly
understood and represents a major avenue for future research
(Ikenouchi, 2018). Moreover, lipids can influence membrane
stiffness, fluidity and mechanosensitivity, which are known to
have an effect on keratinocyte behavior (Mobasseri et al., 2019). An Unbiased Screen for Lipid Regulators
of Differentiation The participation of secreted lipid species of keratinocyte or
sebaceous origin n innate immunity provides a fascinating insight
into how different systems in the body can act in unison to ensure
the preservation of homeostasis and the appropriate response
to external perturbations, and how even foreign micro-organism
colonization can be used as a means to maintain and protect the
outer limit of the body (Gallo and Nakatsuji, 2011; Kobayashi
et al., 2019). Gaining a fuller picture of how antimicrobial lipids
act on their targets, intervene in the inflammatory response and
communicate with the innate and adaptive immune system may
help us in the development of new, more effective, treatments
against pathogens or inflammatory disorders. Frontiers in Physiology | www.frontiersin.org January 2022 | Volume 12 | Article 804824 DISCUSSION However, the extent to which lipids may contribute to the
regulation of these membrane properties, the mechanisms and
species involved, still needs to be fully characterized. There are many factors that can contribute to the biological
activity of lipids. While variation in levels can be a good
indication of the involvement in a biological process – as for
example in the case of cholesterol sulfate in the stratum corneum
(Elias et al., 1984; Sato et al., 1998; Hanyu et al., 2012) – there
are other layers of regulation that likely influence lipid-mediated
regulation of cellular behavior. A characteristic of several lipid
classes is their limited ability, owing to physical and chemical
constraints, to move between different cellular environments or
even across the two leaflets of the same membrane (Hannun and
Obeid, 2008; van Meer et al., 2008). Therefore, cells can selectively
target the activity of specific lipid players to certain subcellular
locales by modulating the expression and activation of transport Far from being only passive building blocks, lipids also
protect the epidermis by way of their chemical features, as in
the fatty acid contribution to the “acid mantle,” and biological
activity against microbes, significantly contributing to shape the
makeup of the commensal microbiota. The crosstalk between
skin surface resident microbes and host factors is extensive. Numerous lines of research have shown that the normal skin
microflora can modulate the expression of antimicrobial peptides
to protect from opportunistic pathogens (Gallo and Nakatsuji,
2011). Lipases of microbial origin are mainly responsible for
the presence of fatty acids in the sebum, and disruption of
the normal microbiome can lead to altered sebum composition. January 2022 | Volume 12 | Article 804824 17 Lipids Roles in Mammalian Epidermis Vietri Rudan and Watt molecules and vesicular trafficking processes. The importance
of this level of regulation in the epidermis is underscored by
the diversity of lipid transporters expressed in keratinocytes and
the emergence of severe pathological conditions associated with
transporter loss or dysfunction (Elias et al., 2008; Khnykin et al.,
2011; Vietri Rudan et al., 2020). membranous organelles may play into their functionality, or
how much individual lipid species or lipid features matter
for epidermal homeostasis in vivo. Technological advances will
be key to answering these complex questions. ACKNOWLEDGMENTS We are grateful for funding from the Department of Health
via the National Institute for Health Research comprehensive
Biomedical Research Centre award to Guy’s and St Thomas’
National Health Service Foundation Trust in partnership with
King’s College London and King’s College Hospital NHS
Foundation Trust. Challenges still remain to explore some of the more
elusive aspects of lipid biology, for example how the cellular
distribution and/or traffic of bioactive lipids among the various Challenges still remain to explore some of the more
elusive aspects of lipid biology, for example how the cellular
distribution and/or traffic of bioactive lipids among the various DISCUSSION In this respect,
implementation of recently developed, sophisticated techniques,
like single-cell lipidomics (Capolupo et al., 2021; Li et al., 2021),
already promises more exciting developments in unraveling the
finer details of the roles played by lipids in the regulation of
cellular behavior (Züllig et al., 2020; Capolupo et al., 2021; Li et al.,
2021). We are confident that in the future the application of these
new technologies will provide a clearer picture of the rich and
essential contribution of lipid molecules to epidermal biology. In the case of the modulation of keratinocyte differentiation, it
often remains unclear to what degree the activity of endogenously
generated individual lipid mediators is important and at what
stage of the in vivo differentiation process they might come into
play. An example of this is the fatty acid-mediated regulation
of PPARs: while it is established that fatty acids can regulate
PPAR activity and that they are abundant in keratinocytes, it is
still not clear whether they do so endogenously, at what stage
of the differentiation process, what PPAR isoforms are involved
and, importantly, what fatty acids. This last question points
toward another key fact to be considered: the cell produces
a vast amount of lipid subspecies (varying for example in
carbon chain length, number, and location of unsaturations, and
hydroxylation profile) whose influence on cellular behavior is still
poorly understood and has only recently begun to be investigated. A combination of lipid enzyme targeting and lipidomics has
identified individual lipid subspecies from multiple classes that
can influence keratinocyte differentiation in vitro, underscoring
the still little-explored regulatory potential of the cellular lipid
repertoire (Vietri Rudan et al., 2020). Such comprehensive
strategies can pinpoint individual lipid actors that can take
part in signaling events or provide indications as to what lipid
properties matter most within a certain process. Indeed, studies
are beginning to shed light on the influence of this diverse
collection of lipid molecules on several cellular and biological
activities, such as cell division or immune cell activation (Atilla-
Gokcumen et al., 2014; Köberlin et al., 2015). FUNDING This work was funded by grants to FW from the United Kingdom
Medical Research Council (MR/PO18823/1) and the Wellcome
Trust (206439/Z/17/Z). AUTHOR CONTRIBUTIONS MV and FW wrote and edited the text. MV designed the
figures. Both authors contributed to the article and approved the
submitted version. REFERENCES langerhans cell migration during schistosomiasis infection. J. Exp. Med. 193,
1135–1148. doi: 10.1084/jem.193.10.1135 Adhikary, G., Chew, Y. C., Reece, E. A., and Eckert, R. L. (2010). PKC-delta and
-eta, MEKK-1, MEK-6, MEK-3, and p38-delta are essential mediators of the
response of normal human epidermal keratinocytes to differentiating agents. J. Invest. Dermatol. 130, 2017–2030. doi: 10.1038/jid.2010.108 Angeli, V., Staumont, D., Charbonnier, A.-S., Hammad, H., Gosset, P., Pichavant,
M., et al. (2004). Activation of the D Prostanoid receptor 1 regulates
immune and skin allergic responses. J. Immunol. 172, 3822–3829. doi: 10.4049/
jimmunol.172.6.3822 J. Invest. Dermatol. 130, 2017–2030. doi: 10.1038/jid.2010.108 Akiyama, M. (2017). Corneocyte lipid envelope (CLE), the key structure for skin
barrier function and ichthyosis pathogenesis. J. Dermatol. Sci. 88, 3–9. doi:
10.1016/j.jdermsci.2017.06.002 Arikawa, J., Ishibashi, M., Kawashima, M., Takagi, Y., Ichikawa, Y., and Imokawa,
G. (2002). Decreased levels of sphingosine, a natural antimicrobial agent, may
be associated with vulnerability of the stratum corneum from patients with
atopic dermatitis to colonization by Staphylococcus aureus. J. Invest. Dermatol. 119, 433–439. doi: 10.1046/j.1523-1747.2002.01846.x Aly, R., Maibach, H. I., Rahman, R., Shinefield, H. R., and Mandel, A. D. (1975). Correlation of human in vivo and in vitro cutaneous antimicrobial factors. J. Infect. Dis. 131, 579–583. doi: 10.1093/infdis/131.5.579 Astudillo, A. M., Meana, C., Guijas, C., Pereira, L., Lebrero, P., Balboa, M. A.,
et al. (2018). Occurrence and biological activity of palmitoleic acid isomers in
phagocytic cells. J. Lipid Res. 59, 237–249. doi: 10.1194/jlr.m079145 Aly, R., Maibach, H. I., Shinefield, H. R., and Strauss, W. G. (1972). Survival of
pathogenic microorganisms on human skin. J. Invest. Dermatol. 58, 205–210. doi: 10.1111/1523-1747.ep12539912 Atilla-Gokcumen, G. E., Muro, E., Relat-Goberna, J., Sasse, S., Bedigian, A.,
Coughlin, M. L., et al. (2014). Dividing cells regulate their lipid composition
and localization. Cell 156, 428–439. doi: 10.1016/j.cell.2013.12.015 Amano, S., Akutsu, N., Ogura, Y., and Nishiyama, T. (2004). Increase of laminin 5
synthesis in human keratinocytes by acute wound fluid, inflammatory cytokines
and growth factors, and lysophospholipids. Br. J. Dermatol. 151, 961–970. doi:
10.1111/j.1365-2133.2004.06175.x Beck, S., Sehl, C., Voortmann, S., Verhasselt, H. L., Edwards, M. J., Buer, J., et al. (2020). Sphingosine is able to prevent and eliminate Staphylococcus epidermidis
biofilm formation on different orthopedic implant materials in vitro. J. Mol. Med. 98, 209–219. doi: 10.1007/s00109-019-01858-x Angeli, V., Faveeuw, C., Roye, O., Fontaine, J., Teissier, E., Capron, A., et al. (2001). REFERENCES Role of the parasite-derived prostaglandin D2 in the inhibition of epidermal January 2022 | Volume 12 | Article 804824 Frontiers in Physiology | www.frontiersin.org 18 Lipids Roles in Mammalian Epidermis Vietri Rudan and Watt Carson, D. D., and Daneo-Moore, L. (1980). Effects of fatty acids on lysis of
Streptococcus faecalis. J. Bacteriol. 141, 1122–1126. doi: 10.1128/jb.141.3.1122-
1126.1980 Beddoes, C. M., Rensen, D. E., Gooris, G. S., Malfois, M., and Bouwstra, J. A. (2021). The importance of free fatty chain length on the lipid organization in the long
periodicity phase. Int. J. Mol. Sci. 22:3679. doi: 10.3390/ijms22073679 Chamberlain, N. R., Mehrtens, B. G., Xiong, Z., Kapral, F. A., Boardman, J. L.,
and Rearick, J. I. (1991). Correlation of carotenoid production, decreased
membrane fluidity, and resistance to oleic acid killing in Staphylococcus aureus
18Z. Infect. Immun. 59, 4332–4337. doi: 10.1128/iai.59.12.4332-4337.1991 Behne, M., Uchida, Y., Seki, T., de Montellano, P. O., Elias, P. M., and Holleran,
W. M. (2000). Omega-Hydroxyceramides are required for Corneocyte Lipid
Envelope (CLE) formation and normal epidermal permeability barrier function. J. Invest. Dermatol. 114, 185–192. doi: 10.1046/j.1523-1747.2000.00846.x membrane fluidity, and resistance to oleic acid killing in Staphylococcus aureu
18Z. Infect. Immun. 59, 4332–4337. doi: 10.1128/iai.59.12.4332-4337.1991 18Z. Infect. Immun. 59, 4332–4337. doi: 10.1128/iai.59.12.4332-4337.1991 J. Invest. Dermatol. 114, 185–192. doi: 10.1046/j.1523-1747.2000.00846.x Bergsson, G., Arfinsson, J., Steingrímsson, Ó, and Thormar, H. (2001). Killing
of Gram-positive cocci by fatty acids and monoglyceridesNote. APMIS 109,
670–678. doi: 10.1034/j.1600-0463.2001.d01-131.x Champagne, A. M., Muñoz-Garcia, A., Shtayyeh, T., Tieleman, B. I., Hegemann,
A., Clement, M. E., et al. (2012). Lipid composition of the stratum corneum
and cutaneous water loss in birds along an aridity gradient. J. Exp. Biol. 215,
4299–4307. doi: 10.1242/jeb.077016 Bibel, D. J., Aly, R., and Shinefield, H. R. (1992). Antimicrobial activity
of sphingosines. J. Invest. Dermatol. 98, 269–273. doi: 10.1111/1523-1747. ep12497842 Cho, Y., and Ziboh, V. A. (1994a). Expression of protein kinase C isozymes
in guinea pig epidermis: selective inhibition of PKC-beta activity by 13-
hydroxyoctadecadienoic acid-containing diacylglycerol. J. Lipid Res. 35, 913–
921. doi: 10.1016/s0022-2275(20)39185-39189 Bibel, D. J., Aly, R., Shah, S., and Shinefield, H. R. (1993). Sphingosines:
antimicrobial barriers of the skin. Acta Derm-venereol. 73, 407–411. Cho, Y., and Ziboh, V. A. (1994b). Incorporation of 13-hydroxyoctadecadienoic
acid (13-HODE) into epidermal ceramides and phospholipids: phospholipase
C-catalyzed release of novel 13-HODE-containing diacylglycerol. J. Lipid Res. 35, 255–262. doi: 10.1016/s0022-2275(20)41214-41213 Black, A., Greaves, M., Hensby, C., and Plummer, N. (1978). REFERENCES Increased
prostaglandins E2 and F2alpha in human skin at 6 and 24 h after ultraviolet
B irradiation (290-320 nm). Br. J. Clin. Pharmacol. 5, 431–436. doi: 10.1111/j. 1365-2125.1978.tb01650.x Cho, Y., and Ziboh, V. A. (1995). Nutritional modulation of guinea pig skin
hyperproliferation by essential fatty acid deficiency is associated with selective
down regulation of protein kinase C-beta. J. Nutrition 125, 2741–2750. Bocos, C., Göttlicher, M., Gearing, K., Banner, C., Enmark, E., Teboul, M., et al. (1995). Fatty acid activation of peroxisome proliferator-activated receptor
(PPAR). J. Steroid Biochem. Mol. Biol. 53, 467–473. doi: 10.1016/0960-0760(95)
00093-f Cho, Y., and Ziboh, V. A. (1997). A novel 15-hydroxyeicosatrienoic acid-
substituted diacylglycerol (15-HETrE-DAG) selectively inhibits epidermal
protein kinase C-β. Biochim. Biophys. Acta 1349, 67–71. doi: 10.1016/s0005-
2760(97)00144-146 Börner, C., Smida, M., Höllt, V., Schraven, B., and Kraus, J. (2009). Cannabinoid
receptor Type 1- and 2-mediated increase in cyclic AMP inhibits T Cell
receptor-triggered signaling∗. J. Biol. Chem. 284, 35450–35460. doi: 10.1074/
jbc.m109.006338 Choi, H., Kim, S., Kim, H.-J., Kim, K.-M., Lee, C.-H., Shin, J. H., et al. (2010). Sphingosylphosphorylcholine down-regulates filaggrin gene transcription
through NOX5-based NADPH oxidase and cyclooxygenase-2 in human
keratinocytes. Biochem. Pharmacol. 80, 95–103. doi: 10.1016/j.bcp.2010. 03.009 Borst, P., Loos, J. A., Christ, E. J., and Slater, E. C. (1962). Uncoupling activity of
long-chian fatty acids. Biochim. Biophys. Acta 62, 509–518. doi: 10.1016/0006-
3002(62)90232-90239 Bouwstra, J. A., Dubbelaar, F. E., Gooris, G. S., and Ponec, M. (2000). The lipid
organisation in the skin barrier. Acta Derm-venereol. 80, 23–30. doi: 10.1080/
000155500750042826 Connelly, J. T., Gautrot, J. E., Trappmann, B., Tan, D. W.-M., Donati, G., Huck,
W. T. S., et al. (2010). Actin and serum response factor transduce physical cues
from the microenvironment to regulate epidermal stem cell fate decisions. Nat. Cell Biol. 12, 711–718. doi: 10.1038/ncb2074 Bouwstra, J. A., Gooris, G. S., Spek, J. A., van der, and Bras, W. (1991). Structural
investigations of human stratum corneum by small-angle x-ray scattering. J. Invest. Dermatol. 97, 1005–1012. doi: 10.1111/1523-1747.ep12492217 Connelly, J. T., Mishra, A., Gautrot, J. E., and Watt, F. M. (2011). Shape-Induced
terminal differentiation of human epidermal stem cells requires p38 and is
regulated by histone acetylation. PLoS One 6:e27259. doi: 10.1371/journal.pone. 0027259 Bouwstra, J. A., Gooris, G. S., Vries, M. A. S., van der Spek, J. A., and Bras, W. (1992). Structure of human stratum corneum as a function of temperature and
hydration: a wide-angle X-ray diffraction study. Int. J. Pharmaceut. REFERENCES 84, 205–216. doi: 10.1016/0378-5173(92)90158-x de Jager, M. W., Gooris, G. S., Dolbnya, I. P., Bras, W., Ponec, M., and Bouwstra,
J. A. (2003). The phase behaviour of skin lipid mixtures based on synthetic
ceramides. Chem. Phys. Lipids 124, 123–134. doi: 10.1016/s0009-3084(03)
00050-51 Bouwstra, J., Gooris, G., and Ponec, M. (2002). The lipid organisation of the skin
barrier: liquid and crystalline domains coexist in lamellar phases. J. Biol. Phys. 28, 211–223. doi: 10.1023/a:1019983715589 Demoyer, J. S., Skalak, T. C., and Durieux, M. E. (2000). Lysophosphatidic
acid
enhances
healing
of
acute
cutaneous
wounds
in
the
mouse. Wound
Repair
Regen. 8,
530–537. doi:
10.1046/j.1524-475x.2000.0
0530.x Bowser, P. A., Nugteren, D. H., White, R. J., Houtsmuller, U. M. T., and Prottey,
C. (1985). Identification, isolation and characterization of epidermal lipids
containing linoleic acid. Biochim. Biophys. Acta 834, 419–428. doi: 10.1016/
0005-2760(85)90016-90015 Breton, J., Woolf, D., Young, P., and Chabot-Fletcher, M. (1996). Human
keratinocytes lack the components to produce leukotriene B4. J. Invest. Dermatol. 106, 162–167. doi: 10.1111/1523-1747.ep12329890 Desbois, A. P., and Smith, V. J. (2010). Antibacterial free fatty acids: activities,
mechanisms of action and biotechnological potential. Appl. Microbiol. Biot. 85,
1629–1642. doi: 10.1007/s00253-009-2355-2353 Brogden, K. A., Drake, D. R., Dawson, D. V., Hill, J. R., Bratt, C. L., and
Wertz, P. W. (2011). “Antimicrobial lipids of the skin and oral mucosa,” in
Innate Immune System of Skin and Oral Mucosa: Properties and Impact in
Pharmaceutics, Cosmetics, and Personal Care Products, eds N. Dayan and P. W. Wertz (Hoboken, NJ: Wiley). Dietrich, C., Bagatolli, L. A., Volovyk, Z. N., Thompson, N. L., Levi, M., Jacobson,
K., et al. (2001). Lipid rafts reconstituted in model membranes. Biophys. J. 80,
1417–1428. doi: 10.1016/s0006-3495(01)76114-76110 Doepping, S., Funk, C. D., Habenicht, A. J. R., and Spanbroek, R. (2007). Selective
5-Lipoxygenase expression in langerhans cells and impaired dendritic cell
migration in 5-LO-Deficient mice reveal leukotriene action in skin. J. Invest. Dermatol. 127, 1692–1700. doi: 10.1038/sj.jid.5700796 Brogden, N. K., Mehalick, L., Fischer, C. L., Wertz, P. W., and Brogden, K. A. (2012). The emerging role of peptides and lipids as antimicrobial epidermal
barriers and modulators of local inflammation. Skin Pharmacol. Phys. 25,
167–181. doi: 10.1159/000337927 Dermatol. 127, 1692–1700. doi: 10.1038/sj.jid.5700796 Dorschner, R. A., Pestonjamasp, V. K., Tamakuwala, S., Ohtake, T., Rudisill, J.,
Nizet, V., et al. (2001). Cutaneous injury induces the release of cathelicidin anti-
microbial peptides active against group a Streptococcus. J. Invest. Dermatol. 117,
91–97. REFERENCES doi: 10.1111/1523-1747.ep12687968 Grayson, S., Johnson-Winegar, A. G., Wintroub, B. U., Isseroff, R. R., Epstein,
E. H., and Elias, P. M. (1985). Lamellar body-enriched fractions from neonatal
mice: preparative techniques and partial characterization. J. Invest. Dermatol. 85, 289–294. doi: 10.1111/1523-1747.ep12276826 Elias, P. M., Mauro, T., Rassner, U., Kömüves, L., Brown, B. E., Menon, G. K., et al. (1998). The secretory granular cell: the outermost granular cell as a specialized
secretory cell. J. Invest. Derm Symp. P 3, 87–100. doi: 10.1038/jidsymp.1998.20 Greenway, D. L. A., and Dyke, K. G. H. (1979). Mechanism of the inhibitory
action of linoleic acid on the growth of Staphylococcus aureus. Microbiology 115,
233–245. doi: 10.1099/00221287-115-1-233 Elias, P. M., Williams, M. L., Holleran, W. M., Jiang, Y. J., and Schmuth, M. (2008). Thematic review series: skin lipids. pathogenesis of permeability barrier
abnormalities in the ichthyoses: inherited disorders of lipid metabolism. J. Lipid
Res. 49, 697–714. doi: 10.1194/jlr.r800002-jlr200 Grice, E. A., and Segre, J. A. (2011). The skin microbiome. Nat. Rev. Microbiol. 9,
244–253. doi: 10.1038/nrmicro2537 Res. 49, 697–714. doi: 10.1194/jlr.r800002-jlr200 Grice, E. A., Kong, H. H., Conlan, S., Deming, C. B., Davis, J., Young, A. C., et al. (2009). Topographical and temporal diversity of the human skin microbiome. Science 324, 1190–1192. doi: 10.1126/science.1171700 Elias, P. M., Williams, M. L., Maloney, M. E., Bonifas, J. A., Brown, B. E., Grayson,
S., et al. (1984). Stratum corneum lipids in disorders of cornification. Steroid
sulfatase and cholesterol sulfate in normal desquamation and the pathogenesis
of recessive X-linked ichthyosis. J. Clin. Invest. 74, 1414–1421. doi: 10.1172/
jci111552 Grond, S., Eichmann, T. O., Dubrac, S., Kolb, D., Schmuth, M., Fischer, J., et al. (2017). PNPLA1 deficiency in mice and humans leads to a defect in the synthesis
of Omega-O-Acylceramides. J. Invest. Dermatol. 137, 394–402. doi: 10.1016/j. jid.2016.08.036 Falconer, A., Ikram, M., Bissett, C. E., Cerio, R., Quinn, A. G., and Ali, R. S. (2001). Expression of the peptide antibiotics human β Defensin-1 and Human
β Defensin-2 in normal human skin. J. Invest. Dermatol. 117, 106–111. doi:
10.1046/j.0022-202x.2001.01401.x Gutknecht, J. (1988). Proton conductance caused by long-chain fatty acids in
phospholipid bilayer membranes. J. Membr. Biol. 106, 83–93. doi: 10.1007/
bf01871769 j
Fischer, C. L. (2020). Antimicrobial activity of host-derived lipids. Antibiotics 9:75. doi: 10.3390/antibiotics9020075 Gutteridge, J. M. C., Lamport, P., and Dormandy, T. L. (1974). Autoxidation as
a cause of antibacterial activity in unsaturated fatty acids. J. Med. Microbiol. 7,
387–389. REFERENCES doi: 10.1046/j.1523-1747.2001.01340.x Bronfman, M., Morales, M. N., and Orellana, A. (1988). Diacylglycerol activation
of protein kinase C is modulated by long-chain acyl-CoA. Biochem. Biophys. Res. Co 152, 987–992. Dowd, P. M., Black, A. K., Woollard, P. M., Camp, R. D. R., and Greaves,
M. W. (1985). Cutaneous responses to 12-Hydroxy-5,8,10,14-eicosatetraenoic
Acid (12-HETE). J. Invest. Dermatol. 84, 537–541. doi: 10.1111/1523-1747. ep12273537 Burtenshaw, J. M. L. (1942). The mechanism of self-disinfection of the human
skin and its appendages. J. Hyg-cambridge 42, 184–210. doi: 10.1017/
s0022172400035373 Capolupo, L., Khven, I., Mazzeo, L., Glousker, G., Russo, F., Montoya, J. P., et al. (2021). Sphingolipid control of fibroblast heterogeneity revealed by single-cell
lipidomics. Biorxiv [preprint] doi: 10.1101/2021.02.23.432420 Downing, D. T., and Strauss, J. S. (1974). Synthesis and composition of surface
lipids of human skin. J. Invest. Dermatol. 62, 228–244. doi: 10.1111/1523-1747. ep12676793 January 2022 | Volume 12 | Article 804824 Frontiers in Physiology | www.frontiersin.org 19 Lipids Roles in Mammalian Epidermis Vietri Rudan and Watt Drake, D. R., Brogden, K. A., Dawson, D. V., and Wertz, P. W. (2008). Thematic
review series: skin lipids. antimicrobial lipids at the skin surface. J. Lipid Res. 49,
4–11. doi: 10.1194/jlr.r700016-jlr200 transformed human keratinocytes. J. Invest. Dermatol. 106, 154–161. doi: 10. 1111/1523-1747.ep12329856 Gomez-Larrauri, A., Presa, N., Dominguez-Herrera, A., Ouro, A., Trueba, M., and
Gomez-Muñoz, A. (2020). Role of bioactive sphingolipids in physiology and
pathology. Essays Biochem. 64, 579–589. doi: 10.1042/ebc20190091 Dréno, B., Dagnelie, M. A., Khammari, A., and Corvec, S. (2020). The skin
microbiome: a new actor in inflammatory acne. Am. J. Clin. Dermatol. 21,
18–24. doi: 10.1007/s40257-020-00531-531 Götz, F., Verheij, H. M., and Rosenstein, R. (1998). Staphylococcal lipases:
molecular characterisation, secretion, and processing. Chem. Phys. Lipids 93,
15–25. doi: 10.1016/s0009-3084(98)00025-25 Drochmans, P., Freudenstein, C., Wanson, J.-C., Laurent, L., Keenan, T. W.,
Stadler, J., et al. (1978). Structure and biochemical composition of desmosomes
and tonofilaments isolated from calf muzzle epidermis. J. Cell Biol. 79, 427–443. doi: 10.1083/jcb.79.2.427 Gray, G. M., King, I. A., and Yardley, H. J. (1980). The plasma membrane
of Malpighian cells from pig epidermis: isolation and lipid and protein
composition. Br. J. Dermatol. 103, 505–516. doi: 10.1111/j.1365-2133.1980. tb01665.x Elias, P. M., and Friend, D. S. (1975). The permeability barrier in mammalian
epidermis. J. Cell Biol. 65, 180–191. doi: 10.1083/jcb.65.1.180 Elias, P. M., Goerke, J., and Friend, D. S. (1977). Mammalian epidermal barrier
layer lipids: composition and influence on structure. J. Invest. Dermatol. 69,
535–546. REFERENCES doi: 10.1099/00222615-7-3-387 Fischer, C. L., Drake, D. R., Dawson, D. V., Blanchette, D. R., Brogden, K. A., and
Wertz, P. W. (2012a). Antibacterial activity of sphingoid bases and fatty acids
against gram-positive and gram-negative bacteria. Antimicrob Agents Ch 56,
1157–1161. doi: 10.1128/aac.05151-5111 Hachem, J.-P., Crumrine, D., Fluhr, J., Brown, B. E., Feingold, K. R., and Elias,
P. M. (2003). pH directly regulates epidermal permeability barrier homeostasis,
and stratum corneum integrity/cohesion. J. Invest. Dermatol. 121, 345–353. doi: 10.1046/j.1523-1747.2003.12365.x Fischer, C. L., Walters, K. S., Drake, D. R., Blanchette, D. R., Dawson, D. V.,
Brogden, K. A., et al. (2012b). Sphingoid bases are taken up by Escherichia coli
and Staphylococcus aureus and induce ultrastructural damage. Skin Pharmacol. Phys. 26, 36–44. doi: 10.1159/000343175 Hanley, K., Jiang, Y., He, S. S., Friedman, M., Elias, P. M., Bikle, D. D., et al. (1998). Keratinocyte differentiation is stimulated by activators of the nuclear hormone
receptor PPARα. J. Invest. Dermatol. 110, 368–375. doi: 10.1046/j.1523-1747. 1998.00139.x Fluhr, J. W., Kao, J., Ahn, S. K., Feingold, K. R., Elias, P. M., and Jain, M. (2001). Generation of free fatty acids from phospholipids regulates stratum corneum
acidification and integrity. J. Invest. Dermatol. 117, 44–51. doi: 10.1046/j.0022-
202x.2001.01399.x Hanley, K., Kömüves, L. G., Ng, D. C., Schoonjans, K., He, S. S., Lau, P., et al. (2000a). Farnesol stimulates differentiation in epidermal keratinocytes via
PPARα∗. J. Biol. Chem. 275, 11484–11491. doi: 10.1074/jbc.275.15.11484 Forslind, B., Lindberg, M., Malmqvist, K. G., Pallon, J., Roomans, G. M., and
Werner-Linde, Y. (1995). Human skin physiology studied by particle probe
microanalysis. Scann. Microscopy 9, 1011–1025. Hanley, K., Ng, D. C., He, S., Lau, P., Min, K., Elias, P. M., et al. (2000b). Oxysterols
induce differentiation in human keratinocytes and increase Ap-1-Dependent
involucrin transcription. J. Invest. Dermatol. 114, 545–553. doi: 10.1046/j.1523-
1747.2000.00895.x Freinkel, R. K., and Traczyk, T. N. (1985). Lipid composition and acid hydrolase
content of lamellar granules of fetal rat epidermis. J. Invest. Dermatol. 85,
295–298. doi: 10.1111/1523-1747.ep12276831 Hannun, Y. A. (1994). The sphingomyelin cycle and the second messenger function
of ceramide. J. Biol. Chem. 269, 3125–3128. doi: 10.1016/s0021-9258(17)41834-
41835 Fulton, C., Anderson, G. M., Zasloff, M., Bull, R., and Quinn, A. G. (1997). Expression of natural peptide antibiotics in human skin. Lancet 350, 1750–
1751. Hannun, Y. A., and Obeid, L. M. (2008). Principles of bioactive lipid signalling:
lessons from sphingolipids. Nat. Rev. Mol. Cell Biol. 9, 139–150. REFERENCES doi: 10.1038/
nrm2329 Galbraith, H., and Miller, T. B. (1973). Effect of long chain fatty acids on bacterial
respiration and amino acid uptake. J. Appl. Bacteriol. 36, 659–675. doi: 10.1111/
j.1365-2672.1973.tb04151.x Hannun, Y. A., Loomis, C. R., Merrill, A. H. Jr., and Bell, R. M. (1986). Sphingosine
inhibition of protein kinase C activity and of phorbol dibutyrate binding in vitro
and in human platelets. J. Biol. Chem. 261, 12604–12609. j
Gallo, R. L., and Nakatsuji, T. (2011). Microbial symbiosis with the innate immune
defense system of the skin. J. Invest. Dermatol. 131, 1974–1980. doi: 10.1038/jid. 2011.182 Hannun, Y. A., Luberto, C., and Argraves, K. M. (2001). Enzymes of sphingolipid
metabolism: from modular to integrative signaling †. Biochemistry 40, 4893–
4903. doi: 10.1021/bi002836k Geraldo, L. H. M., de Spohr, T. C. L. S., do Amaral, R. F., da Fonseca, A. C. C.,
Garcia, C., de Mendes, F. A., et al. (2021). Role of lysophosphatidic acid and its
receptors in health and disease: novel therapeutic strategies. Signal Trans. Target
Ther. 6:45. doi: 10.1038/s41392-020-00367-365 Hanyu, O., Nakae, H., Miida, T., Higashi, Y., Fuda, H., Endo, M., et al. (2012). Cholesterol sulfate induces expression of the skin barrier protein filaggrin in
normal human epidermal keratinocytes through induction of RORα. Biochem. Biophy. Res. Commun. 428, 99–104. doi: 10.1016/j.bbrc.2012.10.013 Cholesterol sulfate induces expression of the skin barrier protein filagg
normal human epidermal keratinocytes through induction of RORα. Bio
Biophy. Res. Commun. 428, 99–104. doi: 10.1016/j.bbrc.2012.10.013 p
y
g
Biophy. Res. Commun. 428, 99–104. doi: 10.1016/j.bbrc.2012.10.013 Gibson, D. F. C., Ratnam, A. V., and Bikle, D. D. (1996). Evidence for separate
control mechanisms at the message, protein, and enzyme activation levels
for transglutaminase during calcium-induced differentiation of normal and Hattori, T., Obinata, H., Ogawa, A., Kishi, M., Tatei, K., Ishikawa, O., et al. (2008). G2A plays proinflammatory roles in human keratinocytes under oxidative January 2022 | Volume 12 | Article 804824 Frontiers in Physiology | www.frontiersin.org 20 Lipids Roles in Mammalian Epidermis Vietri Rudan and Watt Kabara, J. J., Swieczkowski, D. M., Conley, A. J., and Truant, J. P. (1972). Fatty acids
and derivatives as antimicrobial agents. Antimicrob Agents Chemother 2, 23–28. doi: 10.1128/aac.2.1.23 stress as a receptor for 9-hydroxyoctadecadienoic acid. J. Invest. Dermatol. 128,
1123–1133. doi: 10.1038/sj.jid.5701172 stress as a receptor for 9-hydroxyoctadecadienoic acid. J. Invest. Dermatol. 128,
1123–1133. doi: 10.1038/sj.jid.5701172 Higuchi, K., Kawashima, M., Takagi, Y., Kondo, H., Yada, Y., Ichikawa, Y.,
et al. (2001). REFERENCES C., and Nicolaou, A. (2013). Bioactive lipid mediators in skin
inflammation and immunity. Prog. Lipid Res. 52, 141–164. doi: 10.1016/j. plipres.2012.10.003 Khnykin, D., Miner, J. H., and Jahnsen, F. (2011). Role of fatty acid transporters in
epidermis. Dermato-endocrinology 3, 53–61. doi: 10.4161/derm.3.2.14816 Ikenouchi, J. (2018). Roles of membrane lipids in the organization of epithelial
cells: old and new problems. Tissue Barriers 6, 1–8. doi: 10.1080/21688370.2018. 1502531 Kim, D., Kim, S., Moon, S., Chung, J., Kim, K., Cho, K., et al. (2001). Ceramide
inhibits cell proliferation through Akt/PKB inactivation and decreases melanin
synthesis in Mel-Ab Cells. Pigm Cell Res. 14, 110–115. doi: 10.1034/j.1600-0749. 2001.140206.x Imokawa, G., Takagi, Y., Higuchi, K., Kondo, H., and Yada, Y. (1999). Sphingosylphosphorylcholine is a potent inducer of intercellular adhesion
Molecule-1 expression in human keratinocytes. J. Invest. Dermatol. 112, 91–96. doi: 10.1046/j.1523-1747.1999.00462.x Kim, D.-S., Kim, S.-Y., Kleuser, B., Schäfer-Korting, M., Kim, K.-H., and Park, K.-C. (2004). Sphingosine-1-phosphate inhibits human keratinocyte proliferation via
Akt/protein kinase B inactivation. Cell. Signal. 16, 89–95. doi: 10.1016/s0898-
6568(03)00114-111 j
Irving, L., Schmidt-Nielsen, K., and Abrahamsen, N. S. B. (1957). On the melting
points of animal fats in cold climates. Physiol. Zool. 30, 93–105. doi: 10.1086/
physzool.30.2.30155356 Kim, S.-N., Akindehin, S., Kwon, H.-J., Son, Y.-H., Saha, A., Jung, Y.-S.,
et al. (2018). Anti-inflammatory role of 15-lipoxygenase contributes to the
maintenance of skin integrity in mice. Sci. Rep. 8:8856. doi: 10.1038/s41598-
018-27221-27227 Janes, S. M., Ofstad, T. A., Campbell, D. H., Eddaoudi, A., Warnes, G., Davies, D.,
et al. (2009). PI3-kinase-dependent activation of apoptotic machinery occurs on
commitment of epidermal keratinocytes to terminal differentiation. Cell Res. 19,
328–339. doi: 10.1038/cr.2008.281 Kim, Y.-I., Park, K., Kim, J. Y., Seo, H. S., Shin, K.-O., Lee, Y.-M., et al. (2014). An endoplasmic reticulum stress-initiated sphingolipid metabolite,
Ceramide-1-Phosphate, regulates epithelial innate immunity by stimulating
β-Defensin production. Mol. Cell. Biol. 34, 4368–4378. doi: 10.1128/mcb.005
99-514 Janes, S. M., Ofstad, T. A., Campbell, D. H., Watt, F. M., and Prowse, D. M. (2004). Transient activation of FOXN1 in keratinocytes induces a transcriptional
programme that promotes terminal differentiation: contrasting roles of FOXN1
and Akt. J. Cell Sci. 117, 4157–4168. doi: 10.1242/jcs.01302 Janowski, B. A., Willy, P. J., Devi, T. R., Falck, J. R., and Mangelsdorf, D. J. (1996). An oxysterol signalling pathway mediated by the nuclear receptor LXRα. Nature
383, 728–731. doi: 10.1038/383728a0 Klee, S. K., Farwick, M., and Lersch, P. (2007). REFERENCES Sphingosylphosphorylcholine is an activator of transglutaminase
activity in human keratinocytes. J. Lipid Res. 42, 1562–1570. doi: 10.1016/s0022-
2275(20)32209-32204 Kabara, J. J., Vrable, R., and Jie, M. S. F. L. K. (1977). Antimicrobial lipids: natural
and synthetic fatty acids and monoglycerides. Lipids 12, 753–759. doi: 10.1007/
bf02570908 Hirai, H., Tanaka, K., Yoshie, O., Ogawa, K., Kenmotsu, K., Takamori, Y., et al. (2001). Prostaglandin D2 selectively induces chemotaxis in T Helper Type
2 Cells, eosinophils, and basophils via seven-transmembrane receptor Crth2. J. Exp. Med. 193, 255–262. doi: 10.1084/jem.193.2.255 Kabashima, K., Nagamachi, M., Honda, T., Nishigori, C., Miyachi, Y., Tokura,
Y., et al. (2007). Prostaglandin E2 is required for ultraviolet B-induced skin
inflammation via EP2 and EP4 receptors. Lab. Invest. 87, 49–55. doi: 10.1038/
labinvest.3700491 Hiratsuka, T., Bordeu, I., Pruessner, G., and Watt, F. M. (2020). Regulation of
ERK basal and pulsatile activity control proliferation and exit from the stem
cell compartment in mammalian epidermis. Proc. Natl. Acad. Sci. U S A. 117,
17796–17807. doi: 10.1073/pnas.2006965117 Kabashima, K., Sakata, D., Nagamachi, M., Miyachi, Y., Inaba, K., and Narumiya,
S. (2003). Prostaglandin E2-EP4 signaling initiates skin immune responses by
promoting migration and maturation of Langerhans cells. Nat. Med. 9, 744–749. doi: 10.1038/nm872 Kalinin, A. E., Kajava, A. V., and Steinert, P. M. (2002). Epithelial barrier function:
assembly and structural features of the cornified cell envelope. Bioessays 24,
789–800. doi: 10.1002/bies.10144 Hong, J. H., Youm, J.-K., Kwon, M. J., Park, B. D., Lee, Y.-M., Lee, S.-I., et al. (2008). K6PC-5, a direct activator of sphingosine kinase 1, promotes epidermal
differentiation through intracellular Ca2+ signaling. J. Invest. Dermatol. 128,
2166–2178. doi: 10.1038/jid.2008.66 Kanda, N., Ishikawa, T., and Watanabe, S. (2010). Prostaglandin D2 induces
the production of human β-defensin-3 in human keratinocytes. Biochem. Pharmacol. 79, 982–989. doi: 10.1016/j.bcp.2009.11.012 Huang, W.-C., Tsai, T.-H., Chuang, L.-T., Li, Y.-Y., Zouboulis, C. C., and Tsai, P.-J. (2014). Anti-bacterial and anti-inflammatory properties of capric acid against
Propionibacterium acnes: a comparative study with lauric acid. J. Dermatol. Sci. 73, 232–240. doi: 10.1016/j.jdermsci.2013.10.010 Karsak, M., Gaffal, E., Date, R., Wang-Eckhardt, L., Rehnelt, J., Petrosino, S., et al. (2007). Attenuation of allergic contact dermatitis through the endocannabinoid
system. Science 316, 1494–1497. doi: 10.1126/science.1142265 Igawa, S., Choi, J. E., Wang, Z., Chang, Y.-L., Wu, C.-C., Werbel, T., et al. (2019). Human keratinocytes use sphingosine 1-Phosphate and its receptors
to communicate Staphylococcus aureus invasion and activate host defense. J. Invest. Dermatol. 139, 1743–1752.e5. doi: 10.1016/j.jid.2019.02.010. Kendall, A. REFERENCES “Acne and its therapy,” in Basic
and Clinical Dermatology, ed. G. F. Webster and A. V. Rawlings 155–165. doi: 10.3109/9781420018417.013 Janssen-Timmen, U., Vickers, P. J., Wittig, U., Lehmann, W. D., Stark, H.-J.,
Fusenig, N. E., et al. (1995). Expression of 5-lipoxygenase in differentiating
human skin keratinocytes. Proc. Natl. Acad. Sci. U S A. 92, 6966–6970. doi:
10.1073/pnas.92.15.6966 Knapp,
H. R.,
and
Melly,
M. A. (1986). Bactericidal
effects
of
polyunsaturated fatty acids. J. Infect. Dis. 154, 84–94. doi: 10.1093/infdis/15
4.1.84 Jeon, S., Song, J., Lee, D., Kim, G.-T., Park, S.-H., Shin, D.-Y., et al. (2020). Inhibition of sphingosine 1-phosphate lyase activates human keratinocyte
differentiation and attenuates psoriasis in mice. J. Lipid Res. 61, 20–32. doi:
10.1194/jlr.ra119000254 Kobayashi, T., Voisin, B., Kim, D. Y., Kennedy, E. A., Jo, J.-H., Shih, H.-Y.,
et al. (2019). Homeostatic control of sebaceous glands by innate lymphoid cells
regulates commensal bacteria equilibrium. Cell 176, 982–997.e16. doi: 10.1016/
j.cell.2018.12.031. Köberlin, M. S., Snijder, B., Heinz, L. X., Baumann, C. L., Fauster, A., Vladimer,
G. I., et al. (2015). A conserved circular network of coregulated lipids modulates
innate immune responses. Cell 162, 170–183. doi: 10.1016/j.cell.2015. 05.051 Jiang, Y. J., Kim, P., Uchida, Y., Elias, P. M., Bikle, D. D., Grunfeld, C., et al. (2013). Ceramides stimulate caspase-14 expression in human keratinocytes. Exp. Dermatol. 22, 113–118. doi: 10.1111/exd.12079 Jiang, Y. J., Lu, B., Kim, P., Paragh, G., Schmitz, G., Elias, P. M., et al. (2008). PPAR and LXR activators regulate ABCA12 expression in human keratinocytes. J. Invest. Dermatol. 128, 104–109. doi: 10.1038/sj.jid.5700944 Kodicek, E., and Worden, A. N. (1945). The effect of unsaturated fatty acids on
Lactobacillus helveticus and other Gram-positive micro-organisms. Biochem. J. 39, 78–85. doi: 10.1042/bj0390078 Jiang, Y. J., Lu, B., Tarling, E. J., Kim, P., Man, M.-Q., Crumrine, D., et al. (2010). Regulation of ABCG1 expression in human keratinocytes and murine
epidermis[S]. J. Lipid Res. 51, 3185–3195. doi: 10.1194/jlr.m006445 Konger, R. L., Billings, S. D., Thompson, A. B., Morimiya, A., Ladenson, J. H.,
Landt, Y., et al. (2005a). Immunolocalization of low-affinity prostaglandin E2
receptors, EP1 and EP2, in adult human epidermis. J. Invest. Dermatol. 124,
965–970. doi: 10.1111/j.0022-202x.2005.23658.x Jiang, Y. J., Uchida, Y., Lu, B., Kim, P., Mao, C., Akiyama, M., et al. (2009). Ceramide stimulates ABCA12 expression via peroxisome proliferator-activated
receptor δ in human keratinocytes∗. J. Biol. Chem. 284, 18942–18952. doi:
10.1074/jbc.m109.006973 Konger, R. L., Brouxhon, S., Partillo, S., VanBuskirk, J., and Pentland, A. P. (2005b). REFERENCES Comprehensive quantification of ceramide species in human stratum corneum. J. Lipid Res. 50, 1708–1719. doi: 10.1194/jlr.d800055-jlr200 Matoltsy, A. G., and Parakkal, P. F. (1965). Membrane-Coating granules
of keratinizing epithelia. J. Cell Biol. 24, 297–307. doi: 10.1083/jcb.24
.2.297 Lee, D.-Y., Huang, C.-M., Nakatsuji, T., Thiboutot, D., Kang, S.-A., Monestier, M.,
et al. (2009). Histone H4 is a major component of the antimicrobial action of
human sebocytes. J. Invest. Dermatol. 129, 2489–2496. doi: 10.1038/jid.2009. 106 Mazereeuw-Hautier, J., Gres, S., Fanguin, M., Cariven, C., Fauvel, J., Perret, B., et al. (2005). Production of lysophosphatidic acid in blister fluid: involvement of a
lysophospholipase D activity. J. Invest. Dermatol. 125, 421–427. doi: 10.1111/j. 0022-202x.2005.23855.x Lei, L., Su, J., Chen, J., Chen, W., Chen, X., and Peng, C. (2018). The role of
lysophosphatidic acid in the physiology and pathology of the skin. Life Sci. 220,
194–200. doi: 10.1016/j.lfs.2018.12.040 Mishra, A., Oules, B., Pisco, A. O., Ly, T., Liakath-Ali, K., Walko, G., et al. (2017). A protein phosphatase network controls the temporal and spatial dynamics of
differentiation commitment in human epidermis. eLife 6:e27356. doi: 10.7554/
elife.27356 Leonti, M., Casu, L., Raduner, S., Cottiglia, F., Floris, C., Altmann, K.-H., et al. (2010). Falcarinol is a covalent cannabinoid CB1 receptor antagonist and
induces pro-allergic effects in skin. Biochem. Pharmacol. 79, 1815–1826. doi:
10.1016/j.bcp.2010.02.015 Levental, I., Levental, K. R., and Heberle, F. A. (2020). Lipid rafts: controversies
resolved, mysteries remain. Trends Cell Biol. 30, 341–353. doi: 10.1016/j.tcb. 2020.01.009 Mobasseri, S. A., Zijl, S., Salameti, V., Walko, G., Stannard, A., Garcia-Manyes, S.,
et al. (2019). Patterning of human epidermal stem cells on undulating elastomer
substrates reflects differences in cell stiffness. Acta Biomater. 87, 256–264. doi:
10.1016/j.actbio.2019.01.063 Lewis, R. A., Soter, N. A., Diamond, P. T., Austen, K. F., Oates, J. A., and Roberts,
L. J. (1982). Prostaglandin D2 generation after activation of rat and human mast
cells with anti-IgE. J. Immunol. 129, 1627–1631. Molleyres, L. P., and Rando, R. R. (1988). Structural studies on the diglyceride-
mediated activation of protein kinase C. J. Biol. Chem. 263, 14832–14838. Li, Z., Cheng, S., Lin, Q., Cao, W., Yang, J., Zhang, M., et al. (2021). Single-cell
lipidomics with high structural specificity by mass spectrometry. Nat. Commun. 12:2869. doi: 10.1038/s41467-021-23161-23165 Mori, T., Takai, Y., Yu, B., Takahashi, J., Nishizuka, Y., and Fujikura, T. (1982). Specificity of the fatty acyl moieties of diacylglycerol for the activation of
calcium-activated, phospholipid-dependent protein Kinase1. J. Biochem. 91,
427–432. REFERENCES The EP3 receptor stimulates ceramide and diacylglycerol release and inhibits January 2022 | Volume 12 | Article 804824 Frontiers in Physiology | www.frontiersin.org 21 Lipids Roles in Mammalian Epidermis Vietri Rudan and Watt growth of primary keratinocytes. Exp. Dermatol. 14, 914–922. doi: 10.1111/j. 1600-0625.2005.00381.x Madison, K. C., Swartzendruber, D. C., Wertz, P. W., and Downing, D. T. (1987). Presence of intact intercellular lipid lamellae in the upper layers of the stratum
corneum. J. Invest. Dermatol. 88, 714–718. doi: 10.1111/1523-1747.ep12470386 growth of primary keratinocytes. Exp. Dermatol. 14, 914–922. doi: 10.1111/j. 1600-0625.2005.00381.x Konger, R. L., Malaviya, R., and Pentland, A. P. (1998). Growth regulation
of primary human keratinocytes by prostaglandin E receptor EP2 and EP3
subtypes. Biochim. Biophys. Acta 1401, 221–234. Man, M.-Q., Choi, E.-H., Schmuth, M., Crumrine, D., Uchida, Y., Elias, P. M.,
et al. (2006). Basis for improved permeability barrier homeostasis induced by
PPAR and LXR activators: liposensors stimulate lipid synthesis, lamellar body
secretion, and post-secretory lipid processing. J. Invest. Dermatol. 126, 386–392. doi: 10.1038/sj.jid.5700046 Kooyman, D. J. (1932). Lipids of the skin. Some changes in the lipids of the
epidermis during the process of keratinization. Arch. Dermatol. Syph. 25,
444–450. doi: 10.1001/archderm.1932.01450020460003 Mao-Qiang, M., Fowler, A. J., Schmuth, M., Lau, P., Chang, S., Brown, B. E.,
et al. (2004). Peroxisome-Proliferator-Activated Receptor (PPAR)-γ activation
stimulates keratinocyte differentiation. J. Invest. Dermatol. 123, 305–312. doi:
10.1111/j.0022-202x.2004.23235.x Ladd, P. A., Du, L., Capdevila, J. H., Mernaugh, R., and Keeney, D. S. (2003). Epoxyeicosatrienoic acids activate transglutaminases in situ and induce
cornification of epidermal keratinocytes∗. J. Biol. Chem. 278, 35184–35192. doi: 10.1074/jbc.m301666200 Lampe, M. A., Williams, M. L., and Elias, P. M. (1983). Human epidermal
lipids: characterization and modulations during differentiation. J. Lipid Res. 24,
131–140. Marples, R. R., Downing, D. T., and Kligman, A. M. (1971). Control of free fatty
acids in human surface lipids by corynebacterium acnes. J. Invest. Dermatol. 56,
127–131. doi: 10.1111/1523-1747.ep12260695 Marsh, N. L., Elias, P. M., and Holleran, W. M. (1995). Glucosylceramides stimulate
murine epidermal hyperproliferation. J. Clin. Invest. 95, 2903–2909. doi: 10. 1172/jci117997 Landau, J. W. (1983). Commentary: undecylenic acid and fungous infections. Arch. Dermatol. 119, 351–353. doi: 10.1001/archderm.1983.01650280079021 Landmann, L. (1986). Epidermal permeability barrier: transformation of lamellar
granule-disks into intercellular sheets by a membrane-fusion process, a freeze-
fracture study. J. Invest. Dermatol. 87, 202–209. doi: 10.1111/1523-1747. ep12695343 Masukawa, Y., Narita, H., Sato, H., Naoe, A., Kondo, N., Sugai, Y., et al. (2009). REFERENCES Liakath-Ali, K., Vancollie, V. E., Lelliott, C. J., Speak, A. O., Lafont, D., Protheroe,
H. J., et al. (2016). Alkaline ceramidase 1 is essential for mammalian skin
homeostasis and regulating whole-body energy expenditure. J. Pathol. 239,
374–383. doi: 10.1002/path.4737 Müller, H., Hofer, S., Kaneider, N., Neuwirt, H., Mosheimer, B., Mayer, G., et al. (2005). The immunomodulator FTY720 interferes with effector functions of
human monocyte-derived dendritic cells. Eur. J. Immunol. 35, 533–545. doi:
10.1002/eji.200425556 Lichte, K., Rossi, R., Danneberg, K., ter Braak, M., Kürschner, U., Jakobs, K. H.,
et al. (2008). Lysophospholipid receptor-mediated calcium signaling in human
keratinocytes. J. Invest. Dermatol. 128, 1487–1498. doi: 10.1038/sj.jid.5701207 Muro, E., Atilla-Gokcumen, G. E., and Eggert, U. S. (2014). Lipids in cell biology:
how can we understand them better? Mol. Biol. Cell 25, 1819–1823. doi: 10. 1091/mbc.e13-09-0516 Long, V. J. W. (1970). Variations in lipid composition at different depths in the
cow snout epidermis. J. Invest. Dermatol. 55, 269–273. doi: 10.1111/1523-1747. ep12259974 Nakatsuji, T., Kao, M. C., Fang, J.-Y., Zouboulis, C. C., Zhang, L., Gallo, R. L., et al. (2009). Antimicrobial property of lauric acid against propionibacterium acnes:
its therapeutic potential for inflammatory acne vulgaris. J. Invest. Dermatol. 129,
2480–2488. doi: 10.1038/jid.2009.93 Lovászi, M., Szegedi, A., Zouboulis, C. C., and Törõcsik, D. (2017). Sebaceous-
immunobiology is orchestrated by sebum lipids. Dermato-endocrinology
9:e1375636. doi: 10.1080/19381980.2017.1375636 Natsuga, K., Cipolat, S., and Watt, F. M. (2016). Increased bacterial load and
expression of antimicrobial peptides in skin of barrier-deficient mice with
reduced cancer susceptibility. J. Invest. Dermatol. 136, 99–106. doi: 10.1038/jid. 2015.383 Lu, B., Jiang, Y. J., Kim, P., Moser, A., Elias, P. M., Grunfeld, C., et al. (2010). Expression and regulation of GPAT isoforms in cultured human keratinocytes
and rodent epidermis. J. Lipid Res. 51, 3207–3216. doi: 10.1194/jlr.m00
7054 Nekrasova, O., Harmon, R. M., Broussard, J. A., Koetsier, J. L., Godsel, L. M.,
Fitz, G. N., et al. (2018). Desmosomal cadherin association with Tctex-1
and cortactin-Arp2/3 drives perijunctional actin polymerization to promote
keratinocyte delamination. Nat. Commun. 9:1053. doi: 10.1038/s41467-018-
03414-3416 Maccarrone, M., Rienzo, M. D., Battista, N., Gasperi, V., Guerrieri, P., Rossi,
A., et al. (2003). The endocannabinoid system in human keratinocytes -
evidence that anandamide inhibits epidermal differentiation through CB1
receptor-dependent inhibition of Protein Kinase C, Activating Protein-1,
and transglutaminase. J. Biol. Chem. 278, 33896–33903. doi: 10.1074/jbc. m303994200 Nemes, Z., and Steinert, P. M. (1999). Bricks and mortar of the epidermal barrier. Exp. Mol. Med. 31, 5–19. REFERENCES Skin cell heterogeneity in development,
wound healing, and Cancer. Trends Cell Biol. 28, 709–722. doi: 10.1016/j.tcb. 2018.05.002 Paller, A. S., Arnsmeier, S. L., Alvarez-Franco, M., and Bremer, E. G. (1993). Ganglioside GM3 inhibits the proliferation of cultured keratinocytes. J. Invest. Dermatol. 100, 841–845. doi: 10.1111/1523-1747.ep12476755 Rosse, C., Linch, M., Kermorgant, S., Cameron, A. J. M., Boeckeler, K., and Parker,
P. J. (2010). PKC and the control of localized signal dynamics. Nat. Rev. Mol. Cell Biol. 11, 103–112. doi: 10.1038/nrm2847 Pappas, A. (2009). Epidermal surface lipids. Dermato-endocrinology 1, 72–76. doi:
10.4161/derm.1.2.7811 Rothman, S., Smijanic, A. M., and Weitkamp, A. W. (1946). Mechanism of
spontaneous cure in puberty of ringworm of the scalp. Science 104, 201–203. doi: 10.1126/science.104.2696.201 Park, K., Elias, P. M., Shin, K.-O., Lee, Y.-M., Hupe, M., Borkowski, A. W., et al. (2013). A novel role of a lipid species, Sphingosine-1-Phosphate, in epithelial
innate immunity. Mol. Cell. Biol. 33, 752–762. doi: 10.1128/mcb.01103-1112 (2013). A novel role of a lipid species, Sphingosine-1-Phosphate, in epithelial
innate immunity. Mol. Cell. Biol. 33, 752–762. doi: 10.1128/mcb.01103-1112 Russell, L. E., Harrison, W. J., Bahta, A. W., Zouboulis, C. C., Burrin, J. M., and
Philpott, M. P. (2007). Characterization of liver X receptor expression and
function in human skin and the pilosebaceous unit. Exp. Dermatol. 16, 844–852. doi: 10.1111/j.1600-0625.2007.00612.x Park, K., Ikushiro, H., Seo, H. S., Shin, K.-O., Kim, Y., Kim, J. Y., et al. (2016). ER stress stimulates production of the key antimicrobial peptide, cathelicidin,
by forming a previously unidentified intracellular S1P signaling complex. Proc. Natl. Acad. Sci. U S A. 113, E1334–E1342. doi: 10.1073/pnas.1504555113 atl. Acad. Sci. U S A. 113, E1334–E1342. doi: 10.1073/pnas.150455 Sado-Kamdem, S. L., Vannini, L., and Guerzoni, M. E. (2009). Effect of α-linolenic,
capric and lauric acid on the fatty acid biosynthesis in Staphylococcus aureus. Int. J. Food Microbiol. 129, 288–294. doi: 10.1016/j.ijfoodmicro.2008.12.010 Pavicic, T., Wollenweber, U., Farwick, M., and Korting, H. C. (2007). Anti-
microbial and -inflammatory activity and efficacy of phytosphingosine: an
in vitro and in vivo study addressing acne vulgaris. Int. J. Cosmetic Sci. 29,
181–190. doi: 10.1111/j.1467-2494.2007.00378.x Saito, H., Tomioka, H., and Yoneyama, T. (1984). Growth of group IV
mycobacteria on medium containing various saturated and unsaturated fatty
acids. Antimicrob Agents Chemother 26, 164–169. doi: 10.1128/aac.26.2.164 Peters, F., Tellkamp, F., Brodesser, S., Wachsmuth, E., Tosetti, B., Karow, U., et al. (2020). Murine epidermal ceramide synthase 4 is a key regulator of skin barrier
homeostasis. J. Invest. REFERENCES A., and Hunter, I. (2008). The multi-functional role of
sphingosylphosphorylcholine. Prog. Lipid Res. 47, 62–75. doi: 10.1016/j.plipres. 2007.11.001 Rhodes, L. E., Belgi, G., Parslew, R., McLoughlin, L., Clough, G. F., and Friedmann,
P. S. (2001). Ultraviolet-B-Induced erythema is mediated by nitric oxide and
prostaglandin E2 in combination. J. Invest. Dermatol. 117, 880–885. doi: 10. 1046/j.0022-202x.2001.01514.x Norlén, L., Al-Amoudi, A., and Dubochet, J. (2003). A cryotransmission electron
microscopy study of skin barrier formation. J. Invest. Dermatol. 120, 555–560. doi: 10.1046/j.1523-1747.2003.12102.x Odland, G. F. (1960). A submicroscopic granular component in human epidermis∗
∗from the department of anatomy, University of Washington, Seattle,
Washington. J. Invest. Dermatol. 34, 11–15. doi: 10.1038/jid.1960.4 Rhodes, L. E., Gledhill, K., Masoodi, M., Haylett, A. K., Brownrigg, M., Thody,
A. J., et al. (2009). The sunburn response in human skin is characterized by
sequential eicosanoid profiles that may mediate its early and late phases. FASEB
J. 23, 3947–3956. doi: 10.1096/fj.09-136077 Ogawa, E., Owada, Y., Ikawa, S., Adachi, Y., Egawa, T., Nemoto, K., et al. (2011). Epidermal FABP (FABP5) regulates keratinocyte differentiation by
13(S)-HODE-mediated activation of the NF-κB signaling pathway. J. Invest. Dermatol. 131, 604–612. doi: 10.1038/jid.2010.342 Rivier, M., Safonova, I., Lebrun, P., Michel, S., Griffiths, C. E. M., and Ailhaud,
G. (1998). Differential expression of peroxisome proliferator-activated receptor
subtypes during the differentiation of human keratinocytes. J. Invest. Dermatol. 111, 1116–1121. doi: 10.1046/j.1523-1747.1998.00439.x Oizumi, A., Nakayama, H., Okino, N., Iwahara, C., Kina, K., Matsumoto, R., et al. (2014). Pseudomonas-Derived ceramidase induces production of inflammatory
mediators from human keratinocytes via Sphingosine-1-Phosphate. PLoS One
9:e89402. doi: 10.1371/journal.pone.0089402 Rivier, M., Safonova, I., Michel, S., Castiel, I., and Ailhaud, G. (2000). Peroxisome
proliferator-activated receptor-α enhances lipid metabolism in a skin equivalent
model. J. Invest. Dermatol. 114, 681–687. doi: 10.1046/j.1523-1747.2000.0
0939.x Opálka, L., Kovaàˇcik, A., Maixner, J., and Vaìvrovaì, K. (2016). Omega-O-
Acylceramides in skin lipid membranes: effects of concentration, sphingoid
base, and model complexity on microstructure and permeability. Langmuir 32,
12894–12904. doi: 10.1021/acs.langmuir.6b03082 Robbiani, D. F., Finch, R. A., Jäger, D., Muller, W. A., Sartorelli, A. C., and
Randolph, G. J. (2000). The leukotriene C4 transporter MRP1 regulates CCL19
(MIP-3β, ELC)-dependent mobilization of dendritic cells to lymph nodes. Cell
103, 757–768. Oulès, B., Philippeos, C., Segal, J., Tihy, M., Rudan, M. V., Cujba, A.-M.,
et al. (2020). Contribution of GATA6 to homeostasis of the human upper
pilosebaceous unit and acne pathogenesis. Nat. Commun. 11:5067. doi: 10.1038/
s41467-020-18784-z Rognoni, E., and Watt, F. M. (2018). REFERENCES doi: 10.1038/emm.1999.2 Maciejewski-Lenoir, D., Richman, J. G., Hakak, Y., Gaidarov, I., Behan, D. P., and
Connolly, D. T. (2006). Langerhans cells release prostaglandin D2 in response
to nicotinic acid. J. Invest. Dermatol. 126, 2637–2646. doi: 10.1038/sj.jid.570
0586 Nemes, Z., Marekov, L. N., Fésüs, L., and Steinert, P. M. (1999). A novel function
for transglutaminase 1: attachment of long-chain ω-hydroxyceramides to
involucrin by ester bond formation. Proc. Natl. Acad. Sci. U S A. 96, 8402–8407. doi: 10.1073/pnas.96.15.8402 January 2022 | Volume 12 | Article 804824 Frontiers in Physiology | www.frontiersin.org 22 Lipids Roles in Mammalian Epidermis Vietri Rudan and Watt and differentiation in cultured human keratinocytes and mouse skin. Exp. Cell
Res. 216, 51–64. doi: 10.1006/excr.1995.1007 Nicolaides, N. (1974). Skin lipids: their biochemical uniqueness. Science 186, 19–26. doi: 10.1126/science.186.4158.19 Nicolaides, N., and Wells, G. C. (1957). On the biogenesis of the free fatty acids in
human skin surface fat∗. J. Invest. Dermatol. 29, 423–433. doi: 10.1038/jid.1957. 118 Piotrowska, A., Wierzbicka, J., and ˙Zmijewski, M. A. (2016). Vitamin D in the skin
physiology and pathology. Acta Biochim. Pol. 63, 1104–1129. doi: 10.18388/abp. 2015_1104 Nicolaides, N., Fu, H. C., and Rice, G. R. (1968). The skin surface lipids of man
compared with those of eighteen species of animals. J. Invest. Dermatol. 51,
83–89. doi: 10.1038/jid.1968.96 Proksch, E., Brandner, J. M., and Jensen, J. (2008). The skin: an indispensable
barrier. Exp. Dermatol. 17, 1063–1072. doi: 10.1111/j.1600-0625.2008.00786.x Pucci, M., Pasquariello, N., Battista, N., Tommaso, M. D., Rapino, C., Fezza, F.,
et al. (2012). Endocannabinoids stimulate human melanogenesis via Type-1
cannabinoid receptor∗. J. Biol. Chem. 287, 15466–15478. doi: 10.1074/jbc.m111. 314880 Nikaido, H. (1976). Outer membrane of Salmonella typhimurium transmembrane
diffusion of some hydrophobic substances. Biochim. Biophys. Acta 433, 118–
132. Nikkari, T. (1974). Comparative chemistry of sebum. J. Invest. Dermatol. 62,
257–267. doi: 10.1111/1523-1747.ep12676800 Puhvel, S. M., Reisner, R. M., and Sakamoto, M. (1975). Analysis of lipid
composition of isolated human sebaceous gland homogenates after incubation
with cutaneous bacteria. thin-layer chromatography. J. Invest Dermatol. 64,
406–411. doi: 10.1111/1523-1747.ep12512337 Nishizuka, Y. (1992). Intracellular signaling by hydrolysis of phospholipids and
activation of protein kinase C. Science 258, 607–614. doi: 10.1126/science. 1411571 406–411. doi: 10.1111/1523-1747.ep12512337 Reines, I., Kietzmann, M., Mischke, R., Tschernig, T., Lüth, A., Kleuser, B.,
et al. (2009). Topical application of Sphingosine-1-Phosphate and FTY720
attenuate allergic contact dermatitis reaction through inhibition of dendritic
cell migration. J. Invest. Dermatol. 129, 1954–1962. doi: 10.1038/jid.2008.454 Nixon, G. F., Mathieson, F. REFERENCES Dermatol. 140, 1927–1937.e5. doi: 10.1016/j.jid.2020.02. 006. Samsonov, A. V., Mihalyov, I., and Cohen, F. S. (2001). Characterization of
cholesterol-sphingomyelin domains and their dynamics in bilayer membranes. Biophys. J. 81, 1486–1500. Peters, F., Vorhagen, S., Brodesser, S., Jakobshagen, K., Brüning, J. C., Niessen,
C. M., et al. (2015). Ceramide synthase 4 regulates stem cell homeostasis and
hair follicle cycling. J. Invest. Dermatol. 135, 1501–1509. doi: 10.1038/jid.2015. 60 Sato, J., Denda, M., Nakanishi, J., Nomura, J., and Koyama, J. (1998). Cholesterol
sulfate inhibits proteases that are involved in desquamation of stratum
corneum. J. Invest. Dermatol. 111, 189–193. doi: 10.1046/j.1523-1747.1998. 00244.x Piazza, G. A., Ritter, J. L., and Baracka, C. A. (1995). Lysophosphatidic acid
induction of transforming growth factors α and β: modulation of proliferation Sauer, B., Vogler, R., Wenckstern, H., von, Fujii, M., Anzano, M. B., et al. (2004). Involvement of smad signaling in sphingosine 1-Phosphate-mediated biological January 2022 | Volume 12 | Article 804824 Frontiers in Physiology | www.frontiersin.org 23 Lipids Roles in Mammalian Epidermis Vietri Rudan and Watt responses of keratinocytes∗. J. Biol. Chem. 279, 38471–38479. doi: 10.1074/jbc. m313557200 responses of keratinocytes∗. J. Biol. Chem. 279, 38471–38479. doi: 10.1074/jbc. m313557200 Swartzendruber, D. C., Wertz, P. W., Kitko, D. J., Madison, K. C., and Downing,
D. T. (1989). Molecular models of the intercellular lipid lamellae in mammalian
stratum corneum. J. Invest. Dermatol. 92, 251–257. doi: 10.1111/1523-1747. ep12276794 Schade, H., and Marchionini, A. (1928). Der Säuremantel der Haut (Nach
Gaskettenmessungen). Klin Wochenschr 7, 12–14. doi: 10.1007/bf01711684 p
Swartzendruber, D. C., Wertz, P. W., Madison, K. C., and Downing, D. T. (1987). Evidence that the corneocyte has a chemically bound lipid envelope. J. Invest. Swartzendruber, D. C., Wertz, P. W., Madison, K. C., and Downing, D. T. (1987). Evidence that the corneocyte has a chemically bound lipid envelope. J. Invest. Dermatol. 88, 709–713. doi: 10.1111/1523-1747.ep12470383 Scheimann, L. G., Knox, G., Sher, D., and Rothman, S. (1960). The role of bacteria
in the formation of free fatty acids on the human skin surface. J. Invest. Dermatol. 34, 171–174. doi: 10.1038/jid.1960.23 Dermatol. 88, 709–713. doi: 10.1111/1523-1747.ep12470383 Szyma´nski, Ł, Skopek, R., Palusi´nska, M., Schenk, T., Stengel, S., Lewicki, S., et al. Schmitt, T., Lange, S., Sonnenberger, S., Dobner, B., Demé, B., Langner, A., et al. (2019). REFERENCES Endocannabinoids modulate human epidermal keratinocyte
proliferation and survival via the sequential engagement of cannabinoid
Receptor-1 and transient receptor potential Vanilloid-1. J. Invest. Dermatol. 131,
1095 1104 d i 10 1038/jid 2010 421 Selby, C. C. (1957). An electron microscope study of thin sections of human skin
II. superficial cell layers of footpad epidermis 1. J. Invest. Dermatol. 29, 131–149. doi: 10.1038/jid.1957.80 Tsuji, K., Mitsutake, S., Yokose, U., Sugiura, M., Kohama, T., and Igarashi, Y. (2008). Role of ceramide kinase in peroxisome proliferator-activated receptor
beta-induced cell survival of mouse keratinocytes. FEBS J. 275, 3815–3826. doi: 10.1111/j.1742-4658.2008.06527.x Sheu, C. W., and Freese, E. (1972). Effects of fatty acids on growth and envelope
proteins of Bacillus subtilis. J. Bacteriol. 111, 516–524. doi: 10.1128/jb.111.2.516-
524.1972 Shinomura, T., Asaoka, Y., Oka, M., Yoshida, K., and Nishizuka, Y. (1991). Synergistic action of diacylglycerol and unsaturated fatty acid for protein
kinase C activation: its possible implications. Proc. Natl. Acad. Sci. U S A. 88,
5149–5153. doi: 10.1073/pnas.88.12.5149 Uchida, Y. (2014). Ceramide signaling in mammalian epidermis. Biochim. Biophys. Acta 1841, 453–462. doi: 10.1016/j.bbalip.2013.09.003 Uchida, Y., and Park, K. (2021). Ceramides in skin health and disease: an update. Am. J. Clin. Dermatol. 22, 853–866. doi: 10.1007/s40257-021-00619-612 Simons, K., and Sampaio, J. L. (2011). Membrane organization and lipid rafts. Cold
Spring Harb. Perspect. Biol. 3:a004697. doi: 10.1101/cshperspect.a004697 Uchida, Y., Nardo, A. D., Collins, V., Elias, P. M., and Holleran, W. M. (2003). De novo ceramide synthesis participates in the ultraviolet B irradiation-induced
apoptosis in undifferentiated cultured human keratinocytes. J. Invest. Dermatol. 120, 662–669. doi: 10.1046/j.1523-1747.2003.12098.x Školová, B., Kováˇcik, A., Tesaˇr, O., Opálka, L., and Vávrová, K. (2017). Phytosphingosine, sphingosine and dihydrosphingosine ceramides in model
skin lipid membranes: permeability and biophysics. BBA - Biomembranes 1859,
824–834. doi: 10.1016/j.bbamem.2017.01.019 120, 662–669. doi: 10.1046/j.1523-1747.2003.12098.x Vahlquist, A., and Törmä, H. (2020). Ichthyosis: a road model for skin research. Acta Dermato Venereol. 100:adv00097. doi: 10.2340/00015555-13433 Smita, K., Kumar, V. S., and Premendran, J. S. (2007). Anandamide: an update. Fundam. Clin. Pharm. 21, 1–8. doi: 10.1111/j.1472-8206.2006.00454.x van Corven, E. J., van Rijswijk, A., Jalink, K., van der Bend, R. L., van Blitterswijk,
W. J., and Moolenaar, W. H. (1992). Mitogenic action of lysophosphatidic acid
and phosphatidic acid on fibroblasts. dependence on acyl-chain length and
inhibition by suramin. Biochem. J. 281, 163–169. doi: 10.1042/bj2810163 Spik, I., Brénuchon, C., Angéli, V., Staumont, D., Fleury, S., Capron, M., et al. (2005). REFERENCES The long periodicity phase (LPP) controversy part I: the influence of
a natural-like ratio of the CER[EOS] analogue [EOS]-br in a CER[NP]/[AP]
based stratum corneum modelling system: a neutron diffraction study. Biochim. Biophys. Acta 1861, 306–315. doi: 10.1016/j.bbamem.2018.06.008 Szyma´nski, Ł, Skopek, R., Palusi´nska, M., Schenk, T., Stengel, S., Lewicki, S., et al. (2020). Retinoic acid and its derivatives in skin. Cells 9:2660. doi: 10.3390/ Szyma´nski, Ł, Skopek, R., Palusi´nska, M., Schenk, T., Stengel, S., Lewicki, S., et al. (2020). Retinoic acid and its derivatives in skin. Cells 9:2660. doi: 10.3390/
cells9122660 (2020). Retinoic acid and its derivatives in skin. Cells 9:2660. doi: 10.3390/
cells9122660 Takigawa, H., Nakagawa, H., Kuzukawa, M., Mori, H., and Imokawa, G. (2005). Deficient production of hexadecenoic acid in the skin is associated in part with
the vulnerability of atopic dermatitis patients to colonization by Staphylococcus
aureus. Dermatology 211, 240–248. doi: 10.1159/000087018 Schmuth, M., Haqq, C. M., Cairns, W. J., Holder, J. C., Dorsam, S., Chang, S.,
et al. (2004). Peroxisome proliferator-activated receptor (PPAR)-β/δ stimulates
differentiation and lipid accumulation in keratinocytes. J. Invest. Dermatol. 122,
971–983. doi: 10.1111/j.0022-202x.2004.22412.x aureus. Dermatology 211, 240–248. doi: 10.1159/000087018 Theiner, G., Gessner, A., and Lutz, M. B. (2006). The mast cell mediator PGD2
suppresses IL-12 release by dendritic cells leading to Th2 polarized immune
responses in vivo. Immunobiology 211, 463–472. doi: 10.1016/j.imbio.2006.05. 020 Schüppel, M., Kürschner, U., Kleuser, U., Schäfer-Korting, M., and Kleuser,
B. (2008). Sphingosine 1-Phosphate restrains insulin-mediated keratinocyte
proliferation via inhibition of Akt through the S1P2 receptor subtype. J. Invest. Dermatol. 128, 1747–1756. doi: 10.1038/sj.jid.5701259 Thormar, H., and Hilmarsson, H. (2007). The role of microbicidal lipids in host
defense against pathogens and their potential as therapeutic agents. Chem. Phys. Lipids 150, 1–11. doi: 10.1016/j.chemphyslip.2007.06.220 Seitz, A. P., Schumacher, F., Baker, J., Soddemann, M., Wilker, B., Caldwell,
C. C., et al. (2019). Sphingosine-coating of plastic surfaces prevents ventilator-
associated pneumonia. J. Mol. Med. 97, 1195–1211. doi: 10.1007/s00109-019-
01800-1801 Tober, K. L., Thomas-Ahner, J. M., Maruyama, T., and Oberyszyn, T. M. (2007). Possible cross-regulation of the E prostanoid receptors. Mol. Carcinogen. 46,
711–715. doi: 10.1002/mc.20347 Seitz, C. S., Freiberg, R. A., Hinata, K., and Khavari, P. A. (2000). NF-κB determines
localization and features of cell death in epidermis. J. Clin. Invest. 105, 253–260. doi: 10.1172/jci7630 Tóth, B. I., Dobrosi, N., Dajnoki, A., Czifra, G., Oláh, A., Szöll˝osi, A. G.,
et al. (2011). REFERENCES J. Invest. Dermatol. 110, 253–258. doi: 10.1046/j.1523-
1747.1998.00120.x Wille, J. J., and Kydonieus, A. (2003). Palmitoleic acid isomer (C16:116) in human
skin sebum is effective against gram-positive bacteria. Skin Pharmacol. Phys. 16,
176–187. doi: 10.1159/000069757 Wakita, H., Tokura, Y., Yagi, H., Nishimura, K., Furukawa, F., and Takigawa, M. (1994). Keratinocyte differentiation is induced by cell-permeant ceramides and
its proliferation is promoted by sphingosine. Arch. Dermatol. Res. 286, 350–354. doi: 10.1007/bf00402228 Wojtczak, L., and Załuska, H. (1967). The inhibition of translocation of adenine
nucleotides through mitochondrial membranes by oleate. Biochem. Biophy. Res. Commun. 28, 76–81. Wang, J., and Ueda, N. (2009). Biology of endocannabinoid synthesis system. Prostaglandins Other Lipid Mediat. 89, 112–119. doi: 10.1016/j.prostaglandins. 2008.12.002 Yahagi, S., Koike, M., Okano, Y., and Masaki, H. (2011). Lysophospholipids
improve skin moisturization by modulating of calcium-dependent cell
differentiation pathway. Int. J. Cosmetic Sci. 33, 251–256. doi: 10.1111/j.1468-
2494.2010.00625.x Wanner, R., Peiser, M., and Wittig, B. (2004). Keratinocytes rapidly readjust
ceramide-sphingomyelin homeostasis and contain a phosphatidylcholine-
sphingomyelin transacylase. J. Invest. Dermatol. 122, 773–782. doi: 10.1111/j. 0022-202x.2004.22340.x Zheng, C. J., Yoo, J.-S., Lee, T.-G., Cho, H.-Y., Kim, Y.-H., and Kim, W.-
G. (2005). Fatty acid synthesis is a target for antibacterial activity of
unsaturated fatty acids. FEBS Lett. 579, 5157–5162. doi: 10.1016/j.febslet.2005
.08.028 Watt, F. M. (2014). Mammalian skin cell biology: at the interface between
laboratory and clinic. Science 346, 937–940. doi: 10.1126/science.1253734 Zhu, A. J., and Watt, F. M. (1999). beta-catenin signalling modulates
proliferative potential of human epidermal keratinocytes independently of
intercellular adhesion. Development 126, 2285–2298. doi: 10.1242/dev.126.10. 2285 Watt, F. M., and Collins, C. A. (2008). Role of β-catenin in epidermal stem cell
expansion, lineage selection, and Cancer. Cold Spring Harb. Sym. 73, 503–512. doi: 10.1101/sqb.2008.73.011 q
Watt, F. M., Mattey, D. L., and Garrod, D. R. (1984). Calcium-induced
reorganization of desmosomal components in cultured human keratinocytes. J. Cell Biol. 99, 2211–2215. doi: 10.1083/jcb.99.6.2211 Ziboh, V. A., Miller, C. C., and Cho, Y. (2000). Metabolism of polyunsaturated
fatty acids by skin epidermal enzymes: generation of antiinflammatory and
antiproliferative metabolites. Am. J. Clin. Nutrition 71, 361s–366s. doi: 10.1093/
ajcn/71.1.361s Weitkamp, A. W., Smiljanic, A. M., and Rothman, S. (1947). The free fatty acids of
human hair fat. J. Am. Chem. Soc. 69, 1936–1939. doi: 10.1021/ja01200a027 Zouboulis, C. C. (2004). Acne and sebaceous gland function. Clin. Dermatol. 22,
360–366. doi: 10.1016/j.clindermatol.2004.03.004 Wertz, P. (2018). Epidermal lamellar granules. Skin Pharmacol. Phys. 31, 262–268. doi: 10.1159/000491757 Zouboulis, C. REFERENCES C., Seltmann, H., Orfanos, C. E., and Neitzel, H. (1999). Establishment and characterization of an immortalized human sebaceous gland
cell line (SZ95)1. J. Invest. Dermatol. 113, 1011–1020. doi: 10.1046/j.1523-1747. 1999.00771.x Wertz, P. W. (1996). The nature of the epidermal barrier: biochemical aspects. Adv. Drug Deliver Rev. 18, 283–294. doi: 10.1016/0169-409x(95)00077-k Wertz, P. W. (2000). Lipids and barrier function of the skin. Acta Derm-venereol. 80, 7–11. doi: 10.1080/000155500750042790 Züllig, T., Trötzmüller, M., and Köfeler, H. C. (2020). Lipidomics from sample
preparation to data analysis: a primer. Anal. Bioanal. Chem. 412, 2191–2209. doi: 10.1007/s00216-019-02241-y Wertz,
P. W.,
and
Downing,
D. T. (1987). Covalently
bound
ω-
hydroxyacylsphingosine in the stratum corneum. Biochim. Biophys. Acta
917, 108–111. Wertz, P. W., and Downing, D. T. (1990). Free sphingosine in human epidermis. J. Invest. Dermatol. 94, 159–161. doi: 10.1111/1523-1747.ep12874122 Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest. Wertz, P. W., and van den Bergh, B. (1998). The physical, chemical and functional
properties of lipids in the skin and other biological barriers. Chem. Phys. Lipids
91, 85–96. doi: 10.1016/s0009-3084(97)00108-104 Publisher’s Note: All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated organizations, or those of
the publisher, the editors and the reviewers. Any product that may be evaluated in
this article, or claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher. Wertz, P. W., Downing, D. T., Freinkel, R. K., and Traczyk, T. N. (1984). Sphingolipids of the stratum corneum and lamellar granules of fetal rat
epidemis. J. Invest. Dermatol. 83, 193–195. doi: 10.1111/1523-1747.ep12263553 Wertz, P. W., Schwartzendruber, D. C., Madison, K. C., and Downing, D. T. (1987). Composition and morphology of epidermal cyst lipids. J. Invest. Dermatol. 89,
419–425. doi: 10.1111/1523-1747.ep12471781 Copyright © 2022 Vietri Rudan and Watt. This is an open-access article distributed
under the terms of the Creative Commons Attribution License (CC BY). The
use, distribution or reproduction in other forums is permitted, provided the
original author(s) and the copyright owner(s) are credited and that the original
publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with
these terms. 419–425. doi: 10.1111/1523-1747.ep12471781 Wertz, P., and Downing, D. (1982). REFERENCES Activation of the Prostaglandin D2 receptor DP2/CRTH2 increases
allergic inflammation in mouse. J. Immunol. 174, 3703–3708. doi: 10.4049/
jimmunol.174.6.3703 van Meer, G., Voelker, D. R., and Feigenson, G. W. (2008). Membrane lipids:
where they are and how they behave. Nat. Rev. Mol. Cell Biol. 9, 112–124. doi: 10.1038/nrm2330 Stahley, S. N., Saito, M., Faundez, V., Koval, M., Mattheyses, A. L., and
Kowalczyk, A. P. (2014). Desmosome assembly and disassembly are
membrane raft-dependent. PLoS One 9:e87809. doi: 10.1371/journal.pone.00
87809 van Smeden, J., Hoppel, L., Heijden, R., van der, Hankemeier, T., Vreeken, R. J.,
et al. (2011). LC/MS analysis of stratum corneum lipids: ceramide profiling and
discovery. J. Lipid Res. 52, 1211–1221. doi: 10.1194/jlr.m014456 Stewart, M. E., and Downing, D. T. (1991). Chemistry and function of mammalian
sebaceous lipids. Adv. Lipid Res. 24, 263–301. doi: 10.1016/b978-0-12-024924-
4.50013-50014 Vietri Rudan, M., Mishra, A., Klose, C., Eggert, U. S., and Watt, F. M. (2020). Human epidermal stem cell differentiation is modulated by specific lipid
subspecies. Proc. Natl. Acad. Sci. U S A. 117:202011310. doi: 10.1073/pnas. 2011310117 Sugiura, T., Kishimoto, S., Oka, S., and Gokoh, M. (2006). Biochemistry,
pharmacology and physiology of 2-arachidonoylglycerol, an endogenous
cannabinoid receptor ligand. Prog. Lipid Res. 45, 405–446. doi: 10.1016/j.plipres. 2006.03.003 Vogler, R., Sauer, B., Kim, D.-S., Schäfer-Korting, M., and Kleuser, B. (2003). Sphingosine-1-Phosphate and its potentially paradoxical effects on critical
parameters of cutaneous wound healing. J. Invest. Dermatol. 120, 693–700. doi: 10.1046/j.1523-1747.2003.12096.x Sumitomo, A., Siriwach, R., Thumkeo, D., Ito, K., Nakagawa, R., Tanaka, N.,
et al. (2019). LPA induces keratinocyte differentiation and promotes skin
barrier function through the LPAR1/LPAR5-RHO-ROCK-SRF Axis. J. Invest. Dermatol. 139, 1010–1022. doi: 10.1016/j.jid.2018.10.034 Wacnik, P. W., Luhr, K. M., Hill, R. H., Ljunggren, H.-G., Kristensson, K.,
and Svensson, M. (2008). Cannabinoids affect dendritic cell (DC) potassium
channel function and modulate DC T Cell stimulatory capacity. J. Immunol. 181, 3057–3066. doi: 10.4049/jimmunol.181.5.3057 Suzuki, Y., Nomura, J., Hori, J., Koyama, J., Takahashi, M., and Horii, I. (1993). Detection and characterization of endogenous protease associated with
desquamation of stratum corneum. Arch. Dermatol. Res. 285, 372–377. doi:
10.1007/bf00371839 Wakita, H., Matsushita, K., Nishimura, K., Tokura, Y., Furukawa, F., and
Takigawa, M. (1998). Sphingosylphosphorylcholine stimulates proliferation and
upregulates cell surface-associated plasminogen activator activity in cultured January 2022 | Volume 12 | Article 804824 Frontiers in Physiology | www.frontiersin.org 24 Vietri Rudan and Watt Lipids Roles in Mammalian Epidermis human keratinocytes. J. Invest. Dermatol. 110, 253–258. doi: 10.1046/j.1523-
1747.1998.00120.x human keratinocytes. January 2022 | Volume 12 | Article 804824 REFERENCES Glycolipids in mammalian epidermis: structure
and function in the water barrier. Science 217, 1261–1262. doi: 10.1126/science. 7112128 Wilkinson, D. I., and Karasek, M. A. (1966). Skin lipids of a normal and a mutant
(Asebic) mouse strain. J. Invest. Dermatol. 47, 449–455. doi: 10.1038/jid.1966. 168 January 2022 | Volume 12 | Article 804824 Frontiers in Physiology | www.frontiersin.org Frontiers in Physiology | www.frontiersin.org 25
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Hyaluronan Graft Copolymers Bearing Fatty-Acid Residues as Self-Assembling Nanoparticles for Olanzapine Delivery
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Hyaluronan Graft Copolymers Bearing Fatty-Acid
Residues as Self-Assembling Nanoparticles for
Olanzapine Delivery Keywords: hyaluronic acid; ferulic acid; oleic acid; stearic acid; olanzapine; self-assembling
nanocarriers; drug delivery systems Article
Hyaluronan Graft Copolymers Bearing Fatty-Acid
Residues as Self-Assembling Nanoparticles for
Olanzapine Delivery Marco Paolino 1,*
, Mariano Licciardi 2
, Cristina Savoca 2, Gaetano Giammona 2,
Laura Modica De Mohac 3
, Annalisa Reale 1
, Germano Giuliani 1, Hartmut Komber 4,
Alessandro Donati 1, Gemma Leone 1, Agnese Magnani 1
, Maurizio Anzini 1
and
Andrea Cappelli 1 1
Dipartimento di Biotecnologie, Chimica e Farmacia (Dipartimento di Eccellenza 2018-2022), Università degli
Studi di Siena, Via A. Moro 2, 53100 Siena, Italy; reale5@student.unisi.it (A.R.); giuliani5@unisi.it (G.G.);
alessandro.donati@unisi.it (A.D.); gemma.leone@unisi.it (G.L.); agnese.magnani@unisi.it (A.M.);
maurizio.anzini@unisi.it (M.A.); andrea.cappelli@unisi.it (A.C.) 1
Dipartimento di Biotecnologie, Chimica e Farmacia (Dipartimento di Eccellenza 2018-2022), Università degli
Studi di Siena, Via A. Moro 2, 53100 Siena, Italy; reale5@student.unisi.it (A.R.); giuliani5@unisi.it (G.G.);
alessandro.donati@unisi.it (A.D.); gemma.leone@unisi.it (G.L.); agnese.magnani@unisi.it (A.M.);
maurizio.anzini@unisi.it (M.A.); andrea.cappelli@unisi.it (A.C.) 2
Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche (STEBICEF), Università degli
Studi di Palermo, Via Archirafi32, 90123 Palermo, Italy; mariano.licciardi@unipa.it (M.L.);
savocacristina@libero.it (C.S.); gaetano.giammona@unipa.it (G.G.) (
) g
g
p
(
)
3
Dipartimento Promozione della Salute, Materno-Infantile, di Medicina Interna e Specialistica di Eccellenza
“G. D’Alessandro” (PROMISE), Università degli Studi di Palermo, 90100 Palermo, Italy;
laura.modicademohac@unipa.it 4
Leibniz Institute of Polymer Research, Hohe Strasse 6, 01069 Dresden, Germany; komber@ipfdd.de
*
Correspondence: paomar@oneonline.it
Received: 13 November 2019; Accepted: 9 December 2019; Published: 12 December 2019 Abstract: In order to evaluate the potential of a technology platform based on hyaluronan copolymers
grafted with propargylated ferulate fluorophores (HA-FA-Pg) in the development of drug delivery
systems, the propargyl groups of HA-FA-Pg derivatives were employed with oleic acid (OA) or stearic
acid (SA) residues across a biocompatible hexa(ethylene glycol) (HEG) spacer. The designed materials
(i.e., HA-FA-HEG-OA or HA-FA-HEG-SA) showed clear-cut aggregation features in an aqueous
environment, as confirmed by dynamic light scattering (DLS) and transmission electron microscopy
(TEM), generating nanoaggregate systems. In fact, HA-FA-HEG-OA and HA-FA-HEG-SA derivatives
showed the property to create self-assembled cytocompatible nanostructured aggregates in water,
thanks to the simultaneous presence of hydrophilic portions in the polymeric backbone, such as
hyaluronic acid, and hydrophobic portions in the side chains. Furthermore, the designed materials
interact with living cells showing a high degree of cytocompatibility. The potential ability of
nanosystems to load pharmacologically active molecules was assessed by the physical entrapment
of olanzapine into both polymeric systems. The drug loading evaluation demonstrated that the
nanoparticles are able to incorporate a good quantity of olanzapine, as well as improve drug solubility,
release profile, and cytocompatibility. pharmaceutics pharmaceutics pharmaceutics pharmaceutics Pharmaceutics 2019, 11, 675; doi:10.3390/pharmaceutics11120675 1. Introduction Hyaluronic acid (HA, hyaluronan) is a glycosaminoglycan, which possesses important functions
in the human body such as the formation of a pericellular coat having a crucial role in the early stages
of cell adhesion [1]. In fact, HA interacts with the cluster of differentiation 44 (CD44) receptor [2,3] Pharmaceutics 2019, 11, 675; doi:10.3390/pharmaceutics11120675 Pharmaceutics 2019, 11, 675
2 of 20 2 of 20 Pharmaceutics 2019, 11, 675 (a ubiquitous protein expressed in high density in tumor tissues [4]) showing an extracellular
hyaluronan-binding domain. eptor [2,3] (a ubiquitous protein expressed in high density in tumor tissues [4]) showing an
a ellula hyalu o a
bi di
do
ai (a ubiquitous protein expressed in high density in tumor tissues [4]) showing an extracellular
hyaluronan-binding domain. eptor [2,3] (a ubiquitous protein expressed in high density in tumor tissues [4]) showing an
racellular hyaluronan binding domain In our previous studies, a synthetic procedure was developed to functionalize HA with ferulic
acid (FA) residues in order to obtain graft copolymers (i.e., HA-FA derivatives, Figure 1) showing
intriguing gelating and wound healing properties [5,6]. racellular hyaluronan-binding domain. In our previous studies, a synthetic procedure was developed to functionalize HA with ferulic
d (FA) residues in order to obtain graft copolymers (i.e., HA-FA derivatives, Figure 1) showing
riguing gelating and wound healing properties [5 6] Figure 1. Design and development of the technology platform based on hyaluronan copolymers
grafted with propargylated ferulate fluorophores (HA-FA-Pg). 1. Introduction The procedure was optimized by using low-molecular-weight HA (molecular weight (MW) of
out 8.7 kg∙mol−1), which provided hyaluronan copolymers grafted with propargylated ferulate
orophores (HA-FA-Pg) showing grafting degree values in the range from 10% to 35% [11]. Subsequently, the grafting procedure was modified by using a ferulate (FA) derivative bearing a
propargyl group, in order to introduce clickable groups to be employed in click-chemistry coupling
reactions such as copper(I)-catalyzed azide–alkyne 1,3-dipolar cycloaddition (CuAAC) [7–10]. The
procedure was optimized by using low-molecular-weight HA (molecular weight (MW) of about 8.7
kg·mol−1), which provided hyaluronan copolymers grafted with propargylated ferulate fluorophores
(HA-FA-Pg) showing grafting degree values in the range from 10% to 35% [11]. p
(
g)
g g
g
g
g
[
]
HA-FA-Pg derivatives were employed as the basis of a covalent coating technology with HA. In
ticular, an HA-FA-Pg derivative with 2–3 FA residues bearing a propargyl group was
cessfully used in the covalent coating of inorganic magnetite nanoparticles [12]. Moreover, an
A-FA-Pg derivative bearing 4–5 FA residues was used in the covalent coating of a π-stacked
ybenzofulvene derivative [13–19] to prepare the intriguing organic material TCPB (Figure 1) [20]. HA-FA-Pg derivatives were employed as the basis of a covalent coating technology with HA. In
particular, an HA-FA-Pg derivative with 2–3 FA residues bearing a propargyl group was successfully
used in the covalent coating of inorganic magnetite nanoparticles [12]. Moreover, an HA-FA-Pg
derivative bearing 4–5 FA residues was used in the covalent coating of a π-stacked polybenzofulvene
derivative [13–19] to prepare the intriguing organic material TCPB (Figure 1) [20]. In TCPB architecture, HA plays the role of the coating agent for both the polybenzofulvene
indrical brush and the gel nanoparticles generated by TCPB aggregation in the water
vironment [20,21]. Interestingly, the gel nanoparticles were demonstrated to behave as intriguing
riers, capable of transporting the anticancer drug doxorubicin to cancer cells by exploiting the
eraction of HA with the CD44 receptor overexpressed on the cell surface of tumor tissues [21]. The
e of the nanogel systems generated by TCPB is constituted by the highly hydrophobic
ybenzofulvene backbone and by the amphiphilic nona(ethylene glycol) (NEG) side chains that
re assumed to form suitable compartments for drug loading, transport, and delivery. In TCPB architecture, HA plays the role of the coating agent for both the polybenzofulvene
cylindrical brush and the gel nanoparticles generated by TCPB aggregation in the water
environment [20,21]. 1. Introduction HA
HA-FA-Pg
O
O
HO
O
O
HO
NH
O
OH
O
OR
O
x
O
O
HO
O
O
HO
NH
O
OH
O
OR
OH
y
O
O
HO
O
O
HO
NH
O
OH
O
OR
OH
x
HA-FA
O
OH
O
O
HO
O
O
HO
NH
O
OH
O
OR
O
x
O
O
HO
O
O
HO
NH
O
OH
O
OR
OH
y
O
O
O
O
7
O
O
HO
O
O
HO
NH
O
OH
O
OR
O
O
HO
O
O
HO
NH
O
OH
O
OR
O
O
N N
N
N
O
O
O
H
7
OH
O
O
TCPB
HA-FA-NEG-OA
x
y
x
y
O
O
O
N
H
O
O
O
OH
O
O
OH
HN
O
HO
O
RO
HO
O
O
OH
O
O
OH
HN
O
HO
O
RO
O
O
O
N
N
N
O
O
O
O
HO
O
O
HO
NH
O
OH
O
OR
O
O
HO
O
O
HO
NH
O
OH
O
OR
O
O
N N
N
O
OH
O
O
HA-FA-HEG-NP
x
y
HA-based coating technology platform
coating of inorganic
magnetite nanoparticles
coating of organic
hydrophobic polymers
coating of
phospholipid
bilayers
in this work
O y
HA-based coating technology platform 7
TCPB
x
y
O
O
O
N
H
O
O
O
OH
O
O
OH
HN
O
HO
O
RO
HO
O
O
OH
O
O
OH
HN
O
HO
O
RO
O
O
O
N
N
N
O
O
organic
polymers
O in this work O
O
HO
O
O
HO
NH
O
OH
O
OR
x
O
O
HO
O
O
HO
NH
O
OH
O
OR
y
O
O
N N
N
N
O
O
O
H
4
OH
O
O
HA-FA-HEG-SA HA-FA-HEG-OA HA-FA-HEG-SA ure 1. Design and development of the technology platform based on hyaluronan copolymers
fted with propargylated ferulate fluorophores (HA-FA-Pg). Figure 1. Design and development of the technology platform based on hyaluronan copolymers grafted
with propargylated ferulate fluorophores (HA-FA-Pg). Subsequently, the grafting procedure was modified by using a ferulate (FA) derivative bearing
propargyl group, in order to introduce clickable groups to be employed in click-chemistry
upling reactions such as copper(I)-catalyzed azide–alkyne 1,3-dipolar cycloaddition (CuAAC)
10]. 1. Introduction HA
HA-FA-Pg
O
O
HO
O
O
HO
NH
O
OH
O
OR
O
x
O
O
HO
O
O
HO
NH
O
OH
O
OR
OH
y
O
O
HO
O
O
HO
NH
O
OH
O
OR
OH
x
HA-FA
O
OH
O
O
HO
O
O
HO
NH
O
OH
O
OR
O
x
O
O
HO
O
O
HO
NH
O
OH
O
OR
OH
y
O
O
O
O
7
O
O
HO
O
O
HO
NH
O
OH
O
OR
O
O
HO
O
O
HO
NH
O
OH
O
OR
O
O
N N
N
N
O
O
O
H
7
OH
O
O
O
O
HO
O
O
HO
NH
O
OH
O
OR
x
O
O
HO
O
O
HO
NH
O
OH
O
OR
y
O
O
N N
N
N
O
O
O
H
4
OH
O
O
O
O
HO
O
O
HO
NH
O
OH
O
OR
x
O
O
HO
O
O
HO
NH
O
OH
O
OR
y
O
O
N N
N
N
O
O
O
H
4
OH
O
O
TCPB
HA-FA-NEG-OA
HA-FA-HEG-OA
HA-FA-HEG-SA
x
y
x
y
O
O
O
N
H
O
O
O
OH
O
O
OH
HN
O
HO
O
RO
HO
O
O
OH
O
O
OH
HN
O
HO
O
RO
O
O
O
N
N
N
O
O
O
O
HO
O
O
HO
NH
O
OH
O
OR
O
O
HO
O
O
HO
NH
O
OH
O
OR
O
O
N N
N
O
OH
O
O
HA-FA-HEG-NP
x
y
HA-based coating technology platform
coating of inorganic
magnetite nanoparticles
coating of organic
hydrophobic polymers
coating of
phospholipid
bilayers
in this work
O
Figure 1. Design and development of the technology platform based on hyaluronan copolymers grafted
with propargylated ferulate fluorophores (HA-FA-Pg). 1. Introduction Interestingly, the gel nanoparticles were demonstrated to behave as intriguing
carriers, capable of transporting the anticancer drug doxorubicin to cancer cells by exploiting the
interaction of HA with the CD44 receptor overexpressed on the cell surface of tumor tissues [21]. The core of the nanogel systems generated by TCPB is constituted by the highly hydrophobic
polybenzofulvene backbone and by the amphiphilic nona(ethylene glycol) (NEG) side chains that were
assumed to form suitable compartments for drug loading, transport, and delivery. 3 of 20 Pharmaceutics 2019, 11, 675 However, in such architecture, the major potential drawback could be envisaged in the high
hydrophobicity of the covalent backbone of the polybenzofulvene brush. Thus, in order to overcome
this potential drawback, we envisioned the replacement of the covalent synthetic polybenzofulvene
backbone of TCPB with oleic acid (OA), potentially capable of establishing networks in an aqueous
environment by means of non-covalent interactions. These considerations led to the design of the
tetra-component materials HA-FA-NEG-OA (Figure 1), which showed very intriguing features [11]. In particular, HA-FA-NEG-OA derivatives showed the tendency to generate nanostructured
aggregates in water, which were cytocompatible and could be visualized by fluorescence microscopy
techniques by virtue of the AIE (aggregation-induced emission) features of the ferulate luminogen [11]. Moreover, HA-FA-NEG-OA materials are capable of interacting with the phospholipid bilayer of
monolamellar liposomes with an increase in the negative zeta potential without alterations in the
liposome dimensions and the fluorogenic properties of the ferulate fluorophore [11]. On the basis of
these promising results, HA-FA-NEG-OA materials could not only find applications in the coating of
hydrophobic surfaces, but also in the formation of micellar systems potentially useful in the design of
drug delivery systems [22–24]. Because of the multi-component architecture, the features of these materials could be modulated
by the suitable variation of each component: (a) the polysaccharide chain, (b) the fluorophore, (c) the
spacer, and (d) the fatty acid. In the present paper, we describe the effects of the modification of two components of the
previously described HA-FA-NEG-OA materials, namely, the spacer and the fatty acid. In particular,
the nona(ethylene glycol) spacer was replaced with a more readily available hexa(ethylene glycol)
(HEG) spacer, and the monounsaturated oleic acid (OA) residues were replaced with saturated stearic
acid (SA) residues, thus producing HA-FA-HEG-OA and HA-FA-HEG-SA materials (Figure 1). 1. Introduction The
designed materials showed clear-cut aggregation features in an aqueous environment, highlighted by
dynamic light scattering (DLS) and transmission electron microscopy (TEM) analysis, as well as the
potential to act as cytocompatible nanocarriers for active molecules. In this study, olanzapine (OZ) was
tested as a model drug [25,26]. 2. Materials and Methods The reagents and solvents were obtained from Sigma-Aldrich and were used as received. Olanzapine (OZ, MW 312.43 g/mol) was purchased from Merck KGaA (Darmstadt, Germany). Ethanol,
Dulbecco’s phosphate-buffered saline (DPBS), anhydrous dimethyl sulfoxide (DMSO), sodium chloride,
and sodium hydroxide were obtained from Merck KGaA (Darmstadt, Germany). The water used was
produced with Milli-Q ® (Millipore Corporation, Billerica, MA, USA). Merck silica gel 60 (230–400
mesh) was employed for column chromatography, and Merck thin layer chromatography (TLC)
aluminum sheets (silica gel 60 F254) were employed for TLC. NMR spectra were obtained with a Bruker
DRX-400 AVANCE or a Bruker Avance III 500 spectrometer in the indicated solvents. The chemical
shifts are referenced to the solvent signal (CDCl3: δ(1H) = 7.26 ppm, δ(13C) = 77.0 ppm) or to the signal
of a trace acetone for solutions in D2O (δ(1H) = 2.22 ppm, δ(13C)CH3 = 30.9 ppm). Chemical shifts (δ)
are reported in ppm, and the H–H coupling constants (J) are reported in Hz. An Agilent 1100 LC/MSD
running with an electrospray source was used in mass spectrometry measurements. The intermediate
α-azido-ω-amino hexa(ethylene glycol) 3 was synthesized as previously reported in Reference [27]. 2.1. Synthesis and Characterization Preparation of HA-FA-HEG-OA A round-bottom 10-mL flask was charged, under an inert atmosphere, with tert-butanol (2.0
mL), H2O (2.0 mL), and a solution of copper sulfate pentahydrate (12.5 mg, 0.050 mmol in 0.50 mL of
water). Subsequently, a 1 M water solution of sodium ascorbate (0.50 mL) was added. A portion of the
resulting catalyst solution (1.0 mL) was promptly used in the subsequent reaction. To a mixture of HA-FA-Pg graft copolymer bearing a mean distribution of 4–5 propargyl derivatives
for each macromolecule (200 mg, synthetic procedure reported in Reference [11]) in 5.0 mL of water
containing 4a (60 mg, 0.105 mmol), 1.0 mL of the catalyst solution was added, and the resulting mixture
was stirred at room temperature for 1 h and then treated with QUADRASIL MP (200 mg). After
filtration, the resulting mixture was concentrated under reduced pressure. The solid residue was
purified by washing with acetone to obtain pure HA-FA-HEG-OA as a light-brown solid (218 mg). 1H-NMR (500 MHz, D2O): the spectrum is depicted in Figure 3c; 13C-NMR (125 MHz, D2O), selected
signals: 14.6 (18”), 23.2 (17”, Et), 26.4 (3”), 27.8 (8”, 11”), 29–32 (4”–7”, 12”–15”), 32.5 (16”), 36.6 (2”),
39.6 (H), 50.8 (E), 69–72 (F,G,X,Y), 130.0 and 130.4 (9”,10”), 175.6 (1”). For the spectrum and the atom
numbering, compare Figure 4d. N-(17-Azido-3,6,9,12,15-pentaoxaheptadecyl)stearamide (4b) A mixture of stearic acid (1b, 0.18 g, 0.63 mmol) and CDI (0.10 g, 0.62 mmol) in dry THF (10 mL)
was refluxed for 2 h, and then a solution of O-(2-aminoethyl)-O′-(2-azidoethyl)tetraethylene glycol
(3, 0.20 g, 0.65 mmol) in dry THF (5.0 mL) was added. The resulting mixture was stirred overnight
at room temperature and then concentrated under reduced pressure. The resulting oily residue was
purified by flash chromatography with petroleum ether/ethyl acetate (1:1) to obtain 4b as a white solid
(0.33 g, yield 93%, melting point (mp) 48–50 ◦C). 1H-NMR (400 MHz, CDCl3): 0.87 (t, J = 6.7, 3H),
1.17–1.38 (28H), 1.60 (m, 2H), 2.15 (t, J = 7.6, 2H), 3.37 (t, J = 5.1, 2H), 3.43 (q, J = 5.3, 2H), 3.54 (t, J = 5.0,
2H), 3.58–3.72 (18H), 6.02 (br s, 1H). MS (ESI): m/z 595.2 (M + Na+). N-(17-Azido-3,6,9,12,15-pentaoxaheptadecyl)oleamide (4a) A mixture of oleic acid (1a, 0.34 g, 1.20 mmol) and1,1′-carbonyldiimidazole (CDI) (0.20 g,
1.23 mmol) in dry tetrahydrofuran (THF) (5.0 mL) was refluxed for 2 h, and then a solution
O-(2-aminoethyl)-O′-(2-azidoethyl)tetraethylene glycol (3, 0.40 g, 1.31 mmol) in dry THF (5.0 mL) was
added. The resulting mixture was stirred overnight at room temperature and then concentrated under 4 of 20 Pharmaceutics 2019, 11, 675 reduced pressure. The resulting oily residue was purified by flash chromatography with ethyl acetate
to obtain 4a as a pale-yellow oil (0.65 g, yield 95%). 1H-NMR (400 MHz, CDCl3): 0.87 (t, J = 6.6, 3H),
1.24–1.31 (20H), 1.62 (m, 2H), 1.92–2.05 (4H), 2.16 (t, J = 7.6, 2H), 3.37 (t, J = 5.1, 2H), 3.44 (q, J = 5.1, 2H),
3.54 (t, J = 5.1, 2H), 3.59–3.74 (18H), 5.24–5.43 (2H), 6.05 (br s, 1H). MS (ESI): m/z 593.2 (M + Na+). Preparation of HA-FA-HEG-SA Under an inert atmosphere, a round-bottom 10-mL flask was charged with tert-butanol (2.0 mL),
H2O (2.0 mL), and a solution of copper sulfate pentahydrate (12.5 mg, 0.050 mmol in 0.50 mL of
water). Subsequently, a 1 M water solution of sodium ascorbate (0.50 mL) was added. A portion of the
resulting catalyst solution (1.0 mL) was promptly used in the subsequent reaction. To a mixture of HA-FA-Pg graft copolymer bearing a mean distribution of 4–5 propargyl derivatives
for each macromolecule (200 mg, synthetic procedure reported in Reference [11]) in water (5.0 mL)
containing 4b (62 mg, 0.108 mmol), 1.0 mL of the catalyst solution was added, and the resulting mixture
was stirred at room temperature for 1 h and then treated with QUADRASIL MP (200 mg). After
filtration, the resulting mixture was concentrated under reduced pressure. The solid residue was
purified by washing with acetone to obtain HA-FA-HEG-SA as a light-brown solid (230 mg). 1H-NMR
(500 MHz, D2O): the spectrum is depicted in Figure 3d; 13C-NMR (125 MHz, D2O), selected signals:
14.6 (18”), 23.2 (17”,Et), 26.4 (3”), 29–32 (4”–15”), 32.6 (16”), 36.6 (2”), 39.7 (H), 69–72 (F,G,X,Y), 175.6
(1”). For the spectrum and the atom numbering, compare Figure 4c. (E)-Methyl 3-[3-methoxy-4-[[1-[(Z)-19-oxo-3,6,9,12,15-pentaoxa-18-azahexatriacont-27-en-1-yl]-1H-
1,2,3-triazol-4-yl]methoxy]phenyl]acrylate (FE-HEG-OA) Under an inert atmosphere, a round-bottom 10-mL flask was charged with tert-butanol (2.0 mL),
H2O (2.0 mL), and a solution of copper sulfate pentahydrate (12.5 mg, 0.050 mmol in 0.50 mL of water). 5 of 20 Pharmaceutics 2019, 11, 675 Then, a 1 M water solution of sodium ascorbate (0.50 mL) was added. A portion of the resulting
catalyst solution (1.0 mL) was promptly used in the subsequent reaction. A mixture of 4a (250 mg, 0.44
mmol) and 5 [20] (110 mg, 0.45 mmol) in 4.0 mL of tert-butanol and water (2.0 mL) was treated with
the catalyst solution (2.7 mL), and the reaction mixture was stirred overnight at room temperature and
then concentrated under reduced pressure. Purification of the residue by flash chromatography with
ethyl acetate/methanol (9:1) as the eluent afforded FE-HEG-OA as a pale-yellow oil (337 mg, yield
92%). Preparation of HA-FA-HEG-SA 1H-NMR (500 MHz, CDCl3): 0.88 (t, J = 6.9, 3H; 18”), 1.2–1.35 (20H; 4”–7”,12”–17”), 1.62 (m, 2H;
3”), 2.00 (4H; 8”,11”), 2.16 (t, J = 7.6, 2H; 2”), 3.43 (q, J = 5.3, 2H; H), 3.54 (t, J = 5.1, 2H; G), 3.55–3.65
(16H; X,Y), 3.80 (s, 3H; A”), 3.87 (t, J = 5.0, 2H; F), 3.89 (s, 3H; A), 4.53 (t, J = 5.0, 2H; E), 5.3–5.35 (4H;
9”,10”,B), 6.05 (br, 1H; NH), 6.31 (d, J = 16.0, 1H; 2), 7.05–7.10 (3H; 2′,5′,6′), 7.62 (d, J = 16.0, 1H; 3),
7.83 (s, 1H; D). 13C NMR (125 MHz, CDCl3): 14.1 (18”), 22.7 (17”), 25.7 (3”), 27.2 (8”,11”), 29.2–29.7
(4”–7”,12”–15”), 31.9 (16”), 36.7 (2”), 39.1 (H), 50.4 (E), 51.6 (A’), 55.9 (A), 63.0 (B), 69.4 (F), 70.0–70.5
(F,G,X,Y), 110.3 (2′), 113.7 (5′), 115.9 (2), 122.4 (6′), 124.3 (D), 128.1 (1′), 129.7 and 130.0 (9”,10”), 143.5
(C), 144.6 (3), 149.7, and 149.8 (3′,4′), 167.6 (1), 173.2 (1”). For spectra and the atom numbering, compare
Figures 3e and 4e. MS (ESI): m/z 839.5 (M + Na+). (E)-3-[3-Methoxy-4-[[1-[(Z)-19-oxo-3,6,9,12,15-pentaoxa-18-azahexatriacont-27-en-1-yl]-1H-1,2,3-triazol-
4-yl]methoxy]phenyl]acrylic acid (FA-HEG-OA) (E)-3-[3-Methoxy-4-[[1-[(Z)-19-oxo-3,6,9,12,15-pentaoxa-18-azahexatriacont-27-en-1-yl]-1H-1,2,3-triazol-
4-yl]methoxy]phenyl]acrylic acid (FA-HEG-OA) (E)-3-[3-Methoxy-4-[[1-[(Z)-19-oxo-3,6,9,12,15-pentaoxa-18-azahexatriacont-27-en-1-yl]-1H-1,2,3-triazol-
4-yl]methoxy]phenyl]acrylic acid (FA-HEG-OA) A mixture of FE-HEG-OA (0.33 g, 0.40 mmol) in ethanol (7.5 mL) containing a 2 N water solution
of NaOH (4.7 mL) was refluxed for 2 h. The reaction mixture was then cooled at 0 ◦C, acidified with 3
N HCl, and concentrated under reduced pressure. Purification of the residue by flash chromatography
with dichloromethane/methanol (9:1) gave acid FA-HEG-OA as a white solid (0.28 g, yield 87%, mp
67–69 ◦C). 1H-NMR (500 MHz, CDCl3): 0.88 (t, J = 6.9, 3H; 18”), 1.2–1.35 (20H; 4”–7”,12”–17”), 1.62 (m,
2H; 3”), 2.00 (4H; 8”,11”), 2.17 (t, J = 7.6, 2H; 2”), 3.44 (q, J = 5.3, 2H; H), 3.5–3.65 (18H; G,X,Y), 3.86 (t, J
= 5.0, 2H; F), 3.90 (s, 3H; A), 4.53 (t, J = 5.0, 2H; E), 5.3–5.4 (4H; 9”,10”,B), 6.12 (br, 1H; NH), 6.31 (d, J =
16.0, 1H; 2), 7.05–7.10 (3H; 2′,5′,6′), 7.68 (d, J = 16.0, 1H; 3), 7.81 (s, 1H; D), 10.60 (br, 1H; COOH). 13C
NMR (125 MHz, CDCl3): 14.1 (18”), 22.6 (17”), 25.7 (3”), 27.2 (8”,11”), 29.1–29.7 (4”–7”,12”–15”), 31.9
(16”), 36.7 (2”), 39.1 (H), 50.4 (E), 55.9 (A), 62.9 (B), 69.4 (F), 70.0–70.5 (F,G,X,Y), 110.4 (2′), 113.7 (5′),
115.6 (2), 122.7 (6′), 124.3 (D), 127.8 (1′), 129.7 and 130.0 (9”,10”), 143.5 (C), 146.2 (3), 149.7, and 150.0
(3′,4′), 170.9 (1), 173.4 (1”). For spectra and the atom numbering, compare Figure 2. MS (ESI): m/z 825.5
(M + Na+). 6 of 20
of 20 Pharmaceutics 2019, 11, 675
Pha
a euti
2019 11 Figure 2. 1H- and 13C-NMR spectra of model compound FA-HEG-OA in CDCl3. Figure 2. 1H- and 13C-NMR spectra of model compound FA-HEG-OA in CDCl3. 2.2. Particle Size Analysis and Zeta Potential Figure 2. 1H- and 13C-NMR spectra of model compound FA-HEG-OA in CDCl3. Figure 2. 1H- and 13C-NMR spectra of model compound FA-HEG-OA in CDCl3. Figure 2. 1H- and 13C-NMR spectra of model compound FA-HEG-OA in CDCl3. Figure 2. 1H- and 13C-NMR spectra of model compound FA-HEG-OA in CDCl3. Figure 2. 1H- and 13C-NMR spectra of model compound FA-HEG-OA in CDCl3. 2.2. Particle Size Analysis and Zeta Potential 2.2. Particle Size Analysis and Zeta Potential
The size analysis, zeta potential, and polydispersity index of the nanoparticles (NPs) were
determined using a Malvern Zetasizer Nano ZS instrument, fitted with a 532-nm laser at a fixed
scattering angle of 90°. The samples were analyzed before and after drug loading. (E)-3-[3-Methoxy-4-[[1-[(Z)-19-oxo-3,6,9,12,15-pentaoxa-18-azahexatriacont-27-en-1-yl]-1H-1,2,3-triazol-
4-yl]methoxy]phenyl]acrylic acid (FA-HEG-OA) Each sample was
suitably diluted and filtered before the analysis and placed in a disposable sizing micro cuvette
The size analysis, zeta potential, and polydispersity index of the nanoparticles (NPs) were
determined using a Malvern Zetasizer Nano ZS instrument, fitted with a 532-nm laser at a fixed
scattering angle of 90◦. The samples were analyzed before and after drug loading. Each sample was
suitably diluted and filtered before the analysis and placed in a disposable sizing micro cuvette. y
l A
2.3. Critical Aggregation Concentration 2.3. Critical Aggregation Concentration
Critical aggregation concentration (CAC) evaluation was carried out in water and in DPBS by
means of the pyrene fluorescence assay. At the beginning, a solution of pyrene in acetone was
prepared (6.0 × 10−5 M); then, aliquots of 20 μL of this solution were placed in vials and left to
evaporate for 30 min in orbital shaker at 37 °C in order to remove the acetone. Afterward, 2 mL of the
polymer aqueous dispersions were added to the pyrene solid residue. Polymer concentrations were
included in a range from 10 to 0.0005 mg/mL. In this way, the final concentration of pyrene in each
sample was, thus, equal to 6.0 × 10−7 M. The mixtures were kept at 37 °C for 1 h under continuous
agitation, inside an orbital shaker, in order to balance the pyrene with the micelles. The pyrene
excitation and emission spectra were recorded at 25 °C at an excitation wavelength of 333 nm and an
Critical aggregation concentration (CAC) evaluation was carried out in water and in DPBS by
means of the pyrene fluorescence assay. At the beginning, a solution of pyrene in acetone was prepared
(6.0 × 10−5 M); then, aliquots of 20 µL of this solution were placed in vials and left to evaporate for 30
min in orbital shaker at 37 ◦C in order to remove the acetone. Afterward, 2 mL of the polymer aqueous
dispersions were added to the pyrene solid residue. Polymer concentrations were included in a range
from 10 to 0.0005 mg/mL. In this way, the final concentration of pyrene in each sample was, thus, equal
to 6.0 × 10−7 M. The mixtures were kept at 37 ◦C for 1 h under continuous agitation, inside an orbital
shaker, in order to balance the pyrene with the micelles. The pyrene excitation and emission spectra
were recorded at 25 ◦C at an excitation wavelength of 333 nm and an emission wavelength of 373 nm. 2.6. Preparation of Self-Assembling Nanoparticles Olanzapine (OZ, MW 312.43 g/mol) was purchased from Merck KGaA (Darmstadt, Germany). Ethanol, Dulbecco’s phosphate-buffered saline (DPBS), anhydrous dimethyl sulfoxide (DMSO), sodium
chloride, and sodium hydroxide were obtained from Merck KGaA (Darmstadt, Germany). The water
used was produced with Milli-Q ® (Millipore Corporation, Billerica, MA, USA). Aliquots of HA-FA-HEG-OA (30 mg) and HA-FA-HEG-SA (30 mg) were dissolved in 3.0 mL of
Milli-Q water and left, under vigorous stirring, at room temperature overnight. The day after, OZ
(15 mg) was added to polymer dispersions, to get a drug concentration of 5 mg/mL, and the mixture
was left under vigorous stirring, at room temperature overnight. After this time, the dispersions were
centrifuged at 8000 rpm and 25 ◦C for 5 min to remove the drug, which was not incorporated in the
self-assembling nanoparticles (NPs). Supernatant was recovered and then dried by freeze-drying,
thereby obtaining a spongy solid. 2.7. Drug Content and Encapsulation Efficiency The drug content in the NP was determined by dissolving 2.0 mg of the obtained dried products
in 2.0 mL of anhydrous DMSO. Absorbance of the solutions was then measured spectrophotometrically
at 276 nm after appropriate dilution 1:10 in anhydrous DMSO, and the drug content in the NP was
determined. Drug loading (DL) in the NP was calculated by using the calibration curve, built in a
range between 0.05 and 0.001 mg/mL, as follows: DL (%) = A/B × 100, where A is the drug content in the NP, and B is the weight of NP. The results were confirmed taking
into consideration the polymer’s interference in the analysis. where A is the drug content in the NP, and B is the weight of NP. The results were confirmed taking
into consideration the polymer’s interference in the analysis. emission wavelength of 373 nm.
2.4. Transmission Electron Microscopy (TEM) Analysis 2.4. Transmission Electron Microscopy (TEM) Analysis
In order to characterize morphology and size of NPs, a TEM analysis was carried out. Samples
were visualized using an electronic microscope JEOL JEM-2100 at 100 KV, and Digital Micrograph
Gatan (Gatan, Inc., Pleasanton, CA, USA) was used as the software. Samples (1.0 mg) were
solubilized in water (1.0 mL). A drop of each sample was placed in a carbon-coated grid and dried
In order to characterize morphology and size of NPs, a TEM analysis was carried out. Samples
were visualized using an electronic microscope JEOL JEM-2100 at 100 KV, and Digital Micrograph
Gatan (Gatan, Inc., Pleasanton, CA, USA) was used as the software. Samples (1.0 mg) were solubilized
in water (1.0 mL). A drop of each sample was placed in a carbon-coated grid and dried overnight
before analysis. 7 of 20 7 of 20 Pharmaceutics 2019, 11, 675 2.5. Rheological Analysis Firstly, 2% w/v HA-FA-HEG-SA and HA (MW 8700 g·mol−1) solutions were prepared in bi-distilled
water. Flow curves were recorded using an HR-2 Hybrid-Rheometer (TA-Instruments, Leatherhead,
United Kingdom) with a cone-plate measuring device 40 mm in diameter, employing a gap size of 0.1
mm. The temperature was maintained at 25 ◦C. The dynamic viscosity η under a controlled shear rate
ranging from 1 to 100 s−1 was measured [28]. 2.9. Cytotoxicity Assay Cell viability was assessed on human bronchial epithelial cells (16HBE) by the tetrazolium salt
(MTS) assay. Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10% FBS (fetal
bovine serum) and 1% penicillin/streptomycin (100 U·mL−1 penicillin and 100 µg·mL−1 streptomycin),
at 37 ◦C in a 5% CO2 humidified atmosphere after seeding in a 96-well plate at a density of 2 ×
104 cells per well. Different amounts of nanosystems (i.e., empty and OZ-loaded HA-FA-HEG-OA
and HA-FA-HEG-SA) and free OZ as control were weighed in order to obtain drug concentrations
corresponding to 350 µM, 200 µM, 100 µM, and 50 µM. The empty and OZ-loaded systems were
dispersed in DMEM, while OZ was dissolved in DMSO because of the low drug solubility in water. Aliquots of the sample dispersions (200 µL) were added to the cells and incubated for 24 h. After
incubation, in each well, the medium was replaced with 100 µL of fresh DMEM and 20 µL of MTS
(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) reagent
solution. Plates were incubated at 37 ◦C in a 5% CO2 humidified atmosphere for 1 h. Absorbance
was measured by a multi-well plate reader (Eppendorf AF 2200) at 490 nm. All the experiments were
performed in triplicate, and average values were calculated. The equation, (Abs490 treated cells/Abs490
control cells) × 100, was used to calculate the relative cell viability (percentage) as an average of three
values. A positive control was obtained with untreated cells. Pharmaceutics 2019, 11, x
8 of 20
Cell viability was assessed on human bronchial epithelial cells (16HBE) by the tetrazolium salt
(MTS) assay. Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10% FBS
(fetal bovine serum) and 1% penicillin/streptomycin (100 U∙mL−1 penicillin and 100 μg∙mL−1
streptomycin), at 37 °C in a 5% CO2 humidified atmosphere after seeding in a 96-well plate at a
density of 2 × 104 cells per well. Different amounts of nanosystems (i.e., empty and OZ-loaded
HA-FA-HEG-OA and HA-FA-HEG-SA) and free OZ as control were weighed in order to obtain
drug concentrations corresponding to 350 μM, 200 μM, 100 μM, and 50 μM. The empty and
OZ-loaded systems were dispersed in DMEM, while OZ was dissolved in DMSO because of the low
drug solubility in water. Aliquots of the sample dispersions (200 μL) were added to the cells and
incubated for 24 h. 2.8. Drug Release Studies The release of OZ from HA-FA-HEG-OA and HA-FA-HEG-SA NP was evaluated using the
dialysis bag diffusion technique. The release studies were performed in Dulbecco’s phosphate-buffered
saline (DPBS) (pH 7.4) mimicking plasma compartment pH conditions, with 3% (v/v) Tween-80 to
create a perfect sink condition, since OZ has limited solubility in the buffer. The aqueous NP dispersion
equivalent to 0.7 mg of OZ was placed into a dialysis bag (cut-off3.5–5 kDa; Spectra por, Flot-A-Lyzer
G2 Dialysis Device). The dialysis bag was immersed in the acceptor compartment containing 50 mL of
DPBS pH 7.4 plus 3% v/v Tween-80, and incubated under continuous stirring (100 rpm) and maintained
at 37 ◦C in an orbital shaker incubator for 30 h. The acceptor compartment was covered to prevent the
evaporation. At regular time intervals, specific volumes of the external medium were withdrawn, and
the same volume was replaced by fresh buffer. The withdrawn samples were centrifuged and then
freeze-dried. The solid residues were dissolved in DMSO and analyzed spectrophotometrically at 276
nm. The amount of released OZ was determined by using a calibration curve, built in a range between
0.05 and 0.001 mg/mL. The released OZ was determined as a function of incubation time, and it is
reported as the weight percentage of the total amount of drug loaded into the polymer aggregates. The
dilution procedure was considered in the correction of the data. Pharmaceutics 2019, 11, 675 8 of 20 2.9. Cytotoxicity Assay After incubation, in each well, the medium was replaced with 100 μL of fresh
DMEM
and
20
μL
of
MTS
(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)
reagent solution. Plates were incubated at 37 °C in a 5% CO2 humidified atmosphere for 1 h. Absorbance was measured by a multi-well plate reader (Eppendorf AF 2200) at 490 nm. All the
experiments were performed in triplicate, and average values were calculated. The equation,
(Abs490 treated cells/Abs490 control cells) × 100 was used to calculate the relative cell viability 2.10. Statistical Analysis
(percentage) as an av A one-way analysis of variance (ANOVA) was applied to compare different samples. Data were
considered statistically significant with a value of p < 0.05, and differences between groups were
compared using the Bonferroni t-test. Each test was developed in triplicate. 2.10. Statistical Analysis
A one-way analysis of variance (ANOVA) was applied to compare different samples. Data were
considered statistically significant with a value of p < 0.05, and differences between groups were
o
a ed u i
the Bo fe o i t te t Ea h te t
a de elo ed i
t i li ate 3.1. Synthesis of HA-FA-HEG-OA and HA-FA-HEG-SA Materials
3. Results and Discussion
h
f
A
A
E
OA
d
A
A
E
A
l HA-FA-HEG-OA and HA-FA-HEG-SA derivatives were prepared from the previously
published HA-FA-Pg derivative, bearing a mean distribution of 4–5 propargyl derivatives for each
macromolecule [11] by CuAAC coupling [29] with the suitable α-azido-ω-fatty amide hexa(ethylene
glycol) (azido-HEG-OA, 4a or azido-HEG-SA, 4b) as shown in Scheme 1. 3.1. Synthesis of HA-FA-HEG-OA and HA-FA-HEG-SA Materials
HA-FA-HEG-OA and HA-FA-HEG-SA derivatives were prepared from the previously
published HA-FA-Pg derivative, bearing a mean distribution of 4–5 propargyl derivatives for each
macromolecule [11] by CuAAC coupling [29] with the suitable α-azido-ω-fatty amide hexa(ethylene
glycol) (azido-HEG-OA, 4a or azido-HEG-SA, 4b) as shown in Scheme 1. Scheme 1. Synthesis of HA-FA-HEG-OA and HA-FA-HEG-SA materials. Reagents: (i) CDI, THF;
(ii) TEA, THF; (iii) CuSO4, sodium ascorbate, tert-BuOH, H2O. Substituents: R = H or C2H5; R’ =
C17H33 (1a, 2a, 4a, HA-FA-HEG-OA) or C17H35 (1b, 2b, 4b, HA-FA-HEG-SA). Scheme 1. Synthesis of HA-FA-HEG-OA and HA-FA-HEG-SA materials. Reagents: (i) CDI, THF;
(ii) TEA, THF; (iii) CuSO4, sodium ascorbate, tert-BuOH, H2O. Substituents: R = H or C2H5; R’ =
C17H33 (1a, 2a, 4a, HA-FA-HEG-OA) or C17H35 (1b, 2b, 4b, HA-FA-HEG-SA). Scheme 1. Synthesis of HA-FA-HEG-OA and HA-FA-HEG-SA materials. Reagents: (i) CDI, THF;
(ii) TEA, THF; (iii) CuSO4, sodium ascorbate, tert-BuOH, H2O. Substituents: R = H or C2H5; R’ =
C17H33 (1a, 2a, 4a, HA-FA-HEG-OA) or C17H35 (1b, 2b, 4b, HA-FA-HEG-SA). Scheme 1. Synthesis of HA-FA-HEG-OA and HA-FA-HEG-SA materials. Reagents: (i) CDI, THF;
(ii) TEA, THF; (iii) CuSO4, sodium ascorbate, tert-BuOH, H2O. Substituents: R = H or C2H5; R’ =
C17H33 (1a, 2a, 4a, HA-FA-HEG-OA) or C17H35 (1b, 2b, 4b, HA-FA-HEG-SA). 9 of 20
9 of 20
9 of 20 Pharmaceutics 2019, 11, 675
Pharmaceutics 2019, 11, x
Pharmaceutics 2019, 11, x In
particular,
the
appropriate
fatty
acid
(OA,
1a
or
SA,
1b)
was
activated
with
1,1′-carbonyldiimidazole (CDI) in refluxing THF to obtain the corresponding imidazolide (2a,b),
which was promptly reacted with α-azido-ω-amino hexa(ethylene glycol) (3) to obtain the required
azido-HEG-OA (4a) or azido-HEG-SA (4b). The CuAAC coupling was performed under very mild
conditions with the copper(I) catalyst being generated in situ with CuSO4/sodium ascorbate. Moreover,
the coupling reaction was carried out by using HA-FA-Pg samples obtained from low-molecular-weight
HA (MW about 8.7 kg·mol−1) and showing a degree of grafting around 20%, corresponding to four
propargyl groups per HA macromolecule in order to prepare materials bearing about four fatty-acid
side chains pending from the HA backbone [11]. 3.1. Synthesis of HA-FA-HEG-OA and HA-FA-HEG-SA Materials
3. Results and Discussion
h
f
A
A
E
OA
d
A
A
E
A
l In particular, the appropriate fatty acid (OA, 1a or SA, 1b) was activated with
1,1′-carbonyldiimidazole (CDI) in refluxing THF to obtain the corresponding imidazolide (2a,b),
which was promptly reacted with α-azido-ω-amino hexa(ethylene glycol) (3) to obtain the required
azido-HEG-OA (4a) or azido-HEG-SA (4b). The CuAAC coupling was performed under very mild
conditions with the copper(I) catalyst being generated in situ with CuSO4/sodium ascorbate. Moreover, the coupling reaction was carried out by using HA-FA-Pg samples obtained from
low-molecular-weight HA (MW about 8.7 kg∙mol−1) and showing a degree of grafting around 20%,
corresponding to four propargyl groups per HA macromolecule in order to prepare materials
bearing about four fatty-acid side chains pending from the HA backbone [11]. The intermediate α azido ω amino hexa(ethylene glycol) [27] (3) was synthesized as described
1,1′-carbonyldiimidazole (CDI) in refluxing THF to obtain the corresponding imidazolide (2a,b),
which was promptly reacted with α-azido-ω-amino hexa(ethylene glycol) (3) to obtain the required
azido-HEG-OA (4a) or azido-HEG-SA (4b). The CuAAC coupling was performed under very mild
conditions with the copper(I) catalyst being generated in situ with CuSO4/sodium ascorbate. Moreover, the coupling reaction was carried out by using HA-FA-Pg samples obtained from
low-molecular-weight HA (MW about 8.7 kg∙mol−1) and showing a degree of grafting around 20%,
corresponding to four propargyl groups per HA macromolecule in order to prepare materials
bearing about four fatty-acid side chains pending from the HA backbone [11]. The intermediate α-azido-ω-amino hexa(ethylene glycol) [27] (3) was synthesized as described
i
Sche
e 2 a d p e iously epo ted i
Refe e ce [27] The intermediate α-azido-ω-amino hexa(ethylene glycol) [27] (3) was synthesized as described in
Scheme 2 and previously reported in Reference [27]. The intermediate α-azido-ω-amino hexa(ethylene glycol) [27] (3) was synthesized as described
in Scheme 2 and previously reported in Reference [27]. in Scheme 2 and previously reported in Reference [27]. Scheme 2. Synthesis of hexa(ethylene glycol) derivative 3. Reagents: (i) MesCl, TEA, CH2Cl2; (ii)
N N
B
N B
DMF (iii) PPh
l
HCl 3 N
Scheme 2. Synthesis of hexa(ethylene glycol) derivative 3. Reagents: (i) MesCl, TEA, CH2Cl2; (ii) NaN3,
Bu4N+Br−, DMF; (iii) PPh3, toluene, HCl 3 N. Scheme 2. Synthesis of hexa(ethylene glycol) derivative 3. Reagents: (i) MesCl, TEA, CH2Cl2; (ii)
NaN3, Bu4N+Br-, DMF; (iii) PPh3, toluene, HCl 3 N. Scheme 2. Synthesis of hexa(ethylene glycol) derivative 3. 3.2. Structure of HA-FA-HEG-OA and HA-FA-HEG-SA Materials
3.2. Structure of HA-FA-HEG-OA and HA-FA-HEG-SA Materials The conversion of the alkyne moiety of HA-FA-Pg into the triazole one of HA-FA-HEG-OA and The conversion of the alkyne moiety of HA-FA-Pg into the triazole one of HA-FA-HEG-OA and
HA-FA-HEG-SA, indicative of a successful CuAAC coupling reaction, was proven by the disappearance
of the signal of acetylene proton D at 2.98 ppm observed in the 1H-NMR spectrum of HA-FA-Pg
(Figure 3b) and the appearance of a new peak at around 8 ppm in the spectra of HA-FA-HEG-OA and
HA-FA-HEG-SA (Figure 3c,d). Analogously, in the 13C-NMR spectra (Figure 4), the alkyne signal C at
78.5 ppm disappeared. This observation was first evidence of the covalent insertion of components
HEG-OA or HEG-SA in the new materials. Moreover, a significant line broadening was observed for
the signals assigned to the aromatic and acrylic protons of the FA moiety. e co ve sio
o t e a ky e
oiety o
A
A
g i to t e t ia o e o e o
A
A
EG OA a d
HA-FA-HEG-SA, indicative of a successful CuAAC coupling reaction, was proven by the
disappearance of the signal of acetylene proton D at 2.98 ppm observed in the 1H-NMR spectrum of
HA-FA-Pg (Figure 3b) and the appearance of a new peak at around 8 ppm in the spectra of
HA-FA-HEG-OA and HA-FA-HEG-SA (Figure 3c,3d). Analogously, in the 13C-NMR spectra (Figure
4), the alkyne signal C at 78.5 ppm disappeared. This observation was first evidence of the covalent
insertion of components HEG-OA or HEG-SA in the new materials. Moreover, a significant line
broadening was observed for the signals assigned to the aromatic and acrylic protons of the FA
moiety. Figure 3. Comparison of 1H-NMR spectra obtained from HA-FA-HEG-OA (c) and HA-FA-HEG-SA
(d) derivatives measured in D2O and FE-HEG-OA (e) measured in CDCl3 with those obtained from
the starting HA-FA-Pg (b) and HA (a) samples (solvent: D2O; (a) and (b) were recorded with water
suppression). In the spectrum of HA, “Et” labels indicate the signals of ethyl groups of the
monomeric units with R = C2H5. Figure 3. Comparison of 1H-NMR spectra obtained from HA-FA-HEG-OA (c) and HA-FA-HEG-SA
(d) derivatives measured in D2O and FE-HEG-OA (e) measured in CDCl3 with those obtained from
the starting HA-FA-Pg (b) and HA (a) samples (solvent: D2O; (a) and (b) were recorded with water
suppression). 3.2. Structure of HA-FA-HEG-OA and HA-FA-HEG-SA Materials
3.2. Structure of HA-FA-HEG-OA and HA-FA-HEG-SA Materials 3.2. Structure of HA-FA-HEG-OA and HA-FA-HEG-SA Materials
3.2. Structure of HA-FA-HEG-OA and HA-FA-HEG-SA Materials The structure of HA-FA-HEG-OA and HA-FA-HEG-SA derivatives was studied by 1H- and
13C-NMR spectroscopy in D2O as the solvent (Figures 3 and 4). The structure of HA-FA-HEG-OA and HA-FA-HEG-SA derivatives was studied by 1H- and
13C-NMR spectroscopy in D2O as the solvent (Figure 3 and 4). In a first step, the assignment of the 1H- and 13C-NMR spectra of HA-FA-Pg was re-investigated
in order to evaluate previous assignments [11,20]. In particular, we focused on the signals of the
propargyl group, which are considered diagnostic signals for evaluating the functionalization of the
HA-FA-Pg copolymer. Combing one-dimensional (1D) and two-dimensional (2D) NMR methods, the
signals of propargyl protons B and D, as well as of carbons B, C, and D, can be unequivocally assigned
(Figures 3b and 4b)
In a first step, the assignment of the 1H- and 13C-NMR spectra of HA-FA-Pg was re-investigated
in order to evaluate previous assignments [11,20]. In particular, we focused on the signals of the
propargyl group, which are considered diagnostic signals for evaluating the functionalization of the
HA-FA-Pg copolymer. Combing one-dimensional (1D) and two-dimensional (2D) NMR methods,
the signals of propargyl protons B and D, as well as of carbons B, C, and D, can be unequivocally
assigned (Figure 3b,4b)
The intensity of the proton signals allows monitoring their conversion after the CuAAC click The intensity of the proton signals allows monitoring their conversion after the CuAAC click
reaction. Thus, in order to ascertain the effectiveness of the CuAAC coupling reaction producing
HA-FA-HEG-OA and HA-FA-HEG-SA derivatives, the 1H- and 13C-NMR spectra of these materials
were compared with the corresponding spectra of HA and HA-FA-Pg (Figures 3 and 4). The intensity of the proton signals allows monitoring their conversion after the CuAAC click
reaction. Thus, in order to ascertain the effectiveness of the CuAAC coupling reaction producing
HA-FA-HEG-OA and HA-FA-HEG-SA derivatives, the 1H- and 13C-NMR spectra of these materials
were compared with the corresponding spectra of HA and HA-FA-Pg (Figure 3 and 4). 3.1. Synthesis of HA-FA-HEG-OA and HA-FA-HEG-SA Materials
3. Results and Discussion
h
f
A
A
E
OA
d
A
A
E
A
l Reagents: (i) MesCl, TEA, CH2Cl2; (ii)
NaN3 Bu4N+Br- DMF; (iii) PPh3 toluene HCl 3 N
Scheme 2. Synthesis of hexa(ethylene glycol) derivative 3. Reagents: (i) MesCl, TEA, CH2Cl2; (ii) NaN3,
Bu4N+Br−, DMF; (iii) PPh3, toluene, HCl 3 N. Scheme 2. Synthesis of hexa(ethylene glycol) derivative 3. Reagents: (i) MesCl, TEA, CH2Cl2; (ii)
NaN3, Bu4N+Br-, DMF; (iii) PPh3, toluene, HCl 3 N. (
)
Finally, compound FA-HEG-OA and its methyl ester precursor 8 were synthesized from
ferulate derivative 9 [11] and compound 4a as described in Scheme 3 in order to be used as model
Finally, compound FA-HEG-OA and its methyl ester precursor 8 were synthesized from ferulate
derivative 9 [11] and compound 4a as described in Scheme 3 in order to be used as model compounds. Finally, compound FA-HEG-OA and its methyl ester precursor 8 were synthesized from
ferulate derivative 9 [11] and compound 4a as described in Scheme 3 in order to be used as model
compounds mpounds. Scheme 3. Synthesis of model compounds 8 and FA-HEG-OA. Reagents: (i) CuSO4, sodium
ascorbate tert-BuOH H2O; (ii) NaOH H2O C2H5OH
p
Scheme 3. Synthesis of model compounds 8 and FA-HEG-OA. Reagents: (i) CuSO4, sodium
ascorbate, tert-BuOH, H2O; (ii) NaOH, H2O, C2H5OH. Scheme 3. Synthesis of model compounds 8 and FA-HEG-OA. Reagents: (i) CuSO4, sodium ascorbate,
tert-BuOH, H2O; (ii) NaOH, H2O, C2H5OH. Scheme 3. Synthesis of model compounds 8 and FA-HEG-OA. Reagents: (i) CuSO4, sodium
Scheme 3. Synthesis of model compounds 8 and FA-HEG-OA. Reagents: (i) CuSO4, sodium
ascorbate, tert-BuOH, H2O; (ii) NaOH, H2O, C2H5OH. Scheme 3. Synthesis of model compounds 8 and FA-HEG-OA. Reagents: (i) CuSO4, sodium ascorbate,
tert-BuOH, H2O; (ii) NaOH, H2O, C2H5OH. Pharmaceutics 2019, 11, 675 10 of 20 3.2. Structure of HA-FA-HEG-OA and HA-FA-HEG-SA Materials
3.2. Structure of HA-FA-HEG-OA and HA-FA-HEG-SA Materials Comparison of 13C-NMR spectra of HA-FA-HEG-SA (c) and HA-FA-HEG-OA (d) derivatives
measured in D2O and FE-HEG-OA (e) measured in CDCl3 with those obtained from starting HA-FA-Pg
(b) and HA (a) samples in D2O. In the spectrum of HA, “Et” labels indicate the signals of ethyl groups
of the monomeric units with R = C2H5. Owing to their complex structures, the
1H-NMR spectra of HA-FA-HEG-OA and
HA-FA-HEG-SA (Figure 3) were composed by a hard-to-assign set of broad and partially
overlapped signals of the different substructures, representing the hyaluronan backbone, FA triazole
moiety, and PEG fatty acid functional group. However, the 13C-NMR spectra allowed a closer view
of the chemical structures. In this context, two model substances, FE-HEG-OA (Figure 3e,4e) and
FE-HEG-SA (Figure 2), were synthesized and characterized in detail by 1H- and 13C-NMR
spectroscopy. As obvious from the comparison of 13C-NMR spectra in Figure 4, in addition to the
dominating signals of the HA backbone, both the HEG signals in the 72–69 ppm region and the alky
group signals (40 – 25 ppm, CH3 group at 14.6 ppm) appeared. For the OA derivative, the olefinic
carbons resulted in two signals at 130.4 and 130.0 ppm. Unlike the parent HA-FA-Pg the signals of the aromatic carbon atoms of the ferulate triazole
Owing to their complex structures, the 1H-NMR spectra of HA-FA-HEG-OA and HA-FA-HEG-SA
(Figure 3) were composed by a hard-to-assign set of broad and partially overlapped signals of the
different substructures, representing the hyaluronan backbone, FA triazole moiety, and PEG fatty acid
functional group. However, the 13C-NMR spectra allowed a closer view of the chemical structures. In this context, two model substances, FE-HEG-OA (Figures 3e and 4e) and FE-HEG-SA (Figure 2),
were synthesized and characterized in detail by 1H- and 13C-NMR spectroscopy. As obvious from the
comparison of 13C-NMR spectra in Figure 4, in addition to the dominating signals of the HA backbone,
both the HEG signals in the 72–69 ppm region and the alky group signals (40 – 25 ppm, CH3 group at
14.6 ppm) appeared. For the OA derivative, the olefinic carbons resulted in two signals at 130.4 and
130.0 ppm. Unlike the parent HA FA Pg, the signals of the aromatic carbon atoms of the ferulate triazole
structure were almost not detectable. 3.2. Structure of HA-FA-HEG-OA and HA-FA-HEG-SA Materials
3.2. Structure of HA-FA-HEG-OA and HA-FA-HEG-SA Materials In the spectrum of HA, “Et” labels indicate the signals of ethyl groups of the monomeric
units with R = C2H5. Figure 3. Comparison of 1H-NMR spectra obtained from HA-FA-HEG-OA (c) and HA-FA-HEG-SA
(d) derivatives measured in D2O and FE-HEG-OA (e) measured in CDCl3 with those obtained from
the starting HA-FA-Pg (b) and HA (a) samples (solvent: D2O; (a) and (b) were recorded with water
suppression). In the spectrum of HA, “Et” labels indicate the signals of ethyl groups of the
monomeric units with R = C2H5. Figure 3. Comparison of 1H-NMR spectra obtained from HA-FA-HEG-OA (c) and HA-FA-HEG-SA
(d) derivatives measured in D2O and FE-HEG-OA (e) measured in CDCl3 with those obtained from
the starting HA-FA-Pg (b) and HA (a) samples (solvent: D2O; (a) and (b) were recorded with water
suppression). In the spectrum of HA, “Et” labels indicate the signals of ethyl groups of the monomeric
units with R = C2H5. Pharmaceutics 2019, 11, 675
11 of 20
Pharmaceutics 2019, 11, x
11 of 20
Figure 4. Comparison of 13C-NMR spectra of HA-FA-HEG-SA (c) and HA-FA-HEG-OA (d)
derivatives measured in D2O and FE-HEG-OA (e) measured in CDCl3 with those obtained from
starting HA-FA-Pg (b) and HA (a) samples in D2O. In the spectrum of HA, “Et” labels indicate the
signals of ethyl groups of the monomeric units with R = C2H5. Figure 4. Comparison of 13C-NMR spectra of HA-FA-HEG-SA (c) and HA-FA-HEG-OA (d) derivatives
measured in D2O and FE-HEG-OA (e) measured in CDCl3 with those obtained from starting HA-FA-Pg
(b) and HA (a) samples in D2O. In the spectrum of HA, “Et” labels indicate the signals of ethyl groups
of the monomeric units with R = C2H5. Pharmaceutics 2019, 11, 675 11 of 20 ,
,
Pharmaceutics 2019, 11, x
11 of 20
Figure 4
Comparison of 13C-NMR spectra of
HA-FA-HEG-SA (c) and HA-FA-HEG-OA (d)
Figure 4. Comparison of 13C-NMR spectra of HA-FA-HEG-SA (c) and HA-FA-HEG-OA (d) derivatives Figure 4. Comparison of 13C-NMR spectra of HA-FA-HEG-SA (c) and HA-FA-HEG-OA (d)
derivatives measured in D2O and FE-HEG-OA (e) measured in CDCl3 with those obtained from
starting HA-FA-Pg (b) and HA (a) samples in D2O. In the spectrum of HA, “Et” labels indicate the
signals of ethyl groups of the monomeric units with R = C2H5. Figure 4. 3.3. Self-Assembling Properties
Pharmaceutics 2019, 11, x HA-FA-HEG-OA and HA-FA-HEG-SA were designed to be biocompatible and partially
hydrophilic polymers showing clear-cut aggregation features in an aqueous environment. Thanks to
their partially amphiphilic features, they are potentially capable of forming self-assembled nanoparticles
in water dispersions and loading lipophilic drugs such as olanzapine, the solubility of which is rather
negligible in physiological fluids. Moreover, the hydrophilic portion of the copolymer, which we
assumed to be exposed on the surface of the nanosystems, can be used to provide the nanoparticles
with stealth properties for longer circulation times. 3.3. Self-Assembling Properties
HA-FA-HEG-OA and HA-FA-HEG-SA were designed to be biocompatible and partially
hydrophilic polymers showing clear-cut aggregation features in an aqueous environment. Thanks to
their partially amphiphilic features, they are potentially capable of forming self-assembled
nanoparticles in water dispersions and loading lipophilic drugs such as olanzapine, the solubility of
which is rather negligible in physiological fluids. Moreover, the hydrophilic portion of the
copolymer, which we assumed to be exposed on the surface of the nanosystems, can be used to
id
h
i l
i h
l h
i
f
l
i
l
i
i In particular, HA-FA-HEG-OA and HA-FA-HEG-SA derivatives showed a tendency to create
cytocompatible nanostructured aggregates in water, owing to the simultaneous presence of hydrophilic
portions in the polymeric chain (i.e., hyaluronic acid, an essential component of connective tissues in
humans that plays an essential role in the early stages of cell adhesion) and oleic or stearic hydrophobic
portions. The resulting features of the polymeric nanocarriers, such as surface charge, dimensions,
balance between hydrophilic and hydrophobic parts, and biocompatibility, confirmed the possibility of
using these materials as drug delivery systems. provide the nanoparticles with stealth properties for longer circulation times. In particular, HA-FA-HEG-OA and HA-FA-HEG-SA derivatives showed a tendency to create
cytocompatible nanostructured aggregates in water, owing to the simultaneous presence of
hydrophilic portions in the polymeric chain (i.e., hyaluronic acid, an essential component of
connective tissues in humans that plays an essential role in the early stages of cell adhesion) and
oleic or stearic hydrophobic portions. The resulting features of the polymeric nanocarriers, such as
surface charge, dimensions, balance between hydrophilic and hydrophobic parts, and
biocompatibility confirmed the possibility of using these materials as drug delivery systems Polymers were simply dispersed in Milli-Q water in a concentration of 0.5 mg/mL and left to shake
alternating with sonication cycles in order to unpack the macromolecules and favor dissolution. 3.2. Structure of HA-FA-HEG-OA and HA-FA-HEG-SA Materials
3.2. Structure of HA-FA-HEG-OA and HA-FA-HEG-SA Materials In an attempt to explain this result, we assumed that the FA
moiety suffered from reduced mobility, maybe due to poor solvation in D2O producing a
pronounced line broadening as already observed in the 1H-NMR spectra in Figure 3. Here, one has
to recall the disappearance of propargyl signals and the persistence of the signal assigned to the
ferulate methoxy (at about 55.6 ppm). Consequently, by comparison of the NMR spectra of the new
copolymer materials with those of the starting materials and synthesized model molecules, we
successfully assigned all detectable signals of the complex spectra obtained for HA-FA-HEG-OA
and HA-FA-HEG-SA. Unlike the parent HA-FA-Pg, the signals of the aromatic carbon atoms of the ferulate–triazole
structure were almost not detectable. In an attempt to explain this result, we assumed that the FA
moiety suffered from reduced mobility, maybe due to poor solvation in D2O producing a pronounced
line broadening as already observed in the 1H-NMR spectra in Figure 3. Here, one has to recall the
disappearance of propargyl signals and the persistence of the signal assigned to the ferulate methoxy
(at about 55.6 ppm). Consequently, by comparison of the NMR spectra of the new copolymer materials
with those of the starting materials and synthesized model molecules, we successfully assigned all
detectable signals of the complex spectra obtained for HA-FA-HEG-OA and HA-FA-HEG-SA. 12 of 20 12 of 20 Pharmaceutics 2019, 11, 675 3.3. Self-Assembling Properties
Pharmaceutics 2019, 11, x 11, x Moreover, the negative surface charge (in the range −35 to −47 mV) shown by both systems
represents a stabilizing factor owing to repulsion between NPs, preventing their aggregation. Moreover, the negative surface charge (in the range −35 to −47 mV) shown by both systems
represents a stabilizing factor owing to repulsion between NPs, preventing their aggregation. In order to evaluate the potential critical aggregation concentration (CAC) of polymers in aqueous
media, pyrene was used as the fluorescent hydrophobic probe. The high hydrophobicity of the probe
should allow us to predict the ability of the system to internalize active molecules. This method is
based on the difference in pyrene fluorescence when the probe is situated in a water solution or in
the hydrophobic nucleus of micelles [30]. In particular, both the I1/I3 ratio obtained from the pyrene
emission spectra and the I338/I332 ratio obtained from the pyrene excitation spectra, measured at 25 ◦C,
were plotted versus the logarithm of the copolymer concentration. The obtained curves were used
for the evaluation of the critical aggregation concentration (CAC) identifiable with the intersection
of the tangent to the curve at the inflection with the horizontal tangent through the points at lower
concentrations [31]. In order to evaluate the potential critical aggregation concentration (CAC) of polymers in
aqueous media, pyrene was used as the fluorescent hydrophobic probe. The high hydrophobicity of
the probe should allow us to predict the ability of the system to internalize active molecules. This
method is based on the difference in pyrene fluorescence when the probe is situated in a water
solution or in the hydrophobic nucleus of micelles [30]. In particular, both the I1/I3 ratio obtained
from the pyrene emission spectra and the I338/I332 ratio obtained from the pyrene excitation spectra,
measured at 25 °C, were plotted versus the logarithm of the copolymer concentration. The obtained
curves were used for the evaluation of the critical aggregation concentration (CAC) identifiable with
the intersection of the tangent to the curve at the inflection with the horizontal tangent through the
points at lower concentrations [31]. As can be seen in the graph shown in Figure 6A, for the HA-FA-HEG-OA derivative, there was not a
sufficiently significant change in the emission and excitation spectra to justify a concentration-dependent
aggregation phenomenon. 3.3. Self-Assembling Properties
Pharmaceutics 2019, 11, x The
systems were left to agitate overnight and then analyzed by DLS after filtration in order to evaluate both
the presence of dispersed aggregates and the zeta potential of the particulate matter. The results shown
in Table 1 confirmed the ability of HA-FA-HEG-OA and HA-FA-HEG-SA derivatives to self-assemble
into NPs upon dispersion in water. However, DLS curves (Figure 5) revealed the presence of two
heterogeneous populations: one showing a small mean diameter (below 20 nm) that was assumed to
be constituted by small aggregates of a few macromolecules, and a second population with greater
dimensions constituted by a large number of macromolecules. biocompatibility, confirmed the possibility of using these materials as drug delivery systems. Polymers were simply dispersed in Milli-Q water in a concentration of 0.5 mg/mL and left to
shake alternating with sonication cycles in order to unpack the macromolecules and favor
dissolution. The systems were left to agitate overnight and then analyzed by DLS after filtration in
order to evaluate both the presence of dispersed aggregates and the zeta potential of the particulate
matter. The results shown in Table 1 confirmed the ability of HA-FA-HEG-OA and HA-FA-HEG-SA
derivatives to self-assemble into NPs upon dispersion in water. However, DLS curves (Figure 5)
revealed the presence of two heterogeneous populations: one showing a small mean diameter
(below 20 nm) that was assumed to be constituted by small aggregates of a few macromolecules, and
a second population with greater dimensions constituted by a large number of macromolecules. Figure 5. DLS size distribution histograms of HA-FA-HEG-OA (A) and HA-FA-HEG-SA (B) in
Milli-Q water. Figure 5. DLS size distribution histograms of HA-FA-HEG-OA (A) and HA-FA-HEG-SA (B) in
Milli-Q water. Figure 5. DLS size distribution histograms of HA-FA-HEG-OA (A) and HA-FA-HEG-SA (B) in
Milli-Q water. Figure 5. DLS size distribution histograms of HA-FA-HEG-OA (A) and HA-FA-HEG-SA (B) in
Milli-Q water. 13 of 20 Pharmaceutics 2019, 11, 675 Table 1. Size distribution and zeta (Z) potential values of empty systems. Samples
Size (nm)
PDI
Z-potential (mV)
HA-FA-HEG-OA
291
0.424
−47
HA-FA-HEG-SA
364
0.442
−35
11, x
Table 1. Size distribution and zeta (Z) potential values of empty systems. Samples
Size (nm)
PDI
Z-potential (mV)
HA-FA-HEG-OA
291
0.424
− 47
HA FA HEG SA
364
0 442
35 Table 1. Size distribution and zeta (Z) potential values of empty systems. 3.3. Self-Assembling Properties
Pharmaceutics 2019, 11, x Pharmaceutics 2019, 11, 675
HA-FA-HEG-SA (Figu
slightly smaller than th 14 of 20
ensions
regates Also, in the case of the HA-FA-HEG-SA derivative (Figure 6C), we were unable to obtain a
characteristic CAC value, but the results of DLS analysis in the concentration range between 0.5 and 10
mg/mL suggested the formation of colloidal particles of increasing size from 363 to 569 nm. small objects (diameter around 20 nm) and larger nano-aggregates. Self-assembling properties of the polymers were also investigated by evaluating the rheological
behavior of HA-FA-HEG-SA as a model sample. The viscosity trend of HA-FA-HEG-SA vs. shear
d
d
d
d
h
A
h
l
h b
d
h
h The results suggested that the materials do not form so-called micelles, but rather stable
self-assembled nanoparticles. rate is depicted in Figure 8 and compared with native HA. Both solutions exhibited a shear thinning
behavior, since their viscosities decreased with the shear rate increasing. Specifically,
HA FA HEG SA i
i
d
d f
38
3
P
0 13
0 01
P
i
h
h
f TEM observations, reported in Figure 7, showed that HA-FA-HEG-OA (Figure 7A,B) and
HA-FA-HEG-SA (Figure 7C,D) nano-aggregates appeared as spherical objects, showing dimensions
slightly smaller than those obtained by DLS measurements, due to the shrinkage of nano-aggregates
upon dehydration. In line with the DLS results, TEM analysis showed the contemporary presence of
small objects (diameter around 20 nm) and larger nano-aggregates. HA-FA-HEG-SA viscosity decreased from 38 ± 3 mPa∙s to 0.13 ± 0.01 mPa∙s in the shear rate range of
1–100 s−1, whereas native HA viscosity decreased from 14 ± 2 mPa∙s to 1.1 ± 0.1 mPa∙s in the same
range. The obtained results confirmed the capability of functionalized HA to self-assemble into
aggregate structures, whose formation was reflected in the doubled viscosity of the solution in
comparison with bare HA. A
B
C
D
Figure 7. Representative transmission electron micrograph of HA-FA-HEG-OA (A,B) and
HA-FA-HEG-SA (C,D) nanoparticles. B
D B Figure 7. Representative transmission electron micrograph of HA-FA-HEG-OA (A,B) and
HA-FA-HEG-SA (C,D) nanoparticles. Figure 7. Representative transmission electron micrograph of HA-FA-HEG-OA (A and B) and
HA-FA-HEG-SA (C and D) nanoparticles. Self-assembling properties of the polymers were also investigated by evaluating the rheological
behavior of HA-FA-HEG-SA as a model sample. The viscosity trend of HA-FA-HEG-SA vs. shear
rate is depicted in Figure 8 and compared with native HA. 3.3. Self-Assembling Properties
Pharmaceutics 2019, 11, x However, at the concentration of 0.5 mg/mL, DLS analysis (Figure 6B)
showed the presence of colloidal particles with a size of 378 nm, which stabilized to the size of 270 nm
at increasing concentrations between 1 and 10 mg/mL. As can be seen in the graph shown in Figure 6A, for the HA-FA-HEG-OA derivative, there was
not a sufficiently significant change in the emission and excitation spectra to justify a
concentration-dependent aggregation phenomenon. However, at the concentration of 0.5 mg/mL,
DLS analysis (Figure 6B) showed the presence of colloidal particles with a size of 378 nm, which
stabilized to the size of 270 nm at increasing concentrations between 1 and 10 mg/mL. Figure 6. I1(373)/I3(384) intensity ratios obtained from pyrene emission spectra (blue curve) and
I338/I332 intensity ratios obtained from pyrene excitation spectra (orange curve) for
HA-FA-HEG-OA derivative water dispersions (A) or HA-FA-HEG-SA derivative water dispersions
(C) at 25 °C, in the concentration range of 0.0001–10 mg/mL; size distribution values of
HA-FA-HEG-OA (B) or HA-FA-HEG-SA (D) in the same conditions. A)
B)
D)
C)
Figure 6. I1(373)/I3(384) intensity ratios obtained from pyrene emission spectra (blue curve) and I338/I332
intensity ratios obtained from pyrene excitation spectra (orange curve) for HA-FA-HEG-OA derivative
water dispersions (A) or HA-FA-HEG-SA derivative water dispersions (C) at 25 ◦C, in the concentration
range of 0.0001–10 mg/mL; size distribution values of HA-FA-HEG-OA (B) or HA-FA-HEG-SA (D) in
the same conditions. A)
B) B) A) D) D)
C) D) C) Figure 6. I1(373)/I3(384) intensity ratios obtained from pyrene emission spectra (blue curve) and
I338/I332 intensity ratios obtained from pyrene excitation spectra (orange curve) for
HA-FA-HEG-OA derivative water dispersions (A) or HA-FA-HEG-SA derivative water dispersions
(C) at 25 °C, in the concentration range of 0.0001–10 mg/mL; size distribution values of
HA-FA-HEG-OA (B) or HA-FA-HEG-SA (D) in the same conditions. Figure 6. I1(373)/I3(384) intensity ratios obtained from pyrene emission spectra (blue curve) and I338/I332
intensity ratios obtained from pyrene excitation spectra (orange curve) for HA-FA-HEG-OA derivative
water dispersions (A) or HA-FA-HEG-SA derivative water dispersions (C) at 25 ◦C, in the concentration
range of 0.0001–10 mg/mL; size distribution values of HA-FA-HEG-OA (B) or HA-FA-HEG-SA (D) in
the same conditions. (
g
3 4 Ol
i
L
di
d R l
3.4. Olanzapine Loading and Release 3.4. Olanzapine Loading and Release
Due to the potential ability of the nanosystems to load pharmacologically active molecules,
Due to the potential ability of the nanosystems to load pharmacologically active molecules,
olanzapine [32] was selected as a model drug. 3.4. Olanzapine Loading and Release
Due to the potential ability of the nanosystems to load pharmacologically active molecules,
Due to the potential ability of the nanosystems to load pharmacologically active molecules,
olanzapine [32] was selected as a model drug. olanzapine [32] was selected as a model drug. OZ is an antipsychotic agent contained in different pharmaceutical formulations as a free base
or hydrochloride salt. It is used for the treatment of central nervous system disorders and is available
as coated tablets for oral administration. Recently, the injectable form for intramuscular use of 10 mg
was introduced for the emergency treatment of psychomotor agitation in manic crises and
schizophrenic patients. OZ shows highly permeability to biological membranes; however, on the
OZ is an antipsychotic agent contained in different pharmaceutical formulations as a free base or
hydrochloride salt. It is used for the treatment of central nervous system disorders and is available as
coated tablets for oral administration. Recently, the injectable form for intramuscular use of 10 mg was
introduced for the emergency treatment of psychomotor agitation in manic crises and schizophrenic
patients. OZ shows highly permeability to biological membranes; however, on the other hand, it has
very low solubility in water, which limits its bioavailability [25]. other hand, it has very low solubility in water, which limits its bioavailability [25]. In order to increase the bioavailability and reduce its cytotoxic effects, olanzapine was
physically adsorbed into both polymeric systems reported above. After incorporation, the systems
In order to increase the bioavailability and reduce its cytotoxic effects, olanzapine was physically
adsorbed into both polymeric systems reported above. After incorporation, the systems were purified
by centrifugation and filtration in order to remove non-incorporated drug. were purified by centrifugation and filtration in order to remove non-incorporated drug. Subsequent analysis by DLS showed some modifications of the physical parameters upon drug
incorporation, with a consequent lowering of size and the zeta potential with respect to the empty
nanosystems (Table 2). Definitively, the calculated drug loading values demonstrated that the NPs
are capable of incorporating a good quantity of OZ. 3.3. Self-Assembling Properties
Pharmaceutics 2019, 11, x Both solutions exhibited a shear thinning
behavior, since their viscosities decreased with the shear rate increasing. Specifically, HA-FA-HEG-SA
viscosity decreased from 38 ± 3 mPa·s to 0.13 ± 0.01 mPa·s in the shear rate range of 1–100 s−1, whereas
native HA viscosity decreased from 14 ± 2 mPa·s to 1.1 ± 0.1 mPa·s in the same range. The obtained
results confirmed the capability of functionalized HA to self-assemble into aggregate structures, whose
formation was reflected in the doubled viscosity of the solution in comparison with bare HA. 15 of 20
15 of 20 Pharmaceutics 2019, 11, 675
Pharmaceutics 2019, 11, x Figure 8. Viscosity curve of 2% w/v aqueous solution of HA (■) and HA-FA-HEG-SA (●) as a
function of shear rate (range 1–100 s−1). Figure 8. Viscosity curve of 2% w/v aqueous solution of HA (■) and HA-FA-HEG-SA () as a function
of shear rate (range 1–100 s−1). Figure 8. Viscosity curve of 2% w/v aqueous solution of HA (■) and HA-FA-HEG-SA (●) as a
function of shear rate (range 1 100 s−1)
Figure 8. Viscosity curve of 2% w/v aqueous solution of HA (■) and HA-FA-HEG-SA () as a function
of shear rate (range 1–100 s−1). 3 5 I
Vit
Bi l
i
l E
l
ti
3.5. In Vitro Biological Evaluations 3.5. In Vitro Biological Evaluations
In order to evaluate the possibility of using HA-FA-HEG-OA and HA-FA-HEG-SA NP as drug
release systems in vivo, preliminary biological tests were carried out using the MTS assay [34]. The
cytocompatibility of the nanosystems was evaluated using a normal human cell line, namely,
bronchial epithelial cells (16HBE) [35]. 16HBE was chosen because it is one of the most commonly
used model cell lines for evaluating the cytotoxicity of drugs and toxicants, due to its high sensibility
to environmental toxicants [36–38]. The studies were carried out in triplicate by incubating the cells
for 24 h with empty and OZ-loaded nanosystems at increasing drug concentrations. The results in
terms of cell viability (%) are reported in Figure 10. The experiments confirmed that no cytotoxic
effects were generated by the empty polymer aggregates showing, in fact, an average cell viability
higher than 80%, even at the higher tested concentration. Moreover, we can observe a high
cytocompatibility of both OZ-loaded systems at 50 and 100 μM of loaded drug, compared with that
of the pure drug. Although the cytocompatibility of the free drug is very low and consequently
reduces that of the OZ-loaded systems at 200 and 350 μM, the cell toxicity produced by the latter was
d
f
h
b l
f
h
f
f
h
l
In order to evaluate the possibility of using HA-FA-HEG-OA and HA-FA-HEG-SA NP as drug
release systems in vivo, preliminary biological tests were carried out using the MTS assay [34]. The
cytocompatibility of the nanosystems was evaluated using a normal human cell line, namely, bronchial
epithelial cells (16HBE) [35]. 16HBE was chosen because it is one of the most commonly used model cell
lines for evaluating the cytotoxicity of drugs and toxicants, due to its high sensibility to environmental
toxicants [36–38]. The studies were carried out in triplicate by incubating the cells for 24 h with empty
and OZ-loaded nanosystems at increasing drug concentrations. The results in terms of cell viability (%)
are reported in Figure 10. The experiments confirmed that no cytotoxic effects were generated by the
empty polymer aggregates showing, in fact, an average cell viability higher than 80%, even at the higher
tested concentration. Moreover, we can observe a high cytocompatibility of both OZ-loaded systems at
50 and 100 µM of loaded drug, compared with that of the pure drug. (
g
3 4 Ol
i
L
di
d R l
3.4. Olanzapine Loading and Release Subsequently, release studies were carried out to
Subsequent analysis by DLS showed some modifications of the physical parameters upon drug
incorporation, with a consequent lowering of size and the zeta potential with respect to the empty
nanosystems (Table 2). Definitively, the calculated drug loading values demonstrated that the NPs
are capable of incorporating a good quantity of OZ. Subsequently, release studies were carried out to
evaluate the ability of systems to release the incorporated drug. aluate the ability of systems to release the incorporated drug. Table 2. Size distribution, Z-potential values, and drug loading of drug-loaded systems. Samples
Size (nm)
PDI
Z-Potential (mV)
Drug Loading (%)
HA-FA-HEG-OA-OZ
234
0.352
− 31.1
14.0
HA-FA-HEG-SA-OZ
178
0.260
− 24.7
14.2
Table 2. Size distribution, Z-potential values, and drug loading of drug-loaded systems. Samples
Size (nm)
PDI
Z-Potential (mV)
Drug Loading (%)
HA-FA-HEG-OA-OZ
234
0.352
−31.1
14.0
HA-FA-HEG-SA-OZ
178
0.260
−24.7
14.2 y
y
p
g
Table 2. Size distribution, Z-potential values, and drug loading of drug-loaded systems. The release profiles of OZ-loaded NP (Figure 9) suggest that HA-FA-HEG-OA and
HA-FA-HEG-SA aggregates are able to release the loaded active molecules as olanzapine in a
controlled way. The assumption on which the release studies were based was a sufficiently dilute
solution, able to mimic the donor compartment, where drug solubility does not limit the drug
release. However, this situation is difficult to meet with poorly water-soluble drug formulations, as a
very large volume of release medium is required with respect to the formulation making it a
The release profiles of OZ-loaded NP (Figure 9) suggest that HA-FA-HEG-OA and
HA-FA-HEG-SA aggregates are able to release the loaded active molecules as olanzapine in a
controlled way. The assumption on which the release studies were based was a sufficiently dilute
solution, able to mimic the donor compartment, where drug solubility does not limit the drug release. However, this situation is difficult to meet with poorly water-soluble drug formulations, as a very large 16 of 20 16 of 20 Pharmaceutics 2019, 11, 675 volume of release medium is required with respect to the formulation, making it a challenge to measure
the drug concentration accurately [33]. Thus, the low solubility of olanzapine in water required
experimental techniques capable of increasing its solubility (in this case, the role of nanoparticles was
to increase the solubilization of the drug). (
g
3 4 Ol
i
L
di
d R l
3.4. Olanzapine Loading and Release In order to alleviate this difficulty, the sampled solution
compartment was enriched with a surfactant (i.e., Tween-80 at 3%) with the purpose of increasing the
drug solubility. The results shown in the graph of Figure 9 demonstrate the ability of the polymeric
systems to improve the release profile of the loaded drug in a defined time as compared to the release
profile of the drug alone. In particular, the release of OZ loaded into nanosystems was constant in the
first 10 h with a release of about 50% of the encapsulated drug; after 10 h, the release was slower and
almost complete (ca. 80%) after 30 h of incubation. By contrast, the release profile of the free drug
showed only a 30% release within 30 h. Pharmaceutics 2019, 11, x
16 of 20
in water required experimental techniques capable of increasing its solubility (in this case, the role of
nanoparticles was to increase the solubilization of the drug). In order to alleviate this difficulty, the
sampled solution compartment was enriched with a surfactant (i.e., Tween-80 at 3%) with the
purpose of increasing the drug solubility. The results shown in the graph of Figure 9 demonstrate
the ability of the polymeric systems to improve the release profile of the loaded drug in a defined
time as compared to the release profile of the drug alone. In particular, the release of OZ loaded into
nanosystems was constant in the first 10 h with a release of about 50% of the encapsulated drug; after
10 h, the release was slower and almost complete (ca. 80%) after 30 h of incubation. By contrast, the
release profile of the free drug showed only a 30% release within 30 h Figure 9. In vitro OZ release profiles in PBS solution pH 7.4 at 37 °C. The amount of released OZ is
reported as the percentage ratio between the weight of the drug released from the nano-aggregates
and the drug loading. Figure 9. In vitro OZ release profiles in PBS solution pH 7.4 at 37 ◦C. The amount of released OZ is
reported as the percentage ratio between the weight of the drug released from the nano-aggregates and
the drug loading. Figure 9. In vitro OZ release profiles in PBS solution pH 7.4 at 37 °C. (
g
3 4 Ol
i
L
di
d R l
3.4. Olanzapine Loading and Release The amount of released OZ is
reported as the percentage ratio between the weight of the drug released from the nano-aggregates
and the drug loading
Figure 9. In vitro OZ release profiles in PBS solution pH 7.4 at 37 ◦C. The amount of released OZ is
reported as the percentage ratio between the weight of the drug released from the nano-aggregates and
the drug loading. 3 5 I
Vit o Biolo i al E aluatio
3.5. In Vitro Biological Evaluations 3 5 I
Vit o Biolo i al E aluatio
3.5. In Vitro Biological Evaluations 3 5 I
Vit
Bi l
i
l E
l
ti
3.5. In Vitro Biological Evaluations Although the cytocompatibility
of the free drug is very low and consequently reduces that of the OZ-loaded systems at 200 and 350
µM, the cell toxicity produced by the latter was attenuated, confirming the possibility of using these
nanosystems for further evaluation in vivo at concentrations below 200 µM of loaded drug. 17 of 20
17 of 20 Pharmaceutics 2019, 11, 675
Pharmaceutics 2019 11 x Figure 10. Cell viability evaluated by MTS on 16-HBE cells, after 24 h of incubation with
HA-FA-HEG-OA, HA-FA-HEG-SA, HA-FA-HEG-OA-OZ, HA-FA-HEG-SA-OZ, and olanzapine
alone. Figure
10. Cell
viability
evaluated
by
MTS
on
16-HBE
cells,
after
24
h
of
incubation with HA-FA-HEG-OA, HA-FA-HEG-SA, HA-FA-HEG-OA-OZ, HA-FA-HEG-SA-OZ,
and olanzapine alone. Figure 10. Cell viability evaluated by MTS on 16-HBE cells, after 24 h of incubation with
HA-FA-HEG-OA, HA-FA-HEG-SA, HA-FA-HEG-OA-OZ, HA-FA-HEG-SA-OZ, and olanzapine
alone. Figure
10. Cell
viability
evaluated
by
MTS
on
16-HBE
cells,
after
24
h
of
incubation with HA-FA-HEG-OA, HA-FA-HEG-SA, HA-FA-HEG-OA-OZ, HA-FA-HEG-SA-OZ,
and olanzapine alone. Conflicts of In
References References
1. Cohen, M.; Joester, D.; Geiger, B.; Addadi, L. Spatial and Temporal Sequence of Events in Cell Adhesion:
1. Cohen, M.; Joester, D.; Geiger, B.; Addadi, L. Spatial and Temporal Sequence of Events in Cell Adhesion:
From Molecular Recognition to Focal Adhesion Assembly. ChemBioChem 2004, 5, 1393–1399. [CrossRef]
[PubMed] References
1. Cohen, M.; Joester, D.; Geiger, B.; Addadi, L. Spatial and Temporal Sequence of Events in Cell Adhesion:
1. Cohen, M.; Joester, D.; Geiger, B.; Addadi, L. Spatial and Temporal Sequence of Events in Cell Adhesion:
From Molecular Recognition to Focal Adhesion Assembly. ChemBioChem 2004, 5, 1393–1399. [CrossRef]
[PubMed] From Molecular Recognition to Focal Adhesion Assembly. ChemBioChem 2004, 5, 1393–1399. 2. Zhong, Y.; Goltsche, K.; Cheng, L.; Xie, F.; Meng, F.; Deng, C.; Zhong, Z.; Haag, R. Hyaluronic acid-shelled
acid-activatable paclitaxel prodrug micelles effectively target and treat CD44-overexpressing human
2. Zhong, Y.; Goltsche, K.; Cheng, L.; Xie, F.; Meng, F.; Deng, C.; Zhong, Z.; Haag, R. Hyaluronic acid-shelled
acid-activatable paclitaxel prodrug micelles effectively target and treat CD44-overexpressing human breast
tumor xenografts in vivo. Biomaterials 2016, 84, 250–261. [CrossRef] [PubMed] From Molecular Recognition to Focal Adhesion Assembly. ChemBioChem 2004, 5, 1393–1399. 2. Zhong, Y.; Goltsche, K.; Cheng, L.; Xie, F.; Meng, F.; Deng, C.; Zhong, Z.; Haag, R. Hyaluronic acid-shelled
acid-activatable paclitaxel prodrug micelles effectively target and treat CD44-overexpressing human
2. Zhong, Y.; Goltsche, K.; Cheng, L.; Xie, F.; Meng, F.; Deng, C.; Zhong, Z.; Haag, R. Hyaluronic acid-shelled
acid-activatable paclitaxel prodrug micelles effectively target and treat CD44-overexpressing human breast
tumor xenografts in vivo. Biomaterials 2016, 84, 250–261. [CrossRef] [PubMed] breast tumor xenografts in vivo. Biomaterials 2016, 84, 250–261. 3. Dosio, F.; Arpicco, S.; Stella, B.; Fattal, E. Hyaluronic acid for anticancer drug and nucleic acid delivery. 3. Dosio, F.; Arpicco, S.; Stella, B.; Fattal, E. Hyaluronic acid for anticancer drug and nucleic acid delivery. Adv. Drug Deliv. Rev. 2016, 97, 204–236. [CrossRef] [PubMed] breast tumor xenografts in vivo. Biomaterials 2016, 84, 250–261. 3. Dosio, F.; Arpicco, S.; Stella, B.; Fattal, E. Hyaluronic acid for anticancer drug and nucleic acid delivery. 3. Dosio, F.; Arpicco, S.; Stella, B.; Fattal, E. Hyaluronic acid for anticancer drug and nucleic acid delivery. Adv. Drug Deliv. Rev. 2016, 97, 204–236. [CrossRef] [PubMed] p
y
g
y
Adv. Drug Deliv. Rev. 2016, 97, 204–236. 4. Orian-Rousseau, V. CD44, a therapeutic target for metastasising tumours. Eur. J. Cancer 2010, 46,
g
4. Orian-Rousseau, V. 4 Conclusions
4. Conclusions 4. Conclusions
Over the years, pharmaceutical research always showed a great interest in the use of polymeric
materials as drug carriers, capable of improving biocompatibility and bioavailability. Among these
carriers, polymeric nanoparticles have great potential. In this study, hyaluronan copolymers grafted
with propargylated ferulate fluorophores linked to fatty-acid residues by means of hexa(ethylene
glycol) spacers, HA-FA-HEG-OA and HA-FA-HEG-SA, were properly synthesized, characterized,
and tested as biocompatible polymeric materials, potentially useful as self-assembled colloidal
nanosystems for drug delivery. The obtained nanosystems were characterized from the
physico-chemical point of view in terms of average size, zeta potential, and aggregation capacity in
an aqueous environment, showing an average size around 180–360 nm, a narrow dimensional
distribution, and negative surface charge. The results demonstrated the possibility of using
polymeric systems for the production of nano-aggregates capable of delivering hydrophobic drugs
such as OZ (Figure 11). Finally, the results of the cytotoxicity test confirmed the absence of cytotoxic
effects on a normal human cell line i e bronchial epithelial cells at concentrations below 200 μM of
Over the years, pharmaceutical research always showed a great interest in the use of polymeric
materials as drug carriers, capable of improving biocompatibility and bioavailability. Among these
carriers, polymeric nanoparticles have great potential. In this study, hyaluronan copolymers grafted
with propargylated ferulate fluorophores linked to fatty-acid residues by means of hexa(ethylene glycol)
spacers, HA-FA-HEG-OA and HA-FA-HEG-SA, were properly synthesized, characterized, and tested
as biocompatible polymeric materials, potentially useful as self-assembled colloidal nanosystems for
drug delivery. The obtained nanosystems were characterized from the physico-chemical point of
view in terms of average size, zeta potential, and aggregation capacity in an aqueous environment,
showing an average size around 180–360 nm, a narrow dimensional distribution, and negative surface
charge. The results demonstrated the possibility of using polymeric systems for the production of
nano-aggregates capable of delivering hydrophobic drugs such as OZ (Figure 11). Finally, the results
of the cytotoxicity test confirmed the absence of cytotoxic effects on a normal human cell line, i.e.,
bronchial epithelial cells, at concentrations below 200 µM of loaded drug. 18 of 20 18 of 20 Pharmaceutics 2019, 11, 675 Figure 11. Representation of hyaluronan graft copolymer HA-FA-HEG-OA bearing fatty acid
residues as self-assembling nanoparticles for olanzapine delivery. Figure 11. Representation of hyaluronan graft copolymer HA-FA-HEG-OA bearing fatty acid residues
as self-assembling nanoparticles for olanzapine delivery. Figure 11. Conflicts of In
References CD44, a therapeutic target for metastasising tumours. Eur. J. Cancer 2010, 46, 1271–1277. [CrossRef] [PubMed] p
y
g
y
Adv. Drug Deliv. Rev. 2016, 97, 204–236. 4. Orian-Rousseau, V. CD44, a therapeutic target for metastasising tumours. Eur. J. Cancer 2010, 46,
4. Orian-Rousseau, V. CD44, a therapeutic target for metastasising tumours. Eur. J. Cancer 2010, 46, 1271–1277. [CrossRef] [PubMed] p
g
g
1271–1277. 5. Cappelli, A.; Grisci, G.; Paolino, M.; Giuliani, G.; Donati, A.; Mendichi, R.; Artusi, R.; Demiranda, M.;
Zanardi, A.; Giorgi, G.; et al. Hyaluronan derivatives bearing variable densities of ferulic acid residues. J. 5. Cappelli, A.; Grisci, G.; Paolino, M.; Giuliani, G.; Donati, A.; Mendichi, R.; Artusi, R.; Demiranda, M.;
Zanardi, A.; Giorgi, G.; et al. Hyaluronan derivatives bearing variable densities of ferulic acid residues. J. Mater. Chem. B 2014, 2, 4489–4499. [CrossRef] 1271–1277. 5. Cappelli, A.; Grisci, G.; Paolino, M.; Giuliani, G.; Donati, A.; Mendichi, R.; Artusi, R.; Demiranda, M.;
Zanardi, A.; Giorgi, G.; et al. Hyaluronan derivatives bearing variable densities of ferulic acid residues. J. 5. Cappelli, A.; Grisci, G.; Paolino, M.; Giuliani, G.; Donati, A.; Mendichi, R.; Artusi, R.; Demiranda, M.;
Zanardi, A.; Giorgi, G.; et al. Hyaluronan derivatives bearing variable densities of ferulic acid residues. J. Mater. Chem. B 2014, 2, 4489–4499. [CrossRef] g
y
g
Mater. Chem. B 2014, 2, 4489–4499. 6. Valacchi, G.; Grisci, G.; Sticozzi, C.; Lim, Y.; Paolino, M.; Giuliani, G.; Mendichi, R.; Belmonte, G.; Artusi,
R.; Zanardi, A.; et al. Wound healing properties of hyaluronan derivatives bearing ferulate residues. J. 6. Valacchi, G.; Grisci, G.; Sticozzi, C.; Lim, Y.; Paolino, M.; Giuliani, G.; Mendichi, R.; Belmonte, G.; Artusi, R.;
Zanardi, A.; et al. Wound healing properties of hyaluronan derivatives bearing ferulate residues. J. Mater. Chem. B 2015, 3, 7037–7045. [CrossRef] g
y
g
Mater. Chem. B 2014, 2, 4489–4499. 6. Valacchi, G.; Grisci, G.; Sticozzi, C.; Lim, Y.; Paolino, M.; Giuliani, G.; Mendichi, R.; Belmonte, G.; Artusi,
R.; Zanardi, A.; et al. Wound healing properties of hyaluronan derivatives bearing ferulate residues. J. 6. Valacchi, G.; Grisci, G.; Sticozzi, C.; Lim, Y.; Paolino, M.; Giuliani, G.; Mendichi, R.; Belmonte, G.; Artusi, R.;
Zanardi, A.; et al. Wound healing properties of hyaluronan derivatives bearing ferulate residues. J. Mater. Chem. B 2015, 3, 7037–7045. [CrossRef] g p
p
y
g
Mater. Chem. B 2015, 3, 7037–7045. 7. Paolino, M.; Komber, H.; Mennuni, L.; Caselli, G.; Appelhans, D.; Voit, B.; Cappelli, A. 4 Conclusions
4. Conclusions Acknowledgments: Thanks are due to the Italian MIUR (Ministero dell’Istruzione, dell’Università e della
Acknowledgments: Thanks are due to the Italian MIUR (Ministero dell’Istruzione, dell’Università e della Ricerca). Conflicts of Interest: The authors declare no conflict of interest Acknowledgments: Thanks are due to the Italian MIUR (Minis
Ricerca)
Conflicts of Interest: The authors declare no conflict of interest. 4 Conclusions
4. Conclusions Representation of hyaluronan graft copolymer HA-FA-HEG-OA bearing fatty acid
residues as self-assembling nanoparticles for olanzapine delivery. Figure 11. Representation of hyaluronan graft copolymer HA-FA-HEG-OA bearing fatty acid residues
as self-assembling nanoparticles for olanzapine delivery. The combination of all these positive data offers the advantages of a system that can potentially
provide a biocompatible vector for prolonged release of the drug in situ, and it sets the basis for the
exploration of other potential applications in the field of drug release control. The combination of all these positive data offers the advantages of a system that can potentially
provide a biocompatible vector for prolonged release of the drug in situ, and it sets the basis for the
exploration of other potential applications in the field of drug release control. Author Contributions: M.P., A.R., G.Giu., and M.A. performed the synthesis and the preliminary
characterization; H.K. and A.D. performed and analyzed the NMR experiments; G.L. and A.M. performed the
rheological studies; M.L., C.S., G.Gia., and L.M.D.M. performed the DLS and CAC analysis, cytotoxicity
evaluation, and drug release studies; M.P., M.L., and A.C. coordinated the work, analyzed the data, and wrote
the paper
Author Contributions: M.P., A.R., G.G. (Germano Giuliani), and M.A. performed the synthesis and the preliminary
characterization; H.K. and A.D. performed and analyzed the NMR experiments; G.L. and A.M. performed the
rheological studies; M.L., C.S., G.G. (Gaetano Giammona), and L.M.D.M. performed the DLS and CAC analysis,
cytotoxicity evaluation, and drug release studies; M.P., M.L., and A.C. coordinated the work, analyzed the data,
and wrote the paper. the paper. F
di
Thi
h
i
d
t
l f
di
Funding: This research received no external funding. Funding: This research received no external funding. Acknowledgments: Thanks are due to the Italian MIUR (Ministero dell’Istruzione dell’Università e della
Acknowledgments: Thanks are due to the Italian MIUR (Ministero dell’Istruzione, dell’Università e della Ricerca). C
fli t
f I t
t Th
th
d
l
fli t f i t
t unding: This research received no external funding. k
l d
t
Th
k
d
t
th
It li
MIUR (Mi i t
d ll’I t
i
d ll’U i
ità
d
Acknowledgments: Thanks are due to the Italian MIUR (Ministero dell’Istruzione, dell’Università e della Rice Funding: This research received no external funding. Conflicts of In
References Supramolecular
7. Paolino, M.; Komber, H.; Mennuni, L.; Caselli, G.; Appelhans, D.; Voit, B.; Cappelli, A. Supramolecular
glycodendrimer-based hybrid drugs. Biomacromolecules 2014, 15, 3985–3993. [CrossRef] glycodendrimer-based hybrid drugs. Biomacromolecules 2014, 15, 3985–3993. 8. Cappelli, A.; Grisci, G.; Paolino, M.; Castriconi, F.; Giuliani, G.; Donati, A.; Lamponi, S.; Mendichi, R.;
Boccia, A.C.; Samperi, F.; et al. Combining spontaneous polymerization and click reactions for the
8. Cappelli, A.; Grisci, G.; Paolino, M.; Castriconi, F.; Giuliani, G.; Donati, A.; Lamponi, S.; Mendichi, R.;
Boccia, A.C.; Samperi, F.; et al. Combining spontaneous polymerization and click reactions for the synthesis
of polymer brushes: A “grafting onto” approach. Chem. A Eur. J. 2013, 19, 9710–9721. [CrossRef] 19 of 20 19 of 20 Pharmaceutics 2019, 11, 675 9. Paolino, M.; Mennuni, L.; Giuliani, G.; Anzini, M.; Lanza, M.; Caselli, G.; Galimberti, C.; Menziani, M.C.;
Donati, A.; Cappelli, A. Dendrimeric tetravalent ligands for the serotonin-gated ion channel. Chem. Commun. (Camb). 2014, 50, 8582–8585. [CrossRef] 10. Pagano, K.; Paolino, M.; Fusi, S.; Zanirato, V.; Trapella, C.; Giuliani, G.; Cappelli, A.; Zanzoni, S.; Molinari, H.;
Ragona, L.; et al. Bile Acid Binding Protein Functionalization Leads to a Fully Synthetic Rhodopsin Mimic. J. Phys. Chem. Lett. 2019, 10, 2235–2243. [CrossRef] 11. Cappelli, A.; Paolino, M.; Reale, A.; Razzano, V.; Grisci, G.; Giuliani, G.; Donati, A.; Bonechi, C.; Lamponi, S.;
Mendichi, R.; et al. Hyaluronan-based graft copolymers bearing aggregation-induced emission fluorogens. RSC Adv. 2018, 8, 5864–5881. [CrossRef] 12. Atrei, A.; Innocenti, C.; Lamponi, S.; Paesano, S.; Leone, G.; Reale, A.; Paolino, M.; Cappelli, A. Covalent hyaluronic-based coating of magnetite nanoparticles: Preparation, physicochemical and biological
characterization. Mater. Sci. Eng. C 2020, 107, 110271. [CrossRef] [PubMed] 13. Paolino, M.; Grisci, G.; Reale, A.; Razzano, V.; Giuliani, G.; Donati, A.; Mendichi, R.; Piovani, D.; Boccia, A.C.;
Grillo, A.; et al. Structural manipulation of the conjugated phenyl moiety in 3-phenylbenzofulvene monomers:
Effects on spontaneous polymerization. Polymers (Basel). 2018, 10, 752. [CrossRef] [PubMed] 14. Cappelli, A.; Razzano, V.; Paolino, M.; Grisci, G.; Giuliani, G.; Donati, A.; Mendichi, R.; Samperi, F.; Battiato, S.;
Boccia, A.C.; et al. Bithiophene-based polybenzofulvene derivatives with high stacking and hole mobility. Polym. Chem. 2015, 6, 7377–7388. [CrossRef] 15. Villafiorita-Monteleone, F.; Cappelli, A.; Paolino, M.; Colombo, M.; Cariati, E.; Mura, A.; Bongiovanni, G.;
Botta, C. Aggregation-Induced Förster Resonance Energy Transfer in Polybenzofulvene/Dye Nanoparticles. J. Phys. Chem. C 2015, 119, 18986–18991. [CrossRef] 16. Conflicts of In
References Cappelli, A.; Villafiorita-Monteleone, F.; Grisci, G.; Paolino, M.; Razzano, V.; Fabio, G.; Giuliani, G.;
Donati, A.; Mendichi, R.; Boccia, A.C.; et al. Highly emissive supramolecular assemblies based on π-stacked
polybenzofulvene hosts and a benzothiadiazole guest. J. Mater. Chem. C 2014, 2, 7897–7905. [CrossRef] 17. Cappelli, A.; Grisci, G.; Paolino, M.; Razzano, V.; Giuliani, G.; Donati, A.; Bonechi, C.; Mendichi, R.;
Boccia, A.C.; Licciardi, M.; et al. Polybenzofulvene derivatives bearing dynamic binding sites as potential
anticancer drug delivery systems. J. Mater. Chem. B 2015, 3, 361–374. [CrossRef] 18. Mróz, W.; Villafiorita-Monteleone, F.; Pasini, M.; Grisci, G.; Paolino, M.; Razzano, V.; Cappelli, A.; Botta, C. π-stacked polybenzofulvene derivatives as hosts for yellow and red emitting OLEDs. Mater. Lett. 2015, 142,
197–200. [CrossRef] 19. Paolino, M.; Grisci, G.; Castriconi, F.; Reale, A.; Giuliani, G.; Donati, A.; Bonechi, C.; Giorgi, G.; Mendichi, R.;
Piovani, D.; et al. Densely PEGylated polybenzofulvene brushes for potential applications in drug
encapsulation. Pharmaceutics 2018, 10, 234. [CrossRef] 20. Cappelli, A.; Paolino, M.; Grisci, G.; Razzano, V.; Giuliani, G.; Donati, A.; Bonechi, C.; Mendichi, R.; Battiato, S.;
Samperi, F.; et al. Hyaluronan-coated polybenzofulvene brushes as biomimetic materials. Polym. Chem. 2016, 7, 6529–6544. [CrossRef] 21. Licciardi, M.; Scialabba, C.; Giammona, G.; Paolino, M.; Razzano, V.; Grisci, G.; Giuliani, G.; Makovec, F.;
Cappelli, A. Design and development of hyaluronan-functionalized polybenzofulvene nanoparticles as
CD44 receptor mediated drug delivery system. J. Nanoparticle Res. 2017, 19. [CrossRef] p
g
y y
J
p
[
]
22. Licciardi, M.; Scialabba, C.; Sardo, C.; Cavallaro, G.; Giammona, G. Amphiphilic inulin graft co-polymers as
self-assembling micelles for doxorubicin delivery. J. Mater. Chem. B 2014, 2, 4262–4271. [CrossRef] 23. Bongiovì, F.; Di Prima, G.; Palumbo, F.S.; Licciardi, M.; Pitarresi, G.; Giammona, G. Hyaluronic Acid-Based
Micelles as Ocular Platform to Modulate the Loading, Release, and Corneal Permeation of Corticosteroids. Macromol. Biosci. 2017, 17, 1700261. [CrossRef] [PubMed] 24. Di Prima, G.; Bongiovì, F.; Palumbo, F.S.; Pitarresi, G.; Licciardi, M.; Giammona, G. Mucoadhesive PEGylated
inulin-based self-assembling nanoparticles: In vitro and ex vivo transcorneal permeation enhancement of
corticosteroids. J. Drug Deliv. Sci. Technol. 2019, 49, 195–208. [CrossRef] 25. de Mohac, L.M.; de Fátima Pina, M.; Raimi-Abraham, B.T. Solid microcrystalline dispersion films as a new
strategy to improve the dissolution rate of poorly water soluble drugs: A case study using olanzapine. Int. J. Pharm. 2016, 508, 42–50. [CrossRef] [PubMed] 20 of 20 20 of 20 Pharmaceutics 2019, 11, 675 26. Conflicts of In
References Telford, J.E.; Bones, J.; McManus, C.; Saldova, R.; Manning, G.; Doherty, M.; Leweke, F.M.; Rothermundt, M.;
Guest, P.C.; Rahmoune, H.; et al. Antipsychotic Treatment of Acute Paranoid Schizophrenia Patients with
Olanzapine Results in Altered Glycosylation of Serum Glycoproteins. J. Proteome Res. 2012, 11, 3743–3752. [CrossRef] 27. Goswami, L.N.; Houston, Z.H.; Sarma, S.J.; Jalisatgi, S.S.; Hawthorne, M.F. Efficient synthesis of diverse
heterobifunctionalized clickable oligo(ethylene glycol) linkers: Potential applications in bioconjugation and
targeted drug delivery. Org. Biomol. Chem. 2013, 11, 1116–1126. [CrossRef] 28. Leone, G.; Consumi, M.; Franzi, C.; Tamasi, G.; Lamponi, S.; Donati, A.; Magnani, A.; Rossi, C.; Bonechi, C. Development of liposomal formulations to potentiate natural lovastatin inhibitory activity towards
3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase. J. Drug Deliv. Sci. Technol. 2018,
43, 107–112. [CrossRef] 29. Liang, L.; Astruc, D. The copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) “click” reaction and its
applications. An overview. Coord. Chem. Rev. 2011, 255, 2933–2945. [CrossRef] 30. Paolino, M.; Reale, A.; Razzano, V.; Giuliani, G.; Donati, A.; Bonechi, C.; Caselli, G.; Visintin, M.; Makovec, F.;
Scialabba, C.; et al. Nanoreactors for the multi-functionalization of poly-histidine fragments. New J. Chem. 2019, 6834–6837. [CrossRef] 31. Chen, J.; Jiang, M.; Zhang, Y.; Zhou, H. Fluorescence Studies on Hydrophobic Associat
Fluorocarbon-Modified Poly(acrylic acid) Solutions. Macromolecules 1999, 32, 4861–4866. [CrossRef 32. Cavallari, C.; Fini, A.; Ceschel, G. Design of Olanzapine/Lutrol Solid Dispersions of Improved Stability and
Performances. Pharmaceutics 2013, 5, 570–590. [CrossRef] [PubMed] 33. Abouelmagd, S.A.; Sun, B.; Chang, A.C.; Ku, Y.J.; Yeo, Y. Release Kinetics Study of Poorly Water-Soluble
Drugs from Nanoparticles: Are We Doing It Right? Mol. Pharm. 2015, 12, 997–1003. [CrossRef] [PubMed] Drugs from Nanoparticles: Are We Doing It Right? Mol. Pharm. 2015, 12, 997–1003. [CrossRef] [PubMed]
34. Malich, G.; Markovic, B.; Winder, C. The sensitivity and specificity of the MTS tetrazolium assay for detecting
the in vitro cytotoxicity of 20 chemicals using human cell lines. Toxicology 1997, 124, 179–192. [CrossRef] 34. Malich, G.; Markovic, B.; Winder, C. The sensitivity and specificity of the MTS tetrazolium assay for detecting
the in vitro cytotoxicity of 20 chemicals using human cell lines. Toxicology 1997, 124, 179–192. [CrossRef] 35. Sardo, C.; Farra, R.; Licciardi, M.; Dapas, B.; Scialabba, C.; Giammona, G.; Grassi, M.; Grassi, G.; Cavallaro, G. Development of a simple, biocompatible and cost-effective Inulin-Diethylenetriamine based siRNA delivery
system. Eur. J. Pharm. Sci. 2015, 75, 60–71. [CrossRef] [PubMed] 6. Conflicts of In
References Feng, W.; Guo, J.; Huang, H.; Xia, B.; Liu, H.; Li, J.; Lin, S.; Li, T.; Liu, J.; Li, H. Human Normal Bronc
Epithelial Cells: A Novel In Vitro Cell Model for Toxicity Evaluation. PLoS ONE 2015, 10, 1–14. [Cross 37. Xia, B.; Yang, L.; Huang, H.; Pang, L.; Hu, G.; Liu, Q.; Yuan, J.; Liu, J.; Xia, Y.; Zhuang, Z. Chromium(VI) causes
down regulation of biotinidase in human bronchial epithelial cells by modifications of histone acetylation. Toxicol. Lett. 2011, 205, 140–145. [CrossRef] 38. Forbes, B.; Shah, A.; Martin, G.P.; Lansley, A.B. The human bronchial epithelial cell line 16HBE14o−as a
model system of the airways for studying drug transport. Int. J. Pharm. 2003, 257, 161–167. [CrossRef] © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Counteracting gradients of light and soil nutrients in the understorey of Mediterranean oak forests
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Web ecology
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Counteracting gradients of light and soil nutrients in the
understorey of Mediterranean oak forests L. V. García, S. Maltez-Mouro, I. M. Pérez-Ramos, H. Freitas, and T. Marañón García, L.V. et al. 2006. Counteracting gradients of light and soil nutrients in the un-
derstorey of Mediterranean oak forests. – Web Ecol. 6: 67–74. García, L.V. et al. 2006. Counteracting gradients of light and soil nutrients in the un-
derstorey of Mediterranean oak forests. – Web Ecol. 6: 67–74. The forest canopy modifies the availability of resources (light, water, and soil nutrients)
in the understorey. In this paper we analyze the relationships between woody canopy
density, litter accumulation, and topsoil N and P availability in the understorey of two
oak forests: one in southern Portugal and the other in southern Spain. Both forests
persist on low-nutrient soils, particularly poor in P. We hypothesize that direct and
indirect effects of the canopy overstorey cause opposite gradients in the availability of
essential resources (light and key soil nutrients) in the understorey. In both studied
forests we found significant relationships between the overall canopy density, light avail-
ability, topsoil litter accumulation, and the availability of N and P, which frequently
limit plant growth. Path analysis (by Shipley’s d-sep method) showed that the available
data were consistent with the proposed causal model. The average values of soil variables
at the end quartiles of the light-availability gradient were compared. Results showed
large differences in litter accumulation (~30×) and available-N and -P topsoil concen-
trations (~3×) in the Spanish forest (with the wider environmental gradient). Further-
more, P increased from the ‘very low’ range to the ‘low’ or even the ‘optimum’ range of
availability (according to standard plant growth criteria), which suggests potential ef-
fects on the growth of the understorey plant species. We conclude that the counteract-
ing gradients of the essential resources -light and nutrients- in the forest understorey
resulted from direct and indirect effects of the canopy overstorey, respectively. We sug-
gest that these counteracting effects of the woody canopy on essential resources of differ-
ent nature must be considered when interpreting the patterns of understorey plant pop-
ulations and communities. L. V. García (ventura@cica.es), I. M. Pérez-Ramos and T. Marañón, Depto. de Geoecología,
Instituto de Recursos Naturales y Agrobiología (CSIC), P.O. Box 1052, E-41080 Sevilla,
Spain. – S. Maltez-Mouro and H. Freitas, Dept. of Botany, Univ. of Coimbra, Calçada
Martim de Freitas, 3000 Coimbra, Portugal. Web Ecology 6: 67–74. Web Ecology 6: 67–74. Accepted 20 November 2006
Copyright © EEF
ISSN 1399-1183 Counteracting gradients of light and soil nutrients in the
understorey of Mediterranean oak forests The tree canopy modulates the availability and variability
of some key resources for the organisms living in the
understorey, thus affecting its own regeneration (Binkley
& Giardina 1998, Washburn & Arthur 2003, Maltez-
Mouro et al. 2005a, 2005b). Several recent studies have
documented the direct effects of the Mediterranean forest
canopy on the growth and survival of tree seedlings in the understorey through changes in the availability of light
and water (Sack et al. 2003, Quero et al. 2006, Sánchez-
Gómez et al. 2006). However, the indirect effects such as
those mediated by soil and litter have also been proven to
be important for plant growth in the understorey (Rossi
and Villagra 2003, Broncano et al. 2004, Puerta-Piñero et
al. 2006). Accepted 20 November 2006
Copyright © EEF
ISSN 1399-1183 Accepted 20 November 2006
Copyright © EEF
ISSN 1399-1183 67 The spatial and temporal heterogeneity of different re-
sources may induce uncoupled responses by understorey
plants. Tree seedlings can grow faster during spring in open
microsites with higher light availability, but will suffer a
higher mortality during the summer (with lower water
availability) in comparison with seedlings in moderately
shaded microsites (Marañón et al. 2004). We expect that
seedlings in forest gaps will find higher light levels (direct
effect) but lower leaf litter and, in consequence, lower
availability of soil nutrients (indirect effect). noring the role of other more-subtle indirect effects (e.g. changes in soil nutrient availability related to litter accu-
mulation). We analyze the relationships between woody canopy
density and understorey conditions, in particular light
availability, litter accumulation, and changes in soil N and
P concentrations. The aims of our study are 1) to test the
hypothesis that the tree overstorey may induce counteract-
ing gradients of available key resources, such as light and
soil nutrients in the understorey, and through some direct
and indirect effects; 2) to evaluate the potential impact of
the hypothesized canopy indirect effects on the understo-
rey plants; and 3) to analyze the covariation between direct
(light) and indirect (nutrient availability) effects associated
to changes in the canopy density. Detection and evaluation of the indirect effects of the
canopy mediated by litter accumulation and soil changes
on the growth and survival of understorey plants are not
easy. Counteracting gradients of light and soil nutrients in the
understorey of Mediterranean oak forests Firstly, the complex and multivariate nature of the
understorey – and soil in particular (Binkley and Giardina
1998, Washburn and Arthur 2003) – makes it difficult to
distinguish which changes are related to the direct effects
of litter accumulation (e.g. limiting seedling emergence),
which changes result from the indirect effects of litter on
soil properties (e.g. affecting soil chemical composition),
and which changes result from other factors that may af-
fect soil properties such as changes in the parent material. Secondly, covariation exists between gradients of different
essential resources that relate to changes in canopy density. This covariation may lead to the attribution of all the
changes in the understorey to a few well-known and easily
measurable direct effects (e.g. light availability), while ig- Study sites We studied Quercus forests located in two protected areas
in the south of the Iberian Peninsula (Fig. 1): the Sudoeste
Alentejano e Costa Vicentina Natural Park, in southern Por-
tugal (SP, hereafter), and Los Alcornocales Natural Park, in
southern Spain (SS, hereafter). Both forests are dominated Fig. 1. Location of the study areas in the Iberian Peninsula. SS = Spanish site at Los Alcornocales Natural Park; SP = Portuguese site at
Sudoeste Alentejano e Costa Vicentina Natural Park. Fig. 1. Location of the study areas in the Iberian Peninsula. SS = Spanish site at Los Alcornocales Natural Park; SP = Portuguese site at
Sudoeste Alentejano e Costa Vicentina Natural Park. 68 WEB ECOLOGY 6, 2006 by an evergreen oak species (Quercus suber) mixed with a
deciduous one (Q. faginea or Q. canariensis, respectively). The climate is Mediterranean-type, with wet winters
and dry summers. Mean annual rainfall is higher in SS (ca. 1200 mm) than in SP (ca. 600 mm). Average annual tem-
peratures are ca. 16 °C and 15 °C, respectively. Dominant
soils are acidic and usually poor in nutrients in both SP
(Maltez-Mouro et al. 2005a) and SS (Jordan et al. 1998,
Quilchano, C., pers. comm.). by an evergreen oak species (Quercus suber) mixed with a
deciduous one (Q. faginea or Q. canariensis, respectively). Data analysis Relationships between canopy density, litter accumula-
tion, and topsoil nutrient content were analyzed by corre-
lation analyses. To test whether the available data were con-
sistent with the hypothesis of higher canopy density caus-
ing increased accumulation of litter – which decomposes
and is responsible for the increase of N and P availability in
the soil – a d-sep method of path analysis (Shipley 2000,
2003) was used. For both studied nutrients (N and P) and
sites, two separate and one general causal model were
tested. For the general model, two alternatives were con-
sidered: 1) litter accumulation is the immediate cause that
simultaneously explains N and P changes, and 2) litter in-
fluence on N and P availabilities is mediated by soil proc-
esses, which can be summarized considering an
unmeasured (latent) factor (labeled as ‘soil processes’). Al-
though the d-sep method is not designed for testing mod-
els including unmeasured (latent) variables (Shipley
2000), in this special case the d-sep test is still applicable
assuming that N and P have correlated (instead of inde-
pendent) errors (Shipley 2003). Q f g
Q
p
y
The climate is Mediterranean-type, with wet winters
and dry summers. Mean annual rainfall is higher in SS (ca. 1200 mm) than in SP (ca. 600 mm). Average annual tem-
peratures are ca. 16 °C and 15 °C, respectively. Dominant
soils are acidic and usually poor in nutrients in both SP
(Maltez-Mouro et al. 2005a) and SS (Jordan et al. 1998,
Quilchano, C., pers. comm.). Canopy, litter, and soil measurements The soil samples were
dried, crushed, passed through a sieve (2 mm), and then
analyzed for organic N (Kjeldahl method), available P
(Olsen method), and NH4–N (extracted with 2M KCl)
contents. To estimate P availability for plants in leached,
very-poor-in–P, and acidic soils, the Bray and Kurtz meth-
od is recommended (Frank et al. 1998, Anon. 2000) in-
stead of the most frequently used Olsen’s method (Olsen et
al. 1954). Therefore, values of the Bray-1 P-availability in-
dex (besides the Olsen values) were obtained for the SP
samples. All soil analyses were performed according to the
methods prescribed in Sparks (1996). Litter accumulation was evaluated by visual estimates
(percent of the soil surface covered by litter, with agree-
ment of three observers). In the SS plots, litter layer depth
was estimated as the average of ten random measurements
using a metric tape. Litter dry mass accumulation was es-
timated by collecting all the litter included inside a ran-
domly placed quadrat of 30×30 cm; that litter was dried
at 70 °C for three days and weighed to the nearest 0.1 g. Canopy, litter, and soil measurements We sampled 25 plots (4×4 m2) in SP and 60 plots (3×3
m2) in SS distributed along gradients of canopy density in
late fall and beginning of winter (November–December)
of yrs 2003 and 2005, respectively. In each plot we gath-
ered information about three different groups of variables:
canopy density (closely related with light availability), litter
accumulation, and soil nutrients. To quantify the canopy density variables, a spherical
densimeter was used in SP to measure the percent of the
overhead area not covered by the canopy. In the SS plots,
digital hemispherical photographs were analyzed using the
Hemiview software (Anon. 1998) to calculate the Global
Site Factor (GSF, the proportion of total radiation under a
plant canopy relative to that in the open), Leaf Area Index
(LAI, surface area of leaves per ground area unit), and
Ground Cover (GC, vertically projected canopy area per
unit ground area). For comparison purposes, GSF values
for the SP forest were roughly estimated from the GC val-
ues using the GSF-GC regression from SS data (R2 =
0.65). We compared the average values of litter and soil varia-
bles at the end quartiles of the light-availability gradients to
evaluate the magnitude of the increment of the variables
related to litter accumulation and nutrient availability. When necessary, variables were transformed to con-
form to the assumptions of the parametric analysis. Cor-
rections for multiplicity were performed according to
García (2004). All statistical analyses were performed us-
ing the Statistica (Anon. 2001) and Causal Toolbox (Ship-
ley 2000) packages. 5)
Litter accumulation was evaluated by visual estimates
(percent of the soil surface covered by litter, with agree-
ment of three observers). In the SS plots, litter layer depth
was estimated as the average of ten random measurements
using a metric tape. Litter dry mass accumulation was es-
timated by collecting all the litter included inside a ran-
domly placed quadrat of 30×30 cm; that litter was dried
at 70 °C for three days and weighed to the nearest 0.1 g. In each of the studied forests, topsoil (< 25 cm depth)
was sampled using an auger (3 cm in diameter) at 3–7
points located inside each plot, and mixed to produce one
composite soil sample per plot. Results Descriptive values (mean, standard deviation, maximum,
and minimum) of the three groups of forest variables are
shown in Table 1. Ranges of variation were particularly
high in the SS dataset, where the light availability range
was 8–89% of the radiation at an open site. The litter accu-
mulation gradient was also very wide: 0.8–3400 g m–2 that
covered 0.5–100% of the soil surface. Phosphorus avail-
ability ranged from the ‘very low’ (0–3 mg kg–1 Bray 1-P or
0–6 mg kg–1 Olsen-P) to the ‘optimum/normal’ level (8–
20 mg kg–1 Bray 1-P or 13–18 mg kg–1 Olsen-P) – consid-
ering rank values of southern Spanish loamy-textured soils
(Anon. 1992). Ammonium availability increased about
25-fold along the same gradient. The variation ranges for
all measured variables were narrower in the SP forest (Ta-
ble 1). y
g
g
In each of the studied forests, topsoil (< 25 cm depth)
was sampled using an auger (3 cm in diameter) at 3–7
points located inside each plot, and mixed to produce one
composite soil sample per plot. The soil samples were
dried, crushed, passed through a sieve (2 mm), and then
analyzed for organic N (Kjeldahl method), available P
(Olsen method), and NH4–N (extracted with 2M KCl)
contents. To estimate P availability for plants in leached,
very-poor-in–P, and acidic soils, the Bray and Kurtz meth-
od is recommended (Frank et al. 1998, Anon. 2000) in-
stead of the most frequently used Olsen’s method (Olsen et
al. 1954). Therefore, values of the Bray-1 P-availability in-
dex (besides the Olsen values) were obtained for the SP
samples. All soil analyses were performed according to the
methods prescribed in Sparks (1996). The high frequency of soils having P shortage in both
sites is remarkable. In the SS forest, more than 80% of the
studied soils were in the ‘very low’ range, with only 10%
having ‘low availability’ levels (4–7 mg kg–1 Bray 1-P or 7–
12 mg kg–1 Olsen-P), and another 10% having the so- 69 WEB ECOLOGY 6, 2006 Table 1. Mean, standard deviation, minimum, and maximum values for the canopy density, litter accumulation, and topsoil variables
measured in the two studied forests (SS = southern Spain site at Los Alcornocales Natural Park; SP = southern Portugal site at Sudoeste
Alentejano e Costa Vicentina Natural Park). Data estimated using linear regression (see text) are in italics. SS
SP
Mean
SD
Min. Max. Mean
SD
Min. Max. Results Canopy density / light availability
Global Site Factor (GSF, 0–1)
0.32
0.23
0.08
0.89
0.11
0.06
0.05
0.27
Leaf Area Index (LAI, m2 m–1)
1.69
0.71
0.38
3.02
–
–
–
–
Ground Cover (GC, %)
49
34
0
90
85
10
62
95
Litter accumulation
Covered soil (%)
63
38
0.5
100
47
28
5
90
Thickness (cm)
3.1
2.7
0.1
13.7
–
–
–
–
Dry weight (g litter m–2)
557
643
0.8
3399
–
–
–
–
Soil properties
Total N (Kjeldahl, %)
0.33
0.12
0.12
0.75
0.37
0.04
0.23
0.43
Available P (Olsen, mg kg–1)
3.1
2.6
0.0
14.9
3.5
1.3
1.6
5.9
Available N (N–NH4, mg kg–1)
3.2
2.9
0.7
16.1
4.4
2.5
1.6
11.1
Available P (Bray 1, mg kg–1)
2.0
1.8
0.2
8.9
–
–
–
– called ‘normal’ or ‘optimum’ range for most plant species
(Anon. 1992). In the case of the SP forest, all the studied
soil samples had ‘very low’ values of P availability. The light availability (measured as GSF) was very heter-
ogeneous in the SS forest but slightly more homogeneous
in the SP forest, where the estimated GSF (overstorey can-
opy density) ranged from 0.05 to 0.27, with an average
value of 0.11. (Table 2). Finally, as an expected result of the indirect ef-
fects of the canopy on soil enrichment, available N and
available P were highly intercorrelated in both studied for-
ests (Fig. 4). called ‘normal’ or ‘optimum’ range for most plant species
(Anon. 1992). In the case of the SP forest, all the studied
soil samples had ‘very low’ values of P availability. p
y
y
The light availability (measured as GSF) was very heter-
ogeneous in the SS forest but slightly more homogeneous
in the SP forest, where the estimated GSF (overstorey can-
opy density) ranged from 0.05 to 0.27, with an average
value of 0.11. The results of path analyses (using Shipley’s d-sep meth-
od) are shown in Table 3. All the separate (single-nutrient)
linear models tested for both nutrients and sites were
found to be consistent with the observed data (p = 0.35 to
0.96). In contrast, the general (two-nutrient) models as-
suming that the litter accumulation is the direct and im-
mediate cause of the conjoint observed change in the con-
centrations of available N and P in the studied forests soils
were rejected for both sites (Table 3). Relationships between canopy, litter, and soil Scatterplots illustrating the closest linear relationships
found between litter cover (%) and concentration of available P
(upper graph) and available N (lower graph), in the site of south-
ern Portugal. Fig 3. Scatterplots illustrating the closest linear relationships
found between litter cover (%) and concentration of available P
(upper graph) and available N (lower graph), in the site of south-
ern Portugal. Fig 2. Scatterplots illustrating the closest linear relationships
found between the measured canopy density variables and the
litter accumulation variables in the soil of the forest sites located
in southern Spain (SS, upper graph) and southern Portugal (SP,
lower graph). density (× 1.4) was paralleled by marked increases in soil
litter cover (× 4.7) and, to a lesser extent, soil P (× 1.9) and
N-NH4 (× 1.7) availabilities (Table 3). Overall, these results are consistent with the hypothesis
that the overstorey canopy indirectly affects the topsoil N
and P availability through litter accumulation. Relationships between canopy, litter, and soil In both forests, there were highly significant relationships
between variables related to canopy density and variables
related to litter accumulation (Table 2, Fig. 2). Litter ac-
cumulation variables were significantly correlated to top-
soil concentrations of available N and P (Table 2, Fig. 3). In addition, canopy density variables were significantly
correlated with topsoil available-N and -P concentrations j
However, the general model assuming that an unmeas-
ured latent factor (‘soil processes’) is the immediate com-
mon cause for the observed conjoint increase of N and P
availabilities along the canopy-density/litter-accumulation
gradients was consistent with the empirical data (p > 0.70,
Table 3). In this model, errors of N and P are correlated. Table 2. Summary of the correlation values between canopy density, litter accumulation, and topsoil parameters in the two studied
forests. For litter accumulation and canopy density, only the highest correlation found is shown. The per-test significance of the results
is indicated by ns p>0.05, *: p≤0.05, **: 0.05 < p ≤ 0.005, ***: p < 0.005. Values in italics did not remain significant after controlling
the familywise error rate (FWER) in the entire correlation matrix at the 0.05 level. SS = Spanish site at Los Alcornocales Natural Park;
SP = Portuguese site at Sudoeste Alentejano e Costa Vicentina Natural Park. Canopy density
Litter accumulation
SS
SP
SS
SP
Litter accumulation
0.77***
0.85***
–
–
P availability
0.49***
0.56***
0.51***
0.70***
N availability
0.47***
0.49*
0.47***
0.59***
Total N
0.41***
0.24 ns
0.32*
0.12 ns 70 WEB ECOLOGY 6, 2006 density (× 1.4) was paralleled by marked increases in soil
litter cover (× 4.7) and, to a lesser extent, soil P (× 1.9) and
N NH (
)
il bili i
(T bl
)
Fig 2. Scatterplots illustrating the closest linear relationships
found between the measured canopy density variables and the
litter accumulation variables in the soil of the forest sites located
in southern Spain (SS, upper graph) and southern Portugal (SP,
lower graph). Fig 3. Scatterplots illustrating the closest linear relationships
found between litter cover (%) and concentration of available P
(upper graph) and available N (lower graph), in the site of south-
ern Portugal. Fig 3. Scatterplots illustrating the closest linear relationships
found between litter cover (%) and concentration of available P
(upper graph) and available N (lower graph), in the site of south-
ern Portugal. Fig 3. Discussion However, Gallardo (
were differentially affected by
ish dehesa: only the N distrib
the tree canopies, while P wa
canopies due to geochemical
The plants’ growth and sur
ing gradients depend on their
light versus soil nutrient avail
tions of the illumination grad
vere light limitations) one wo
nutrient availability – related
erate amounts of litter – wou
the composition of the under
understorey plants having a c
ance may take advantage of th
i
h
N
d P
Fig. 4. Relationships between P and N available concentrations
in topsoil of the forest sites located in southern Spain (upper
graph) and southern Portugal (lower graph). Fig. 4. Relationships between P and N available concentrations
in topsoil of the forest sites located in southern Spain (upper
graph) and southern Portugal (lower graph). light gradient (with values about 1/10 of full sun) in both
studied forests. At the same time, except in very low light conditions,
forest plants usually respond positively to soil nutrient in-
creases, especially in poor soils (Walters and Reich 2000). Therefore, soil nutrients may condition plant growth and
survival in the SS forest, where a significant proportion of
microsites have enough light availability (Table 4). g
g
In both forests, there is correlational support for the ex-
istence of counteracting trends between nutrient and light
availability. This pattern is particularly significant for P
availability, which reached extremely low values of availa-
ble concentration at the brightest end of the canopy densi-
ty gradient, while increasing to 300% and reaching non-
limiting levels for most plant species at the darkest end. g
p
p
The patterns for P and N availability in relation to light
were similar: both reached very low values for the sparsest
canopy and increased towards the darker end of the light
gradient. In semi-arid ecosystems, isolated trees induce a
joint decrease in light availability and an increase in organ-
ic matter and nutrients under the canopy (Mordelet et al. 1993). However, Gallardo (2003) found that N and P
were differentially affected by the oak canopies in a Span-
ish dehesa: only the N distributions were coincident with
the tree canopies, while P was distributed beyond the oak
canopies due to geochemical processes. p
g
p
The plants’ growth and survival along these counteract-
ing gradients depend on their ability to cope with limiting
light versus soil nutrient availability. Discussion At intermediate posi-
tions of the illumination gradient (i.e. in sites without se-
vere light limitations) one would expect that the increased
nutrient availability – related to the accumulation of mod-
erate amounts of litter – would have significant effects on
the composition of the understorey communities. In fact,
understorey plants having a certain degree of shade toler-
ance may take advantage of the soil enrichment in essential
nutrients such as N and P as the canopy density increases. In contrast, plants being more efficient in the use of N and Fig. 4. Relationships between P and N available concentrations
in topsoil of the forest sites located in southern Spain (upper
graph) and southern Portugal (lower graph). Table 3. D-sep test results of different causal models explaining the observed covariation between canopy density (“Cover”), litter
accumulation (“Litter”), and nutrient (N and/or P) availability in the two studied sites (SS = Spanish site, SP = Portuguese site). The
upper panel shows the results related to models including only one nutrient (N or P). The lower panel refers to models including both
nutrients (N and P). These models consider either litter accumulation the immediate cause of their variation (upper line, correspond-
ing to a model with independent errors) or assuming that litter influence on the availability of the two nutrients is mediated by an
unmeasured, latent factor related to soil processes (lower line, corresponding to a model with correlated errors). Table 3. D-sep test results of different causal models explaining the observed covariation between canopy density (“Cover”), litter
accumulation (“Litter”), and nutrient (N and/or P) availability in the two studied sites (SS = Spanish site, SP = Portuguese site). The
upper panel shows the results related to models including only one nutrient (N or P). The lower panel refers to models including both
nutrients (N and P). These models consider either litter accumulation the immediate cause of their variation (upper line, correspond-
ing to a model with independent errors) or assuming that litter influence on the availability of the two nutrients is mediated by an
unmeasured, latent factor related to soil processes (lower line, corresponding to a model with correlated errors). Discussion Conjoint analysis of the canopy density gradients and
changes in litter accumulation and nutrient availability
showed that these variables covariate. Moreover, the results
support the hypothesis of counteracting effects from the
woody canopy on two different resources – light and nutri-
ents – that are essential for plants living in the understorey. Th d
l
f l h
l b l
h
d y
g
There were marked differences in litter accumulation
and soil nutrient contents between the plots in contrasting
light environments (end quartiles, Q1–Q4, of the light-
availability gradients) in both studied sites (Table 4). In the
SS forest, the decrease in light availability along the gradi-
ent (5.6 times) was paralleled by a notable increase in LAI
(× 3.5). This change in canopy density was amplified by its
direct effects on the proportion of soil covered by litter (×
15), litter thickness (× 19), and accumulated litter mass (×
29). The consequent increase in soil nutrient concentra-
tions (indirect effects) was notable for both the available N
(× 2.7) and Bray 1-P (× 3.2). A similar pattern was found
in the SP forest, where a moderate increase in the canopy The direct limitation of light availability in the under-
storey is the best-known and most-conspicuous effect of
increased canopy density, and plays a major role in struc-
turing the understorey communities in Mediterranean for-
ests (Pérez-Ramos et al. 2006, Sánchez-Gómez et al. 2006,
Valladares and Guzmán 2006). Therefore, light must be
the most-limiting ecological factor at the darkest end of the 71 WEB ECOLOGY 6, 2006 light gradient (with values ab
studied forests. At the same time, except
forest plants usually respond
creases, especially in poor soil
Therefore, soil nutrients may
survival in the SS forest, wher
microsites have enough light
In both forests, there is cor
istence of counteracting trend
availability. This pattern is p
availability, which reached ex
ble concentration at the brigh
ty gradient, while increasing
limiting levels for most plant
The patterns for P and N a
were similar: both reached ve
canopy and increased toward
gradient. In semi-arid ecosys
joint decrease in light availabi
ic matter and nutrients under
1993). Discussion SS
SP
Chi-sq
DF
p
Chi-sq
DF
p
One-nutrient models
Cover – Litter – N availability
0.11
2
0.94
0.08
2
0.96
Cover – Litter – P availability
2.09
2
0.35
0.78
2
0.67
Two-nutrient models
Cover – Litter – N & P availabilities
41.35
6
0.00
13.01
6
0.04
Cover – Litter – (Soil) – N & P availabilities
2.20
4
0.70
0.86
4
0.93 72 WEB ECOLOGY 6, 2006 Table 4. Comparison of litter accumulation and soil properties between the end quartiles of the studied light-availability gradients. For
each variable, the value of Mean±Standard Deviation and of the ratio between means of end quartiles (Q1/Q4) are shown. each variable, the value of Mean±Standard Deviation and of the ratio between means of end quartiles (Q1/Q4) are shown. Light availability in the understorey
Low (Q1)
High (Q4)
Q1/Q4
South Spain’s forest (SS)
Litter layer
Covered soil (%)
91 ± 10
6 ± 8
15.3
Thickness (cm)
5.6 ± 2.1
0.3 ± 0.4
18.6
Dry weight (g litter m–2)
917 ± 720
31.8 ± 36.7
28.8
Soil properties
Total NKjeldahl (%)
0.43 ± 0.16
0.29 ± 0.10
1.5
Available N (N–NH4) (mg kg–1)
5.1 ± 4.7
1.9 ± 1.6
2.7
Available POlsen (mg kg–1)
5.4 ± 3.9
3.0 ± 1.0
1.8
Available PBray (mg kg–1)
2.9 ± 2.4
0.9 ± 0.7
3.2
South Portugal’s forest (SP)
Litter cover (%)
75 ± 17
16 ± 9
4.7
Total NKjeldahl (%)
0.38 ± 0.06
0.35 ± 0.03
1.1
Available N (N–NH4)
4.7 ± 1.0
2.7 ± 0.8
1.7
Available POlsen (mg kg–1)
4.3 ± 1.3
2.3 ± 0.4
1.9 P may be favored in sites having higher light availability
(Walters and Reich 2000). P may be favored in sites having higher light availability
(Walters and Reich 2000). There are marked changes in density, composition, and
diversity of the understorey communities growing along
the studied combined gradients of light and nutrients
(Maltez-Mouro et al. 2005a, 2005b, García, unpubl.). For
a mechanistic explanation, we must know the response of
existing species to light and nutrient gradients, or at least
some index of their ecological preferences such as the indi-
ces defined by Ellenberg (1988) for Central Europe. Fur-
ther studies on the ecophysiology of Mediterranean spe-
cies, together with field manipulative experiments, will al-
low evaluating the relative importance of these covariating
factors for forest regeneration and dynamics. Discussion Interactions between light, soil resources, and the over-
storey canopy species will affect regeneration dynamics of
the forest ecosystem. Therefore, the complex covariation of
different resources must be fully understood to optimize
management or conservation actions in Mediterranean
forests. In particular, some soil chemical properties with
ecological relevance (such as soil N and P availabilities) in
forest regeneration may change in a close relationship with
other better-known ecological factors (such as light, mois-
ture, and temperature). Thus, attention should be given to
separating direct (e.g. light limitation) versus indirect (litter
and soil mediated) effects of the woody canopy on ecosys-
tem processes. Acknowledgements – We thank Ana Polo, Eduardo Gutiérrez,
Antonio Jordán, and Lorena Martínez-Zavala for field and labo-
ratory assistance. The Spanish MEC (CGL2005-05830-C03-
01-BOS, DINAMED project) and the Portuguese FCT (SFRH/
BD/8322/2002 grant to SMM) supported the research. Thanks
to Felipe Oliveros (Director) and the staff of Los Alcornocales Nat-
ural Park for their support with fieldwork. Covariation of different essential resources depending
(direct or indirectly) on canopy density tends to obscure
the role of more-complex, indirect, and subtle effects (e.g. soil nutrients) against those that are more conspicuous and
direct (e.g. light) when interpreting the patterns of under-
storey plant populations and communities. The indirect
effects may influence (in some sites or moments) regenera-
tion processes as much as those directly depending on can-
opy density (e.g. light). Based on our results, it can be pre-
dicted that the understorey communities at the recent gaps
(natural or artificially opened) in dense canopies will in-
clude new species with higher light requirements than
those formerly living in deep shade, but also with higher
nutrient requirements than those usually living in the N-
and P-depleted older gaps. Thus, periodic disturbances
would reduce the usual negative correlation between light
and nutrients (typical of unmanaged forests), inducing
higher habitat and species diversity (Spies and Turner
1999). References Anonymous 1992. Interpretación de análisis de suelo, foliar y
agua de riego. Consejo de abonado (normas básicas). – Mun-
di-Prensa, Madrid. Anonymous 1998. HemiView canopy analysis software Version
2.1 of Delta-T Devices Ltd. – Cambridge. Anonymous 2000. Soil and Plant Analysis Council, Inc. Soil
Analysis. Handbook of Reference Methods. – CRC Press,
Boca Raton. Anonymous 2001. STATISTICA (data analysis software), ver-
sion 6. http://www.statsoft.com/ Binkley, D. and Giardina, C. 1998. Why do trees species affect
soils? The warp and woof of tree-soil interactions. – Biogeo-
chemistry 42: 89–106 73 WEB ECOLOGY 6, 2006 Vázquez, J. (ed.). Universidad de Huelva. Huelva, Spain (in
press). Broncano, M. J., Riba, M. and Retana, J. 2004. Seed germina-
tion and seedling performance of two Mediterranean tree
species, holm oak (Quercus ilex L.) and Aleppo pine (Pinus
halepensis Mill.): a multifactor experimental approach. –
Plant Ecol. 138: 17–26 p
Puerta-Piñero, C., Gómez, J. M. and Zamora, R. 2006. Species-
specific effects on topsoil development affect Quercus ilex
seedling performance. – Acta Oecol. 29: 65–71. Ellenberg, H. 1988. Vegetation ecology of central Europe. 4th
edition. – Cambridge University Press, Cambridge. g
Quero J. L. et al. 2006. Interactions of drought and shade effects
on seedlings of four Quercus species: physiological and struc-
tural leaf responses. – New Phytol. 170: 819–834. Frank, K., Beegle, D. and Denning, J. 1998. Phosphorus. – In:
Recommended Chemical Soil Test Procedures for the North
Central Region. North Central Regional Research Publica-
tion No. 221. Missouri Agricultural Experiment Station, pp. 21–30. p
y
Rossi, B.E. and Villagra, P.E. 2003. Effects of Prosopis flexuosa on
soil properties and the spatial pattern of understorey species
in Arid Argentina. – J. Veg. Sci. 14: 543–550. g
g
Sack, L., Grubb, P. J., and Marañón, T. 2003. The functional
morphology of juvenile plants tolerant of strong summer
drought in shaded forest understories in southern Spain. –
Plant Ecol. 168: 139–163. García, L. V. 2004. Escaping the Bonferroni iron claw in ecolog-
ical studies. – Oikos 105: 657–663. Gallardo, A. 2003. Effects of tree canopy on the spatial distribu-
tion of soil nutrients in a Mediterranean Dehesa. – Pedobio-
logia 47: 117–125. Sánchez-Gómez, D., Valladares, F. and Zavala, M. A. 2006. Per-
formance of seedlings of Mediterranean woody species under
experimental gradients of irradiance and water availability:
trade-offs and evidence for niche differentiation. – New Phy-
tol. 170: 795–806. g
Jordán, A. et al. 1998. References Principales tipos de suelos asociados al
bosque de Quercus sp. y brezal en el Parque Natural “Los Al-
cornocales” (Cádiz, Málaga). – Almoraima 19: 231–240. Shipley, B. 2000. Cause and correlation in biology. A user’s guide
to path analysis, structural equations and causal inference. –
Cambridge University Press, Cambridge. g
Maltez-Mouro, S. et al. 2005a. The combined role of topography
and overstorey tree composition in promoting edaphic and
floristic variation in a Mediterranean forest. – Ecol. Res. 20:
668–677. g
y
g
Shipley, B. 2003. Testing recursive path models with correlated
errors using d-separation. – Struct. Eq. Model. 10: 214–221. Maltez-Mouro, S. et al. 2005b. Understory floristic heterogenei-
ty within a Mediterranean oak forest: how much is explained
by edaphic, spatial and overstory components? – Rev. Biol. (Lisbon) 23: 121–133. g
Sparks, D. L. (ed.) 1996. Methods of Soil Analysis. Part 3. Chemical Methods. – Soil Science Society of America, Mad-
ison, USA. Spies, T. A. and Turner, M. G. 1999. Dynamic forest mosaics. –
In: Hunter ML (ed) Maintaining biodiversity in forest eco-
systems. Cambridge University Press, Cambridge, U.K., pp. 95–160. Marañón, T. et al. 2004. Regeneration of tree species and restora-
tion under contrasted Mediterranean habitats: field and
glasshouse experiments. – Int. J. Ecol. Env. Sci. 30: 187–
196. Mordelet, P., Abbadie, L. and Menaut, J. C. 1993. Effects of tree
clumps on soil characteristics in a humid savanna of West
Africa (Lamto, Cote d’Ivoire). – Plant Soil 153: 103–111. Valladares, F. and Guzmán, B. 2006. Canopy structure and spa-
tial heterogeneity of understory in an abandoned Holm oak
woodland. – Ann. For. Sci. 63: 1–13. Walters, M. B. and Reich, P. B. 2000. Seed size, nitrogen supply,
and growth rate affect tree seedling survival in deep shade. –
Ecology 81: 1887–1901. Olsen, S. R. et al. 1954. Estimation of available phosphorus in
soils by extraction with sodium bicarbonate. USDA Circular
939. – U.S. Government Printing Office, Washington D.C. gy
Washburn, C. S. M. and Arthur, M. A. 2003. Spatial variability
in soil nutrient availability in an oak-pine forest: potential
effects of tree species. – Can. J. For. Res. 33: 2321–2330. g
g
Pérez-Ramos, I. M. et al. 2006. Regeneration ecology of Quercus
suber (cork oak) in Southern Spain. – In: Suberwood: New
challenges for integration of cork oak forests and products, 74 WEB ECOLOGY 6, 2006 WEB ECOLOGY 6, 2006
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Driving cancer evolution
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eLife
| 2,017
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cc-by
| 2,439
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Copyright Fitzgerald and
Rosenberg. This article is distributed
under the terms of the Creative
Commons Attribution License, which
permits unrestricted use and
redistribution provided that the
original author and source are
credited. Driving cancer evolution Tumor-growth-factor-beta signaling helps cancer cells to evolve and
become resistant to drugs by down-regulating accurate DNA repair. Tumor-growth-factor-beta signaling helps cancer cells to evolve and
become resistant to drugs by down-regulating accurate DNA repair. DEVON M FITZGERALD AND SUSAN M ROSENBERG A signaling pathway known as tumor growth
factor (TGF)-beta signaling promotes cell growth
and is often up-regulated in cancer cells. Now,
in eLife, Raffaella Sordella of Cold Spring Harbor
Laboratory and colleagues – including Debjani
Pal as first author – report that TGF-beta signal-
ing in cancer cells temporarily down-regulates
high-fidelity DNA repair, leading to populations
of cells that are genetically diverse (Pal et al.,
2017). These populations harbor more cells that
are resistant to a broad range of chemotherapy
drugs. Related research article Pal D, Pertot A,
Shirole NH, Yao Z, Anaparthy N, Garvin T,
Cox H, Chang K, Rollins F, Kendall J,
Edwards L, Singh VA, Stone GC, Schatz MC,
Hicks J, Hannon GJ, Sordella R. 2017. TGF-b
reduces DNA ds-break repair mechanisms to
heighten genetic diversity and adaptability
of CD44+/CD24 cancer cells. eLife 6:
e21615. doi: 10.7554/eLife.21615 INSIGHT GENETIC DIVERSITY
Driving cancer evolution GENETIC DIVERSITY Related research article Pa Pal et al. focused on a subpopulation of cells
known as CD44+/CD24 cells, which arise ran-
domly within many types of tumor and in cancer
cell lines (reviewed by Polyak and Weinberg,
2009). They behave like stem cells thanks to the
TGF-beta signaling pathway being continuously
active, and are linked to drug-resistance, metas-
tasis and other poor outcomes for patients
(Shipitsin et al., 2007). T
umors
can
form
when
cells
acquire
mutations that allow them to grow and
divide rapidly. Further mutations and
the selection of successful cell clones allow the
tumor to evolve and drive the progression of
the
disease
with
deadly
consequences
(reviewed by Gerlinger et al., 2014). Many
tumors have unstable genomes with lots of
small
mutations
and/or
genome
rearrange-
ments, and this makes it more likely that the
cancer will become progressively worse and
evolve
resistance
to
chemotherapy
drugs
(reviewed by Lee et al., 2016). T Cells generally use a process called homol-
ogy-directed repair to mend double-stranded
breaks in DNA. This process is mostly accurate
and can heal DNA breaks without rearranging
the chromosome. Pal et al. found that RNAs
that encode proteins used in homology-directed
repair and other types of DNA repair are less
abundant in CD44+/CD24 cells. In these cells,
DNA damage accumulates and genome re-
arrangements occur, with the cells appearing to
repair DNA breaks by other, less accurate,
means. Historically, mutations were assumed to arise
randomly, at constant rates. More recent discov-
eries indicate
that cells
and organisms
can
increase the rate at which they acquire new
mutations (a process known as mutagenesis)
when they activate cellular stress responses –
that is, when they are stressed and poorly
adapted to their environments (reviewed by
Fitzgerald et al., 2017). Mutagenesis induced
by stress has been predicted to speed up evolu-
tion (Ram and Hadany, 2012). TGF-beta signaling was necessary for homol-
ogy-directed repair to be reduced in CD44+/
CD24 cells, and sufficient for it to be reduced
in other types of cells. Sequencing the genomes
of individual cells demonstrated that TGF-beta
signaling induces copy-number alterations – that
is, insertions or deletions of gene-fragments to Fitzgerald and Rosenberg. eLife 2017;6:e25431. DOI: 10.7554/eLife.25431 1 of 3 Fitzgerald and Rosenberg. eLife 2017;6:e25431. DOI: 10.7554/eLife.25431 Insight Insight Genetic diversity
Driving cancer evolution Figure 1. TGF-beta signaling promotes a form of DNA repair that accelerates cancer evolution. Related research article Pa used a clever model of short-term
exposure to TGF-beta to show that the heritable
genomic changes that occurred, not the TGF-
beta signaling itself, help the cancer cells
adapt. Cell populations previously exposed
TGF-beta harbored more cells that were res
tant to three chemotherapy drugs than popu
tions that had not been exposed. Although
et al. did not test the tendency of these cells
become more dangerous, the increased ada
ability of cells exposed to TGF-beta proba
speeds
the
progression
of
cancer
as
w
(Figure 1). The consequences are important. We pre
ously suggested that a new class of “anti-evo
ability” drugs should be developed to target t
processes
of
cancer
evolution,
rather
th
targeting the products of evolution (that is, t
disease
characteristics;
Rosenberg
a
Queitsch, 2014). Current anti-cancer drugs a
to reduce the ability of cancer cells to grow a
divide,
and
some
induce
mutagene
(Fitzgerald et al., 2017). Inhibiting evoluti
itself might reduce the emergence of dr
resistance. Several stress-related proteins that promo
mutagenesis and other mechanisms that chan
cell characteristics are promising targets
these new drugs (Rosenberg and Queitsc beta signaling itself, help the cancer cells to
adapt. Cell populations previously exposed to
TGF-beta harbored more cells that were resis-
tant to three chemotherapy drugs than popula-
tions that had not been exposed. Although Pal
et al. did not test the tendency of these cells to
become more dangerous, the increased adapt-
ability of cells exposed to TGF-beta probably
speeds
the
progression
of
cancer
as
well
(Figure 1). many neighboring genes – causing heritable
genetic changes and populations of cells to
become genetically diverse. Furthermore, Pal
et al. used various cancer cell lines, primary
tumor samples and published cancer genome
datasets to demonstrate that TGF-beta signaling
often decreases accurate DNA repair in cancer
cells, which can cause copy-number alterations
(Figure 1). A gene called PMS1 – which operates in
another DNA repair process called mismatch-
repair – was also less active in CD44+/CD24 cells. Mismatch repair corrects small errors in
DNA replication, and more mutations accumu-
late when it is down-regulated (reviewed by
Scanlon and Glazer, 2015; Fitzgerald et al.,
2017). Although Pal et al. did not examine
whether small mutations are increased, we sus-
pect that, like copy-number alterations, TGF-
beta may have increased the production of small
mutations as well. The consequences are important. Related research article Pa Cells generally
repair spontaneous DNA breaks by a process called homology-directed (HD) repair (left). TGF-beta signaling
down-regulates this process, which leads to the DNA breaks being repaired by other, less accurate, forms of
repair that destabilize chromosomes and generate large DNA deletions and duplications known as copy-number
alterations (CNAs). This leads to populations of cells that are genetically diverse (middle). Subpopulations of
cancer cells known as CD44+/CD24 cells often have over-active TGF-beta signaling: this leads these cells to
acquire high levels of CNAs and to rapidly evolve resistance to chemotherapy. Treating other cells with TGF-beta
has a similar effect. Cells with red nuclei have undergone genetic changes; the other colors represent the resultin
diverse cell characteristics. Figure 1. TGF-beta signaling promotes a form of DNA repair that accelerates cancer evolution. Cells generally
repair spontaneous DNA breaks by a process called homology-directed (HD) repair (left). TGF-beta signaling
down-regulates this process, which leads to the DNA breaks being repaired by other, less accurate, forms of
repair that destabilize chromosomes and generate large DNA deletions and duplications known as copy-number
alterations (CNAs). This leads to populations of cells that are genetically diverse (middle). Subpopulations of
cancer cells known as CD44+/CD24 cells often have over-active TGF-beta signaling: this leads these cells to
acquire high levels of CNAs and to rapidly evolve resistance to chemotherapy. Treating other cells with TGF-beta
has a similar effect. Cells with red nuclei have undergone genetic changes; the other colors represent the resulting
diverse cell characteristics. many neighboring genes – causing heritable
genetic changes and populations of cells to
become genetically diverse. Furthermore, Pal
et al. used various cancer cell lines, primary
tumor samples and published cancer genome
datasets to demonstrate that TGF-beta signaling
often decreases accurate DNA repair in cancer
cells, which can cause copy-number alterations
(Figure 1). A gene called PMS1 – which operates in
another DNA repair process called mismatch-
repair – was also less active in CD44+/CD24 cells. Mismatch repair corrects small errors in
DNA replication, and more mutations accumu-
late when it is down-regulated (reviewed by
Scanlon and Glazer, 2015; Fitzgerald et al.,
2017). Although Pal et al. did not examine
whether small mutations are increased, we sus-
pect that, like copy-number alterations, TGF-
beta may have increased the production of small
mutations as well. Does the genome instability caused by TGF-
beta signaling drive the evolution of cancers? Pal et al. Related research article Pa We previ-
ously suggested that a new class of “anti-evolv-
ability” drugs should be developed to target the
processes
of
cancer
evolution,
rather
than
targeting the products of evolution (that is, the
disease
characteristics;
Rosenberg
and
Queitsch, 2014). Current anti-cancer drugs act
to reduce the ability of cancer cells to grow and
divide,
and
some
induce
mutagenesis
(Fitzgerald et al., 2017). Inhibiting evolution
itself might reduce the emergence of drug
resistance. Does the genome instability caused by TGF-
beta signaling drive the evolution of cancers? Pal et al. used a clever model of short-term
exposure to TGF-beta to show that the heritable
genomic changes that occurred, not the TGF- Several stress-related proteins that promote
mutagenesis and other mechanisms that change
cell characteristics are promising targets for
these new drugs (Rosenberg and Queitsch, Fitzgerald and Rosenberg. eLife 2017;6:e25431. DOI: 10.7554/eLife.25431 2 of 3 Insight Genetic diversity
Driving cancer evolution 2014; Fitzgerald et al., 2017). The findings of
Pal et al. suggest that proteins of the TGF-
beta signaling pathway are potential targets for
new
“anti-evolvability”
drugs
that
could
be
effective against many different types of cancer. Several drugs that target the TGF-beta pathway
are
already
in
clinical
trials
(reviewed
by
Herbertz et al., 2015). References doi: 10.1038/nrc2620, PMID: 19262571
Ram Y Hadany L 2012 The evolution of stress Pal D, Pertot A, Shirole NH, Yao Z, Anaparthy N,
Garvin T, Cox H, Chang K, Rollins F, Kendall J,
Edwards L, Singh VA, Stone GC, Schatz MC, Hicks J,
Hannon GJ, Sordella R. 2017. TGF-b reduces DNA ds-
break repair mechanisms to heighten genetic diversity
and adaptability of CD44+/CD24 cancer cells. eLife
6:21615. doi: 10.7554/eLife.21615, PMID: 28092266
Polyak K, Weinberg RA. 2009. Transitions between Edwards L, Singh VA, Stone GC, Schatz MC, Hicks J,
Hannon GJ, Sordella R. 2017. TGF-b reduces DNA ds-
break repair mechanisms to heighten genetic diversity
and adaptability of CD44+/CD24 cancer cells. eLife
6:21615. doi: 10.7554/eLife.21615, PMID: 28092266 Devon M Fitzgerald is in the Department of
Molecular and Human Genetics, Baylor College
of Medicine, Houston, United States, the
Department of Biochemistry and Molecular
Biology, Baylor College of Medicine, Houston,
United States, the Department of Molecular
Virology and Microbiology, Baylor College of
Medicine, Houston, United States, and the Dan
L Duncan Comprehensive Cancer Center, Baylor
College of Medicine, Houston, United States
http://orcid.org/0000-0002-2480-7269
Susan M Rosenberg is in the Department of
Molecular and Human Genetics, Baylor College
of Medicine, Houston, United States, the
Department of Biochemistry and Molecular
Biology, Baylor College of Medicine, Houston,
United States, the Department of Molecular
Virology and Microbiology, Baylor College of
Medicine, Houston, United States, and the Dan
L Duncan Comprehensive Cancer Center, Baylor
College of Medicine, Houston, United States
smr@bcm.edu Polyak K, Weinberg RA. 2009. Transitions between
epithelial and mesenchymal states: acquisition of
malignant and stem cell traits. Nature Reviews Cancer
9:265–273. doi: 10.1038/nrc2620, PMID: 19262571 Ram Y, Hadany L. 2012. The evolution of stress-
induced hypermutation in asexual populations. Evolution 66:2315–2328. doi: 10.1111/j.1558-5646. 2012.01576.x, PMID: 22759304 Rosenberg SM, Queitsch C. 2014. Combating
evolution to fight disease. Science 343:1088–1089. doi: 10.1126/science.1247472, PMID: 24604189
Scanlon SE, Glazer PM. 2015. Multifaceted control of
DNA repair pathways by the hypoxic tumor
microenvironment. DNA Repair 32:180–189. doi: 10. 1016/j.dnarep.2015.04.030, PMID: 25956861 Rosenberg SM, Queitsch C. 2014. Combating
evolution to fight disease. Science 343:1088–1089. Scanlon SE, Glazer PM. 2015. Multifaceted control of
DNA repair pathways by the hypoxic tumor Shipitsin M, Campbell LL, Argani P, Weremowicz S,
Bloushtain-Qimron N, Yao J, Nikolskaya T,
Serebryiskaya T, Beroukhim R, Hu M, Halushka MK,
Sukumar S, Parker LM, Anderson KS, Harris LN, Garber
JE, Richardson AL, Schnitt SJ, Nikolsky Y, Gelman RS,
et al. 2007. Fitzgerald and Rosenberg. eLife 2017;6:e25431. DOI: 10.7554/eLife.25431 References Fitzgerald DM, Hastings PJ, Rosenberg SM. 2017. Stress-induced mutagenesis: implications in cancer and
drug resistance. Annual Review of Cancer Biology 1:
119–140. doi: 10.1146/annurev-cancerbio-050216-
121919 Gerlinger M, McGranahan N, Dewhurst SM, Burrell
RA, Tomlinson I, Swanton C. 2014. Cancer: evolution
within a lifetime. Annual Review of Genetics 48:215–
236. doi: 10.1146/annurev-genet-120213-092314,
PMID: 25292359 Gerlinger M, McGranahan N, Dewhurst SM, Burrell
RA, Tomlinson I, Swanton C. 2014. Cancer: evolution
within a lifetime. Annual Review of Genetics 48:215–
236 doi: 10 1146/annurev genet 120213 092314 This study also suggests the possibility that
organisms may increase mutagenesis and evolu-
tion in the normal course of development. Pro-
grammed mutagenesis and evolution drive our
adaptive immune systems, but might also under-
lie other complex developmental programs that
require more flexibility and responsiveness than
our
genomes
encode
(discussed
by
Fitzgerald et al., 2017; Pal et al., 2017). TGF-
beta signaling controls how wounds heal, certain
aspects of embryo development, and how the
nervous system forms. Future work may indicate
that TGF-beta and other inducible mutagenesis
responses allow multicellular complexity and
flexibility in ways not yet appreciated. Herbertz S, Sawyer JS, Stauber AJ, Gueorguieva I,
Driscoll KE, Estrem ST, Cleverly AL, Desaiah D, Guba
SC, Benhadji KA, Slapak CA, Lahn MM. 2015. Clinical
development of galunisertib (LY2157299
monohydrate), a small molecule inhibitor of
transforming growth factor-beta signaling pathway. Drug Design, Development and Therapy 9:4479–4499. doi: 10.2147/DDDT.S86621, PMID: 26309397
Lee JK, Choi YL, Kwon M, Park PJ. 2016. Mechanisms
and consequences of Cancer genome instability:
lessons from genome sequencing studies. Annual
Review of Pathology: Mechanisms of Disease 11:283–
312. doi: 10.1146/annurev-pathol-012615-044446,
PMID: 26907526 Pal D, Pertot A, Shirole NH, Yao Z, Anaparthy N,
Garvin T, Cox H, Chang K, Rollins F, Kendall J, Pal D, Pertot A, Shirole NH, Yao Z, Anaparthy N,
Garvin T, Cox H, Chang K, Rollins F, Kendall J,
Edwards L, Singh VA, Stone GC, Schatz MC, Hicks J,
Hannon GJ, Sordella R. 2017. TGF-b reduces DNA ds-
break repair mechanisms to heighten genetic diversity
and adaptability of CD44+/CD24 cancer cells. eLife
6:21615. doi: 10.7554/eLife.21615, PMID: 28092266
Polyak K, Weinberg RA. 2009. Transitions between
epithelial and mesenchymal states: acquisition of
malignant and stem cell traits. Nature Reviews Cancer
9:265–273. References Molecular definition of breast tumor
heterogeneity. Cancer Cell 11:259–273. doi: 10.1016/j. ccr.2007.01.013, PMID: 17349583 http://orcid.org/0000-0003-1444-473X http://orcid.org/0000-0003-1444-473X
Competing interests: The authors declare that
no competing interests exist. Competing interests: The authors declare that
no competing interests exist. 3 of 3 Fitzgerald and Rosenberg. eLife 2017;6:e25431. DOI: 10.7554/eLife.25431
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https://openalex.org/W4246160266
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https://figshare.com/articles/journal_contribution/Escher--a_geometrical_layout_system_for_recursively_defined_circuits/6605264/1/files/12095705.pdf
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English
| null |
Escher---a geometrical layout system for recursively defined circuits
|
Proceedings of the 23rd ACM/IEEE conference on Design automation - DAC '86
| 1,986
|
cc-by
| 9,103
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NOTICE WARNING CONCERNING COPYRIGHT RESTRICTIONS:
The copyright law of the United States (title 17, U.S. Code) governs the making
of photocopies or other reproductions of copyrighted material. Any copying of this
document without permission of its author may be prohibited by law. NOTICE WARNING CONCERNING COPYRIGHT RESTRICTIONS:
The copyright law of the United States (title 17, U.S. Code) governs the making
of photocopies or other reproductions of copyrighted material. Any copying of this
document without permission of its author may be prohibited by law. CMU-CS-85-150 CMU-CS-85-150 This research was supported by NSF. Grant Number MCS-82-16706 • -
t • 2- Escher--A Geometrical Layout System
For Recursively Defined Circuits Edmund Clarke, Yulin Feng Department of Computer Science
Carnegie-Mellon University
Pittsburgh, Pennsylvania 15213 Department of Computer Science
Carnegie-Mellon University
Pittsburgh, Pennsylvania 15213
July 1985 ABSTRACT: An Kschcr circuit description is a hierarchical structure composed of cells,
wires, connectors between wires, and pins that connect wires to cells. Cells may
correspond to primitive circuit elements, or they may be defined in terms of lower level
subcells. Unlike other geometrical layout systems, a subccll may be instance of die cell
being defined. When such a recursive cell definition is instantiated, the recursion is
unwound in a manner reminiscent of the procedure call copy rule in Algol-like
programming languages. Cell specifications may have parameters that are used to
control the unwinding of recursive cells and to provide for cell families with varying
numbers of pins and other internal components. We illustrate how die Escher layout
system might be used with several nontrivial examples, including a parallel sorting
network and a FFf implementation. We also briefly describe the unwinding algorithm. UNIVERSITY LIBRARIES
CARNEGIE-MELLON UNIVERSITY
PITTSBURGH, PENNSYLVANIA 15213 2 ESCHER--A Geometrical Layout System
For Recursively Defined Circuits Edmund Clarke, Yulin Feng 1. Introduction Many circuits such as sorting networks, hardware multipliers, and FFT implementations can be described
by recursive geometrical patterns. Some layout languages provide support for recursion ([2], [6], [7] and [10]);
however, in all such systems familiar to us the circuit description is textual rather than geometrical. We
believe that it is more natural to describe complicated circuits geometrically, rather than by giving a textual
description and requiring that a program figure out the details of the layout. Some circuit editors have
powerful iteration operators that can be viewed as implementing a form of tail recursion [3], but none allow
full recursion. We have implemented a geometrical layout system (called the Escher System) in which
recursive patterns can be specified directly and then instantiated to obtain layouts for complex circuits
automatically. An Escher circuit description is a hierarchical structure in which the basic building blocks are cells, wires,
connectors between wires, and pins that connect wires to cells. Cells may correspond to primitive circuit
elements such as NAND gates and latches, or they may be defined in terms of lower level subcells, which are
defined in terms of even lower level subcells, etc. By using the Escher system, a number of primitive cells can
be connected together in complex geometrical pattern to describe the layout for a large and intricate circuit. Designers do not need to worry about the absolute sizes and positions of various circuit components; only the
topological relationships are important. Moreover, the system is completely interactive. Circuit diagrams are
constructed using a pointing device ("mouse") and tablet. Although many circuit editors provide a set of features similar to the ones that we have just listed, our
system is unique in that a subccll may, in fact, be instance of the cell being defined. When a recursive cell
definition is instantiated, the recursion is unwound in a manner reminiscent of the procedure call copy rule in
Algol-like programming languages. Cell specifications may have non-negative integer parameters that are 3 used to control the unwinding of recursive cells and to provide for cell families with varying numbers of pins
and other internal components. While the notion of parameterized cell specifications is quite common in
textual hardware description languages, we believe that it has not been previously used with graphical circuit
editors and, therefore, may be of independent interest. 1. Introduction Our paper is organized as follows: Section 2 describes the various notational conventions that the Escher
system uses for specifying recursive circuits. Since recursive cells arc usually parameterized by some integer
variable, special conventions are needed for describing groups of subcells that depend on die parameter. In
section 3 we give two examples of how the Kscher System might be used with recursive circuits Uiat are based
on parallel divide and conquer strategics. We believe that the Escher system will prove most useful for laying
out circuits with this type of structure. Section 4 shows how the Escher System might be used for laying out a
more complicated example, the parallel prefix circuit originally described by Fisher and Ladner ([I]). In
sections 5 and 6 we discuss how the Escher system works. Section 5 briefly describes how various circuit
components are represented in the system. This section also addresses the question of how much circuit
components may be moved around in obtaining a layout. The algorithm that unwinds and lays out a
recursive diagram is outlined in section 6. Since basic subcells must occupy a fixed area, the algorithm must
proceed bottom up, expanding each higher level cell so that all of its lower level subcells will fit. The paper
concludes in section 7 with summary and discussion of ways in which die Escher system might be extended to
produce better layouts. 2. Conventions for Specifying Recursive Circuit Diagrams As an example of how die Escher system might be used, we consider die problem of laying out the Tally
circuit described in [8] and also in [9], This circuit has n inputs and n + 1 outputs. The k-th output will be high
and all other outputs low, if exacdy k of the inputs are high. Figure 2-1 gives the Escher version of a recursive
definition for die Tally circuit. In die specification diere are two kinds of cells: Basic cells that cannot be refined further (like the two input
multiplexers), and Composite cells that contain other cells, wires, and connectors (like the recursive
occurrence of Tally(n-l) ). The cells that are directly contained within a composite cell are its subcells. Sometimes several subcells S r S 2, ..., S n are instances of the same cell C. In thr case we say that C is the
source of each of the S.'s. Since the specification is parameterized by n, some abbreviations are needed to represent groups of lines
and subcells that depend on n. When a definite value is provided for n, each such abbreviation in the
specification may be evaluated. 4 gnc
vd(
t a l l y ( n - l )
command: d e f c e l l
t a l l y ( n )
|b
mux[0]
b
mux[*]
b
m u x [ n - l ]
c
d
b
mux[n]
r
gnd
o u t
o u t
o u t
Figure 2-1: Recursive Pattern for Tally(n) gnc
vd(
t a l l y ( n - l )
command: d e f c e l l
t a l l y ( n )
|b
mux[0]
b
mux[*]
b
m u x [ n - l ]
c
d
b
mux[n]
r
gnd
o u t
o u t
o u t
Figure 2-1: Recursive Pattern for Tally(n) Figure 2-1: Recursive Pattern for Tally(n) Figure 2-1: Recursive Pattern for Tally(n) Groups in Escher are somewhat like one dimensional arrays in programming languages. A group is a
horizontal or vertical array of identical cells with the appropriate interconnecting wires. The subcells of a
group may be either basic cells or composite cells. They are distinguished from one another by an integer
index, which increases from left to right in the case of a horizontal array or from top to bottom in the case of
an vertical array. The initial and final values of the index may depend on a parameter of the cell containing
the group; however, the increment must be a fixed positive integer. A g-uip whose lengdi depends on an
undetermined parameter is represented by three subcells, one for the first subcell, one for die last subcell, and
one in die middle with index "*" to represent all of the remaining subcells. Thus, the "*" serves exactly the
same function in our formal specification that the ellipsis
serves in an informal specification. A number
appearing after the
represents an index increment; when the "*" appears alone, the default value for the
increment is 1. In the Tally example (Figure 2-1) there are a total of (n + 1) multiplexers, the MUX[n] and 5 MUX'S with indices from 0 to n-1 in a group. When a group of subcells is specified, it is only necessary to
give the the position of the first and the last subcells of the group with respect to some other part of the
circuit. When the containing cell is instantiated and all of the parameters of the group arc fixed, this
information is sufficient to determine the position of each of the subcells of the group. Finally, Escher uses a short diagonal mark on a wire to represent a group of wires. An expression associated
with die mark indicates how many lines arc in the group. We call such groups of wires, buses, and the
associated expression, the bus width. In Figure 2-1 there arc two buses, and each represents n-1 wires. We also
use die convention that a wire connected to a subcell with index "*" actually represents the same number of
wires as the number of omitted subcells. The circuit for the base case, Tally(l), is shown in Figure 2-2. After we instantiate the TALLY circuit with a given value, for example, n = 6, the Escher system will 3. Divide and Conquer Circuits The simplest recursive circuits have only a single recursive sub-circuit. This case is somewhat like tail-
recursion in programming languages and is relatively easy to implement. The Tally circuit in Figure 2-1 is an
example of such a recursion. Unfortunately, not all recursive circuits have such a simple structure. Many
interesting circuits are based on a divide and conquer strategy in which a complicated task is realized by a
number of subcircuits each of which is a recursive instance of die circuit being defined. Adders, multipliers,
sorters, FFT circuits, etc., can all be structured in this manner. Figuring out by hand an appropriate layout for
an instance of such a circuit can be quite tricky. Once the recursive structure of the circuit has been
determined, die Fscher system may be used to unwind a particular instance of the circuit. We illustrate below
how Escher might be used with two well known examples of recursive divide and conquer circuits. Figure 2-1: Recursive Pattern for Tally(n) Figure 2-2: TALLY(l), a base case for TALLY recursive specification Figure 2-2: TALLY(l), a base case for TALLY recursive specification Examination of the recursive specification for the TALLY circuit immediately shows how it works. Each
multiplexer has three inputs labeled a, 6, c and one output labeled d. If b is high, die output dselects the value
c; otherwise, it selects the value a. It is easy to see that die base case is correct. We assume diat TALLY(n-l) is
correct and that k of die first n-1 inputs are high. By the induction hypothesis, the k-th output of TALLY(n-l)
is high. If the
input is also high, then all of the selector inputs of the multiplexers will be high, so each
MUX's with index in the range from 0 to n-1 will select as its output the value of its c input, while the output
of MUX[n] will be low. Thus, the (k-hl)* output (counting from bottom to top) of TALLY(n) w;
1l be high
and the other outputs will be low. A similar discussion can be used for the case in which die n
t h input of
TALLY(n) is low. After we instantiate the TALLY circuit with a given value, for example, n = 6, the Escher system will 6 automatically unwind the recursive specification into the circuit diagram shown in Figure 2-3. A final phase
(that has not been completed) will compact the circuit diagram produced by the Escher system in accordance
with a set of design rules appropriate to the transistor technology used to fabricate the chip. 7 Figure 2-3: instantiation for Tally(6) Figure 2-3: instantiation for Tally(6) 3.1. Example 1: Parallel Sorting Our first example is a network for sorting a sequence of n k bit numbers into increasing order, where n is
assumed a power of 2 [5]. The standard divide and conquer approach is to sort the first half and the second
half in parallel and then merge the two sorted sequences. The Escher specification for such a circuit is shown
in Figure 3-1. Note that every bus width number here means the number of k-bit wires. r—
1
/ 'n/2
/ 'n/2
s o r t ( n / 2 )
so r t ( n / 2 )
/ 'n/2
/ 'n/2
m e r g e ( n / 2 )
/ n
command: d e f c e l l
s o r t ( n )
Figure 3-1: Recursive pattern for Sort(n) r—
1
/ 'n/2
/ 'n/2
s o r t ( n / 2 )
so r t ( n / 2 )
/ 'n/2
/ 'n/2
m e r g e ( n / 2 )
/ n
command: d e f c e l l
s o r t ( n )
Figure 3-1: Recursive pattern for Sort(n) Figure 3-1: Recursive pattern for Sort(n) 9 The Merge cell can also be defined recursively. To merge two sequences "a" and "b", we merge the
even-indexed elements of "a" with the odd-indexed elements of "b", and the odd-indcxed elements of "a"
with the even-indexed elements of "b". The outputs of the two half-size merging circuits arc sent through an
array of comparators. Each comparator "CMP" sorts two k-bit numbers in order. Figure 3-2 gives the
recursive definition of Mcrge(n). Pass(n), shown in Figure 3-3, contains only wires and is used to separate the
even-indexed inputs and the odd-indexed inputs. A*
p a s s ( n )
A n/2
A
4"
p a s s ( n )
n/2
/
n/2
m e r g e ( n / 2 )
m e r g e ( n / 2 )
cmp[0]
r
cmp[»]
c m p [ n - l ]
command: d e f c e l l
merge(n)
Figure 3-2: Recursive pattern for Merge(n) command: d e f c e l l
merge(n) Figure 3-2: Recursive pattern for Merge(n) 10 Figure 3-3: Recursive pattern for connections Pass(n) Figure 3-3: Recursive pattern for connections Pass(n) If we instantiate the recursive specification shown in Figure 3-1 with n = 16, our system automatically
generates the pattern shown in Figure 3-4. 11 11 Figure 3-4: Instantiation for SORT(16) Figure 3-4: Instantiation for SORT(16) 12 3.2. Example 2: Fast Fourier Transform The second example is a circuit for computing the Fast Fourier Transform ([4], [11J). Let u = Q-"
]/n. The
Fast Fourier Transform (FIT)of .v(0)
x(n-1)
is defined for k = 0,1,2
n-1 by The second example is a circuit for computing the Fast Fourier Transform ([4], [11J). Let u = Q-"
]/n. The
Fast Fourier Transform (FIT)of .v(0)
x(n-1)
is defined for k = 0,1,2
n-1 by The second example is a circuit for computing the Fast Fourier Transform ([4], [11J). Let u = Q-"
]/n. The
Fast Fourier Transform (FIT)of .v(0)
x(n-1)
is defined for k = 0,1,2
n-1 by This equation can be "folded" to obtain for j = 0,1,2
n/2-1:
A2j)= 2 1 = 7
^
k(Ak)+Jik+n/2)), This equation can be "folded" to obtain for j = 0,1,2
n/2-1:
A2j)= 2 1 = 7
^
k(Ak)+Jik+n/2)), This equation can be "folded" to obtain for j = 0,1,2
n/2-1:
A2j)= 2 1 = 7
^
k(Ak)+Jik+n/2)), This equation can be "folded" to obtain for j = 0,1,2
n/2-1: }{2j+1 )= 2 1 = 7 »
2ik(o>
kMk)-x(k
+ ii/2))). Fork = 0,1,2,...,n/2-1, let W)=x(k)
+
4k+n/2), v(k+ n/2)=a
k(x(k)-x(k+
n/2)). v(k+ n/2)=a
k(x(k)-x(k+
n/2)). If we express y in terms of v, we obtain
}{2j+D=^l=7W
2f<k+n/2) If we express y in terms of v, we obtain
}{2j+D=^l=7W
2f<k+n/2) If we express y in terms of v, we obtain for j = 0,1,2
n/2-1. This series of equations can be expressed in matrix form as in Figure 3-5. for j = 0,1,2
n/2-1. This series of equations can be expressed in matrix form as in Figure 3-5. "y(o>
i
1
1
1
i
2
4
n
y ( 2 )
i
w
w
w
y(n-z)
i
*
w
*
w
*
w
y ( i )
0
0
0
•
•
0
y ( 3 )
0
0
0
•
•
0
_ y ( n - i ) _
0
0
0
•
•
0
n-2
0
0
0
0
0
0
0
0
0
1
1
1
I
2
4
w
w
1
w
w
n-2
v ( 0 )
v ( l )
v ( n / 2 - l )
v ( n / 2 )
v ( n - l )
(
Figure 3-5: Block Diagonal Matrix for Computing FFT "y(o>
i
1
1
1
i
2
4
n
y ( 2 )
i
w
w
w
y(n-z)
i
*
w
*
w
*
w
y ( i )
0
0
0
•
•
0
y ( 3 )
0
0
0
•
•
0
_ y ( n - i ) _
0
0
0
•
•
0
n-2
0
0
0
0
0
0
0
0
0
1
1
1
I
2
4
w
w
1
w
w
n-2
v ( 0 )
v ( l )
v ( n / 2 - l )
v ( n / 2 )
v ( n - l )
(
Figure 3-5: Block Diagonal Matrix for Computing FFT Figure 3-5: Block Diagonal Matrix for Computing FFT Examination of the block diagonal matrix suggests a recursive circuit for computing the FFT. First, we use a
group of multiplier-adder cells MAC[0], MAC[1], • • • ,MAC[n/2-l] to transform x[0],x[l]f • • •, x[n-l] into
v[0],v[l], • • • ,v[n-l]. Each multiplier-adder cell will have two inputs and two outputs. In the case of cell 13 MAC[k] the two inputs arc x[k] and x[k + n/2|, and the two outputs arc v[k] and v[k + n/2]. v(k+ n/2)=a
k(x(k)-x(k+
n/2)). In general, die
behavior of each MAC[k] cell will depend on the value of k-cach must be provided with register holding its
particular value of <o
k. For simplicity, however, we will neglect this difference, and assume diat MAC is dicir
common source. MAC[k] the two inputs arc x[k] and x[k + n/2|, and the two outputs arc v[k] and v[k + n/2]. In general, die
behavior of each MAC[k] cell will depend on the value of k-cach must be provided with register holding its
particular value of <o
k. For simplicity, however, we will neglect this difference, and assume diat MAC is dicir
common source. We eventually obtain two half size FFY problems: One on v[0],
v[n/2-l] and one on v[n/2],.. .,v[n-l]. The Fschcr specification of die FFY circuit will contain two recursive instances of FFT(n/2) as shown in
Figure 3-6. The cell labeled RPS is just the reverse of the cell PASS defined in die previous example. It takes
two sets of n/2 inputs and merges diem into n outputs, so that the first set of inputs corresponds to the
even-indexed outputs and the second set of inputs to die odd-indexed outputs. If we instantiate the circuit with n = 16, Escher generates the network shown in Figure 3-7. The eight MACs
in the first row have registers holding co°, co
1, <o
2, • • •, <JP in sequence from left to right; the eight MACs
the second row are divided into two groups, the left four in one group and die right four in another. In each
group of four, the registers hold <o°, co
2,co
4 and o
8 , respectively. The eight MACs in the third row arc divided
into four groups, each of which contains two MACs, one storing the value <o° and one storing the value co
4. Each MAC in die last row has co° in its register. Although we will not address the problem of inidalizing the
registers, it is not difficult to solve. v(k+ n/2)=a
k(x(k)-x(k+
n/2)). 14 14 n/2
/ I n / 2
r p s ( n )
J
^
/
n
j
command: d e f c e l l
f f t ( n )
Figure 3-6: Recursive pattern for FFf(n) Figure 3-6: Recursive pattern for FFf(n) 15 15 15 r
r
r
1
1
command: eval
f f t
n=16 r
r
r
1
1
command: eval
f f t
n=16
Figure 3-7: Instantiation for FFT(16) Figure 3-7: Instantiation for FFT(16) Figure 3-7: Instantiation for FFT(16) 16 4. A More Complicated Example In this section wc show how to layout die parallel prefix circuit described by Fischer and Ladner in [1]. We
assume ® is an associative binary operator that is implemented by a cell named "OP
M with two inputs and
one output. Hie parallel prefix circuit has n inputs and n outputs. The n outputs are the successive partial
products obtained by using ® to combine the inputs. Thus, if the inputs are
then the outputs are
x ,(x <8> x2),... ,(((* ® * 2)® . . . ) ® xj. Figure 4-1 shows the clever recursive circuit suggested by Fischer
and Ladner for computing the partial products in parallel. op
op
op
HE
op
op
p f x ( n / 2 )
op
op
op
op
op
op
Figure 4-1: Parallel Prefix Circuit Figure 4-1: Parallel Prefix Circuit How do we specify the parallel prefix circuit with the Escher system? It is convenient to split the circuit into
5 parts as shown by the dotted lines in Figure 4-1. Each of these parts will correspond to an Escher cell that
can easily be defined recursively. See Figure 4-2. DPASS(n) and UPASS(n) contain only connection wires and are defined in Figure 4-3 and 4-4,
respectively. DPART(n) can be split again into two parts, a left part DLEFT and a right part DRIGHT. Each
of these parts can, in turn, be defined recursively. See Figures 4-5,4-6 and 4-7. The definition of UPART is
similar to that of DPART and will be omitted. If we instantiate PFX(n) with n = 16, Escher will unwind the recursive specification and compact the
unwound layout to produce the circuit shown in Figure 4-8. Representation of Circuit Components and Structural Elasticity A coll is represented in the Kscher system by a record structure consisting of three Holds, die AttributeList,
the PointNct, and die SubCcllList. The AttributeList contains die name of die cell, its parameter list, and its
position (TopY, BottomY, RightX, LcftX) with respect to a fixed coordinate system. The PointNct is used to
keep track of the different kinds of points (pins, bends, connectors, vias, transistors, etc.) and their locations. Kach point is represented by a record structure that specifies its type, its coordinates PosX and PosY, and how
it is connected to the other components of the cell. All of the points in a cell arc linked together in an
undirected graph structure called die PointNct. From each point in die cell it is possible to find die next
connected point in a vertical or horizontal direction by following die appropriate link in die PointNct. The
SubCcllList contains a descriptor for each component subcell. A subcell descriptor has a pointer to the source
of the subcell, an assignment of symbolic expressions for any parameters of the source cell, and information
on the position and orientation of the subcell (/.e., whether it has been flipped or rotated). Subcells in a group
are linked together in a circular list. Some information in the AttributeList of the source cell, like the cell
name, is also duplicated to prevent unnecessary searching. Only the relative sizes and positions of the various cell components are important. Cells may be expanded
or shrunk, points may be moved around, and wires may be lengthened or shortened, provided that the
underlying topological structure of the circuit does not change. This structural elasticity is exploited by the
Escher system to obtain a good layout and is discussed in more detail below. For simplicity, we initially
assume that all subcells, points and wires are at the same level. We say that subcell SCL1 is Above another subcell SCL2, if SCLl.BottomY > SCL2.TopY. Similarly, we
define the Below, Rightof and Leftof relations between pairs of subcells. If two subcells are not related by
either the Above relation or the Below reladon, they are Beside one another. We say SCL1 Precedes SCL2 in
position order if and only if 4. A More Complicated Example 17 17 n/4
u p a r t ( n )
/ I n
u p a s s ( n )
An/2
p f x ( n / 2 )
/ I n / 2
d p a s s ( n )
/ l n - 2
d p a r t ( n - 2 )
/ f n - 2
A n/4
command: d e f c e l l
pfx(n)
Figure 4-2: Recursive Specification for Parallel Prefix Circuit command: d e f c e l l
pfx(n) Figure 4-2: Recursive Specification for Parallel Prefix Circuit 18 18 A
A
n / 4 - l
n/2-2
d p a s s ( n - 4 )
n-4
A n / 4 - l
Figure 4-3: Recursive Definition of Subcell DPASS(n)
Figure 4-4: Recursive Definition of Subcell UPASS(n) A
A
n / 4 - l
n/2-2
d p a s s ( n - 4 )
n-4
A n / 4 - l
Figure 4-3: Recursive Definition of Subcell DPASS(n) A
A
n / 4 - l
n/2-2
d p a s s ( n - 4 )
n-4
A n / 4 - l
Figure 4-3: Recursive Definition of Subcell DPASS(n) A
A
n / 4 - l
n/2-2
d p a s s ( n - 4 )
n-4
A n / 4 - l Figure 4-3: Recursive Definition of Subcell DPASS(n) Figure 4-4: Recursive Definition of Subcell UPASS(n) Figure 4-4: Recursive Definition of Subcell UPASS(n) Figure 4-4: Recursive Definition of Subcell UPASS(n) 19 / 'n/2
/ 'n/2
d l e f t ( n / 2 )
d r i g h t (n/2)
/ 'n/2
/ 'n/2
Figure 4-5: Recursive Definition of Subcell DPART(n) Figure 4-5: Recursive Definition of Subcell DPART(n) Figure 4-5: Recursive Definition of Subcell DPART(n) Figure 4-6: Recursive Definition of Subcell DLEFT(n) Figure 4-6: Recursive Definition of Subcell DLEFT(n) Figure 4-6: Recursive Definition of Subcell DLEFT(n) / i n - 2
d r i g h t ( n - 2 )
A n-2
op
Figure 4-7: Recursive Definition of Subcell DRIGHT(n) Figure 4-7: Recursive Definition of Subcell DRIGHT(n) 20 20 Figure 4-8: PFX(16) Figure 4-8: PFX(16) Figure 4-8: PFX(16) 21 (SCL1 AboveSCLl)
Or((SCLl BesideSCU)
And (SOLI Leftof SCL2)). In the Tally example in Figure 2-1, each of the subcells MUX[0] ... MUX[n-l] is Rightof the subcell tal(n-l)
and Above the subcell MUX[n]. Similar definitions may be given to describe the relative ordering of pins. In
this case, however, the corresponding pardal order relations only hold between pins on the sairr. side of a cell. So far, we have only defined the position order relation between circuit components at the same level. We
can extend die reladon to apply to components at different levels by requiring that if subcell SCL1 precedes
SCL2 in position order, then each subcell of SCL1 must precede each subcell of SCL2, etc. A recursive circuit specification may be unwound into a tree structure in which nodes correspond to cells, A recursive circuit specification may be unwound into a tree structure in which nodes correspond to cells, 22 and one node is a son of another if the cell corresponding to the first node is a subccll of the cell
corresponding to the second. Thus, a cell will appear at level i in the tree if it is a subccll of a cell that appears
at level i-1. A layout is generated from die tree in a bottom-up fashion in which layouts are determined for all
of the sons of a node before laying out the node itself. To accomplish this task it may be necessary to move
various circuit components in order to make room for components generated at lower levels. The algorithms
that Escher uses for this purpose are discussed in detail in the next section. To insure correctness we must
guarantee that as the program transforms a geometrical pattern, the hierarchical position order among
components remains unchanged, even though the absolute size and position of the components may change
frequently. We give below some basic rules for deciding when points and subcclls may be moved. and one node is a son of another if the cell corresponding to the first node is a subccll of the cell
corresponding to the second. Thus, a cell will appear at level i in the tree if it is a subccll of a cell that appears
at level i-1. A layout is generated from die tree in a bottom-up fashion in which layouts are determined for all
of the sons of a node before laying out the node itself. (SCL1 AboveSCLl)
Or((SCLl BesideSCU)
And (SOLI Leftof SCL2)). To accomplish this task it may be necessary to move
various circuit components in order to make room for components generated at lower levels. The algorithms
that Escher uses for this purpose are discussed in detail in the next section. To insure correctness we must
guarantee that as the program transforms a geometrical pattern, the hierarchical position order among
components remains unchanged, even though the absolute size and position of the components may change
frequently. We give below some basic rules for deciding when points and subcclls may be moved. • Subcclls of a cell may be moved provided that their relative position order remains invariant. • Subcclls of a cell may be moved provided that their relative position order remains invari • Each pin on a basic subccll has a fixed position relative to the subccll and can not be moved. • Each pin on a basic subccll has a fixed position relative to the subccll and can not be mov • Pins on some side of a composite subcell may be moved as long as they remain on the same side
and their relative order does not change. • Pins on some side of a composite subcell may be moved as long as they remain on the same side
and their relative order does not change. • Any non-pin point (Le.y a bend or connector) may be moved, provided all the points whose
positions depend on diat point are moved accordingly and the move does not violate one of the
first three ailes. 23 6. How To Unwind a Recursive Circuit Specification Wc begin by describing algorithms for expanding and shrinking cells. There arc two instances when this
may be necessary: The first occurs when die omitted subcclls of a group arc filled in. The second instance
occurs when a subccll is replaced by a copy of its source. For simplicity, our algorithms for these two cases are
only given for die vertical direction. The horizontal direction can be obtained by rotation and need not be
given here. When expanding in the vertical direction some parts of the cell must be moved upward, while
other parts must be moved downward. However, as long as die guidelines in the previous section are
followed, wc do not have to worry about changing the behavior of the circuit. Let C1(N) be a parameterized cell. Suppose that C1(V) is the cell obtained from C1(N) by replacing each
expression with its value at N = V. Suppose also that Scl[Vl], Scl[*l], Scl[V2] is a subcell group in C1(V). In
order to make room for all of subcclls represented by Scl[*I], we must expand (or shrink) C1(V) as shown in
Figure 6-1. O r i
S C L [ v t ]
S C L [ v 2 ]
b e f o r e
e x p a n s i o n
a f t e r
e x p a n s i o n
Figure 6-1: Expanding a Group b e f o r e
e x p a n s i o n a f t e r
e x p a n s i o n Figure 6-1: Expanding a Group Figure 6-1: Expanding a Group 24 Vdist is the vertical distance that must be allocated for each subccll in the group, including the space
between consecutive subcclls. K is the total number of subcclls in die group; if K is greater than 3, wc must
enlarge die space allocated to the group by (K-3) * Vdist units in the vertical direction. Kach object in the cell
will be moved either up or down according to whether it is above or below the vertical midpoint of the region
occupied by die subccll group. (K-2) new subcclls must be created to fill out die group. Points and wires will
be added so diat each of these subcclls has die same set of attachments as Scl[*l], procedure ExpandingGroup; [(
)
]
;
Vdist
:=
(Sc1[Vl] TopY
-
Scl[V2] TopY)
div
2; Vdist
:= (Sc1[Vl].TopY - Scl[V2].TopY) div 2;
i f
K>3 then
Voff
:= [(K-3)*Vdist]
div
2 else
Voff
:=
0; i f K>3 then Voff
:= [(K 3)
Vdist] div 2 else Voff
OrigY := (Scl[VI].TopY + Scl[V2].BottomY) div 2; Record a l l wires and points associated with S c l [ * I ] ,
then delete Record a l l wires and points associated with S c l [ * I ] ,
then delete Record a l l wires and points associated with S c l [ * I ]
a l l of them along with the subcell for
S c l [ * I ] ; a l l of them along with the subcell for
S c l [ * I ] ; a l l of them along with the subcell for
S c l [ * I ] ; i f Voff>0 then
move the part of C1(V) that is above OrigY up by Voff;
move the part of C1(V) that is below OrigY down by Voff;
endif; i f Voff>0 then
move the part of C1(V) that is above OrigY up by Voff;
move the part of C1(V) that is below OrigY down by Voff;
endif; create (K-2) new subcells that are copies of S c l [ * I ] ; align the
subcells
in the space allocated for the group, making sure that
the top of successive subcells are separated by Vdist units
in
the vertical
direction; Connect up the points and wires associated with the
individual
subcells so that each is a copy Scl[*I] in the original
diagram; endproc; endproc; Figure 6-3: Expanding A Subcell procedure
ExpandingSubcell; Voff
:= (CU.Height - SCI.Height) div 2; Voff
:= (CU.Height - SCI.Height) div 2; i f Voff<0 then
for each pin P on S c l ' s North side. P.PosY := P.PosY-Voff;
for each pin P on S c l ' s South side. P.PosY := P.PosY+Voff;
endif; i f Voff<0 then
for each pin P on S c l ' s North side. P.PosY := P.PosY-Voff;
for each pin P on S c l ' s South side. P.PosY := P.PosY+Voff;
endif; i f Voff>0 then CI.TopY
:= CI.TopY - Voff; . Cl.BottomY
Cl.BottomY + Voff; i f P.PosY>=Scl.BottomY
then P.PosY :» P.PosY + Voff;
endfor Figure 6-2: Algorithm for Expanding a Group During the unwinding phase we replace each recursive subcell by an instance of its source cell. For
example, when unwinding the TALLY circuit in Figure 2-1 with N = 6 we must first replace the recursive
subcell TALLY(5) by a copy of the source cell. When we unwind TALLY(5) we need to replace subcell
TALLY(4) by another copy of die source cell. This process continues until a base case is reached. When we replace a subcell with the body of its source cell, it may be nccessary*to expand (or shrink) the
subcell so that it is the same size as its source. Suppose that CI is a cell, that Scl is one of its subcells, and that
Cll is the source of Scl. Let Voff be half the difference in size in the vertical direction between Scl and its
source Cll. When Voff < 0, Scl must be bigger than Cll, so Scl should be shrunk. If Voff > 0, then Scl is
smaller than Cll, so Scl should be expanded. When the expansion is made, every object in CI that is above Scl
in position order must be moved up by Voff. Likewise, every object in CI that is below Scl must be moved
down by Voff. The positions of objects that are beside Scl do not need to be changed. See Figure 6-3. 25 N
I
I
1
1
'.I
SCL
1
r
.1
s
.1
b e f o r e
e x p a n s i o n
a f t e r
e x p a n s i o n b e f o r e
e x p a n s i o n a f t e r
e x p a n s i o n a f t e r
e x p a n s i o n for each subcell
S e l l , i f
(Sell.TopY<=Scl.TopY)
and (Scl1.BottomY>=Scl.BottomY)
then
Cll.TopY
:» Sell.TopY
- Voff;
Scll.BottomY
:= Scl1.BottomY + Voff;
else
i f Scll.TopY<=Scl.TopY
then
Sell.TopY
:= Sell.TopY - Voff;
Scll.BottomY
:= Scll.BottomY - Voff;
endif;
i f Scll.BottomY>=Scl.BottomY
then
Sell.TopY
:= Sell.TopY + Voff;
Scll.BottomY
:= Scll.BottomY + Voff;
endif;
endif;
endfor;
endif;
endproc;
Figure 6-4: Algorithm for Expanding a Subcell i f
(Sell.TopY<=Scl.TopY)
and (Scl1.BottomY>=Scl.BottomY)
then Y
:» Sell.TopY
- Voff; Scll.BottomY
:= Scl1.BottomY + Voff; i f Scll.TopY<=Scl.TopY
then Sell.TopY
:= Sell.TopY - Voff; Scll.BottomY
:= Scll.BottomY - Voff;
endif; i f Scll.BottomY>=Scl.BottomY
then Sell.TopY
:= Sell.TopY + Voff; Scll.BottomY
:= Scll.BottomY + Voff;
endif; Figure 6-4: Algorithm for Expanding a Subcell 26 Once Scl and its source cell Cll have the same size, wc must connect up the pins of Scl. Although
corresponding sides of Cll and Scl will have exactly the same number of pins, corresponding pins on the same
side will, in general, have different offsets with respect to die center of die side. Let P be a pin on Scl, and let
PI be the corresponding pin on Cll. Wc assume without loss of generality that both pins are on the South
side of their respective cells. Let P2 be diat point on the South side of Scl that has the same position with
respect to the center of the side that PI has with respect to the center of die corresponding side of CM. In
general, P and P2 will not coincide and it will be necessary to introduce a jog (P,P2) in order to connect them. Frequently, diis type of jog can be eliminated by moving some pins or subcclls. This problem is addressed by
the next algoridim. In order to explain how the algoridim works we consider two cases as illustrated in Figure
6-5. Wc say that a point U is rigidly connected to pin V if U is connected to V by a path consisting of
horizontal and vertical wire segments. c a s e
1
c a s e
2
Figure 6-5: Eliminating Jogs (Two Cases). Figure 6-5: Eliminating Jogs (Two Cases). In the first case, all of the points rigidly connected with P (/.&, points Q and R in Figure 6-5) are movable,
so we move all of them right by distance D so that P and P2 coincide. for each subcell
S e l l , let SclOut be the set of a l l
points r i g i d l y connected to S e l l ;
i f every point in SclOut is movable,
then
for each P3 in SclOut,
P3.PosX := P3.PosX+D;
Scll.RightX
:= Scll.RightX + D;
S c l l . L e f t X
;= S c l l . L e f t X + D;
endif;
endfor;
endif;
endfor
endproc;
Figure 6-6: Algoridim for Eliminating Jogs. procedure
EliminateJogs; add
new point
P2 at
(NewX NewY); let SclOut be the set of a l l
points r i g i d l y connected to S e l l ; S c l l . L e f t X
;= S c l l . L e f t X + D; Figure 6-6: Algoridim for Eliminating Jogs. for each subcell
S e l l , We must be careful not to change the
position order among pins on the South side of Scl when we move R. In the second case, it appears that some
point Q, rigidly connected widi P, is not movable, because it is a pin on the boundary of another subcell Sell*
However, subccll Sell and all of the points rigidly connected with its pins are movable. Thus, we can move
Sc
1! and all of the points rigidly connected to it right by distance D so that P coincides with P2. 27 procedure
EliminateJogs;
for every pin P on the South side of S c l ,
find the corresponding pin PI on C l l ;
let C,C1 be the centers for Scl and C l l ;
NewX := C.PosX + (Pl.PosX - Cl.PosX);
NewY := P.PosY;
add new point P2 at (NewX.NewY);
D
NewX - P.PosX;
let Pout be the set of a l l points r i g i d l y
connected to P;
i f every point in Pout is movable,
then
for each P3 in Pout. P3.PosX := P3.PosX+D
el se
for every subcell S e l l connected to P. let SclOut be the set of a l l
points r i g i d l y connected to S e l l ;
i f every point in SclOut is movable,
then
for each P3 in SclOut,
P3.PosX := P3.PosX+D;
Scll.RightX
:= Scll.RightX + D;
S c l l . L e f t X
;= S c l l . L e f t X + D;
endif;
endfor;
endif;
endfor
endproc;
Figure 6-6: Algoridim for Eliminating Jogs. procedure
EliminateJogs;
for every pin P on the South side of S c l ,
find the corresponding pin PI on C l l ;
let C,C1 be the centers for Scl and C l l ;
NewX := C.PosX + (Pl.PosX - Cl.PosX);
NewY := P.PosY;
add new point P2 at (NewX.NewY);
D
NewX - P.PosX;
let Pout be the set of a l l points r i g i d l y
connected to P;
i f every point in Pout is movable,
then
for each P3 in Pout. P3.PosX := P3.PosX+D
el se
for every subcell S e l l connected to P. Figure 6-6: Algoridim for Eliminating Jogs. Finally, we show how to put the previous algorithms together and actually do the unwinding. Our major
concern at this point in the algorithm is efficiency. When we unwind a recursive Escher specification, we
obtain a tree in which the nodes represent subcells and a directed arc exists between two nodes when the head
is a subcell of the tail. We must be careful not to duplicate steps if we encounter the same cell more dian once
when we traverse the tree. For example, when we instantiate SORT with n = 4, we obtain the tree structure
shown in Figure 6-7. S 0 R T ( 4 )
MERGE(2)
S 0 R T ( 2 ) )
S 0 R T ( 2 )
CMP
CMP
M E R G E ( l )
M E R G E ( l )
CMP
CMP
P A S S ( 2 )
P A S S ( 2 )
CMP
CMP
Figure 6-7: Traversal Tree for SORT(4) Figure 6-7: Traversal Tree for SORT(4) 28 In this case there arc several duplicates among the 8 terminal nodes and 4 non-terminal nodes. In order to
unwind SORT(4), wc have to unwind SORT(2) twice and MERGE(2) once; when wc unwind MERGE(2),
wc must unwind PASS(2) twice, MERGE(l) twice, and CMP twice. In fact, if diis representation is used, it is
possible to create examples in which the number of duplicated steps will be exponential in the size of the
original Escher specification. Instead, Escher uses a directed acyclic graph structure to represent the nesting of subcells. We call diis data
structure the Subcell Nesting Graph orSNG. Since each subcell corresponds to at most one node in die SNG,
it is only necessary to unwind a given subcell once. The graph for SOR 1(4) is shown in Figure 6-8. Note that
each of the subcells SORT(4), SORT(2), CMP, MERGE(2), MERGE(l), and PASS(2) is represented
uniquely this time. S 0 R T ( 4 )
S 0 R T ( 2 )
P A S S ( 2 )
CMP
Figure 6-8: Directed Acyclic Graph for SORT(4) Figure 6-8: Directed Acyclic Graph for SORT(4) The unwinding algorithm consists of two phases. In the first phase we evaluate all of those expressions that
depend on the parameters of the cell and create the SNG. Figure 6-6: Algoridim for Eliminating Jogs. Expressions may appear in the specifications of
groups and buses, and they may be used as parameters of lower level subcells. After we have figured out the
exact number of subcells in a subcell group, we use the algorithm in Figure 6-2 to obtain enough space for the
omitted subcells in the group; then we copy the subcells into the cell. After a cell has been evaluated it will be
linked to its source cell in the SNG. The SNG for cell CL(V) will not be complete undl all of its descendant
subcells have been processed in this manner. The second phase in the unwinding process is a depth first traversal of the SNG. When all of the subcells of
a cell in the SNG have been unwound, we replace each subcell with its source body and mark the cell as
unwound. The algorithm in Figure 6-4 is used to obtain enough space for filling in the subcell bodies. The
algorithm in Figure 6-6 is used for eliminadng jogs in wires that result from these substitutions. 29 procedure Eval(CI,OrigCI, N, V);
name N;
if C1(V) is already in the SNG
then return
e l s e i f CI is a basic cell
then add a Cl-node into SNG
el se
evaluate a l l of the expressions
in C1(N), replacing N by its
value V;
expand a l l
subcell
groups using the algorithm in Figure 6-2;
replace each bus by a number of wires equal to the bus width;
for each subcell S e l l do
let the source of S e l l be CU(V1);
E v a l ( C U , C l ,N,V1);
endfor;
add a Cl(v)-node
into SNG;
endif;
set link from OrigCl
to C1(V);
endproc; procedure Eval(CI,OrigCI, N, V); procedure Eval(CI,OrigCI, N, V); if C1(V) is already in the SNG
then return e l s e i f CI is a basic cell
then add a Cl-node into SNG
el se evaluate a l l of the expressions
in C1(N), replacing N by its
value V; for each subcell S e l l do let the source of S e l l be CU(V1); E v a l ( C U , C l ,N,V1); endfor; add a Cl(v)-node
into SNG; endif; set link from OrigCl
to C1(V); endproc; endproc; Figure 6-9: Algorithm for constructing Subcell Nesting Graph. 7. Conclusion and Directions for Future Research We believe diat ultimately recursion will play much the same role in hardware design that it has in software
design. Although recursion has always been an indispcnsiblc tool for theoretical investigations in algoridim
design, only in the last few years has it become respectable to write application programs that use recursive
procedures. The acceptance of recursion is a result of two factors: First, many software designers have come to
realize diat it natural to express certain algorithms recursively- particularly those that access recursive data
structures. Secondly, advances in computer architecture, like hardware stacks and displays, have decreased die
overhead associated with recursive procedure calls. We believe that an analogous process will occur in
hardware design. When design environments routinely provide support for recursion, designers will begin to
find elegant recursive solutions for problems that they currently must solve in an awkward manner using
iteration alone. Since recursive hardware designs are implemented by unwinding die recursion, the overhead
in efficiency that is associated widi the use of recursion in software will not be a problem. We further believe
that our use of parameterized subcell and group specifications will be of practical importance in any
completely general graphical design system, even if ftill recursion is not supported. Finally, we list below some of the problems witii the current system that we hope to address in a future
version: Finally, we list below some of the problems witii the current system that we hope to address in a futur
version: • Multiple parameters. As currently implemented, the Escher system only permits cell
specifications widi a single recursive parameter. A number of interesting examples can be
specified most naturally by using multiple recursive parameters. It should be fairly easy to modify
die current implementation so diat multiple parameters are permitted. • Compaction and optimization. The layouts produced by our system frequently contain long wires
and have area that grows more rapidly with the recursion depdi than necessary. Although we
have implemented some simple compaction algorithms, we believe that this problem requires
much more thought. It may be possible to design compaction algorithms diat take advantage of
the hierarchical structure of Escher specifications. However, the simple algoridims that have
already been implemented do not make use of diis information. • Combined textual and geometric description. For certain applications like simulation a textual
circuit description may be quite useful. procedure Unwind(Cl(v)); procedure Unwind(Cl(v)); for each descendant C U ( v l )
of Cl(v) do
i f C U ( v l )
is not unwound then Unwind(Cl 1 ( v l ) ) ;
endfor; for each descendant C U ( v l )
of Cl(v) do for each subcell Scl of C l ( v ) ,
expand Scl to be the same size as i t s
source;
map the pins of C l l onto Scl and minimize the number of jogs
using
the procedure described in Figure 6-6; expand Scl to be the same size as i t s
source;
map the pins of C l l onto Scl and minimize the number of jogs
using map the pins of C l l onto Scl and minimize the number of jogs
using p
p
the procedure described in Figure 6-6; copy C U ( v l )
into S c l ; endfor; mark Cl(v) as unwound; endproc; Figure 6-10: Algorithm for Unwinding Recursive Cell Specifications). 30 Finally, some simple compaction algorithms arc used to shorten wires and move subcclls closer together. It
should be noted that these algorithms may violate the position order relation among subcells that is described
in section 5. For example, the compaction algorithms were used to obtain Figure 4-8. The position order
among subccll OFs is not the same as diat given in the original specification of the parallel prefix circuit. 31 verification, etc. A change in the geometrical description would be automatically reflected by a
corresponding change in the HDL representation. The dual representation would provide access
to die best features of both types of design systems. verification, etc. A change in the geometrical description would be automatically reflected by a
corresponding change in the HDL representation. The dual representation would provide access
to die best features of both types of design systems. verification, etc. A change in the geometrical description would be automatically reflected by a
corresponding change in the HDL representation. The dual representation would provide access
to die best features of both types of design systems. 7. Conclusion and Directions for Future Research We envision a VLSI design system with multiple windows
which would permit both textual and geometric descriptions of circuit components. One window
would contain a geometrical representation of the circuit like the one described in this paper. Another window would contain a representation of the circuit in an appropriate (textual)
hardware description language. The textual description could be used directly for simulation, REFERENCES 1. M. Fischer and R. Ladner. "Paralllel prefix computation". Journal of the ACM 27,4 (1980). 2. S.M.Gcrman, K.J.Licbcrhcrr. "Zeus: a language for expressing algorithms in hardware". Computers
(1985). 3. J.Oustcrhout. "Caesar: An interactive layout editor for VLSI design". VLSI design (Fourth Quarter
1981), 34-38. 4. L Johnson, U. Weiser et tal. Towards a formal treatment of VLSI arrays. Caltech conference on VLSI,
January, 1981, pp. 375-398. 5. D.E.Knuth. The art of computer programming. Volume: Sorting and searching. Addison-Wesley, 1973. 6. R.J.Lipton, S.C.Nordi, R.Sedgewick et tal. ALI: a procedural language to describe VLSI layouts. 19th
design automation conference, IEEE, 1982, pp. 467-474. 7. W.K.Luk, J.E.Vuillemin. Recursive implementation of optimal dme VLSI integer multipliers. VLSI
design of digital systems, ed. F.Anceau & E.J.Aas, 1983, pp. 155-168. 8. CA..Mcad, L.A.Conway. Introduction to VLSI systems. Addison-Wesley, 1980. 9. Mary Sheeran. muFP-- An algebraic VLSI design language. PRG-39, Oxford University Computing Lab.,
November, 1984. 10. P.Henderson. Functional geometry. Symposium on LISP and functional programming, ACM, 1982, pp. 179-187. 11. C.D.Thompson. "Fourier transforms in VLSI". IEEE transaction on computers c32,11 (1983),
1047-1057.
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https://scholarlypublications.universiteitleiden.nl/access/item%3A2944204/view
|
English
| null |
The tumor suppressor LKB1 regulates starvation-induced autophagy under systemic metabolic stress
|
Scientific reports
| 2,017
|
cc-by
| 10,886
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The tumor suppressor LKB1
regulates starvation-induced
autophagy under systemic
metabolic stress Received: 22 November 2016
Accepted: 21 June 2017
Published: xx xx xxxx Laurie A. Mans1, Laia Querol Cano1,3, Jason van Pelt1, Panagiota Giardoglou1,
Willem-Jan Keune2 & Anna-Pavlina G. Haramis 1 Autophagy is an evolutionarily conserved process that degrades cellular components to restore energy
homeostasis under limited nutrient conditions. How this starvation-induced autophagy is regulated
at the whole-body level is not fully understood. Here, we show that the tumor suppressor Lkb1, which
activates the key energy sensor AMPK, also regulates starvation-induced autophagy at the organismal
level. Lkb1-deficient zebrafish larvae fail to activate autophagy in response to nutrient restriction upon
yolk termination, shown by reduced levels of the autophagy-activating proteins Atg5, Lc3-II and Becn1,
and aberrant accumulation of the cargo receptor and autophagy substrate p62. We demonstrate that
the autophagy defect in lkb1 mutants can be partially rescued by inhibiting mTOR signaling but not
by inhibiting the PI3K pathway. Interestingly, mTOR-independent activation of autophagy restores
degradation of the aberrantly accumulated p62 in lkb1 mutants and prolongs their survival. Our data
uncover a novel critical role for Lkb1 in regulating starvation-induced autophagy at the organismal
level, providing mechanistic insight into metabolic adaptation during development. Autophagy is a highly conserved, multi-step intracellular process of self-degradation. Under basal conditions,
autophagy eliminates damaged proteins and organelles from cells, serving a housekeeping/recycling function. However, upon metabolic stress, starvation–induced autophagy serves to provide substrates for biosynthesis and
energy production in order to maintain cellular homeostasis1, 2. The importance of autophagy for cellular and
organismal health is showcased by the fact that defects in autophagy have been linked to neurodegeneration,
cancer, aging and metabolic syndrome1. Starvation-induced autophagy promotes survival in the Drosophila fat body3 and in Caenorhabditis elegans4. A critical role for autophagy in surviving the metabolic stress at birth has also been demonstrated in mammals:
mice deficient in Atg5 (autophagy protein 5, an E3 ubiquitin ligase necessary for autophagosomal elongation)
survive foetal development, but die within one day after birth, exhibiting severe hypoglycaemia and hypolipidae-
mia2. However, the regulation of systemic starvation-induced autophagy is not well understood. Autophagy-induction in response to energetic stress is triggered by the activation of AMPK-activated protein
kinase (AMPK)5, 6, a key, evolutionarily conserved energy sensor. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 22 November 2016
Accepted: 21 June 2017
Published: xx xx xxxx The tumor suppressor LKB1
regulates starvation-induced
autophagy under systemic
metabolic stress AMPK activation restores energy homeostasis
at the cellular and organismal levels7 by many different pathways, including via inhibition of the mechanistic tar-
get of rapamycin (mTOR)8, a conserved serine-threonine kinase involved in nutrient sensing, growth and prolif-
eration9, 10. AMPK itself is activated by multiple mechanisms including phosphorylation by the tumor suppressor
LKB1/STK11 kinase in response to increased AMP or ADP levels in the cell11, 12. Because of the role of AMPK as a
central energy checkpoint in the cell, these findings link LKB1 signaling to energy metabolism control, position-
ing LKB1 as a critical mediator of the effects of low energy on cell viability11, 13. Accordingly, cells lacking LKB1
undergo apoptosis under metabolic stress as they are unable to respond to energy deficiency and restore home-
ostasis11. LKB1/AMPK signaling is also important for long-term survival under nutrient-limiting conditions 1Institute of Biology, Leiden University, Sylviusweg 72, 2333 BE, Leiden, The Netherlands. 2Department of
Biochemistry, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands. 3Present
address: Radboud Institute for Molecular Life Sciences - Tumour immunology department, Geert Grooteplein
28, 6525 GA, Nijmegen, The Netherlands. Laurie A. Mans and Laia Querol Cano contributed equally to this work. Correspondence and requests for materials should be addressed to A.-P.G.H. (email: a.haramis@biology.leidenuniv.nl) Scientific Reports | 7: 7327 | DOI:10.1038/s41598-017-07116-9 1 www.nature.com/scientificreports/ during C. elegans dauer (diapause) stage14. However, the early embryonic lethality of both Lkb1 mutant15 and
Ampka1/a2 double mutant mice16 has precluded analysis of the in vivo role of LKB1/AMPK in physiological pro-
cesses occurring at later developmental stages in vertebrates, such as during metabolic stress at birth.h during C. elegans dauer (diapause) stage14. However, the early embryonic lethality of both Lkb1 mutant15 and
Ampka1/a2 double mutant mice16 has precluded analysis of the in vivo role of LKB1/AMPK in physiological pro-
cesses occurring at later developmental stages in vertebrates, such as during metabolic stress at birth.h g
p
g
g
The LKB1/AMPK axis is a negative regulator of mTOR signaling8 and mTOR signaling is a known inhibitor of
autophagy17. However, LKB1 also activates 12 other AMPK-related kinases18, and many mTOR-dependent and
mTOR-independent autophagy regulators exist19.f p
p
gy
g
LKB1/AMPK regulation of mTOR has been linked to the regulation of autophagy in different settings, for
example in autophagy stimulated by fluid flow over the primary cilium of epithelial cells20, and in cancer cells21. Furthermore, AMPK directly stimulates autophagy via the ULK1/Atg1 phosphorylation22, 23. The tumor suppressor LKB1
regulates starvation-induced
autophagy under systemic
metabolic stress And LKB1 may also
stimulate autophagy by stabilizing p27, thereby linking nutrient sensing to cell-cycle progression13. However,
whether and how LKB1 signaling regulates systemic starvation-induced autophagy in vertebrates is currently
unknown. The regulation of systemic metabolism and autophagy are often studied in zebrafish because of its small
size and vertebrate physiology24, 25. Importantly, fundamental principles of energy homeostasis are highly con-
served between humans and zebrafish26, 27. Autophagy has critical functions during zebrafish embryonic devel-
opment28, with autophagy-defective animals displaying abnormal heart development29, which is also seen in
mice30. Zebrafish are also a valuable model for studying tissue regeneration, and autophagy has been shown to
be required for the regeneration of amputated caudal fins31. Like mammals, zebrafish also experience metabolic
stress at birth, when the maternal nutrient supply (yolk) is depleted. The metabolic stress at birth in mammals is
accompanied by induction of gluconeogenesis32, 33 and of autophagy2, 34. While induction of gluconeogenesis also
serves as a mechanism to restore energy homeostasis in zebrafish35, a role for autophagy during this metabolic
transition has not been investigated.i g
We previously used TILLING36 to generate zebrafish mutants that carried a point mutation leading to a stop
codon in the kinase domain of Lkb1 (stk11hu1968). These mutants survived gastrulation and early embryonic devel-
opment but died prematurely from starvation at 7 to 8 days post-fertilization (dpf). Our experiments, impossible
to conduct in mice due to the early embryonic lethality of Lkb1 knock-out mice15, established Lkb1 as a critical
regulator of whole-body energy homeostasis37. Zebrafish lkb1 (stk11hu1968) mutants are unable to cope with the
energetic stress induced upon yolk depletion and fail to adapt their metabolism to lower nutrient levels. lkb1
mutants are indistinguishable from wild type (wt) siblings while their maternal nutrient supply is still present,
until 5–6 dpf. However, they die within 1–2 days following yolk depletion whereas wt larvae can survive without
food until 13–14 dpf38.hi p
The zebrafish lkb1 mutant phenotype is reminiscent of the Atg5 knock-out mice that appear normal until birth
but die soon after, due to their inability to cope with the metabolic stress at birth2. This resemblance prompted
us to investigate the autophagy status in the lkb1 mutants to study the role of Lkb1 in regulation of systemic
starvation-induced autophagy.i p
gy
We show that Lkb1-deficient larvae fail to activate autophagy in response to nutrient restriction. Results
lkb1
t lkb1 mutants fail to activate autophagy under nutrient limitation. To determine if autophagy ini-
tiation and maintenance is affected in the lkb1 mutants we previously generated37, we analyzed wt and lkb1 larvae
between 5–7 dpf during the metabolic transition following yolk depletion. lkb1 mutants are indistinguishable
from wt larvae up to day 5–6 dpf37 (while there is still yolk). We chose this time window also because the mor-
phological lkb1 phenotype of flattened intestine and darkened liver is apparent at 7 dpf37 and the majority of lkb1
mutants die at 8 dpf (Supplementary Fig. S1). Autophagic activity is commonly monitored by accumulation of
the membrane-bound form of MAP1LC3B (microtubule-associated proteins 1 A/1B light chain 3B, Atg8 in yeast;
Lc3B in zebrafish), Lc3-II, which is a ubiquitin-like protein39 that localizes in autophagosomal membranes upon
induction of autophagy. To enable visualization of Lc3-II accumulation in autophagosomes, we first blocked the
fusion of autophagosomes with lysosomes by treating the larvae with 2.5 μM chloroquine40 for 14 hours (h) before
analysis.t y
We found that Lc3-II protein levels were lower in lkb1 mutant larvae compared to their wt siblings after yolk
depletion at 6 and 7 dpf (Fig. 1A). Note that the Lc3 antibody in zebrafish recognizes predominantly the cleaved
Lc3B-II form, which still accurately reflects autophagic activity41 (Supplementary Fig. S2A), and we were only
able to detect a faint signal for Lc3B-I in zebrafish lysates (Fig. 1A). To verify this result, we used an alternative
marker of autophagy by analyzing the expression of Atg5-containing protein complexes during development. We
found that while the expression of the common ~56 KD complex was unaffected, the ~47 KD complex, which is
indicative of autophagy induction31 was undetectable in the lkb1 mutants (Fig. 1A). Finally, we also assessed the
levels of Beclin 1 (Becn1), a protein involved in autophagosome nucleation30, 42 and also commonly used as an
autophagy indicator. Becn1 expression was also strongly reduced in the lkb1 mutants (Supplementary Fig. S2B). While Lc3-II and Becn1 levels progressively increased in wt larvae between 5–7 dpf, indicating upregulation of
autophagy upon yolk termination, lkb1 mutants did not show such an increase, suggesting they fail to activate
autophagy under nutrient limiting conditions (Fig. 1A, and Supplementary Fig. S2B).l To examine the spatial distribution of autophagy, we performed immunofluorescence analysis with antibod-
ies against Lc3B on transverse liver and intestine sections of lkb1 and wt larvae at 7 dpf. The tumor suppressor LKB1
regulates starvation-induced
autophagy under systemic
metabolic stress Furthermore,
we demonstrate aberrant accumulation of the autophagy adaptor and substrate p62 in lkb1 mutants, confirming
impaired autophagy. Genetic or chemical induction of autophagy in lkb1 mutants prolongs their survival, while
suppression of autophagy shortens it. Survival prolongation only occurs when degradation of p62 is restored. We
therefore show that autophagy is essential to survive the feeding-fasting transition in zebrafish, and identify Lkb1
as a critical regulator of whole-body starvation-induced autophagy in vertebrates. Scientific Reports | 7: 7327 | DOI:10.1038/s41598-017-07116-9 Results
lkb1
t Lc3B expression was
strongly reduced in lkb1 intestines and livers as compared to their wt counterparts (Fig. 1B–E and Supplementary Scientific Reports | 7: 7327 | DOI:10.1038/s41598-017-07116-9 2 www.nature.com/scientificreports/ Figure 1. lkb1 mutant larvae show impaired activation of autophagy following yolk depletion. (A) Representative
Western blot analysis of Lc3-II, Atg5, p62 and Tubulin (loading control) in total protein lysates of wt and lkb1 trunks
between 5–7 dpf. Larvae were treated with chloroquine (2.5 μM) for 14 h prior to processing. The marked decrease
in Lc3-II and Atg5-containing complexes together with the p62 accumulation indicate impaired autophagy in
lkb1 larvae. Uncropped images of the blots are shown in Supplementary Fig. S7A–C. (B–E) Transverse vibratome
sections (150 μm) of intestine of 7 dpf wt and lkb1 mutants stained with anti-LC3B antibody (green), rhodamine-
phalloidin to detect F-actin (red) and DAPI to detect nuclei (blue). Lc3B staining in the lkb1 intestine is barely
detectable (C,E) and more foci of intense staining were visible in wt sections compared to sections from lkb1
mutants. (F,G) Immunohistochemical analysis of transverse paraffin sections (5 μm) of liver and intestine of 7
dpf wt and lkb1 larvae reveals high levels of p62 accumulation in lkb1 liver and intestine. Magnification: 40×. PD:
pronephric ducts; L: liver; SI: intestine. Figure 1. lkb1 mutant larvae show impaired activation of autophagy following yolk depletion. (A) Representative
Western blot analysis of Lc3-II, Atg5, p62 and Tubulin (loading control) in total protein lysates of wt and lkb1 trunks
between 5–7 dpf. Larvae were treated with chloroquine (2.5 μM) for 14 h prior to processing. The marked decrease
in Lc3-II and Atg5-containing complexes together with the p62 accumulation indicate impaired autophagy in
lkb1 larvae. Uncropped images of the blots are shown in Supplementary Fig. S7A–C. (B–E) Transverse vibratome
sections (150 μm) of intestine of 7 dpf wt and lkb1 mutants stained with anti-LC3B antibody (green), rhodamine-
phalloidin to detect F-actin (red) and DAPI to detect nuclei (blue). Lc3B staining in the lkb1 intestine is barely
detectable (C,E) and more foci of intense staining were visible in wt sections compared to sections from lkb1
mutants. (F,G) Immunohistochemical analysis of transverse paraffin sections (5 μm) of liver and intestine of 7
dpf wt and lkb1 larvae reveals high levels of p62 accumulation in lkb1 liver and intestine. Magnification: 40×. PD:
pronephric ducts; L: liver; SI: intestine. Fig. S2E,F), confirming and supporting the immunoblotting results. Results
lkb1
t Furthermore, immunohistochemistry (IHC)
against Becn1 on transverse sections of wt and lkb1 mutants showed markedly reduced Becn1 staining in the lkb1
mutants compared to their wt counterparts (Supplementary Fig. S2C,D).i To further confirm that activation of autophagy is impaired in lkb1 mutants, we monitored expression levels
of the p62 protein (also known as sequestosome 1, (SQSTM1). p62 is an adaptor protein that targets ubiquiti-
nated proteins or organelles that bind to it for selective autophagy43. Accumulation of p62 has been observed in
mouse AMPK-deficient fibroblasts44, and is associated with liver toxicity in autophagy-deficient mouse liver45. p62 itself is also an autophagy substrate, thus accumulation of p62 levels is a marker for impaired autophagy46. In
agreement with our model that lkb1 mutants have impaired autophagy, Western blot analysis of p62 levels at 5, 6
and 7 dpf showed progressive accumulation of p62 specifically in the lkb1 mutants (Fig. 1A). IHC performed on
transverse sections of lkb1 intestine and liver confirmed a marked accumulation of p62 in lkb1 larvae, whereas wt
siblings were devoid of staining (Fig. 1F,G).i g
g
g
Collectively, these findings demonstrate that whole-body autophagy is impaired in lkb1 mutants during the
eeding-fasting transition in zebrafish, which could contribute to their premature death. Abrogation of autophagy further decreases survival of lkb1 mutants. To investigate the effect
of inhibiting autophagy on lkb1 larvae survival prior to yolk depletion, we blocked autophagosome formation
using an antisense morpholino oligonucleotide (MO), that targets the translational start-site of atg5 mRNA28, 47,
atg5MO. We confirmed that injection of atg5MO abolishes Atg5 protein expression (Supplementary Fig. S3). Atg5-knockdown led to a reduction in Lc3-II levels compared to the negative control in both wt and lkb1 mutants
at 4 dpf, before yolk depletion, confirming autophagy suppression upon atg5MO injection (Fig. 2A). While all
un-injected wt and lkb1 larvae were alive at 4 dpf, a significant number of atg5MO-injected embryos were found
dead at that time point. Genotyping all larvae at 4 dpf revealed that 75% of atg5MO-injected lkb1 larvae had died
compared to only 25% of atg5MO-injected wt larvae (Fig. 2B). Thus, lkb1 mutants, which fail to induce autophagy
at the metabolic transition, are also more sensitive to autophagy inhibition at earlier embryonic stages. The autophagy defect in lkb1 mutants can be ameliorated by mTOR-dependent and
-independent mechanisms. We next investigated the mechanism behind the impaired autophagy
observed in the lkb1 mutants. Results
lkb1
t g
p
y
g (
p
)
nor restore p62 degradation in lkb1 mutants. (A) Representative Western blot analysis of Ribosomal protein S6
(RS6), Phospho-RS6 and Tubulin (loading control) in total protein lysates of wt and lkb1 trunks at 6 dpf that
were treated with either 10 μM rapamycin from 24 hpf onwards, or with DMSO (negative control). Increased
levels of RS6 and P-RS6 are observed in rapamycin-treated wt samples. Total RS6 levels did not change in
lkb1 samples but P-RS6 decreased upon rapamycin treatment. (B) Representative Western blot analysis of
p62, Lc3-II, and histone H3 (loading control). Larvae were treated with chloroquine (2.5 μM) for 14 h prior
to processing. Rapamycin treatment leads to increased Lc3-II levels in both wt and lkb1 larvae, while p62
accumulation remained high in rapamycin-treated lkb1 mutants. (C) Representative Western blot analysis of
Atg5 and Tubulin (loading control). Rapamycin treatment leads to an increase in the amount of the ~47 KD
Atg5-containing complex in wt larvae and to a lesser extent in lkb1 mutants. Uncropped images of the blots are
shown in Supplementary Fig. S9A–C. birth enables activation of autophagy49. We hypothesized that mTOR inactivation was defective in the absence
of Lkb1. Therefore, we first assessed the status of mTOR signaling in lkb1 mutants at the metabolic transition (6
dpf) by analyzing phosphorylation of the mTOR-substrate ribosomal protein S6 (RS6) by Western blot. Total RS6
levels were almost undetectable in wt larvae at this stage, consistent with our previous report37. However, both
total and phospho- RS6 levels were high in lkb1 mutants (Fig. 3A), indicating active mTOR signaling, which was
inhibited by rapamycin treatment. In comparison, in rapamycin-treated 6 dpf wt larvae, we observed increased
phospho-RS6 expression (Fig. 3A). This could be explained by a known developmental delay caused by chronic
mTOR inhibition during development50. Consistent with this, rapamycin-treated wt larvae retained significant
amounts of yolk at 7 dpf, demonstrating a delay in larval development (Supplementary Fig. S4C). y
g
y
y
g
To determine whether mTOR signaling mediates the inhibition of autophagy seen in the lkb1 mutants at
6 dpf, we examined whether rapamycin treatment could restore autophagy in these mutants. We treated wt
and lkb1 embryos with rapamycin from 1 dpf onwards. We have previously reported that rapamycin-treated
lkb1 larvae survive until 9 dpf, but still have a considerable amount of yolk, demonstrating a developmental
delay37. Results
lkb1
t Signaling through mTOR is known to inhibit autophagy, and we and others have
previously reported that mTOR activity is high in wt larvae between 2 and 5 dpf and is downregulated at later
stages of larval development37, 47, 48. This suggests that at the time of yolk depletion, mTOR activity is switched
off, enabling the activation of autophagy. It has also been shown in mice that suppression of mTOR activity at Scientific Reports | 7: 7327 | DOI:10.1038/s41598-017-07116-9 3 www.nature.com/scientificreports/ Figure 2. Inhibition of autophagy shortens survival of lkb1 larvae. (A) Representative Western blot analysis of
Lc3-II and Actin (loading control) in total protein lysates of trunks of surviving wt and lkb1 larvae at 4 dpf that
were injected with an atg5MO at the one-cell stage, and controls. Larvae were previously treated with 2.5 μM
chloroquine for 14 h. Atg5 knock-down led to downregulation of Lc3-II levels in both wt and lkb1 mutants. Uncropped images of the blots are shown in Supplementary Fig. S8. (B) Graph depicting mortality percentages
of atg5MO-injected wt and lkb1 larvae at 4 dpf. Data represent the means ± standard errors of the means (SEM)
and are pooled from two independent experiments (n = 80/experiment). *P value < 0.05. Figure 2. Inhibition of autophagy shortens survival of lkb1 larvae. (A) Representative Western blot analysis of
Lc3-II and Actin (loading control) in total protein lysates of trunks of surviving wt and lkb1 larvae at 4 dpf that
were injected with an atg5MO at the one-cell stage, and controls. Larvae were previously treated with 2.5 μM
chloroquine for 14 h. Atg5 knock-down led to downregulation of Lc3-II levels in both wt and lkb1 mutants. Uncropped images of the blots are shown in Supplementary Fig. S8. (B) Graph depicting mortality percentages
of atg5MO-injected wt and lkb1 larvae at 4 dpf. Data represent the means ± standard errors of the means (SEM)
and are pooled from two independent experiments (n = 80/experiment). *P value < 0.05. hloroquine for 14 h. Atg5 knock-down led to downregulation of Lc3-II levels in both wt and lkb1 mutants. Uncropped images of the blots are shown in Supplementary Fig. S8. (B) Graph depicting mortality percentages
f atg5MO-injected wt and lkb1 larvae at 4 dpf. Data represent the means ± standard errors of the means (SEM)
nd are pooled from two independent experiments (n = 80/experiment). *P value < 0.05. Figure 3. Results
lkb1
t Rapamycin treatment leads to increased Lc3-II accumulation but does not increase Atg5 (complexes)
nor restore p62 degradation in lkb1 mutants. (A) Representative Western blot analysis of Ribosomal protein S6
(RS6), Phospho-RS6 and Tubulin (loading control) in total protein lysates of wt and lkb1 trunks at 6 dpf that
were treated with either 10 μM rapamycin from 24 hpf onwards, or with DMSO (negative control). Increased
levels of RS6 and P-RS6 are observed in rapamycin-treated wt samples. Total RS6 levels did not change in
lkb1 samples but P-RS6 decreased upon rapamycin treatment. (B) Representative Western blot analysis of
p62, Lc3-II, and histone H3 (loading control). Larvae were treated with chloroquine (2.5 μM) for 14 h prior
to processing. Rapamycin treatment leads to increased Lc3-II levels in both wt and lkb1 larvae, while p62
accumulation remained high in rapamycin-treated lkb1 mutants. (C) Representative Western blot analysis of
Atg5 and Tubulin (loading control). Rapamycin treatment leads to an increase in the amount of the ~47 KD
Atg5-containing complex in wt larvae and to a lesser extent in lkb1 mutants. Uncropped images of the blots are
shown in Supplementary Fig. S9A–C. igure 3. Rapamycin treatment leads to increased Lc3-II accumulation but does not increase Atg5 (complexes) Figure 3. Rapamycin treatment leads to increased Lc3-II accumulation but does not increase Atg5 (complexes)
nor restore p62 degradation in lkb1 mutants. (A) Representative Western blot analysis of Ribosomal protein S6
(RS6), Phospho-RS6 and Tubulin (loading control) in total protein lysates of wt and lkb1 trunks at 6 dpf that
were treated with either 10 μM rapamycin from 24 hpf onwards, or with DMSO (negative control). Increased
levels of RS6 and P-RS6 are observed in rapamycin-treated wt samples. Total RS6 levels did not change in
lkb1 samples but P-RS6 decreased upon rapamycin treatment. (B) Representative Western blot analysis of
p62, Lc3-II, and histone H3 (loading control). Larvae were treated with chloroquine (2.5 μM) for 14 h prior
to processing. Rapamycin treatment leads to increased Lc3-II levels in both wt and lkb1 larvae, while p62
accumulation remained high in rapamycin-treated lkb1 mutants. (C) Representative Western blot analysis of
Atg5 and Tubulin (loading control). Rapamycin treatment leads to an increase in the amount of the ~47 KD
Atg5-containing complex in wt larvae and to a lesser extent in lkb1 mutants. Uncropped images of the blots are
shown in Supplementary Fig. S9A–C. Results
lkb1
t Embryos were treated with 1 μM AR-12 or DMSO from 1 dpf, collected
at 9 dpf, and genotyped for the lkb1 gene. Data represent the means ± standard errors of the means (SEM) and
are pooled from three independent experiments P value > 0.05, ns: not statistically significant. We next analyzed the pro-survival PI3K pathway, which in response to external stimuli (growth factors, insu-
lin) also suppresses autophagy, acting upstream of mTOR signaling52. To this end, we used the small molecule
AR-12, an inhibitor of phosphoinositide-dependent kinase (PDK)-1, a component of the PI3K pathway53, which
has been shown to activate autophagy in zebrafish54. Treatment of wt and lkb1 siblings with AR-12 from 1 dpf
onwards, led to accumulation of Lc3-II protein levels in wt but not in lkb1 larvae at 6 dpf (Fig. 4A). Interestingly,
AR-12 treatment resulted in upregulation of Atg5 expression and formation of Atg5/12 complexes in wt lar-
vae indicating autophagy induction, but no changes in Atg5 expression were observed in lkb1 larvae (Fig. 4B). Furthermore, while p62 protein expression was diminished in wt larvae upon AR-12-mediated activation of
autophagy, accumulation of p62 remained unchanged in AR-12-treated lkb1 larvae (Fig. 4A). This indicates that
inhibition of the PI3K pathway fails to induce autophagy in mutant larvae. We next assessed phosphorylation
of the mTOR-substrate RS6 upon AR-12 treatment in wt and lkb1 larvae at 6 dpf. RS6 and phosphorylated RS6
(P-RS6) were not detectable in wt larvae at 6 dpf (Fig. 4C), consistent with downregulation of mTOR activity at
later developmental stages (here and refs 37, 47 and 48). AR-12 treatment did not affect the high protein levels
of RS6 or P-RS6 seen in the lkb1 mutants, indicating that they are unsusceptible to PI3K pathway inhibition. In line with the lack of autophagy induction, AR-12 treatment did not enhance lkb1 survival, as no statistically
significant differences were observed in the percentage of AR-12-treated lkb1 larvae alive at 9 dpf compared to
DMSO-treated controls (Fig. 4D). Activation of autophagy by an mTOR-independent pathway can be achieved using calpeptin, which inhibits
the autophagy inhibitors calpain proteases55. Calpeptin treatment of lkb1 and wt embryos from 1 dpf onwards, in
the presence or absence of chloroquine, enhanced Lc3-II levels in both wt and lkb1 larvae at 6 dpf (Fig. 5A and
Supplementary Fig. S5) without any effects on development or yolk absorption. Results
lkb1
t (A) Representative Western blot analysis of Lc3-II, p62 and Histone H3 and Tubulin
(loading controls) in total protein lysates of wt and lkb1 trunks at 6 dpf. Larvae were treated with 1 μM AR-12
or DMSO (negative control) from 1 dpf, and with 2.5 μM chloroquine for 14 h prior to processing. AR-12
treatment leads to upregulation of Lc3-II levels in wt larvae but not in lkb1 mutants. p62 levels remain high in
AR-12-treated lkb1 larvae. (B) AR-12 treatment leads to strong increase in the amounts of Atg5 and complexed
Atg5 in wt larvae but not in lkb1 mutants. Uncropped images of the blots are shown in Supplementary
Fig. S10A,B. (C) Representative Western blot analysis of Ribosomal protein S6 (RS6), Phospho-RS6 and
Tubulin (loading control). Protein levels of total RS6 and of P-RS6 were not affected by AR-12 treatment in lkb1
mutants. Uncropped images of the blots are shown in Supplementary Fig. S9A–C. (D) Graph depicting survival
percentage of lkb1 larvae alive at 9 dpf. Embryos were treated with 1 μM AR-12 or DMSO from 1 dpf, collected
at 9 dpf, and genotyped for the lkb1 gene. Data represent the means ± standard errors of the means (SEM) and
are pooled from three independent experiments P value > 0.05, ns: not statistically significant. Figure 4. The PI3K-inhibitor AR-12 does not rescue the autophagy defect in lkb1 mutants and does not
prolong their survival. (A) Representative Western blot analysis of Lc3-II, p62 and Histone H3 and Tubulin
(loading controls) in total protein lysates of wt and lkb1 trunks at 6 dpf. Larvae were treated with 1 μM AR-12
or DMSO (negative control) from 1 dpf, and with 2.5 μM chloroquine for 14 h prior to processing. AR-12
treatment leads to upregulation of Lc3-II levels in wt larvae but not in lkb1 mutants. p62 levels remain high in
AR-12-treated lkb1 larvae. (B) AR-12 treatment leads to strong increase in the amounts of Atg5 and complexed
Atg5 in wt larvae but not in lkb1 mutants. Uncropped images of the blots are shown in Supplementary
Fig. S10A,B. (C) Representative Western blot analysis of Ribosomal protein S6 (RS6), Phospho-RS6 and
Tubulin (loading control). Protein levels of total RS6 and of P-RS6 were not affected by AR-12 treatment in lkb1
mutants. Uncropped images of the blots are shown in Supplementary Fig. S9A–C. (D) Graph depicting survival
percentage of lkb1 larvae alive at 9 dpf. Results
lkb1
t Rapamycin-treatment resulted in elevated Lc3-II levels in both wt and lkb1 larvae, at 6 dpf (Fig. 3B),
indicating that autophagy in the lkb1 mutants is inhibited, at least in part, by mTOR signaling. The same was
also observed when blocking autophagosome-lysosome fusion by chloroquine, which prevents degradation
of autophagosome-associated Lc3, allowing monitoring of the autophagic flux51 (Supplementary Fig. S4A,B). However, rapamycin-treatment led to only slight upregulation of Atg5 and complexed Atg5 in lkb1 mutants
(Fig. 3C), and was not sufficient to decrease the marked p62 accumulation (Fig. 3B). These results suggest that
while mTOR inhibition can at least partially restore autophagy in lkb1 mutant larvae, it cannot entirely alleviate
the observed phenotype. Scientific Reports | 7: 7327 | DOI:10.1038/s41598-017-07116-9 4 www.nature.com/scientificreports/ Figure 4. The PI3K-inhibitor AR-12 does not rescue the autophagy defect in lkb1 mutants and does not
prolong their survival. (A) Representative Western blot analysis of Lc3-II, p62 and Histone H3 and Tubulin
(loading controls) in total protein lysates of wt and lkb1 trunks at 6 dpf. Larvae were treated with 1 μM AR-12
or DMSO (negative control) from 1 dpf, and with 2.5 μM chloroquine for 14 h prior to processing. AR-12
treatment leads to upregulation of Lc3-II levels in wt larvae but not in lkb1 mutants. p62 levels remain high in
AR-12-treated lkb1 larvae. (B) AR-12 treatment leads to strong increase in the amounts of Atg5 and complexed
Atg5 in wt larvae but not in lkb1 mutants. Uncropped images of the blots are shown in Supplementary
Fig. S10A,B. (C) Representative Western blot analysis of Ribosomal protein S6 (RS6), Phospho-RS6 and
Tubulin (loading control). Protein levels of total RS6 and of P-RS6 were not affected by AR-12 treatment in lkb1
mutants. Uncropped images of the blots are shown in Supplementary Fig. S9A–C. (D) Graph depicting survival
percentage of lkb1 larvae alive at 9 dpf. Embryos were treated with 1 μM AR-12 or DMSO from 1 dpf, collected
at 9 dpf, and genotyped for the lkb1 gene. Data represent the means ± standard errors of the means (SEM) and
are pooled from three independent experiments P value > 0.05, ns: not statistically significant. Figure 4. The PI3K-inhibitor AR-12 does not rescue the autophagy defect in lkb1 mutants and does not Figure 4. The PI3K-inhibitor AR-12 does not rescue the autophagy defect in lkb1 mutants and does not
prolong their survival. Results
lkb1
t Calpeptin treatment does not affect RS6 or P-RS6 levels in wt or
lkb1 mutants. Uncropped images of the blots are shown in Supplementary Fig. S11A–C. (D) Graph depicting
survival percentage of lkb1 mutants alive at 9 dpf. Embryos were treated with 50 μM calpeptin or DMSO from 1
dpf, collected at 9 dpf, and genotyped for the lkb1 gene. 17,5% out of a total 25% (70% of calpeptin-treated lkb1
larvae) are alive at 9 dpf. Only 1% of DMSO-treated lkb1 larvae are alive at 9 dpf. Data depicted in (D) represen
the means ± standard errors of the means (SEM) and are pooled from three independent experiments (n = 100
experiment). **P value < 0.05. Accumulation of p62 is an important regulator of autophagy in lkb1 mutants. Aberrant p62
accumulation appeared as a hallmark of impaired autophagy in lkb1 mutants, and strongly correlated with sur-
vival. While p62 is primarily thought of as a receptor delivering cargo proteins to autophagosomes for degrada-
tion, it has also been implicated in enhancing mTOR activity56, thereby regulating autophagy as well. Loss of p62
function led to increased autophagy in mammalian cells and in C. elegans56. We thus set out to determine whether
reducing p62 levels in larvae would affect autophagy and survival. To this end, we injected a sqstm1/p62 MO, tar-
geting splicing of sqstm1/p62 mRNA54, into 1–2-cell stage embryos. RT-PCR confirmed that the sqstm1/p62 MO
blocked sqstm1/p62 mRNA splicing until at least 5 dpf (Supplementary Fig. S6). Western blot analysis of 6 dpf
larvae showed decreased p62 expression compared to un-injected controls in both wt and lkb1 lysates (Fig. 6A). This was coupled with increased Lc3-II protein levels, suggestive of autophagy induction. Knockdown of p62
significantly prolonged lkb1 survival up to 9 dpf: Approximately 70% of sqstm1/p62 MO-injected lkb1 mutants
survived to 9 dpf, whereas less than 5% of un-injected lkb1 larvae were alive at this time-point (Fig. 6B). Thus,
depleting p62 is sufficient to activate impaired autophagy in lkb1 mutants and extend survival. Results
lkb1
t (A) Representative Figure 5. Calpeptin treatment induces autophagy and prolongs survival of lkb1 larvae. (A) Representative
Western blot analysis of Lc3-II, p62 and Histone H3 (loading control) in total protein lysates of wt and lkb1
trunks at 6 dpf. The embryos were treated with 50 μM calpeptin or DMSO (negative control) from 1 dpf
onwards, and with 2.5 μM chloroquine for 14 h prior to lysing. Calpeptin treatment leads to upregulation
of Lc3-II levels in both wt and lkb1 larvae. Induction of autophagy by calpeptin also leads to robust
downregulation of p62 accumulation in lkb1 larvae. (B) Representative Western blot analysis of Atg5 and
Tubulin (loading control). Calpeptin treatment leads stark increase in the amounts of Atg5 and of complexed
Atg5 in both wt and lkb1 larvae (C) Representative Western blot analysis of Ribosomal protein S6 (RS6),
Phospho-RS6 and Tubulin (loading control). Calpeptin treatment does not affect RS6 or P-RS6 levels in wt or
lkb1 mutants. Uncropped images of the blots are shown in Supplementary Fig. S11A–C. (D) Graph depicting
survival percentage of lkb1 mutants alive at 9 dpf. Embryos were treated with 50 μM calpeptin or DMSO from 1
dpf, collected at 9 dpf, and genotyped for the lkb1 gene. 17,5% out of a total 25% (70% of calpeptin-treated lkb1
larvae) are alive at 9 dpf. Only 1% of DMSO-treated lkb1 larvae are alive at 9 dpf. Data depicted in (D) represent
the means ± standard errors of the means (SEM) and are pooled from three independent experiments (n = 100/
experiment). **P value < 0.05. Figure 5. Calpeptin treatment induces autophagy and prolongs survival of lkb1 larvae. (A) Representative
Western blot analysis of Lc3-II, p62 and Histone H3 (loading control) in total protein lysates of wt and lkb1
trunks at 6 dpf. The embryos were treated with 50 μM calpeptin or DMSO (negative control) from 1 dpf
onwards, and with 2.5 μM chloroquine for 14 h prior to lysing. Calpeptin treatment leads to upregulation
of Lc3-II levels in both wt and lkb1 larvae. Induction of autophagy by calpeptin also leads to robust
downregulation of p62 accumulation in lkb1 larvae. (B) Representative Western blot analysis of Atg5 and
Tubulin (loading control). Calpeptin treatment leads stark increase in the amounts of Atg5 and of complexed
Atg5 in both wt and lkb1 larvae (C) Representative Western blot analysis of Ribosomal protein S6 (RS6),
Phospho-RS6 and Tubulin (loading control). Results
lkb1
t Calpeptin treatment also resulted
in upregulation of the amounts of Atg5 and complexed-Atg5 in both wt and lkb1 mutants (Fig. 5B). In contrast
to rapamycin treatment, treatment with calpeptin restored p62 degradation in lkb1 mutants (Fig. 5A). Calpeptin
treatment had no effect on RS6 phosphorylation in lkb1 mutants (Fig. 5C), consistent with calpeptin being an
mTOR-independent autophagy activator55. Moreover, calpeptin-mediated activation of autophagy prolonged
survival in 70% of the treated lkb1 larvae. Specifically, 17.5% of calpeptin-treated lkb1 larvae survived until 9
dpf, whereas only 1% of vehicle-treated lkb1 larvae were alive at this point (Fig. 5D). Therefore, we conclude that
induction of mTOR-independent autophagy results in a more complete restoration of the lkb1 mutant phenotypes
compared to that obtained upon inhibition of mTOR signaling. Scientific Reports | 7: 7327 | DOI:10.1038/s41598-017-07116-9 5 www.nature.com/scientificreports/ Figure 5. Calpeptin treatment induces autophagy and prolongs survival of lkb1 larvae. (A) Representative
Western blot analysis of Lc3-II, p62 and Histone H3 (loading control) in total protein lysates of wt and lkb1
trunks at 6 dpf. The embryos were treated with 50 μM calpeptin or DMSO (negative control) from 1 dpf
onwards, and with 2.5 μM chloroquine for 14 h prior to lysing. Calpeptin treatment leads to upregulation
of Lc3-II levels in both wt and lkb1 larvae. Induction of autophagy by calpeptin also leads to robust
downregulation of p62 accumulation in lkb1 larvae. (B) Representative Western blot analysis of Atg5 and
Tubulin (loading control). Calpeptin treatment leads stark increase in the amounts of Atg5 and of complexed
Atg5 in both wt and lkb1 larvae (C) Representative Western blot analysis of Ribosomal protein S6 (RS6),
Phospho-RS6 and Tubulin (loading control). Calpeptin treatment does not affect RS6 or P-RS6 levels in wt or
lkb1 mutants. Uncropped images of the blots are shown in Supplementary Fig. S11A–C. (D) Graph depicting
survival percentage of lkb1 mutants alive at 9 dpf. Embryos were treated with 50 μM calpeptin or DMSO from 1
dpf, collected at 9 dpf, and genotyped for the lkb1 gene. 17,5% out of a total 25% (70% of calpeptin-treated lkb1
larvae) are alive at 9 dpf. Only 1% of DMSO-treated lkb1 larvae are alive at 9 dpf. Data depicted in (D) represent
the means ± standard errors of the means (SEM) and are pooled from three independent experiments (n = 100/
experiment). **P value < 0.05. Figure 5. Calpeptin treatment induces autophagy and prolongs survival of lkb1 larvae. Discussion p62 knock-down leads to increased Lc3-II levels in both wt and lkb1 larvae. p62
expression in lkb1 larvae is reduced upon p62 knock-down. Uncropped images of the blots are shown in
Supplementary Fig. S12. (B) Graph depicting survival percentage of lkb1 larvae alive at 9 dpf. Embryos were
injected with 0,5 mM sqstm1MO at the one cell stage, collected at 9 dpf, and genotyped for the lkb1 gene. Data
represent the means ± standard errors of the means (SEM) and are pooled from three independent experiments
***P value ≤ 0.0001. Figure 6. p62 knock-down extends lkb1 mutants’ survival. (A) Representative Western blot analysis of p62, occurred even while the yolk is not yet consumed, suggesting that even though the larvae do not show a mor-
phological phenotype at this embryonic stage, the loss of Lkb1 appears to sensitize them to additional stress. This
stress may be specifically autophagy inhibition, or related to alternative mechanisms, as autophagy-independent
functions have been reported for several of the autophagy-related genes57, 58, including Atg559. p
p
gy
g
g
g
Various mechanisms, including mTOR and PI3K signaling, as well as calpains, are known to regulate auto-
phagy19, and likely interact at multiple levels. Indeed, our results, together with published work, indicate that all
these influence the energy-sensing defect we observe in lkb1 mutants. We show that activating autophagy by cal-
peptin, which inhibits the action of the general autophagy inhibitors calpains55, led to robust upregulation of Atg5
expression and restored degradation of p62 in lkb1 mutants. Thus, calpeptin fully rescued the autophagy defect of
the lkb1 larvae and prolonged their survival. In contrast, while the mTOR-inhibitor rapamycin increased Lc3-II
accumulation in lkb1 larvae, autophagy was not completely restored since p62 still accumulated. This may be due
to the high mTOR activity in the mutants that could not be fully blocked by rapamycin treatment under these
experimental conditions. In addition, although rapamycin treatment also prolonged lkb1 survival, we believe this
was likely due to a generalized growth delay, evidenced by the presence of a considerable amount of yolk at 7 dpf
(this study and refs 37 and 50), rather than due to partial restoration of autophagy. Discussion A developmental delay caused
by rapamycin is further supported by the persistence of RS6 expression in rapamycin-treated wt larvae at 7 dpf
when mTOR would normally be suppressed (mTOR signaling is suppressed in wt larvae upon yolk depletion at
5–6 dpf37, 47, 48).hi p
)
The autophagy receptor and substrate p62 aberrantly accumulates in lkb1 mutants indicating deficient auto-
phagy. p62 is also a regulator of autophagy, as it participates in a feed-forward loop in which p62 enhances mTOR
activity resulting in reduced autophagy, in turn leading to higher p62 levels in mice60. Here we also show that
depletion of p62 in lkb1 larvae leads to activation of autophagy and prolonged survival. This implies that as the
amount of p62 decreases due to autophagosomal clearance, its effect on mTOR activity is also reduced, and thus
autophagy can be maintained. Furthermore, the aberrant accumulation of p62 in lkb1 larvae may in itself con-
tribute to their premature lethality, as it has been shown that increased levels of p62 in autophagy-deficient mouse
livers cause hepatotoxicity (reviewed in ref. 60). Further supporting our hypothesis, in apoptosis-impaired tumor
cells with deficient autophagy, p62 accumulation triggers a positive feedback loop for the generation of reactive
oxygen species (ROS) leading to enhanced genomic instability and tumorigenesis61. yg
g
g
y
g
PI3K signaling is a nutrient-sensing pathway that is also implicated in starvation-induced autophagy. Inhibition of the PI3K pathway activated autophagy in wt larvae, but not in lkb1 mutants, and did not prolong
their survival. This is consistent with our previous findings that PI3K signaling is compromised in lkb1 mutants37. We postulate that defective PI3K signaling may contribute to the autophagy defect seen in these mutants. While
AMPK is considered a major regulator of metabolism and has an important role in induction of autophagy under
energetic stress23, 44, it is not overtly activated in wt larvae at 7 dpf37; in agreement with these data, studies in
mice have also reported that 24 hours of fasting did not lead to significant AMPK activation62, 63. Thus, the auto-
phagy defect we describe in lkb1 mutants is unlikely to be solely attributable to impaired AMPK signaling, and
deregulation of additional pathways, such as PI3K signaling and AMPK/mTOR-independent pathways may also
be involved. Discussion Organisms adapt their metabolism in response to nutrient limitation to restore energy homeostasis and ensure
survival. Here, we identify a novel link between metabolic adaptation during development and induction and
maintenance of autophagy, mediated by the tumor suppressor Lkb1. Specifically, we use metabolically compro-
mised Lkb1-deficient zebrafish larvae to show that Lkb1 is crucial in the induction of autophagy in response to
the metabolic challenge accompanying depletion of the maternal nutrient supply. Our data therefore reveal an
essential function for Lkb1 in controlling starvation-induced autophagy at the organismal level in vertebrates. Overall autophagy levels in lkb1 mutants are lower compared to those of wt siblings: while expression of auto-
phagy–related proteins is progressively upregulated following yolk depletion in wt larvae, induction of autophagy
in lkb1 mutants is strongly attenuated. Importantly, we demonstrate that genetic and chemical manipulation
of autophagy levels significantly impacts lkb1 larvae survival: inducing autophagy by mTOR-dependent and –
independent mechanisms prolongs survival, and suppressing autophagy by Atg5 depletion leads to premature
death selectively of the mutants. The increased susceptibility of lkb1 larvae to Atg5 depletion during development Scientific Reports | 7: 7327 | DOI:10.1038/s41598-017-07116-9 6 www.nature.com/scientificreports/ Figure 6. p62 knock-down extends lkb1 mutants’ survival. (A) Representative Western blot analysis of p62,
Lc3-II, and histone H3 (loading control) in total protein lysates of wt and lkb1 trunks at 6 dpf that were either
injected with an sqstm1MO at the one-cell stage or controls. The larvae were treated with 2.5 μM chloroquine
for 14 h prior to processing. p62 knock-down leads to increased Lc3-II levels in both wt and lkb1 larvae. p62
expression in lkb1 larvae is reduced upon p62 knock-down. Uncropped images of the blots are shown in
Supplementary Fig. S12. (B) Graph depicting survival percentage of lkb1 larvae alive at 9 dpf. Embryos were
injected with 0,5 mM sqstm1MO at the one cell stage, collected at 9 dpf, and genotyped for the lkb1 gene. Data
represent the means ± standard errors of the means (SEM) and are pooled from three independent experiments. ***P value ≤ 0.0001. Figure 6. p62 knock-down extends lkb1 mutants’ survival. (A) Representative Western blot analysis of p62,
Lc3-II, and histone H3 (loading control) in total protein lysates of wt and lkb1 trunks at 6 dpf that were either
injected with an sqstm1MO at the one-cell stage or controls. The larvae were treated with 2.5 μM chloroquine
for 14 h prior to processing. Materials and Methods
b
fi h
i
d Zebrafish strains and Screening Methods. Zebrafish were handled in compliance with the local animal
welfare regulations and were maintained according to standard protocols (zfin.org). Their culture was approved
by the local animal welfare committee (DEC) of the University of Leiden and all protocols adhered to the inter-
national guidelines specified by the EU Animal Protection Directive 2010/63/EU. Genotype analysis for lkb1
mutants embryos was performed as previously described37. Longitudinal analysis of survival of lkb1 mutants. Larvae obtained from single matings of heterozy-
gous lkb1 adults were analyzed over time. 48–95 larvae were genotyped on 6, 7, 8, 9, 10 and 11 dpf to assess the
numbers of lkb1 mutants alive. Western Blot analysis. Approximately 20 larvae/sample were lysed (3 μl per larva) in cold lysis
buffer (50 mM Hepes, pH 7.6, 50 mM KCl, 50 mM NaF, 5 mM NaPPi, 1 mM EGTA, 1 mM EDTA, 1 mM
beta-Glycerophosphate, 1 mM DTT, 1 mM Vanadate, 1% NP40) containing phosphatase and proteinase inhibi-
tors. Lysates were pestled for 5 min, sonicated for 30 seconds at 30 seconds intervals for 5 min and centrifuged at
13.000 rpm for 15 min at 4 °C to pellet nuclei and cell debris. Protein lysates were boiled for 10 min and BCA assay
was performed to measure protein concentration. Samples containing 12–30 μg of protein were heated at 95 °C
for 5 min with 4× Bolt LDS sample buffer (Thermofisher, #B0007), supplemented with 5% beta-mercaptoethanol,
and loaded onto a 12% Bis-Tris plus gel (Thermofisher, #NW00122). The protein marker used was Precision
Plus ProteinTM Dual Color Standards, #1610374 (BioRad). Proteins were transferred onto a nitrocellulose mem-
brane (Thermofisher, #88018) using a wet transfer system (Bio-Rad) according to manufacturer’s instructions. Subsequent blocking and antibody incubation were performed in 5% skimmed milk powder (#115363, Merck
Millipore) in PBS containing 0.1% Tween-20. For the anti-p62 antibody, blocking was performed in 10% milk
powder and antibody incubation in 1% milk powder in PBS containing 0.1% Tween-20. Antibodies used were:
rabbit anti-LC3B (1:1000, Abcam, #ab51520), rabbit anti-p62 (1:1000, MBL, #PM045), rabbit anti-BECN1
(1:500, Santa Cruz, #sc-11427), rabbit anti-H3 (1:5000, Santa Cruz, #sc-10809), mouse anti-beta-actin (1:5000,
Sigma, #A5441), mouse anti-Tubulin (1:500, Sigma, #T9026), rabbit anti-Atg5 (1:500, Novus, #NB110-53818). Secondary antibodies used were goat Anti-Mouse IgG (H + L)-HRP (1:10.000, BioRad, #1721011) and Goat
Anti-Rabbit IgG (H + L)-HRP Conjugate (1:10.000, BioRad, #17210191). Materials and Methods
b
fi h
i
d Membranes were developed using ECL
(BioRad, #1705060), followed by chemiluminescence detection with a gel doc system (BioRad). Immunohistochemistry and Immunofluorescence. For transverse sections, larvae were fixed in 40%
ethanol, 5% acetic acid and 10% formalin for 3 h at room temperature followed by three washes in 70% ethanol
before being dehydrated following serial washes in Histoclear and reducing ethanol concentrations. Larvae were
then sectioned at 5 μm intervals using a Reichert-Jung 2050 microtome (Leica). Sections were deparaffinized and
hydrated following by 20 min of antigen retrieval in sodium citrate buffer pH 6.0 at 100 °C. Sections were blocked
in 5% BSA in PBS − 0.1% Tween-20 for 1 h at room temperature and incubated overnight with sheep anti-p62
(1:200, Abcam, #ab31545) and rabbit anti-BECN1 (1:150, Santa Cruz, #sc-11427). Endogenous peroxidase activ-
ity was blocked in 0.3% H2O2 for 20 min at room temperature followed by incubation with rabbit anti-sheep anti-
body (1:800, Abcam, #ab6747) for 1 h at room temperature. Sections were incubated with 0.1 M imidazole prior
to detection with 3,3′-diaminobenzidine (DAB) substrate and counterstaining with hematoxylin.li For immunofluorescence, larvae were fixed in 4% PFA overnight at 4 °C, embedded vertically in a 0.5% gela-
tin/30% albumin mixture and sectioned at 120 μm intervals using a VT1000S vibratome (Leica). Sections were
transferred to the wells of a 24-well plate containing PBD (PBS + 0.1% Tween-20 and 0.5% Triton-X-100), which
was then replaced with blocking solution (PBD + 1% BSA) for 1 h at RT. Sections were incubated with rabbit
anti-LC3B (1:1000, Abcam, #ab51520) in blocking solution overnight at 4 °C. Sections were washed three times
for 15 min in PBS-0.1% Tween-20 and incubated with secondary anti-rabbit 488 green fluorescent antibody
(1:100, Thermofisher, #A11008) for 2 h at room temperature. Sections were then washed three times for 15 min in
PBS-0.1% Tween-20 prior to be incubated with phalloidin-Alexa 588 (1:25, Thermofisher, #A12380) and DAPI
(1:200, Thermofisher, #62248) for 30 min at room temperature in the dark and rinsed three times for 5 min with
dH2O. Sections were then imaged using the Zeiss LSM5 Exciter confocal laser-scanning microscope. Equipment and settings. For immunohistochemistry, the sections were imaged on an upright compound
Nikon Eclipse E800 microscope. The images were captured using a Nikon Digital Sight camera unit, equipped
with a DS-Fi1 digital camera head and a DS-L2 camera controller. Discussion Hence, nutrient-sensing pathways (like the PI3K pathway) and energy-sensing pathways (like the
AMPK pathway) are likely in close cross-talk with each other, not only through their convergence on mTOR sig-
naling but also through different, mTOR-independent mechanisms. g
gf
p
Together, our data indicate that Lkb1 plays an important role in the regulation of autophagy at the
whole-organism level, and confirm that autophagy is critical for survival during the metabolic transition in Scientific Reports | 7: 7327 | DOI:10.1038/s41598-017-07116-9 7 www.nature.com/scientificreports/ development. Since defects in autophagy are implicated in a plethora of diseases, a better understanding of the
upstream regulatory pathways could provide new insights into their pathophysiology. development. Since defects in autophagy are implicated in a plethora of diseases, a better understanding of the
upstream regulatory pathways could provide new insights into their pathophysiology. Materials and Methods
b
fi h
i
d Pixel dimensions of the acquired images were
W2584 X H1936 pixels, at 150 pixels/inch.hii p
p
The magnification used was either 40×/0,75 magnification for anti-p62 staining (Fig. 1) or 100×/1,4 magni-
fication for anti-Becn1 staining (Supplementary Fig. S2). Th i
d
i
Ph
h
CS6
ft
Th
i i
l i
l d d
i i
The images were processed using Photoshop CS6 software. The original images were scaled-down constrain-
ing proportions, and cropped to the area of interest. Adjustment of Image Levels was applied on whole images. Assembly of the composite figures and labeling was done on Illustrator CC2015. i
Confocal images were obtained in a sequential manner using a Zeiss LSM5 Exciter Confocal Laser Scanning
Microscope equipped with Argon (458, 488, 514 nm), and 405, 450 and 635 diode excitation lasers and a 40×
water immersion objective (C-APOCHROMAT 40×/1.2 Water). Emission ranges were set at 420–480, 505–550
and 560–615 nm in separate channels to prevent bleeding. Images were obtained using the Leica application X
software (Leica, Wetzlar, Germany) and post-acquisition data analysis was performed using ImageJ software. Scientific Reports | 7: 7327 | DOI:10.1038/s41598-017-07116-9 8 www.nature.com/scientificreports/ Morpholino injections. Translation-blocking morpholino (MO) directed against atg5
(CATCCTTGTCATCTGCCATTATCAT) was obtained from Gene-Tools. The splice-blocking MO
against Sqstm1/p62 (CTTCATCTAGAGACAAAGTTCAGGA) was a kind gift from Prof. AM Meijer. Splice efficiency of sqstm1 mRNA was tested in RT-PCR using a specific primer-set (Forward primer: 5′
ATTTGCAGCGAAAAGTGCTC 3′; Reverse primer: ;
p
5′ AGTGAACGGAAACCCAGGAA 3′). Embryos were injected at the 1–2-cell stage with either 2 ng (atg5)
r 4 ng (Sqstm1/p62) of MO. Drug treatments. Wild type or lkb1 mutant zebrafish embryos were treated from 1 dpf in embryo-medium
at 28 °C with either of the following treatments: 50 μM calpeptin (Abcam, #4ab120804), 1 μM AR-12 (Medkoo
Biosciences, #200272), or 10 μM rapamycin (Sigma, #R0395). Stock solutions of AR-12, rapamycin and calpeptin
were prepared in DMSO and diluted in embryo medium for treatment (final concentration of DMSO, 0.2%). Other treatments were prepared in embryo medium. All treatments were refreshed every 2–3 days, larvae col-
lected at the specified time points and genotyped for the lkb1 gene. For Western Blotting, embryos were exposed
to 2,5 μM chloroquine (Sigma, #C6628) for 14 h prior to lysing. Statistics and quantification. Statistical significance was determined using Fisher’s exact test in GraphPad
software. Materials and Methods
b
fi h
i
d Error bars represent the means ± standard errors of the means (SEM) and are pooled from a minimum
of two independent experiments. A p-value of <0.05 was used to define statistical significance. References 1. Rabinowitz, J. D. & White, E. Autophagy and metabolism. Science 330, 1344–1348 (2010).h gy
2. Kuma, A. et al. The role of autophagy during the early neonatal starvation period. Nature 432, 1032–1036 (2004). 3. Scott, R. C., Schuldiner, O. & Neufeld, T. P. Role and regulation of starvation-induced autophagy in the Drosophila fat body. Developmental cell 7, 167–178 (2004). p
4. Kang, C., You, Y. J. & Avery, L. Dual roles of autophagy in the survival of Caenorhabditis elegans during starvation. Genes Dev 21
2161–2171 (2007). (
)
5. Fleming, A., Noda, T., Yoshimori, T. & Rubinsztein, D. C. Chemical modulators of autophagy as biological probes and potentia
therapeutics. Nature chemical biology 7, 9–17 (2011). p
gy
. Galluzzi, L., Pietrocola, F., Levine, B. & Kroemer, G. Metabolic co 6. Galluzzi, L., Pietrocola, F., Levine, B. & Kroemer, G. Metabolic control of autophagy. Cell 159, 1263–1276 (2014). p
gy
(
)
7. Hardie, D. G., Ross, F. A. & Hawley, S. A. AMPK: a nutrient and energy sensor that maintains energy homeostasis. Nature reviews
Molecular cell biology 13, 251–262 (2012).h gy
(
)
8. Shaw, R. J. et al. The LKB1 tumor suppressor negatively regulates mTOR signaling. Cancer Cell 6, 91–99 (2004). gy
8. Shaw, R. J. et al. The LKB1 tumor suppressor negatively regul ressor negatively regulates mTOR signaling. Cancer Cell 6, 91–99 ( h
pp
g
y
g
g
g
9. Laplante, M. & Sabatini, D. M. mTOR signaling in growth control and disease. Cell 149, 274–293 (2012). g
g
g
10. Polak, P. & Hall, M. N. mTOR and the control of whole body metabolism. Current opinion in cell biology 21, 209–218 (2009).h 11. Shaw, R. J. et al. The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in respon
energy stress. Proc Natl Acad Sci USA 101, 3329–3335 (2004). 12. Hawley, S. A. et al. Complexes between the LKB1 tumor suppressor, STRAD alpha/beta and MO25 alpha/beta are upstream kinases
in the AMP-activated protein kinase cascade. Journal of biology 2, 28 (2003).h p
f
gy
13. Liang, J. et al. The energy sensing LKB1-AMPK pathway regulates p27(kip1) phosphorylation mediating the decision to
autophagy or apoptosis. Nature cell biology 9, 218–224 (2007). h
autophagy or apoptosis. Nature cell biology 9, 218–224 (2007). gy
gy
14. Narbonne, P. & Roy, R. Caenorhabditis elegans dauers need LKB1/AMPK to ration lipid reserves and ensure long-term survival. References Nature 457, 210–214 (2009). ,
(
)
5. Ylikorkala, A. et al. Vascular abnormalities and deregulation of VEGF in Lkb1-deficient mice. Science 293, 1323–1326 (2001). 6 L d
K R
l 5′ AMP
i
d
i ki
(AMPK) i i d
d b l
d l
d
i
i
di i
f 15. Ylikorkala, A. et al. Vascular abnormalities and deregulation of i
16. Laderoute, K. R. et al. 5′-AMP-activated protein kinase (AMPK) is induced by low-oxygen and glucose deprivation conditions f
in solid-tumor microenvironments. Mol Cell Biol 26, 5336–5347 (2006).i 17. Mizushima, N., Levine, B., Cuervo, A. M. & Klionsky, D. J. Autophagy fights disease through cellular self-digestion. Nature
1069–1075 (2008).h 18. Lizcano, J. M. et al. LKB1 is a master kinase that activates 13 kinases of the AMPK subfamily, including MARK/PAR-1. The E
journal 23, 833–843 (2004). j
(
)
19. Kroemer, G., Marino, G. & Levine, B. Autophagy and the integrated stress response. Molecular cell 40, 280–293 (2010).ll 19. Kroemer, G., Marino, G. & Levine, B. Autophagy and the integrated stress response. Molecular cell 40, 280–293 (2010). 20 O h
I
t l P i
ili
d
d
t
t
h
t
l
ith li l
ll
l
i
t fl id fl
N t
ll bi l
1 20. Orhon, I. et al. Primary-cilium-dependent autophagy controls epithelial cell volume in response to fluid flow. Nature cell b
657–667 (2016).f 21. Maiuri, M. C. et al. Control of autophagy by oncogenes and tumor suppressor genes. Cell Death Differ 16, 87–93 (2009).h f
22. Egan, D., Kim, J., Shaw, R. J. & Guan, K. L. The autophagy initiating kinase ULK1 is regulated via opposing phosphorylat
AMPK and mTOR. Autophagy 7, 643–644 (2011). p
gy
23. Kim, J., Kundu, M., Viollet, B. & Guan, K. L. AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nature
cell biology 13, 132–141 (2011).li 3. Kim, J., Kundu, M., Viollet, B. & Guan, K. L. AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nature
cell biology 13, 132–141 (2011). 4 S hl
l A & St i i
D Y L
f
“l
”
i
h t
fli
d
b
fi h
t
h
b
t h gy
24. Schlegel, A. & Stainier, D. Y. Lessons from “lower” organisms: what worms, flies, and zebrafish can teach us about human en
metabolism. PLoS Genet 3, e199 (2007).i 25. Scientific Reports | 7: 7327 | DOI:10.1038/s41598-017-07116-9 References Eradication of intracellular Francisella tularensis in THP-1 human macrophages with a novel autophagy inducing
agent. Journal of biomedical science 16, 110 (2009).h agent. Journal of biomedical science 16, 110 (2009) g
f
4. van der Vaart, M. et al. The DNA damage-regulated autophagy modulator DRAM1 links mycobacterial recognition via TLP-MYD88
to authophagic defense. Cell host & microbe 15, 753–767 (2014). 54. van der Vaart, M. et al. The DNA damage-regulated h
to authophagic defense. Cell host & microbe 15, 753–767 p
g
55. Williams, A. et al. Novel targets for Huntington’s disease in an mTOR-independent autophagy pathway. Nature chemical biolo
295–305 (2008). 56. Duran, A. et al. p62 is a key regulator of nutrient sensing in the mTORC1 pathway. Molecular cell 44, 134–146 (2011). p
y
g
g
p
y
57. Subramani, S. & Malhotra, V. Non-autophagic roles of autophagy-related proteins. EMBO Rep 14, 143–151 (2013). bramani, S. & Malhotra, V. Non-autophagic roles of autophagy-rela g
gy
58. Boya, P., Reggiori, F. & Codogno, P. Emerging regulation and functions of autophagy. Nature cell biology 15, 713–720 (2013).i i ,
p
g
g
p
gy
p p
gy ,
(
0. Moscat, J. & Diaz-Meco, M. T. Feedback on fat: p62-mTORC1-autophagy connections. Cell 147, 724–727 (2011). i
60. Moscat, J. & Diaz-Meco, M. T. Feedback on fat: p62-mTORC1-autophagy connections. Cell 147, 724–727 (2011). z-Meco, M. T. Feedback on fat: p62-mTORC1-autophagy connecti 61. Moscat, J. & Diaz-Meco, M. T. p62 at the crossroads of autophagy, apoptosis, and cancer. Cell 137, 1001–1004 (2009). p
p
gy p p
62. Gonzalez, A. A. et al. Metabolic adaptations to fasting and chronic caloric restriction in heart, muscle, and liver do not in
changes in AMPK activity. American journal of physiology. Endocrinology and metabolism 287, E1032–1037 (2004). 63. Viollet, B. et al. AMP-activated protein kinase in the regulation of hepatic energy metabolism: from physiology to therap
perspectives. Acta physiologica 196, 81–98 (2009). Author Contributions Conceived and designed the experiments: A.-P.G.H., L.A.M. and L.Q.C. Performed the experiments and collected
data: L.A.M., L.Q.C., J.v.P., P.G. Analyzed the data: L.Q.C., L.A.M., W.-J.K., A.-P.G.H. Wrote the manuscript
L.Q.C., A.-P.G.H. Reviewed and edited the manuscript: L.A.M., L.Q.C., W.-J.K., A.-P.G.H. References Aquatic toxicology 105, 270–278 (2011).h 9. Sugawara, K. et al. The crystal structure of microtubule-associated protein light chain 3, a mammalian homologue of Saccharomyce
cerevisiae Atg8. Genes Cells 9, 611–618 (2004). gh
y
cerevisiae Atg8. Genes Cells 9, 611–618 (2004). Klionsky, D. J. Autophagy in health and disease: a double-edged sw 41. Mizushima, N. & Yoshimori, T. How to interpret LC3 immunoblotting. Autophagy 3, 542–545 (2007). 42. Mizushima, N. & Klionsky, D. J. Protein turnover via autophagy: implications for metabolism. Annual review of nutrition 27, 1
(2007). 3. Lamark, T., Kirkin, V., Dikic, I. & Johansen, T. NBR1 and p62 as cargo receptors for selective autophagy of ubiquitinated targets. Cel
Cycle 8, 1986–1990 (2009). y
4. Egan, D. F. et al. Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy
Science 331, 456–461 (2011). 45. Komatsu, M. et al. The selective autophagy substrate p62 activates the stress responsive transcription factor Nrf2 through
inactivation of Keap1. Nature cell biology 12, 213–223 (2010). p
gy
(
)
46. Rusten, T. E. & Stenmark, H. p62, an autophagy hero or culprit? Nature cell biology 12, 207–209 (2010). 47. Boglev, Y. et al. Autophagy induction is a Tor- and Tp53-independent cell survival response in a zebrafish model of disrupted
ribosome biogenesis. PLoS Genet 9, e1003279 (2013). 48. Marshall, K. E., Tomasini, A. J., Makky, K., S., N. K. & Mayer, A. N. Dynamic Lkb1-TORC1 signaling as a possible mechanism for
regulating the endoderm-intestine transition. Dev Dyn 239, 3000–3012 (2010). g
g
y
9. Efeyan, A. et al. Regulation of mTORC1 by the Rag GTPases is necessary for neonatal autophagy and survival. Nature 493, 679–683
(2013). 50. Makky, K., Tekiela, J. & Mayer, A. N. Target of rapamycin (TOR) signaling controls epithelial morphogenesis in the vertebrate
intestine. Dev Biol 303, 501–513 (2007). 1. Chittaranjan, S., Bortnik, S. & Gorski, S. M. Monitoring Autophagic Flux by Using Lysosomal Inhibitors and Western Blotting o
Endogenous MAP1LC3B Cold Spring Harb Protoc 2015 743 750 (2015) 51. Chittaranjan, S., Bortnik, S. & Gorski, S. M. Monitoring Autophagic Flux by Using Lysosomal Inhibitors and Western Blottin
Endogenous MAP1LC3B. Cold Spring Harb Protoc 2015, 743–750 (2015). g
p
g
52. Dibble, C. C. & Cantley, L. C. Regulation of mTORC1 by PI3K signaling. Trends in cell biology 25, 545–555 (2015). y
g
y
g
g
gy
53. Chiu, H. C. et al. References Varga, M., Fodor, E. & Vellai, T. Autophagy in zebrafish. Methods 75, 172–180 (2015).i 25. Varga, M., Fodor, E. & Vellai, T. Autophagy in zebrafish. Methods 75, 172–180 (2015).i i
26. Schlegel, A. & Gut, P. Metabolic insights from zebrafish genetics, physiology, and chemical biology. Cellular and molecula
sciences: CMLS (2015).hi 27. Seth, A., Stemple, D. L. & Barroso, I. The emerging use of zebrafish to model metabolic disease. Disease models & mechanisms 6,
1080–1088 (2013). (
)
28. Hu, Z., Zhang, J. & Zhang, Q. Expression pattern and functions of autophagy-related gene atg5 in zebrafish organogenesis. Autophagy 7, 1514–1527 (2011). p
gy
29. Lee, E. et al. Autophagy is essential for cardiac morphogenesis during vertebrate development. Autophagy 10, 572–587 (2014) 30. Meyer, G. et al. The cellular autophagy markers Beclin-1 and LC3B-II are increased during reperfusion in fibrillated mouse hearts. Current pharmaceutical design 19, 6912–6918 (2013).iif p
g
31. Varga, M. et al. Autophagy is required for zebrafish caudal fin regeneration. Cell Death Differ 21, 547–556 (2014).h iif
32. Gustafsson, J. Neonatal energy substrate production. The Indian journal of medical research 130, 618–623 (2009). 33. Pilkis, S. J. & Granner, D. K. Molecular physiology of the regulation of hepatic gluconeogenesis and glycolysis. Annual review of
physiology 54, 885–909 (1992). p y
gy
34. Heintz, N. Developmental biology: survival by self-digestion. Nature 432, 963 (2004).i p y
gy
34. Heintz, N. Developmental biology: survival by self-digestion. Nature 432, 963 (2004).i gy
y
g
35. Gut, P. et al. Whole-organism screening for gluconeogenesis identifies activators of fasting metabolism. Nature chemical biology 9,
97–104 (2013).fii (
)
36. Wienholds, E. et al. Efficient target-selected mutagenesis in zebrafish. Genome Res 13, 2700–2707 (2003). (
)
36. Wienholds, E. et al. Efficient target-selected mutagenesis in zebrafish. Genome Res 13, 2700–2707 (2003). Scientific Reports | 7: 7327 | DOI:10.1038/s41598-017-07116-9 9 www.nature.com/scientificreports/ 7. van der Velden, Y. U. et al. The serine-threonine kinase LKB1 is essential for survival under energetic stress in zebrafish. Proc Nat
Acad Sci USA 108, 4358–4363 (2011).i Acad Sci USA 108, 4358–4363 (2011). 38. Daouk, T. et al. Long-term food-exposure of zebrafish to PCB mixtures mimicking some environmental situations induces ovary
h l
d i
i
d
i
bili
A
i
i
l
105 270 278 (2011) 38. Daouk, T. et al. Long-term food-exposure of zebrafish to PCB mixtures mimicking some environmental situations induces o
pathology and impairs reproduction ability. Acknowledgements We thank Prof. Annemarie Meijer (IBL) for the sqstm1/p62 morpholino and critical advice on the manuscript,
Gerda Lamers (IBL) for advice with confocal microscopy, the DNA-Markerpoint facility (IBL) for genotyping,
and the animal caretakers for excellent care of the fish. We also thank Dr. Anastassis Perrakis (NKI) for critical
reading of the manuscript and the Life Science Editors for editorial assistance. The work was supported by a grant
from the Dutch Cancer Society (KWF UL 2012–5395) to A.-P.G.H. Additional Information Supplementary information accompanies this paper at doi:10.1038/s41598-017-07116-9 Supplementary information accompanies this paper at doi:10.1038/s41598-017-07116-9 Competing Interests: The authors declare that they have no competing interests. Competing Interests: The authors declare that they have no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps an
institutional affiliations. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and
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Disrupting neuronal transmission: mechanism of DBS?
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INTRODUCTION GPi neurons during GPi-DBS (Bar-Gad et al., 2004; Erez et al.,
2009; McCairn and Turner, 2009; Leblois et al., 2010). In this
article, we critically review recent studies, and discuss the possible
mechanism of effectiveness of DBS. Applying high-frequency electrical stimulation (HFS) to a specific
target in subcortical structures, known as deep brain stimulation
(DBS), was introduced as a surgical treatment for movement
disorders in early 1990s (Benabid et al., 1991, 1994; Siegfried
and Lippitz, 1994a,b; Limousin et al., 1995). Since then, DBS
has been widely accepted as an effective therapeutic option. DBS
targeting the ventral thalamus dramatically alleviates essential and
resting tremor (Benabid et al., 1991, 1996; Siegfried and Lippitz,
1994b; Koller et al., 1997; Rehncrona et al., 2003). DBS targeting
the subthalamic nucleus (STN) and the internal segment of the
globus pallidus (GPi) has been largely used for treatment of
Parkinson’s disease, and GPi-DBS has marked effects on improve-
ment of dystonic symptoms (Limousin et al., 1995; Deep-Brain
Stimulation for Parkinson’s Disease Study Group, 2001; Coubes
et al., 2004; Wichmann and Delong, 2006; Kringelbach et al.,
2007; Ostrem and Starr, 2008; Vitek, 2008; Vidailhet et al., 2013). However, the exact mechanism of the effectiveness remains to be
elucidated. SYSTEMS NEUROSCIENCE REVIEW ARTICLE *Correspondence: *Correspondence:
Atsushi Nambu, Division of System
Neurophysiology, National Institute for
Physiological Sciences and
Department of Physiological Sciences,
Graduate University for Advanced
Studies, 38 Nishigonaka, Myodaiji,
Okazaki 444-8585, Japan
e-mail: nambu@nips.ac.jp Keywords: deep brain stimulation, basal ganglia, subthalamic nucleus, globus pallidus, cortico-basal ganglia loop,
electrophysiology Satomi Chiken and Atsushi Nambu* Division of System Neurophysiology, National Institute for Physiological Sciences and Department of Physiological Sciences, Graduate University for Advanced
Studies, Myodaiji, Okazaki, Japan National Institute for Physiological Sciences and Department of Physiological Sciences, Graduate University for Advanced Division of System Neurophysiology, National Institute for Physiological Sciences and Department of Physiological Scie
Studies, Myodaiji, Okazaki, Japan Applying high-frequency stimulation (HFS) to deep brain structure, known as deep
brain stimulation (DBS), has now been recognized an effective therapeutic option for a
wide range of neurological and psychiatric disorders. DBS targeting the basal ganglia
thalamo-cortical loop, especially the internal segment of the globus pallidus (GPi),
subthalamic nucleus (STN) and thalamus, has been widely employed as a successful
surgical therapy for movement disorders, such as Parkinson’s disease, dystonia and
tremor. However, the neurophysiological mechanism underling the action of DBS remains
unclear and is still under debate: does DBS inhibit or excite local neuronal elements? In
this review, we will examine this question and propose the alternative interpretation: DBS
dissociates inputs and outputs, resulting in disruption of abnormal signal transmission. Keywords: deep brain stimulation, basal ganglia, subthalamic nucleus, globus pallidus, cortico-basal ganglia loop,
electrophysiology REVIEW ARTICLE
published: 14 March 2014
doi: 10.3389/fnsys.2014.00033 REVIEW ARTICLE
published: 14 March 2014
doi: 10.3389/fnsys.2014.00033 Frontiers in Systems Neuroscience Edited by: y
Ahmed A. Moustafa, University of
Western Sydney, Australia y
Ahmed A. Moustafa, University of
Western Sydney, Australia
Reviewed by:
Alessandro Stefani, University of
Rome, Italy
Robert S. Turner, University of
Pittsburgh, USA Ahmed A. Moustafa, University of
Western Sydney, Australia DEEP BRAIN STIMULATION (DBS) INHIBITS LOCAL
NEURONAL ELEMENTS Chiken and Nambu FIGURE 1 | Deep brain stimulation (DBS) inhibits local neuronal firing. (A) Responses of an internal pallidal (GPi) neuron to local GPi repetitive
high-frequency stimulation (HFS; 30 µA, 100 Hz, 10 pulses) in a normal
monkey. Raw traces of spike discharges after removing the stimulus artifacts
by the template subtraction method (1) and raster and peristimulus time
histogram (PSTHs; 100 trials; binwidth, 1 ms) (2) are shown. Arrows indicate the timing of local stimulation. Spontaneous discharges of the GPi neuron
were completely inhibited by the stimulation. (B) Effect of local injection of
gabazine (GABAA receptor antagonist) in the vicinity of the recorded GPi
neuron on inhibition of spontaneous activity induced by GPi-HFS. The
inhibition was abolished after gabazine injection. Modified from Chiken and
Nambu (2013). FIGURE 1 | Deep brain stimulation (DBS) inhibits local neuronal firing. (A) Responses of an internal pallidal (GPi) neuron to local GPi repetitive
high-frequency stimulation (HFS; 30 µA, 100 Hz, 10 pulses) in a normal
monkey. Raw traces of spike discharges after removing the stimulus artifacts
by the template subtraction method (1) and raster and peristimulus time
histogram (PSTHs; 100 trials; binwidth, 1 ms) (2) are shown. Arrows indicate
the timing of local stimulation. Spontaneous discharges of the GPi neuron
were completely inhibited by the stimulation. (B) Effect of local injection of
gabazine (GABAA receptor antagonist) in the vicinity of the recorded GPi
neuron on inhibition of spontaneous activity induced by GPi-HFS. The
inhibition was abolished after gabazine injection. Modified from Chiken and
Nambu (2013). he timing of local stimulation. Spontaneous discharges of the GPi neuron
were completely inhibited by the stimulation. (B) Effect of local injection of
abazine (GABAA receptor antagonist) in the vicinity of the recorded GPi
euron on inhibition of spontaneous activity induced by GPi-HFS. The
nhibition was abolished after gabazine injection. Modified from Chiken and
Nambu (2013). the timing of local stimulation. Spontaneous discharges of the GPi neuron
were completely inhibited by the stimulation. (B) Effect of local injection of
gabazine (GABAA receptor antagonist) in the vicinity of the recorded GPi
neuron on inhibition of spontaneous activity induced by GPi-HFS. The
inhibition was abolished after gabazine injection. Modified from Chiken and
Nambu (2013). FIGURE 1 | Deep brain stimulation (DBS) inhibits local neuronal firing. DEEP BRAIN STIMULATION (DBS) INHIBITS LOCAL
NEURONAL ELEMENTS (A) Responses of an internal pallidal (GPi) neuron to local GPi repetitive
high-frequency stimulation (HFS; 30 µA, 100 Hz, 10 pulses) in a normal
monkey. Raw traces of spike discharges after removing the stimulus artifacts
by the template subtraction method (1) and raster and peristimulus time
histogram (PSTHs; 100 trials; binwidth, 1 ms) (2) are shown. Arrows indicate abnormal firings, such as bursting and oscillatory activity in
Parkinson’s disease and dystonia as described below. STN firing was observed in a limited number of neurons. STN-
HFS at 140 Hz reduced mean firing rate of STN neurons by 77%
in Parkinsonian patients, and among them, 71% of STN neurons
exhibited residual neuronal activity, while only 29% of STN
neurons exhibited total inhibition (Welter et al., 2004). Similar
results were also observed in Parkinsonian monkeys (Meissner
et al., 2005), and Parkinsonian and normal rats (Tai et al., 2003). Decreased abnormal oscillatory activity in the STN was also
observed during STN-DBS in Parkinsonian monkeys (Meissner
et al., 2005). Inhibitory effects sometimes outlasted the stimulus
period (Tai et al., 2003; Filali et al., 2004; Welter et al., 2004). MECHANISM OF INHIBITION Several possible mechanisms account for the inhibitory responses
have been proposed, including depolarization-block and inacti-
vation of voltage-gated currents (Beurrier et al., 2001; Shin et al.,
2007). However, these are less probable, because both single-pulse
and low-frequency stimulation in the GPi evoked intense short
latency inhibition in neighboring neurons (Dostrovsky et al.,
2000; Dostrovsky and Lozano, 2002; Chiken and Nambu, 2013). Another possible mechanism is that the inhibition is caused
by activation of GABAergic afferents in the stimulated nucleus
(Boraud et al., 1996; Dostrovsky et al., 2000; Dostrovsky and
Lozano, 2002; Meissner et al., 2005; Johnson et al., 2008; Liu
et al., 2008; Deniau et al., 2010). A recent study confirmed
that inhibitory responses induced by GPi-HFS were mediated
by GABAA and GABAB receptors (Chiken and Nambu, 2013;
Figure 1B). GABAergic inhibition is strong and inhibits even
directly evoked spikes by GPi stimulation, which is characterized
constant- and short latency (Figures 2A, B; Chiken and Nambu,
2013). Inhibitory effects of GPi-DBS on firing of the neighboring
neurons were also reported (Boraud et al., 1996; Dostrovsky et al.,
2000; Wu et al., 2001; McCairn and Turner, 2009). Complete
inhibition of local neuronal firing was more commonly induced
by GPi-DBS than by STN-DBS (Figure 1A). GPi-HFS at 100 Hz
induced complete inhibition of 76% of neighboring neurons in
normal monkeys (Chiken and Nambu, 2013), and the inhibi-
tion outlasted the stimulus period, sometimes over 100 ms after
the end of stimulation. Similar post-train inhibition was also
observed in Parkinsonian patients (Lafreniere-Roula et al., 2010). Inhibitory effects of GPi-DBS on firing of the neighboring
neurons were also reported (Boraud et al., 1996; Dostrovsky et al.,
2000; Wu et al., 2001; McCairn and Turner, 2009). Complete
inhibition of local neuronal firing was more commonly induced
by GPi-DBS than by STN-DBS (Figure 1A). GPi-HFS at 100 Hz
induced complete inhibition of 76% of neighboring neurons in
normal monkeys (Chiken and Nambu, 2013), and the inhibi-
tion outlasted the stimulus period, sometimes over 100 ms after
the end of stimulation. Similar post-train inhibition was also
observed in Parkinsonian patients (Lafreniere-Roula et al., 2010). To the contrary, multiphasic responses consisting of the exci-
tation and inhibition during GPi-HFS were recently observed in
GPi neurons of Parkinsonian monkeys (Bar-Gad et al., 2004; Erez
et al., 2009; McCairn and Turner, 2009) and dystonic hamsters
(Leblois et al., 2010). DEEP BRAIN STIMULATION (DBS) INHIBITS LOCAL
NEURONAL ELEMENTS Both DBS and lesion were found to produce similar benefits
on alleviation of symptoms. For example, STN-DBS has similar
effects on Parkinsonian motor signs (Benazzouz et al., 1993;
Benabid et al., 1994; Limousin et al., 1995) to the STN-lesion
(Bergman et al., 1990; Aziz et al., 1991; Levy et al., 2001) and
blockade of synaptic transmission from the STN to the GPi
(Graham et al., 1990; Brotchie et al., 1991). Thus, DBS was
originally assumed to inhibit local neuronal elements. Actually,
the most common effect of STN- or GPi-HFS on neighboring
neurons was reduction of the firing rates. g
Distinct suppression of neuronal activity was recorded during
STN-DBS around the stimulating sites in Parkinsonian patients
during stereotactic surgery (Filali et al., 2004; Welter et al., 2004). Similar results were also obtained in animal models, such as
Parkinsonian monkeys (Meissner et al., 2005; Moran et al., 2011)
and rats (Tai et al., 2003; Shi et al., 2006). Stimulus artifacts hinder
detection of spikes during 2–3 ms after stimulus pulses and some
spikes may be obscured when neuronal activities are recorded
nearby the stimulating electrodes. Recent studies enabled detec-
tion of spikes just after stimulus pulses by removal of stimulus
artifacts using the template subtraction method (Wichmann,
2000; Hashimoto et al., 2002) and confirmed that STN-DBS
decreased firing of neighboring neurons (Meissner et al., 2005;
Moran et al., 2011). Although STN-HFS much decreased neu-
ronal firing around the stimulation site, complete cessation of Since DBS gives rise to similar effects to those of lesions, it
was originally considered to inhibit local neuronal elements. In
fact, neuronal firings of neighboring neurons were inhibited by
STN- or GPi-DBS (Boraud et al., 1996; Dostrovsky et al., 2000;
Wu et al., 2001; Filali et al., 2004; Lafreniere-Roula et al., 2010). On the other hand, recent studies have emphasized activation of
neuronal elements. Actually, STN-DBS increased activity of GPi
neurons through the excitatory STN-GPi projections (Hashimoto
et al., 2003; Galati et al., 2006; Reese et al., 2011), and GPi-
DBS reduced activity of thalamic neurons through the inhibitory
GPi-thalamic projections (Anderson et al., 2003; Pralong et al.,
2003; Montgomery, 2006). In addition, recent studies reported
multi-phasic responses consisting of excitation and inhibition in March 2014 | Volume 8 | Article 33 | 1 Frontiers in Systems Neuroscience www.frontiersin.org www.frontiersin.org www.frontiersin.org Disrupting neuronal transmission: mechanism of DBS? MECHANISM OF INHIBITION The discrepant results may be due to dif-
ferences in stimulus parameters used in these experiments: larger
axons are easily activated by electrical stimulation than smaller
ones (Ranck, 1975), and continuous repetitive stimulation might
cause failure of postsynaptic events due to receptor desensitization
and/or transmitter depletion (Wang and Kaczmarek, 1998; Zucker
and Regehr, 2002). Such multiphasic responses may normalize To the contrary, multiphasic responses consisting of the exci-
tation and inhibition during GPi-HFS were recently observed in
GPi neurons of Parkinsonian monkeys (Bar-Gad et al., 2004; Erez
et al., 2009; McCairn and Turner, 2009) and dystonic hamsters
(Leblois et al., 2010). The discrepant results may be due to dif-
ferences in stimulus parameters used in these experiments: larger
axons are easily activated by electrical stimulation than smaller
ones (Ranck, 1975), and continuous repetitive stimulation might
cause failure of postsynaptic events due to receptor desensitization
and/or transmitter depletion (Wang and Kaczmarek, 1998; Zucker
and Regehr, 2002). Such multiphasic responses may normalize The GPi receives excitatory glutamatergic inputs from the STN
as well as inhibitory GABAergic inputs from the striatum and
GPe (Smith et al., 1994; Shink and Smith, 1995). Afferent axon
terminals from the STN are also activated by the stimulation,
but the glutamatergic excitation is probably overwhelmed because
of predominance of GABAergic inputs in the GPi (Shink and
Smith, 1995). On the other hand, many GPe neurons exhibited
complex responses composed of both excitation and inhibition
during GPe-HFS (Chiken and Nambu, 2013). The density of GPe
terminals on GPi neurons is higher than those on GPe neurons March 2014 | Volume 8 | Article 33 | 2 Frontiers in Systems Neuroscience www.frontiersin.org www.frontiersin.org Disrupting neuronal transmission: mechanism of DBS? Chiken and Nambu FIGURE 2 | Directly evoked spikes of GPi neurons were inhibited
during GPi-HFS. (A) Raw traces showing directly evoked spikes of a
GPi neuron by stimulus pulses during GPi-HFS (40 µA, 100 Hz, 10
pulses) in a normal monkey. Traces with long (top) and short (bottom)
time scales are shown. Arrows with dotted lines indicate the timing of
local stimulation (time 0 in the bottom traces). Filled arrowheads
indicate directly evoked spikes. GPi-HFS failed to evoke spikes (open
arrowheads; from 6th to 10th stimuli). (B) Effects of local gabazine
injection on the inhibition of direct evoked GPi responses. Gabazine
injection decreased failure rate, and each stimulus successfully evoked
spikes (5th, 9th, and 10th stimuli). MECHANISM OF INHIBITION Modified from Chiken and Nambu
(2013). indicate directly evoked spikes. GPi-HFS failed to evoke spikes (open
arrowheads; from 6th to 10th stimuli). (B) Effects of local gabazine
injection on the inhibition of direct evoked GPi responses. Gabazine
injection decreased failure rate, and each stimulus successfully evoked
spikes (5th, 9th, and 10th stimuli). Modified from Chiken and Nambu
(2013). FIGURE 2 | Directly evoked spikes of GPi neurons were inhibited
during GPi-HFS. (A) Raw traces showing directly evoked spikes of a
GPi neuron by stimulus pulses during GPi-HFS (40 µA, 100 Hz, 10
pulses) in a normal monkey. Traces with long (top) and short (bottom)
time scales are shown. Arrows with dotted lines indicate the timing of
local stimulation (time 0 in the bottom traces). Filled arrowheads 2006). GPi activity was increased during STN-DBS through exci-
tatory STN-GPi projections (Hashimoto et al., 2003; Galati et al.,
2006; Reese et al., 2011). STN-DBS increased both glutamate
and GABA levels in the substantia nigra pars reticulata (SNr)
of normal rats in microdialysis studies (Windels et al., 2000; see
also Windels et al., 2005). An intraoperative microdialysis study
revealed that STN-DBS produced significant increase in extracel-
lular concentration of cyclic guanosine monophosphate (cGMP)
in the GPi (Stefani et al., 2005). Functional magnetic resonance
imaging (MRI) and positron emission tomography (PET) studies
in humans indicated that efferent outputs from the stimulated
nucleus are excited during DBS (Jech et al., 2001; Hershey et al.,
2003; Boertien et al., 2011). Changes of the firing rates and
patterns of target nuclei may normalize abnormal firings, such
as bursting and oscillatory activity, which are observes in the
cortico-basal ganglia loop of Parkinson’s disease and dystonia
(Anderson et al., 2003; Hashimoto et al., 2003; Hammond et al.,
2007; Johnson et al., 2008; Vitek, 2008; Deniau et al., 2010). (Shink and Smith, 1995), and the balance between GABAergic and
glutamatergic inputs may explain the different effects between
GPe-HFS and GPi-HFS. Similarly, STN-HFS stimulated both
glutamatergic and GABAergic afferents and generated both exci-
tatory and inhibitory post-synaptic potentials (EPSPs and IPSPs)
in the STN neurons (Lee et al., 2004). Thus, HFS activates afferent
axons in the stimulated nucleus, and the effects vary depending on
the composition of the inhibitory and excitatory axon terminals. Frontiers in Systems Neuroscience DEEP BRAIN STIMULATION (DBS) EXCITES LOCAL
NEURONAL ELEMENTS (A)
Schematic diagram showing the cortico-basal ganglia pathway and stimulating
(Stim.) and recording (Rec.) sites in the electrophysiological experiments (left),
along with a typical response pattern (right) in the (GPi) to cortical stimulation
(Cx Stim.) with early excitation, inhibition, and late excitation, which are
mediated by the (1) cortico-subthalamo (STN)-GPi hyperdirect, (2) striato-GPi
direct, and (3) striato-external pallido (GPe)-STN-GPi indirect pathways, FIGURE 3 | GPi-DBS disrupts information flow through the GPi. (A)
Schematic diagram showing the cortico-basal ganglia pathway and stimulating
(Stim.) and recording (Rec.) sites in the electrophysiological experiments (left),
along with a typical response pattern (right) in the (GPi) to cortical stimulation
(Cx Stim.) with early excitation, inhibition, and late excitation, which are
mediated by the (1) cortico-subthalamo (STN)-GPi hyperdirect, (2) striato-GPi
direct, and (3) striato-external pallido (GPe)-STN-GPi indirect pathways, respectively. (B) Effects of local GPi-HFS on cortically evoked responses of a
GPi neuron in a normal monkey. PSTH (100 trials) in response to the single
pulse stimulation (arrowhead with dotted line) of the primary motor cortex
(Cx) (1) and PSTH in response to Cx stimulation (arrowhead with dotted line)
during GPi-HFS (arrows) (2) are shown. Cortical stimulation was applied 50
ms after the initiation of GPi-HFS. The cortically evoked responses were
entirely inhibited during GPi-HFS. Modified from Chiken and Nambu (2013). FIGURE 3 | GPi-DBS disrupts information flow through the GPi. (A) suprathreshold HFS generated efferent outputs at the stimulus
frequency in the axon without representative activation of the
cell bodies. Thus, although stimulation may fail to activate cell
bodies of GPi neurons due to strong GABAergic inhibition, it can
still excite the efferent axons and provide inhibitory inputs to the
thalamus at the stimulus frequency. axons can robustly ameliorate symptoms in Parkinsonian rats
without activation of STN efferent axons (Gradinaru et al.,
2009), suggesting that therapeutic effects of STN-DBS may
be exclusively accounted for activation of cortico-STN afferent
axons. It is also probable that STN-DBS induces dopamine release
through STN-SNc projections. STN-DBS induced dopamine
release by activation of nigrostriatal dopaminergic neurons in
rats (Meissner et al., 2003) and pigs (Shon et al., 2010), however
it did not increase dopamine level of the striatum in human
patients (Abosch et al., 2003; Hilker et al., 2003). DBS may also
affect neurons whose axons pass nearby the stimulating site. DEEP BRAIN STIMULATION (DBS) EXCITES LOCAL
NEURONAL ELEMENTS It is rational that local stimulation excites local neuronal elements. Actually, directly evoked spikes, which are characterized by short-
and constant latency, are induced in GPi neurons by GPi-HFS
(Johnson and McIntyre, 2008; McCairn and Turner, 2009). Such
excitation may propagate through efferent projections. Thalamic
activity was reduced during GPi-HFS through inhibitory GPi-
thalamic projections in Parkinsonian monkeys (Anderson et al.,
2003) and dystonia patients (Pralong et al., 2003; Montgomery, According to the modeling study (McIntyre et al., 2004), sub-
threshold HFS suppressed intrinsic firings in the cell bodies, while March 2014 | Volume 8 | Article 33 | 3 Frontiers in Systems Neuroscience www.frontiersin.org www.frontiersin.org Disrupting neuronal transmission: mechanism of DBS? Chiken and Nambu respectively. (B) Effects of local GPi-HFS on cortically evoked responses of a
GPi neuron in a normal monkey. PSTH (100 trials) in response to the single
pulse stimulation (arrowhead with dotted line) of the primary motor cortex
(Cx) (1) and PSTH in response to Cx stimulation (arrowhead with dotted line)
during GPi-HFS (arrows) (2) are shown. Cortical stimulation was applied 50
ms after the initiation of GPi-HFS. The cortically evoked responses were
entirely inhibited during GPi-HFS. Modified from Chiken and Nambu (2013). FIGURE 3 | GPi-DBS disrupts information flow through the GPi. (A)
Schematic diagram showing the cortico-basal ganglia pathway and stimulating
(Stim.) and recording (Rec.) sites in the electrophysiological experiments (left),
along with a typical response pattern (right) in the (GPi) to cortical stimulation
(Cx Stim.) with early excitation, inhibition, and late excitation, which are
mediated by the (1) cortico-subthalamo (STN)-GPi hyperdirect, (2) striato-GPi
direct, and (3) striato-external pallido (GPe)-STN-GPi indirect pathways,
respectively. (B) Effects of local GPi-HFS on cortically evoked responses of a
GPi neuron in a normal monkey. PSTH (100 trials) in response to the single
pulse stimulation (arrowhead with dotted line) of the primary motor cortex
(Cx) (1) and PSTH in response to Cx stimulation (arrowhead with dotted line)
during GPi-HFS (arrows) (2) are shown. Cortical stimulation was applied 50
ms after the initiation of GPi-HFS. The cortically evoked responses were
entirely inhibited during GPi-HFS. Modified from Chiken and Nambu (2013). FIGURE 3 | GPi-DBS disrupts information flow through the GPi. Frontiers in Systems Neuroscience DEEP BRAIN STIMULATION (DBS) EXCITES LOCAL
NEURONAL ELEMENTS (B) Effects of STN blocking on cortically evoked responses of a GPi FIGURE 4 | Both STN-DBS and STN blocking disrupt information flow
through the STN (A) Effects of local STN DBS on cortically evoked neuron in a normal monkey. PSTH (100 trials) in response to the single pulse
stimulation of the Cx (arrow with dotted line) (1) and PSTH in response to Cx
stimulation after blocking STN activity by muscimol (GABAA receptor agonist)
injection into the STN (2) are shown. The cortically evoked early and late
excitation was abolished after injection of muscimol into the STN, while
cortically evoked inhibition was preserved. Modified from Nambu et al. (2000). Note that the pattern of cortically evoked responses of a SNr neuron
during STN-DBS is similar to that of a GPi neuron after STN blocking. neuron in a normal monkey. PSTH (100 trials) in response to the single pulse
stimulation of the Cx (arrow with dotted line) (1) and PSTH in response to Cx
stimulation after blocking STN activity by muscimol (GABAA receptor agonist)
injection into the STN (2) are shown. The cortically evoked early and late
excitation was abolished after injection of muscimol into the STN, while
cortically evoked inhibition was preserved. Modified from Nambu et al. (2000). Note that the pattern of cortically evoked responses of a SNr neuron
during STN-DBS is similar to that of a GPi neuron after STN blocking. g
y
responses of a substantia nigra pars reticulata (SNr) neuron in a normal rat. PSTH (50 trials) in response to the single pulse stimulation of the Cx (arrow)
(1) and PSTH in response to Cx stimulation during STN-DBS (2) are shown. The cortically evoked early and late excitation was abolished during STN-DBS,
while cortically evoked inhibition was preserved. Modified from Maurice et al. (2003). (B) Effects of STN blocking on cortically evoked responses of a GPi Chiken and Nambu (2013) recently examined responses of GPi
neurons evoked by motor cortical stimulation during GPi-HFS in
normal monkeys. In that study, both cortically evoked responses
and spontaneous discharges were completely inhibited during
GPi-HFS by strong GABAergic inhibition (Figure 3B), suggesting
that GPi-HFS blocks information flow through the GPi. DEEP BRAIN STIMULATION (DBS) EXCITES LOCAL
NEURONAL ELEMENTS A
model-based study showed that clinically effective STN-DBS also
activated the lenticular fasciculus, which is composed of GPi-
thalamic fibers, in addition to STN neurons temselves (Mioci-
novic et al., 2006). Actually, STN-DBS induced direct excitation
of GPi neurons through activation of the lenticular fasciculus
(Moran et al., 2011). DBS also antidromically excites afferent axons. Actually,
antidromic activation of GPi neurons induced by STN-DBS was
observed in Parkinsonian monkeys (Moran et al., 2011), and
antidromic activation of thatamic (Vop) neurons induced by
GPi-DBS was observed in Parkinsonian patients (Montgomery,
2006). Low intensity STN-HFS induced GABAergic inhibition
in the SNr through antidromic activation of GPe neurons pro-
jecting to both the STN and SNr (Maurice et al., 2003; see
also Moran et al., 2011), whereas higher intensity stimulation
induced glutamatergic excitation in the SNr through activation
of STN-SNr projections. STN-HFS also activated motor cortical
neurons antidromically and suppressed abnormal low frequency
synchronization including beta band oscillation in Parkinso-
nian rats (Li et al., 2007, 2012; Degos et al., 2013). Recent
development of optogenetics has enabled selective stimulation
of afferent inputs or efferent outputs, and contribute to ana-
lyzing the mechanism of effectiveness of DBS. A recent study
has shown that selective stimulation of cortico-STN afferent Participation of non-neuronal glial tissues should also be
considered as one of possible mechanisms of DBS effectiveness. DBS induced glutamate and adenosine triphosphate (ATP) release
from astrocytes (Fellin et al., 2006; Tawfik et al., 2010). A recent
study revealed that HFS applied to the thalamus induced abrupt
increase in extracellular ATP and adenosine (Bekar et al., 2008). March 2014 | Volume 8 | Article 33 | 4 Frontiers in Systems Neuroscience Frontiers in Systems Neuroscience www.frontiersin.org www.frontiersin.org www.frontiersin.org Disrupting neuronal transmission: mechanism of DBS? Chiken and Nambu neuron in a normal monkey. PSTH (100 trials) in response to the single pulse
stimulation of the Cx (arrow with dotted line) (1) and PSTH in response to Cx
stimulation after blocking STN activity by muscimol (GABAA receptor agonist)
injection into the STN (2) are shown. The cortically evoked early and late
excitation was abolished after injection of muscimol into the STN, while
cortically evoked inhibition was preserved. Modified from Nambu et al. (2000). Note that the pattern of cortically evoked responses of a SNr neuron
during STN-DBS is similar to that of a GPi neuron after STN blocking. DEEP BRAIN STIMULATION (DBS) EXCITES LOCAL
NEURONAL ELEMENTS Since
abnormal cortically evoked responses (Chiken et al., 2008; Kita
and Kita, 2011; Nishibayashi et al., 2011) and abnormal bursts and
oscillatory activity (Wichmann et al., 1994; Bergman et al., 1998;
Starr et al., 2005; Brown, 2007; Chiken et al., 2008; Nishibayashi
et al., 2011; Tachibana et al., 2011) were observed in GPi neurons
in Parkinson’s disease and dystonia, signal transmission of such
abnormal activities to the thalamus and motor cortex would
be responsible for motor symptoms. Thus, disruption of the
abnormal information flow could suppress expression of motor
symptoms. This mechanism may explain the paradox that GPi-
DBS produces similar therapeutic effects to lesions of the GPi:
both GPi-DBS and GPi-lesion interrupt abnormal information
flow through the GPi. Adenosine activation of A1 receptors depressed excitatory trans-
mission in the thalamus, and alleviated tremor in a mouse model. Thus, it is possible that ATP and glutamate are released from
astrocytes triggered by DBS and modulate neuronal activity in the
stimulated nucleus (Vedam-Mai et al., 2012; Jantz and Watanabe,
2013). Adenosine activation of A1 receptors depressed excitatory trans-
mission in the thalamus, and alleviated tremor in a mouse model. Thus, it is possible that ATP and glutamate are released from
astrocytes triggered by DBS and modulate neuronal activity in the
stimulated nucleus (Vedam-Mai et al., 2012; Jantz and Watanabe,
2013). Thus, it is possible that ATP and glutamate are released from
astrocytes triggered by DBS and modulate neuronal activity in the
stimulated nucleus (Vedam-Mai et al., 2012; Jantz and Watanabe,
2013). DEEP BRAIN STIMULATION (DBS) EXCITES LOCAL
NEURONAL ELEMENTS FIGURE 4 | Both STN-DBS and STN blocking disrupt information flow
through the STN. (A) Effects of local STN-DBS on cortically evoked
responses of a substantia nigra pars reticulata (SNr) neuron in a normal rat. PSTH (50 trials) in response to the single pulse stimulation of the Cx (arrow)
(1) and PSTH in response to Cx stimulation during STN-DBS (2) are shown. The cortically evoked early and late excitation was abolished during STN-DBS,
while cortically evoked inhibition was preserved. Modified from Maurice et al. (2003). (B) Effects of STN blocking on cortically evoked responses of a GPi
neuron in a normal monkey. PSTH (100 trials) in response to the single pulse
stimulation of the Cx (arrow with dotted line) (1) and PSTH in response to Cx
stimulation after blocking STN activity by muscimol (GABAA receptor agonist)
injection into the STN (2) are shown. The cortically evoked early and late
excitation was abolished after injection of muscimol into the STN, while
cortically evoked inhibition was preserved. Modified from Nambu et al. (2000). Note that the pattern of cortically evoked responses of a SNr neuron
during STN-DBS is similar to that of a GPi neuron after STN blocking. FIGURE 4 | Both STN-DBS and STN blocking disrupt information flow
through the STN. (A) Effects of local STN-DBS on cortically evoked
responses of a substantia nigra pars reticulata (SNr) neuron in a normal rat. PSTH (50 trials) in response to the single pulse stimulation of the Cx (arrow)
(1) and PSTH in response to Cx stimulation during STN-DBS (2) are shown. The cortically evoked early and late excitation was abolished during STN-DBS
while cortically evoked inhibition was preserved. Modified from Maurice et al
(2003). (B) Effects of STN blocking on cortically evoked responses of a GPi FIGURE 4 | Both STN-DBS and STN blocking disrupt information flow
through the STN. (A) Effects of local STN-DBS on cortically evoked
responses of a substantia nigra pars reticulata (SNr) neuron in a normal rat. PSTH (50 trials) in response to the single pulse stimulation of the Cx (arrow)
(1) and PSTH in response to Cx stimulation during STN-DBS (2) are shown. The cortically evoked early and late excitation was abolished during STN-DBS,
while cortically evoked inhibition was preserved. Modified from Maurice et al. (2003). REFERENCES FIGURE 5 | Mechanism underling effectiveness of deep brain
stimulation (DBS). DBS activates axon terminals in the stimulated nucleus
and induces release of large amount of neurotransmitters, such as GABA
and glutamate, and dissociates inputs and outputs in the stimulated
nucleus, resulting in disruption of abnormal information flow through the
cortico-basal ganglia loop. GURE 5 | Mechanism underling effectiveness of deep brain FIGURE 5 | Mechanism underling effectiveness of deep brain
stimulation (DBS). DBS activates axon terminals in the stimulated nu
and induces release of large amount of neurotransmitters, such as GA
and glutamate, and dissociates inputs and outputs in the stimulated
nucleus, resulting in disruption of abnormal information flow through th
cortico-basal ganglia loop. Abosch, A., Kapur, S., Lang, A. E., Hussey, D., Sime, E., Miyasaki, J., et al. (2003). Stimulation of the subthalamic nucleus in Parkinson’s disease does not produce
striatal dopamine release. Neurosurgery 53, 1095–1105. doi: 10.1227/01.neu. 0000088662.69419.1b Anderson, T. R., Hu, B., Iremonger, K., and Kiss, Z. H. (2006). Selective atten-
uation of afferent synaptic transmission as a mechanism of thalamic deep
brain stimulation-induced tremor arrest. J. Neurosci. 26, 841–850. doi: 10. 1523/jneurosci.3523-05.2006 Anderson, M. E., Postupna, N., and Ruffo, M. (2003). Effects of high-frequency
stimulation in the internal globus pallidus on the activity of thalamic neurons in
the awake monkey. J. Neurophysiol. 89, 1150–1160. doi: 10.1152/jn.00475.2002 Cortically evoked early and late excitation was totally abolished
during high intensity STN-HFS, and much reduced during
low intensity STN-DBS, while cortically evoked inhibition
was preserved (Figure 4A), suggesting that information flow
through the trans-STN pathway was blocked by STN-DBS
without interrupting other pathways. The response patterns
of SNr neurons during STN-DBS are similar to those of GPi
neurons during STN blockade by muscimol in normal monkeys
(Nambu et al., 2000; Figure 4B). Thus it is rational that STN-
DBS has similar effect to lesion or silencing of the STN. In
Parkinson’s disease, due to the loss of dopaminergic modulation,
the information flow through the striato-GPi direct pathway is
weakened, whereas the information flow through the striato-GPe
indirect pathway is facilitated. Both STN-DBS and STN lesioning
may alter the balance of inhibitory inputs through the direct
pathway and excitatory inputs through the hyperdirect and
indirect pathways to the GPi by disrupting information flow
through the STN, and effectively alleviate bradykinesia seen in
Parkinson’s disease. ACKNOWLEDGMENTS This work was supported by a Grant-in-Aid for Scientific
Research (A) 21240039, the Core Research for Evolutional Sci-
ence and Technology, Strategic Japanese-German Cooperative
Programme, and Brain Machine Interface Development under the
Strategic Research Program for Brain Sciences to Atsushi Nambu,
and a Grant-in-Aid for Scientific Research (C) 25430021 to
Satomi Chiken. We thank Kana Miyamoto, Shigeki Sato, Hitomi
Isogai, and Keiko Matsuzawa for technical assistance. DEEP BRAIN STIMULATION (DBS) DISRUPTS NEURONAL
TRANSMISSION The striatum and STN are input stations of the basal ganglia and
receive inputs from a wide area of the cerebral cortex (Mink, 1996;
Nambu et al., 2002). The information is processed through the
hyperdirect, direct, and indirect pathways and reaches the GPi/SNr,
the output station of the basal ganglia (Figure 3A). During
voluntary movements, neuronal signals originating in the cortex
are considered to be transmitted through these pathways, reach
the GPi/SNr and control movements (Mink, 1996; Nambu et al.,
2002). Signal transmission through the direct pathway reduces
GPi activity and facilitates movements by disinhibiting the tha-
lamus, whereas the hyperdirect and indirect pathways increase GPi
activity and suppress movements (Nambu et al., 2002; Nambu,
2007; Kravitz et al., 2010; Sano et al., 2013). STN-DBS may also interrupt neurotransmission of abnormal
signals. Maurice et al. (2003) examined the effects of STN-DBS
on cortically evoked responses of SNr neurons in normal rats. March 2014 | Volume 8 | Article 33 | 5 Frontiers in Systems Neuroscience www.frontiersin.org Disrupting neuronal transmission: mechanism of DBS? Chiken and Nambu DBS produces similar therapeutic effects to lesions or silencing of
the nucleus. FIGURE 5 | Mechanism underling effectiveness of deep brain
stimulation (DBS). DBS activates axon terminals in the stimulated nucleus
and induces release of large amount of neurotransmitters, such as GABA
and glutamate, and dissociates inputs and outputs in the stimulated
nucleus, resulting in disruption of abnormal information flow through the
cortico-basal ganglia loop. REFERENCES Similar idea, a functional disconnection
of the stimulated elements, has also proposed by other groups
(Anderson et al., 2006; Deniau et al., 2010; Moran et al., 2011). Aziz, T. Z., Peggs, D., Sambrook, M. A., and Crossman, A. R. (1991). Lesion
of the subthalamic nucleus for the alleviation of 1-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine (MPTP)-induced parkinsonism in the primate. Mov. Disord. 6, 288–292. doi: 10.1002/mds.870060404 Bar-Gad, I., Elias, S., Vaadia, E., and Bergman, H. (2004). Complex locking
rather than complete cessation of neuronal activity in the globus pallidus of
a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated primate in response to
pallidal microstimulation. J. Neurosci. 24, 7410–7419. doi: 10.1523/jneurosci. 1691-04.2004 Bekar, L., Libionka, W., Tian, G. F., Xu, Q., Torres, A., Wang, X., et al. (2008). Adenosine is crucial for deep brain stimulation-mediated attenuation of tremor. Nat. Med. 14, 75–80. doi: 10.1038/nm1693 Benabid, A. L., Pollak, P., Gao, D., Hoffmann, D., Limousin, P., Gay, E., et al. (1996). Chronic electrical stimulation of the ventralis intermedius nucleus of
the thalamus as a treatment of movement disorders. J. Neurosurg. 84, 203–214. doi: 10.3171/jns.1996.84.2.0203 Benabid, A. L., Pollak, P., Gervason, C., Hoffmann, D., Gao, D. M., Hommel, M.,
et al. (1991). Long-term suppression of tremor by chronic stimulation of the
ventral intermediate thalamic nucleus. Lancet 337, 403–406. doi: 10.1016/0140-
6736(91)91175-t Benabid, A. L., Pollak, P., Gross, C., Hoffmann, D., Benazzouz, A., Gao, D. M.,
et al. (1994). Acute and long-term effects of subthalamic nucleus stimula-
tion in Parkinson’s disease. Stereotact. Funct. Neurosurg. 62, 76–84. doi: 10. 1159/000098600 Benazzouz, A., Gross, C., Féger, J., Boraud, T., and Bioulac, B. (1993). Reversal
of rigidity and improvement in motor performance by subthalamic high-
frequency stimulation in MPTP-treated monkeys. Eur. J. Neurosci. 5, 382–389. doi: 10.1111/j.1460-9568.1993.tb00505.x Frontiers in Systems Neuroscience CONCLUSION M., Degos, B., Bosch, C., and Maurice, N. (2010). Deep brain stimulation
mechanisms: beyond the concept of local functional inhibition. Eur. J. Neurosci. 32, 1080–1091. doi: 10.1111/j.1460-9568.2010.07413.x Kravitz, A. V., Freeze, B. S., Parker, P. R. L., Kay, K., Thwin, M. T., Deisseroth,
K., et al. (2010). Regulation of parkinsonian motor behaviours by optogenetic
control of basal ganglia circuitry. Nature 466, 622–626. doi: 10.1038/nature0
9159 Dostrovsky, J. O., Levy, R., Wu, J. P., Hutchison, W. D., Tasker, R. R., and Lozano,
A. M. (2000). Microstimulation-induced inhibition of neuronal firing in human
globus pallidus. J. Neurophysiol. 84, 570–574. Dostrovsky, J. O., and Lozano, A. M. (2002). Mechanisms of deep brain stimulation. Mov. Disord. 17, S63–S68. doi: 10.1002/mds.10143 Kringelbach, M. L., Jenkinson, N., Owen, S. L., and Aziz, T. Z. (2007). Translational
principles of deep brain stimulation. Nat. Rev. Neurosci. 8, 623–635. doi: 10. 1038/nrn2196 Erez, Y., Czitron, H., McCairn, K., Belelovsky, K., and Bar-Gad, I. (2009). Short-term depression of synaptic transmission during stimulation in the
globus
pallidus
of
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated
primates. J. Neurosci. 29,
7797–7802. doi:
10.1523/jneurosci.0401-09. 2009 Lafreniere-Roula, M., Kim, E., Hutchison, W. D., Lozano, A. M., Hodaie, M., and
Dostrovsky, J. O. (2010). High-frequency microstimulation in human globus
pallidus and substantia nigra. Exp. Brain Res. 205, 251–261. doi: 10.1007/s00221-
010-2362-8 Fellin, T., Pascual, O., and Haydon, P. G. (2006). Astrocytes coordinate synaptic
networks: balanced excitation and inhibition. Physiology (Bethesda) 21, 208–
215. doi: 10.1152/physiol.00161.2005 Leblois, A., Reese, R., Labarre, D., Hamann, M., Richter, A., Boraud, T., et al. (2010). Deep brain stimulation changes basal ganglia output nuclei firing pattern in
the dystonic hamster. Neurobiol. Dis. 38, 288–298. doi: 10.1016/j.nbd.2010. 01.020 Filali, M., Hutchison, W. D., Palter, V. N., Lozano, A. M., and Dostrovsky,
J. O. (2004). Stimulation-induced inhibition of neuronal firing in human
subthalamic nucleus. Exp. Brain Res. 156, 274–281. doi: 10.1007/s00221-003-
1784-y Lee, K. H., Chang, S. Y., Roberts, D. W., and Kim, U. (2004). Neurotransmitter
release from high-frequency stimulation of the subthalamic nucleus. J. Neuro-
surg. 101, 511–517. doi: 10.3171/jns.2004.101.3.0511 Galati, S., Mazzone, P., Fedele, E., Pisani, A., Peppe, A., Pierantozzi, M., et al. (2006). Biochemical and electrophysiological changes of substantia nigra
pars reticulata driven by subthalamic stimulation in patients with Parkin-
son’s disease. Eur. J. Neurosci. 23, 2923–2928. doi: 10.1111/j.1460-9568.2006. 04816.x Levy, R., Lang, A. E., Dostrovsky, J. O., Pahapill, P., Romas, J., Saint-Cyr, J., et al. (2001). CONCLUSION DBS has variety of effects on neurons in the stimulated nucleus
of the cortico-basal ganglia loop, though transmitter release,
orthodromic activation of efferent axons, antidromic activation
of afferent axons, and direct stimulation of passing axons nearby
the stimulating electrode. The effects vary depending on the
neural composition of the stimulated nucleus, and the effects
extend much wider than originally expected. However, a common
mechanism would underlie the effectiveness of DBS: DBS dissoci-
ates inputs and outputs in the stimulated nucleus and disrupts
abnormal information flow through the cortico-basal ganglia
loop (Figure 5). The mechanism may explain the paradox that Bergman, H., Feingold, A., Nini, A., Raz, A., Slovin, H., Abeles, M., et al. (1998). Physiological aspects of information processing in the basal ganglia of normal
and parkinsonian primates. Trends Neurosci. 21, 32–38. doi: 10.1016/S0166-
2236(97)01151-X Bergman, H., Wichmann, T., and DeLong, M. R. (1990). Reversal of experimental
parkinsonism by lesions of the subthalamic nucleus. Science 249, 1436–1438. doi: 10.1126/science.2402638 Beurrier, C., Bioulac, B., Audin, J., and Hammond, C. (2001). High-frequency stim-
ulation produces a transient blockade of voltage-gated currents in subthalamic
neurons. J. Neurophysiol. 85, 1351–1356. Boertien, T., Zrinzo, L., Kahan, J., Jahanshahi, M., Hariz, M., Mancini, L., et al. (2011). Functional imaging of subthalamic nucleus deep brain stimulation in
Parkinson’s disease. Mov. Disord. 26, 1835–1843. doi: 10.1002/mds.23788 Frontiers in Systems Neuroscience March 2014 | Volume 8 | Article 33 | 6 www.frontiersin.org Disrupting neuronal transmission: mechanism of DBS? Chiken and Nambu Hashimoto, T., Elder, C. M., and Vitek, J. L. (2002). A template subtrac-
tion method for stimulus artifact removal in high-frequency deep brain
stimulation. J. Neurosci. Methods 113, 181–186. doi: 10.1016/s0165-0270(01)
00491-5 Boraud, T., Bezard, E., Bioulac, B., and Gross, C. (1996). High frequency stimula-
tion of the internal globus pallidus (GPi) simultaneously improves parkinsonian
symptoms and reduces the firing frequency of GPi neurons in the MPTP-treated
monkey. Neurosci. Lett. 215, 17–20. doi: 10.1016/s0304-3940(96)12943-8 Hershey, T., Revilla, F. J., Wernle, A. R., McGee-Minnich, L., Antenor, J. V.,
Videen, T. O., et al. (2003). Cortical and subcortical blood flow effects of
subthalamic nucleus stimulation in PD. Neurology 61, 816–821. doi: 10.1212/01. wnl.0000083991.81859.73 Brotchie, J. M., Mitchell, I. J., Sambrook, M. A., and Crossman, A. R. (1991). Alle-
viation of parkinsonism by antagonism of excitatory amino acid transmission
in the medial segment of the globus pallidus in rat and primate. Mov. Disord. 6,
133–138. doi: 10.1002/mds.870060208 Brown, P. (2007). CONCLUSION Abnormal oscillatory synchronisation in the motor system
leads to impaired movement. Curr. Opin. Neurobiol. 17, 656–664. doi: 10. 1016/j.conb.2007.12.001 Hilker, R., Voges, J., Ghaemi, M., Lehrke, R., Rudolf, J., Koulousakis, A., et al. (2003). Deep brain stimulation of the subthalamic nucleus does not increase
the striatal dopamine concentration in parkinsonian humans. Mov. Disord. 18,
41–48. doi: 10.1002/mds.10297 Chiken, S., and Nambu, A. (2013). High-frequency pallidal stimulation disrupts
information flow through the pallidum by GABAergic inhibition. J. Neurosci. 33, 2268–2280. doi: 10.1523/jneurosci.4144-11.2013 Jantz, J. J., and Watanabe, M. (2013). Pallidal deep brain stimulation modulates
afferent fibers, efferent fibers and glia. J. Neurosci. 33, 9873–9875. doi: 10. 1523/jneurosci.1471-13.2013 Chiken, S., Shashidharan, P., and Nambu, A. (2008). Cortically-evoked long-lasting
inhibition of pallidal neurons in a transgenic mouse model of dystonia. J. Neurosci. 28, 13967–13977. doi: 10.1523/jneurosci.3834-08.2008 Jech, R., Urgosík, D., Tintera, J., Nebuzelský, A., Krásenský, J., Liscák, R., et al. (2001). Functional magnetic resonance imaging during deep brain stimulation:
a pilot study in four patients with Parkinson’s disease. Mov. Disord. 16, 1126–
1132. doi: 10.1002/mds.1217 Coubes, P., Cif, L., El Fertit, H., Hemm, S., Vayssiere, N., Serrat, S., et al. (2004). Electrical stimulation of the globus pallidus internus in patients with primary
generalized dystonia: long-term results. J. Neurosurg. 101, 189–194. doi: 10. 3171/jns.2004.101.2.0189 Johnson, M. D., and McIntyre, C. C. (2008). Quantifying the neural elements acti-
vated and inhibited by globus pallidus deep brain stimulation. J. Neurophysiol. 100, 2549–2563. doi: 10.1152/jn.90372.2008 Deep-Brain Stimulation for Parkinson’s Disease Study Group. (2001). Deep-
brain stimulation of the subthalamic nucleus or the pars interna of the
globus pallidus in Parkinson’s disease. N. Engl. J. Med. 345, 956–963. doi: 10. 1056/nejmoa000827 Johnson, M. D., Miocinovic, S., McIntyre, C. C., and Vitek, J. L. (2008). Mecha-
nisms and targets of deep brain stimulation in movement disorders. Neurother-
apeutics 5, 294–308. doi: 10.1016/j.nurt.2008.01.010 Degos, B., Deniau, J. M., Chavez, M., and Maurice, N. (2013). Subthalamic nucleus
high-frequency stimulation restores altered electrophysiological properties of
cortical neurons in parkinsonian rat. PLoS One 8:e83608. doi: 10.1371/journal. pone.0083608 Kita, H., and Kita, T. (2011). Cortical stimulation evokes abnormal responses in
the dopamine-depleted rat basal ganglia. J. Neurosci. 31, 10311–10322. doi: 10. 1523/jneurosci.0915-11.2011 Koller, W., Pahwa, R., Busenbark, K., Hubble, J., Wilkinson, S., Lang, A., et al. (1997). High-frequency unilateral thalamic stimulation in the treatment of
essential and parkinsonian tremor. Ann. Neurol. 42, 292–299. doi: 10.1002/ana. 410420304 Deniau, J. CONCLUSION Effects of GPi stimulation on human thalamic
neuronal activity. Clin. Neurophysiol. 117, 2691–2702. doi: 10.1016/j.clinph. 2006.08.011 Starr, P. A., Rau, G. M., Davis, V., Marks, W. J. Jr., Ostrem, J. L., Simmons, D., et al. (2005). Spontaneous pallidal neuronal activity in human dystonia: comparison
with Parkinson’s disease and normal macaque. J. Neurophysiol. 93, 3165–3176. doi: 10.1152/jn.00971.2004 Moran, A., Stein, E., Tischler, H., Belelovsky, K., and Bar-Gad, I. (2011). Dynamic
stereotypic responses of Basal Ganglia neurons to subthalamic nucleus high-
frequency stimulation in the parkinsonian primate. Front. Syst. Neurosci. 5:21. doi: 10.3389/fnsys.2011.00021 Stefani, A., Fedele, E., Galati, S., Pepicelli, O., Frasca, S., Pierantozzi, M., et al. (2005). Subthalamic stimulation activates internal pallidus: evidence from
cGMP microdialysis in PD patients. Ann. Neurol. 57, 448–452. doi: 10.1002/ana. 20402 Nambu, A. (2007). Globus pallidus internal segment. Prog. Brain. Res. 160, 135–
150. doi: 10.1016/s0079-6123(06)60008-3 Nambu, A., Tokuno, H., and Takada, M. (2002). Functional significance of the
cortico-subthalamo-pallidal ‘hyperdirect’ pathway. Neurosci. Res. 43, 111–117. doi: 10.1016/s0168-0102(02)00027-5 Tachibana, Y., Iwamuro, H., Kita, H., Takada, M., and Nambu, A. (2011). Subthalamo-pallidal interactions underlying parkinsonian neuronal oscillations
in the primate basal ganglia. Eur. J. Neurosci. 34, 1470–1484. doi: 10.1111/j.1460-
9568.2011.07865.x Nambu, A., Tokuno, H., Hamada, I., Kita, H., Imanishi, M., Akazawa, T., et al. (2000). Excitatory cortical inputs to pallidal neurons via the subthalamic
nucleus in the monkey. J. Neurophysiol. 84, 289–300. Tai, C. H., Boraud, T., Bezard, E., Bioulac, B., Gross, C., and Benazzouz, A. (2003). Electrophysiological and metabolic evidence that high-frequency stimulation of
the subthalamic nucleus bridles neuronal activity in the subthalamic nucleus
and the substantia nigra reticulata. FASEB J. 17, 1820–1830. doi: 10.1096/fj.03-
0163com Nishibayashi, H., Ogura, M., Kakishita, K., Tanaka, S., Tachibana, Y., Nambu, A.,
et al. (2011). Cortically evoked responses of human pallidal neurons recorded
during stereotactic neurosurgery. Mov. Disord. 26, 469–476. doi: 10.1002/mds. 23502 Ostrem, J. L., and Starr, P. A. (2008). Treatment of dystonia with deep
brain stimulation. Neurotherapeutics 5, 320–330. doi: 10.1016/j.nurt.2008. 01.002 Tawfik, V. L., Chang, S. Y., Hitti, F. L., Roberts, D. W., Leiter, J. C., Jovanovic, S., et al. (2010). Deep brain stimulation results in local glutamate and adenosine release:
investigation into the role of astrocytes. Neurosurgery 67, 367–375. doi: 10. 1227/01.neu.0000371988.73620.4c Pralong, E., Debatisse, D., Maeder, M., Vingerhoets, F., Ghika, J., and Villemure,
J. G. (2003). Effect of deep brain stimulation of GPI on neuronal activity of the
thalamic nucleus ventralis oralis in a dystonic patient. CONCLUSION Lidocaine and muscimol microinjections in subthalamic nucleus reverse
Parkinsonian symptoms. Brain 124, 2105–2118. doi: 10.1093/brain/124.10. 2105 Gradinaru, V., Mogri, M., Thompson, K. R., Henderson, J. M., and Deisseroth,
K. (2009). Optical deconstruction of parkinsonian neural circuitry. Science 324,
354–359. doi: 10.1126/science.1167093 Li, S., Arbuthnott, G. W., Jutras, M. J., Goldberg, J. A., and Jaeger, D. (2007). Resonant antidromic cortical circuit activation as a consequence of high-
frequency subthalamic deep-brain stimulation. J. Neurophysiol. 98, 3525–3537. doi: 10.1152/jn.00808.2007 Graham, W. C., Robertson, R. G., Sambrook, M. A., and Crossman, A. R. (1990). Injection of excitatory amino acid antagonists into the medial pallidal segment
of a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treated primate
reverses motor symptoms of parkinsonism. Life Sci. 47, PL91–PL97. doi: 10. 1016/0024-3205(90)90376-3 j
Li, Q., Ke, Y., Chan, D. C., Qian, Z. M., Yung, K. K., Ko, H., et al. (2012). Therapeutic
deep brain stimulation in Parkinsonian rats directly influences motor cortex. Neuron 76, 1030–1041. doi: 10.1016/j.neuron.2012.09.032 Limousin, P., Pollak, P., Benazzouz, A., Hoffmann, D., Le Bas, J. F., Broussolle,
E., et al. (1995). Effect of parkinsonian signs and symptoms of bilateral sub-
thalamic nucleus stimulation. Lancet 345, 91–95. doi: 10.1016/s0140-6736(95)
90062-4 Hammond, C., Bergman, H., and Brown, P. (2007). Pathological synchronization
in Parkinson’s disease: networks, models and treatments. Trends Neurosci. 30,
357–364. doi: 10.1016/j.tins.2007.05.004 Hashimoto, T., Elder, C. M., Okun, M. S., Patrick, S. K., and Vitek, J. L. (2003). Stimulation of the subthalamic nucleus changes the firing pattern of pallidal
neurons. J. Neurosci. 23, 1916–1923. Liu, Y., Postupna, N., Falkenberg, J., and Anderson, M. E. (2008). High frequency
deep brain stimulation: what are the therapeutic mechanisms? Neurosci. Biobe-
hav. Rev. 32, 343–351. doi: 10.1016/j.neubiorev.2006.10.007 March 2014 | Volume 8 | Article 33 | 7 Frontiers in Systems Neuroscience www.frontiersin.org Disrupting neuronal transmission: mechanism of DBS? Chiken and Nambu Maurice, N., Thierry, A. M., Glowinski, J., and Deniau, J. M. (2003). Sponta-
neous and evoked activity of substantia nigra pars reticulata neurons during
high-frequency stimulation of the subthalamic nucleus. J. Neurosci. 23, 9929–
9936. Shi, L. H., Luo, F., Woodward, D. J., and Chang, J. Y. (2006). Basal ganglia
neural responses during behaviorally effective deep brain stimulation of the
subthalamic nucleus in rats performing a treadmill locomotion test. Synapse 59,
445–457. doi: 10.1002/syn.20261 McCairn, K. W., and Turner, R. S. (2009). Deep brain stimulation of the globus pal-
lidus internus in the parkinsonian primate: local entrainment and suppression
of low-frequency oscillations. J. CONCLUSION Neurophysiol. 101, 1941–1960. doi: 10.1152/jn. 91092.2008 Shin, D. S., Samoilova, M., Cotic, M., Zhang, L., Brotchie, J. M., and Carlen, P. L. (2007). High frequency stimulation or elevated K+ depresses neuronal activity
in the rat entopeduncular nucleus. Neuroscience 149, 68–86. doi: 10.1016/j. neuroscience.2007.06.055 McIntyre, C. C., Savasta, M., Walter, B. L., and Vitek, J. L. (2004). How
does deep brain stimulation work? Present understanding and future ques-
tions. J. Clin. Neurophysiol. 21, 40–50. doi: 10.1097/00004691-200401000-
00006 Shink, E., and Smith, Y. (1995). Differential synaptic innervation of neurons in
the internal and external segments of the globus pallidus by the GABA- and
glutamate-containing terminals in the squirrel monkey. J. Comp. Neurol. 358,
119–141. doi: 10.1002/cne.903580108 Meissner, W., Harnack, D., Reese, R., Paul, G., Reum, T., Ansorge, M., et al. (2003). High-frequency stimulation of the subthalamic nucleus enhances stri-
atal dopamine release and metabolism in rats. J. Neurochem. 85, 601–609. doi: 10.1046/j.1471-4159.2003.01665.x Shon, Y. M., Lee, K. H., Goerss, S. J., Kim, I. Y., Kimble, C., Van Gompel,
J. J., et al. (2010). High frequency stimulation of the subthalamic nucleus
evokes striatal dopamine release in a large animal model of human
DBS neurosurgery. Neurosci. Lett. 475, 136–140. doi: 10.1016/j.neulet.2010. 03.060 Meissner, W., Leblois, A., Hansel, D., Bioulac, B., Gross, C. E., Benazzouz,
A., et al. (2005). Subthalamic high frequency stimulation resets subthala-
mic firing and reduces abnormal oscillations. Brain 128, 2372–2382. doi: 10. 1093/brain/awh616 Siegfried, J., and Lippitz, B. (1994a). Bilateral chronic electrostimulation of
ventroposterolateral pallidum: a new therapeutic approach for alleviating all
parkinsonian symptoms. Neurosurgery 35, 1126–1130. doi: 10.1227/00006123-
199412000-00016 Mink, J. W. (1996). The basal ganglia: focused selection and inhibition of com-
peting motor programs. Prog. Neurobiol. 50, 381–425. doi: 10.1016/s0301-
0082(96)00042-1 Siegfried, J., and Lippitz, B. (1994b). Chronic electrical stimulation of the VL-
VPL complex and of the pallidum in the treatment of movement disorders:
personal experience since 1982. Stereotact. Funct. Neurosurg. 62, 71–75. doi: 10. 1159/000098599 Miocinovic, S., Parent, M., Butson, C. R., Hahn, P. J., Russo, G. S., Vitek, J. L., et al. (2006). Computational analysis of subthalamic nucleus and lenticular fasciculus
activation during therapeutic deep brain stimulation. J. Neurophysiol. 96, 1569–
1580. doi: 10.1152/jn.00305.2006 Smith, Y., Wichmann, T., and DeLong, M. R. (1994). Synaptic innervation of
neurones in the internal pallidal segment by the subthalamic nucleus and the
external pallidum in monkeys. J. Comp. Neurol. 343, 297–318. doi: 10.1002/cne. 903430209 Montgomery, E. B. Jr. (2006). CONCLUSION Neurophysiol. Clin. 33,
169–173. doi: 10.1016/j.neucli.2003.07.001 Vedam-Mai, V., van Battum, E. Y., Kamphuis, W., Feenstra, M. G., Denys, D.,
Reynolds, B. A., et al. (2012). Deep brain stimulation and the role of astrocytes. Mol. Psychiatry 17, 124–131. doi: 10.1038/mp.2011.61 Vidailhet, M., Jutras, M. F., Roze, E., and Grabli, D. (2013). Deep brain stimulation
for dystonia. Handb. Clin. Neurol. 116, 167–187. doi: 10.1016/B978-0-444-
53497-2.00014-0 Ranck, J. B. Jr. (1975). Which elements are excited in electrical stimulation of
mammalian central nervous system: a review. Brain Res. 98, 417–440. doi: 10. 1016/0006-8993(75)90364-9 Vitek, J. L. (2008). Deep brain stimulation: how does it work? Cleve. Clin. J. Med. 75(Suppl. 2), S59–S65. doi: 10.3949/ccjm.75.suppl_2.s59 Reese, R., Leblois, A., Steigerwald, F., Potter-Nerger, M., Herzog, J., Mehdorn,
H. M., et al. (2011). Subthalamic deep brain stimulation increases pallidal firing
rate and regularity. Exp. Neurol. 229, 517–521. doi: 10.1016/j.expneurol.2011. 01.020 Wang, L. Y., and Kaczmarek, L. K. (1998). High-frequency firing helps replenish
the readily releasable pool of synaptic vesicles. Nature 394, 384–388. doi: 10. 1038/28645 Rehncrona, S., Johnels, B., Widner, H., Törnqvist, A. L., Hariz, M., and
Sydow, O. (2003). Long-term efficacy of thalamic deep brain stimulation for
tremor: double-blind assessments. Mov. Disord. 18, 163–170. doi: 10.1002/mds. 10309 Welter, M. L., Houeto, J. L., Bonnet, A. M., Bejjani, P. B., Mesnage, V., Dormont,
D., et al. (2004). Effects of high-frequency stimulation on subthalamic neuronal
activity in parkinsonian patients. Arch. Neurol. 61, 89–96. doi: 10.1001/archneur. 61.1.89 Sano, H., Chiken, S., Hikida, T., Kobayashi, K., and Nambu, A. (2013). Signals
through the striatopallidal indirect pathway stop movements by phasic excita-
tion in the substantia nigra. J. Neurosci. 33, 7583–7594. doi: 10.1523/jneurosci. 4932-12.2013 Wichmann, T. (2000). A digital averaging method for removal of stimulus artifacts
in neurophysiologic experiments. J. Neurosci. Methods 98, 57–62. doi: 10. 1016/s0165-0270(00)00190-4 March 2014 | Volume 8 | Article 33 | 8 Frontiers in Systems Neuroscience www.frontiersin.org Disrupting neuronal transmission: mechanism of DBS? Chiken and Nambu Zucker, R. S., and Regehr, W. G. (2002). Short-term synaptic plasticity. Annu. Rev. Physiol. 64, 355–405. doi: 10.1146/annurev.physiol.64.092501.11
4547 Wichmann, T., Bergman, H., and DeLong, M. R. (1994). The primate subthalamic
nucleus. III. Changes in motor behavior and neuronal activity in the internal
pallidum induced by subthalamic inactivation in the MPTP model of parkin-
sonism. J. Neurophysiol. 72, 521–530. March 2014 | Volume 8 | Article 33 | 9 CONCLUSION Conflict of Interest Statement: The authors declare that the research was conducted
in the absence of any commercial or financial relationships that could be construed
as a potential conflict of interest. Wichmann, T., and Delong, M. R. (2006). Deep brain stimulation for neurologic
and neuropsychiatric disorders. Neuron 52, 197–204. doi: 10.1016/j.neuron. 2006.09.022 Windels, F., Bruet, N., Poupard, A., Urbain, N., Chouvet, G., Feuerstein, C.,
et al. (2000). Effects of high frequency stimulation of subthalamic nucleus on
extracellular glutamate and GABA in substantia nigra and globus pallidus in
the normal rat. Eur. J. Neurosci. 12, 4141–4146. doi: 10.1046/j.1460-9568.2000. 00296.x Received: 26 August 2013; accepted: 19 February 2014; published online: 14 March
2014. Received: 26 August 2013; accepted: 19 February 2014; published online: 14 March
2014. Citation: Chiken S and Nambu A (2014) Disrupting neuronal transmission:
mechanism of DBS? Front. Syst. Neurosci. 8:33. doi: 10.3389/fnsys.2014.00033
This article was submitted to the journal Frontiers in Systems Neuroscience. Citation: Chiken S and Nambu A (2014) Disrupting neuronal transmission:
mechanism of DBS? Front. Syst. Neurosci. 8:33. doi: 10.3389/fnsys.2014.00033
This article was submitted to the journal Frontiers in Systems Neuroscience. Windels, F., Carcenac, C., Poupard, A., and Savasta, M. (2005). Pallidal origin of
GABA release within the substantia nigra pars reticulata during high-frequency
stimulation of the subthalamic nucleus. J. Neurosci. 25, 5079–5086. doi: 10. 1523/jneurosci.0360-05.2005 Copyright © 2014 Chiken and Nambu. This is an open-access article distributed under
the terms of the Creative Commons Attribution License (CC BY). The use, distribution
or reproduction in other forums is permitted, provided the original author(s) or licensor
are credited and that the original publication in this journal is cited, in accordance with
accepted academic practice. No use, distribution or reproduction is permitted which
does not comply with these terms. Wu, Y. R., Levy, R., Ashby, P., Tasker, R. R., and Dostrovsky, J. O. (2001). Does
stimulation of the GPi control dyskinesia by activating inhibitory axons? Mov. Disord. 16, 208–216. doi: 10.1002/mds.1046 March 2014 | Volume 8 | Article 33 | 9 Frontiers in Systems Neuroscience Frontiers in Systems Neuroscience www.frontiersin.org www.frontiersin.org
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Comparison of Volatile and Nonvolatile Compounds in Rice Fermented by Different Lactic Acid Bacteria
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Molecules/Molecules online/Molecules annual
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Academic Editors: Michael C. Qian and Yanping L. Qian Received: 15 February 2019; Accepted: 24 March 2019; Published: 26 March 2019 Abstract: The production of rice-based beverages fermented by lactic acid bacteria (LAB) can
increase the consumption of rice in the form of a dairy replacement. This study investigated
volatile and nonvolatile components in rice fermented by 12 different LABs. Volatile compounds
of fermented rice samples were analyzed using gas chromatography-mass spectrometry (GC-MS)
combined with solid-phase microextraction (SPME), while nonvolatile compounds were determined
using gas chromatography-time-of-flight/mass spectrometry (GC-TOF/MS) after derivatization. The 47 identified volatile compounds included acids, aldehydes, esters, furan derivatives, ketones,
alcohols, benzene and benzene derivatives, hydrocarbons, and terpenes, while the 37 identified
nonvolatile components included amino acids, organic acids, and carbohydrates. The profiles
of volatile and nonvolatile components generally differed significantly between obligatorily
homofermentative/facultatively heterofermentative LAB and obligatorily heterofermentative
LAB. The rice sample fermented by Lactobacillus sakei (RTCL16) was clearly differentiated from
the other samples on principal component analysis (PCA) plots. The results of PCA revealed that the
rice samples fermented by LABs could be distinguished according to microbial strains. Keywords: lactic acid bacteria; volatile compounds; nonvolatile compounds; fermented rice molecules molecules molecules Comparison of Volatile and Nonvolatile Compounds
in Rice Fermented by Different Lactic Acid Bacteria Sang Mi Lee 1, Young Rim Hwang 1, Moon Seok Kim 2, Myung Sub Chung 3 and
1 Sang Mi Lee 1, Young Rim Hwang 1, Moon Seok Kim 2, Myung Sub Chung 3 and
Young-Suk Kim 1,* 1
Department of Food Science and Engineering, Ewha Womans University, Seoul 120-750, Korea;
smlee78@ewha.ac.kr (S.M.L.); hhyr9@naver.com (Y.R.H.) 1
Department of Food Science and Engineering, Ewha Womans University, Seoul 120-750, Korea;
smlee78@ewha.ac.kr (S.M.L.); hhyr9@naver.com (Y.R.H.) p
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;
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3
Department of Food Science and Technology, Chung Ang University, 4726 Seodongdae-ro, Daeduk-myun
Ansung, Gyungki-do 17546, Korea; chungms@cau.ac.kr g,
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,
;
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*
Correspondence: yskim10@ewha.ac.kr; Tel.: +82-2-32773091; Fax: +82-2-32774213 *
Correspondence: yskim10@ewha.ac.kr; Tel.: +82-2-32773091; Fax: +82-2-32774213 Molecules 2019, 24, 1183; doi:10.3390/molecules24061183 www.mdpi.com/journal/molecules 1. Introduction The grains such as rice, wheat, oats, malt, and barley are widely used as ingredients for improving
the functional properties in diverse foods [1,2]. In particular, grain-based probiotic functional foods
are becoming increasingly popular and have considerable potential as dairy replacements [2,3]. Grains can be used as substrates to produce fermented foods by lactic acid bacteria (LAB) [4,5]. Several studies have demonstrated the fermentation of LAB in grain substrates [4–8]. Marklinder
and Johansson found that grain-based nondairy products were suitable for the growth of probiotic
LAB strains for fermentation [6]. Angelov et al. utilized whole grains as a substrate for fermenting
LAB to produce a grain-based beverage [4]. The role of LAB in rice fermentation has also been
reported [8–11]. Steamed breads such as idli and puto, which are made with rice, are fermented by
Leuconostoc strains, with Leuconostoc mesenteroides in particular initiating relatively rapid growth in
rice [8,9]. Chicha and Haria, which are rice-based ethnic fermented beverages of east and central India
and Brazil, are fermented simultaneously by LAB and yeast [10,11]. Fermentation by LAB can change the taste and aroma of substrates and has been used to improve
the organoleptic quality of fermented foods [2]. LAB can produce large amounts of lactic acid via Molecules 2019, 24, 1183; doi:10.3390/molecules24061183 www.mdpi.com/journal/molecules 2 of 15 Molecules 2019, 24, 1183 diverse metabolic pathways and form volatile compounds derived from amino acids, peptides, and
fatty acids upon further bioconversions [12,13]. Some studies have reported the formation of volatile
compounds in grain substrates and grain-based products by LAB [1,14,15]. Salmerón et al. demonstrated
the formation of volatile compounds by Lactobacillus plantarum NCIMB 8826 (NCIMB 8826; National
Collection of Industrial and Marine Bacteria, UK) grown in grain substrates (wheat, oats, malt,
and barley) [14]. Those authors identified 60 compounds, with the most abundant in all grain
substrates (wheat, oats, malt, and barley) being linoleic acid, oleic acid, 5-hydroxymethylfurfural, and
acetic acid, respectively [14]. In addition, Salmerón et al. employed headspace gas chromatography
analysis to determine volatile compounds formed from barley and malt fermented by Lactobacillus
reuteri, Lac. plantarum, and Lactobacillus acidophilus [1]. They found that these Lactobacillus strains
can produce large amounts of certain major flavor compounds (acetaldehyde, ethanol, acetone,
diacetyl, and ethyl acetate), which affects the organoleptic qualities of the fermented products [1]. 1. Introduction In particular, fermentation by LAB generates characteristic aroma compounds such as acetaldehyde,
acetone, butan-2-one, butane-2,3-dione (diacetyl), and 3-hydroxybutan-2-one (acetoin) in fermented
products [15]. Butane-2,3-dione and 3-hydroxybutan-2-one, which have creamy and buttery aroma
notes, can serve as major flavor compounds in various dairy products fermented by LAB [13,16,17]. j
p
y p
y
LAB can also contribute to the formation of nonvolatile compounds that can act as precursors of
characteristic aroma compounds as well as tastants themselves in fermented products such as ripened
cheese, sourdough, butter, buttermilk, fermented vegetables, and yogurt [18]. Drinan et al. investigated
the effects of citrate on the formation of butane-2,3-dione and 3-hydroxybutan-2-one by different LAB
strains [19]. The heterofermentative LAB did not produce butane-2,3-dione or 3-hydroxybutan-2-one
in the absence of citrate, whereas all of the homofermentative LAB produced significant amounts of
butane-2,3-dione and 3-hydroxybutan-2-one in the presence of citrate. In addition, lactones can be
formed from unsaturated fatty acids such as oleic acid and linoleic acid during fermentation [13,20]. It has been reported that these lactones can contribute significantly to the flavor characteristics of
fermented rice foods such as rice beer and Chinese rice wine [13,21,22]. Rice is widely consumed as the staple food in many Asian countries and has considerable potential
as a dairy replacement via the application of fermentation. However, no study has compared the
formation of volatile and nonvolatile compounds in rice fermented by various LABs, which have
different metabolic activities. Accordingly, the present study investigated and compared the formation
of volatile and nonvolatile compounds in rice fermented by 12 different LABs. 2.1. Volatile Compounds in Fermented Rice Samples The volatile compounds in the fermented rice samples inoculated by LAB are listed in Table 1. The 45 identified volatile compounds were comprised of three acids, five aldehydes, four esters,
four furan derivatives, four ketones, 13 alcohols, 10 benzene and benzene derivatives, one hydrocarbon,
and one terpene. Esters, which have floral and fruity odor notes, are formed by esterification between alcohols and
acids during LAB metabolism [23]. The present study detected four esters (butyl acetate, ethyl acetate,
hexyl acetate, and methyl butanoate) in the fermented rice samples, although they were present at only
low levels. In particular, ethyl acetate and methyl butanoate were found in all of the samples studied. These compounds, which were formed from pyruvate metabolism, were previously identified as key
aroma compounds in fermentation products by Lactobacillus [13]. On the other hand, butyl acetate was
detected in only five samples (KR7, KR10, JKA1-6, JFK2-2, and RTCL9), whereas hexyl acetate was
observed only in samples KR7 and KR10; both of these LABs are Lactobacillus brevis strains. Esters were
generally most abundant in JKA1-6, with the largest amounts in samples fermented by obligatorily
heterofermentative LAB except for sample RTCL16. It was considered that heterofermentative LABs
produce more ethanol and acetic acid [19], which can be better used for esterification compared to
facultatively hetero-/homo- ones. 3 of 15 Molecules 2019, 24, 1183 Table 1. Volatile compounds identified in fermented rice according to strains of lactic acid bacteria (LABs). No. Compounds
RI 1
Relative Peak Area 2
ID 3
RTJL3
LPC
KR10
KR7
RTCL16
RTCL79
RTJL4
RTCL3
RTCL31
JFK2-2
JKA1-6
RTCL
ACIDS
1
acetic acid
1457
4.39 ± 0.69 ab 4
1.45 ± 0.42 a
9.99 ± 1.85 ab
13.42 ± 8.76 ab
80.20 ± 19.99 c
11.25 ± 8.77 ab
8.48 ± 4.02 ab
14.36 ± 4.06 ab
1.47 ± 0.71 a
18.36 ± 8.57 b
14.87 ± 2.23 ab
18.39 ± 6.29 b
A
2
hexanoic acid
1860
0.95 ± 0.06 g
0.45 ± 0.08 cd
0.61 ± 0.08 de
N.D. 2.1. Volatile Compounds in Fermented Rice Samples a
2.17 ± 0.29 h
0.36 ± 0.14 bc
0.48 ± 0.10 cd
0.89 ± 0.12 f
0.24 ± 0.08 b
0.78 ± 0.10 efg
0.59 ± 0.04 de
0.70 ± 0.06 ef
A
3
octanoic acid
2065
0.14 ± 0.01 b
0.34 ± 0.02 d
0.04 ± 0.01 a
0.14 ± 0.03 b
0.17 ± 0.02 bc
0.16 ± 0.05 bc
0.22 ± 0.04 c
0.49 ± 0.12 e
0.14 ± 0.01 b
0.05 ± 0.01 a
0.03 ± 0.01 a
0.43 ± 0.04 e
A
ALDEHYDES
4
2-methylbutanal
911
N.D. 5 a
N.D. a
N.D. a
N.D. a
0.64 ± 0.08 d
N.D. a
N.D. a
0.34 ± 0.02 c
0.07 ± 0.02 b
N.D. a
N.D. a
N.D. a
A
5
3-methylbutanal
915
0.42 ± 0.04 c
0.34 ± 0.05 b
N.D. a
N.D. a
N.D. a
N.D. a
N.D. a
0.58 ± 0.03 d
N.D. a
N.D. a
N.D. a
N.D. a
A
6
hexanal
1078
N.D. a
N.D. a
0.52 ± 0.34 b
0.64 ± 0.21 b
4.08 ± 0.11 d
N.D. a
N.D. a
3.02 ± 0.10 c
0.72 ± 0.04 b
0.51 ± 0.12 b
0.22 ± 0.01 a
0.19 ± 0.01 a
A
7
nonanal
1397
N.D. a
N.D. a
N.D. a
N.D. a
N.D. a
0.168 ± 0.018 b
N.D. a
N.D. a
0.218 ± 0.060 c
N.D. a
N.D. a
N.D. a
A
8
benzaldehyde
1530
2.68 ± 0.22 c
2.72 ± 0.02 c
0.40 ± 0.05 a
0.52 ± 0.06 a
6.94 ± 0.54 e
0.47 ± 0.05 a
0.24 ± 0.05 a
3.19 ± 0.04 d
2.13 ± 0.12 b
0.28 ± 0.05 a
0.33 ± 0.03 a
0.38 ± 0.03 a
A
ESTERS
9
ethyl acetate
885
0.33 ± 0.03 a
0.25 ± 0.06 a
0.66 ± 0.02 bc
0.97 ± 0.13 d
0.72 ± 0.09 c
0.34 ± 0.12 a
0.96 ± 0.02 d
0.37 ± 0.12 a
0.25 ± 0.03 a
0.70 ± 0.04 bc
1.71 ± 0.07 e
0.56 ± 0.05 b
A
10
methyl
butanoate
984
0.13 ± 0.02 a
0.13 ± 0.01 a
0.21 ± 0.02 bc
0.27 ± 0.02 d
0.43 ± 0.06 e
0.19 ± 0.02 bc
0.19 ± 0.03 bc
0.23 ± 0.02 cd
0.18 ± 0.01 ab
0.18 ± 0.04 ab
0.22 ± 0.02 bcd
0.21 ± 0.01 bc
B
11
butyl acetate
1070
N.D. a
N.D. 2.1. Volatile Compounds in Fermented Rice Samples a
0.09 ± 0.05 b
0.13 ± 0.01 c
N.D. a
N.D. a
N.D. a
N.D. a
N.D. a
0.13 ± 0.01 c
0.18 ± 0.02 d
0.16 ± 0.03 cd
B
12
hexyl acetate
1274
N.D. a
N.D. a
0.07 ± 0.01 b
0.12 ± 0.01 c
N.D. a
N.D. a
N.D. a
N.D. a
N.D. a
N.D. a
N.D. a
N.D. a
A
KETONES
13
propan-2-one
816
2.14 ± 0.43 cd
1.93 ± 0.15 c
2.69 ± 0.29 def
N.D. a
6.90 ± 0.17 g
2.45 ± 0.26 de
N.D. a
2.84 ± 0.18 ef
1.93 ± 0.14 c
1.00 ± 0.20 b
1.36 ± 0.06 b
1.13 ± 0.11 b
A
14
butan-2-one
901
0.52 ± 0.08 d
0.55 ± 0.03 d
N.D. a
N.D. a
1.57 ± 0.05 f
0.59 ± 0.03 d
N.D. a
0.69 ± 0.03 e
0.56 ± 0.02 d
0.39 ± 0.02 c
0.38 ± 0.08 c
0.29 ± 0.07 b
A
15
butane-2,3-dione
975
67.10 ± 3.89 e
29.33 ± 1.30 d
0.08 ± 0.01 a
0.19 ± 0.04 a
7.04 ± 0.64 c
2.66 ± 0.22 ab
N.D. a
3.77 ± 0.21 b
1.98 ± 0.02 ab
0.44 ± 0.03 a
0.14 ± 0.02 a
0.09 ± 0.01 a
A
16
3-hydroxybutan-
2-one
1284
8.80 ± 0.35 c
3.12 ± 0.33 b
N.D. a
N.D. a
8.64 ± 0.96 c
2.88 ± 0.42 b
N.D. a
2.70 ± 0.35 b
3.15 ± 0.42 b
N.D. a
N.D. a
N.D. a
A
17
6-methylhept-5-
en-2-one
1340
0.15 ± 0.03 c
0.29 ± 0.02 d
N.D. N.D. a
N.D. a
0.13 ± 0.01 b
N.D. a
0.31 ± 0.02 e
0.11 ± 0.01 b
N.D. a
N.D. a
N.D. a
A
18
nonan-2-one
1390
0.22 ± 0.01 d
0.14 ± 0.03 c
0.34 ± 0.03 e
0.42 ± 0.05 f
0.37 ± 0.05 e
0.16 ± 0.01 a
N.D. a
0.15 ± 0.01 c
0.06 ± 0.01 b
N.D. a
N.D. a
N.D. 2.1. Volatile Compounds in Fermented Rice Samples a
A
ALCOHOLS
19
ethanol
938
7.28 ± 0.87 a
7.46 ± 0.42 a
148.13 ± 6.73 d
171.54 ± 24.01
e
20.79 ± 1.95 a
7.29 ± 0.40 a
88.28 ± 14.58 bc
9.52 ± 1.64 a
7.39 ± 0.46 a
101.64 ± 7.36 c
82.42 ± 1.90 b
85.02 ± 8.11 b
A
20
butan-1-ol
1151
0.24 ± 0.02 a
0.18 ± 0.02 a
0.36 ± 0.02 c
0.19 ± 0.01 a
0.90 ± 0.09 d
0.35 ± 0.03 bc
0.19 ± 0.03 a
0.38 ± 0.03 c
0.21 ± 0.01 a
0.34 ± 0.02 bc
0.38 ± 0.01 c
0.30 ± 0.01 b
A
21
3-methylbutan-1-ol
1212
0.60 ± 0.04 ab
0.49 ± 0.03 a
1.11 ± 0.03 f
1.36 ± 0.21 g
1.78 ± 0.12
0.73 ± 0.08 bc
0.76 ± 0.05 cd
0.68 ± 0.04 bc
0.57 ± 0.02 ab
0.99 ± 0.01 ef
0.90 ± 0.01 de
0.93 ± 0.15 e
A
22
pentan-1-ol
1255
1.32 ± 0.28 ab
1.47 ± 0.15 abc
1.42 ± 0.05 ab
1.73 ± 0.11 c
4.04 ± 0.13 d
1.18 ± 0.20 a
1.36 ± 0.06 ab
1.56 ± 0.14 bc
1.55 ± 0.20 bc
1.36 ± 0.21 ab
1.29 ± 0.04 ab
1.23 ± 0.08 a
A
23
heptan-2-ol
1325
N.D. a
N.D. a
0.44 ± 0.02 d
0.68 ± 0.10 e
N.D. a
N.D. a
0.31 ± 0.04 b
N.D. a
N.D. a
0.39 ± 0.08 cd
0.41 ± 0.04 d
0.33 ± 0.06 bc
A
24
hexan-1-ol
1358
4.18 ± 0.26 a
4.25 ± 0.19 a
8.04 ± 0.45 c
10.17 ± 0.19 d
20.11 ± 1.36 e
6.91 ± 0.24 b
7.51 ± 0.38 bc
7.55 ± 0.49 bc
7.58 ± 0.45 bc
7.22 ± 0.43 bc
6.92 ± 0.06 b
7.20 ± 0.05 bc
A
25
oct-1-en-3-ol
1454
0.76 ± 0.10 b
0.89 ± 0.03 b
0.91 ± 0.26 b
1.27 ± 0.35 c
N.D. a
0.66 ± 0.22 b
0.77 ± 0.16 b
0.82 ± 0.28 b
0.84 ± 0.03 b
N.D. a
N.D. a
N.D. 2.1. Volatile Compounds in Fermented Rice Samples a
A
26
2-ethylhexan-1-ol
1495
0.09 ± 0.01 a
0.08 ± 0.01 a
0.20 ± 0.01 cd
0.44 ± 0.03 e
0.39 ± 0.07 e
0.09 ± 0.01 a
0.12 ± 0.02 ab
0.24 ± 0.05 d
0.08 ± 0.01 a
0.16 ± 0.04 bc
0.26 ± 0.01 d
0.21 ± 0.03 cd
A
27
(E)-hept-2-en-1-ol
1517
N.D. a
N.D. a
0.11 ± 0.01 b
0.15 ± 0.03 c
N.D. a
N.D. a
0.15 ± 0.01 c
N.D. a
N.D. a
0.13 ± 0.01 b
0.12 ± 0.01 b
0.1 ± 0.02 b
A
28
octan-1-ol
1564
0.26 ± 0.03 ab
0.24 ± 0.02 a
0.37 ± 0.02 c
0.45 ± 0.10 d
0.75 ± 0.05 e
0.28 ± 0.04 ab
0.25 ± 0.02 ab
0.33 ± 0.02 bc
0.25 ± 0.04 ab
0.32 ± 0.01 bc
0.32 ± 0.04 bc
0.29 ± 0.03 ab
A
29
(E)-oct-2-en-1-ol
1616
N.D. a
N.D. a
0.05 ± 0.01 b
0.06 ± 0.01 c
N.D. a
N.D. a
N.D. a
N.D. a
N.D. a
N.D. a
N.D. a
N.D. 2.1. Volatile Compounds in Fermented Rice Samples a
A
30
nonan-1-ol
1667
0.33 ± 0.04 ab
0.30 ± 0.02 a
0.40 ± 0.05 bc
0.59 ± 0.09 d
1.03 ± 0.07 e
0.36 ± 0.04 abc
0.34 ± 0.04 ab
0.43 ± 0.03 c
0.31 ± 0.04 a
0.40 ± 0.01 bc
0.41 ± 0.02 bc
0.3 ± 0.03 abc
A
31
butane-1,4-diol
1928
0.19 ± 0.01 ab
0.22 ± 0.03 ab
0.49 ± 0.07 e
0.77 ± 0.10 f
0.54 ± 0.04 e
0.19 ± 0.03 ab
0.14 ± 0.01 a
0.31 ± 0.06 cd
0.18 ± 0.01 a
0.31 ± 0.05 cd
0.3 ± 0.04 d
0.27 ± 0.02 bc
A
FURAN DERIVATIVES
32
2-ethylfuran
950
1.23 ± 0.34 d
1.16 ± 0.13 cd
0.57 ± 0.09 ab
0.90 ± 0.10 bc
2.40 ± 0.08 f
1.16 ± 0.11 cd
0.84 ± 0.08 ab
1.54 ± 0.38 e
0.58 ± 0.17 ab
0.55 ± 0.11 a
0.56 ± 0.11 a
0.58 ± 0.07 ab
A
33
2-propylfuran
1030
1.59 ± 0.22 e
1.45 ± 0.11 cde
1.32 ± 0.08 bcd
1.88 ± 0.10 f
3.15 ± 0.19 g
1.51 ± 0.17 de
1.32 ± 0.08 bcd
2.02 ± 0.14 f
0.86 ± 0.19 a
1.25 ± 0.09 bc
1.33 ± 0.08 bcd
1.10 ± 0.05 b
A
34
2-butylfuran
1130
3.48 ± 0.30 b
3.06 ± 0.09 ab
3.07 ± 0.06 ab
4.20 ± 0.47 c
8.93 ± 0.17 d
3.40 ± 0.43 b
3.24 ± 0.08 ab
4.41 ± 0.16 c
3.27 ± 0.72 ab
2.94 ± 0.02 ab
3.22 ± 0.19 ab
2.76 ± 0.05 a
A
35
2-pentylfuran
1231
16.15 ± 1.55 bc
13.51 ± 0.78 ab
12.82 ± 0.46 a
16.97 ± 1.75 c
49.08 ± 0.93 e
16.05 ± 2.59 bc
14.77 ± 1.06 abc
20.05 ± 1.59 d
13.32 ± 2.52 a
12.93 ± 0.37 a
14.32 ± 0.82 abc
12.69 ± 0.59 a
B able 1. Volatile compounds identified in fermented rice according to strains of lactic acid bacteria (LABs). 4 of 15 Molecules 2019, 24, 1183 Table 1. Cont. Table 1. Cont. No. 2.1. Volatile Compounds in Fermented Rice Samples Compounds
RI 1
Relative Peak Area 2
ID 3
RTJL3
LPC
KR10
KR7
RTCL16
RTCL79
RTJL4
RTCL3
RTCL31
JFK2-2
JKA1-6
RTCL
BENZENE AND BENZENE DERIVATIVES
36
toluene
1036
1.32 ± 0.12 abc
1.01 ± 0.01 a
2.06 ± 0.25 de
2.39 ± 0.10 e
2.33 ± 0.26 e
1.11 ± 0.15 a
2.33 ± 0.18 e
1.73 ± 0.50 bcd
1.28 ± 0.16 ab
1.79 ± 0.18 cd
1.8 ± 0.09 d
2.00 ± 0.60 de
A
37
ethylbenzene
1120
2.23 ± 0.37 ab
1.93 ± 0.12 ab
2.10 ± 0.13 ab
2.47 ± 0.40 ab
4.51 ± 0.07 d
2.03 ± 0.19 ab
2.34 ± 0.32 ab
2.52 ± 0.05 b
3.88 ± 0.78 c
1.93 ± 0.12 ab
1.96 ± 0.15 ab
1.90 ± 0.15 a
B
38
1,4-xylene
1136
1.63 ± 0.15 ab
1.45 ± 0.05 a
1.82 ± 0.13 abc
2.17 ± 0.41 cd
3.55 ± 0.20 e
1.52 ± 0.07 a
1.69 ± 0.09 ab
1.97 ± 0.11 bc
2.49 ± 0.45 d
1.68 ± 0.15 ab
1.67 ± 0.08 ab
1.63 ± 0.08 ab
A
39
1,2-xylene
1179
N.D. a
N.D. a
0.80 ± 0.05 b
1.14 ± 0.11 c
N.D. a
N.D. a
N.D. a
N.D. a
N.D. a
0.61 ± 0.49 b
0.83 ± 0.04 b
0.89 ± 0.18 bc
A
40
propylbenzene
1205
N.D. a
0.04 ± 0.01 ab
0.05 ± 0.01 bc
0.04 ± 0.00 ab
0.15 ± 0.01 e
0.06 ± 0.01
0.10 ± 0.02 d
0.07 ± 0.01 bcd
0.09 ± 0.01 cd
N.D. bcd
0.09 ± 0.06 cd
N.D. a
B
41
1-ethyl-2-
methylbenzene
1222
0.04 ± 0.01 a
0.0 ± 0.01 a
0.14 ± 0.02 d
0.09 ± 0.02 c
0.30 ± 0.01 e
0.04 ± 0.01 a
0.06 ± 0.01 ab
0.05 ± 0.01 ab
0.09 ± 0.01 c
0.07 ± 0.01 bc
0.06 ± 0.01 ab
0.16 ± 0.01 d
B
42
1-ethyl-3-
methylbenzene
1223
0.13 ± 0.00 bc
0.16 ± 0.05 c
0.07 ± 0.00 a
0.17 ± 0.01 c
0.13 ± 0.02 bc
0.10 ± 0.01 ab
0.16 ± 0.04 c
0.14 ± 0.02 bc
0.23 ± 0.06 d
0.14 ± 0.01 bc
0.14 ± 0.01 bc
0.07 ± 0.01 a
A
43
1,3,5-
trimethylbenzene
1242
0.13 ± 0.00 c
0.12 ± 0.02 c
0.19 ± 0.02 d
N.D. a
0.13 ± 0.01 c
N.D. 2.1. Volatile Compounds in Fermented Rice Samples p
y
1-Octen-3-ol, which can be produced from unsaturated fatty acids with some microbial
involvement via an enzymatic reaction [26], was found in samples RTCL16, JFK2-2, JKA1-6, and
RTCL9. Matsui et al. reported that 1-octen-3-ol could be formed from 10-hydroperoxide of linoleic
acid [26]. Linoleic acid would be hydroperoxided by lipoxygenase, producing 10-hydroperoxylinoleic
acid, and then hydroperoxide lyase would decompose 10-hydroperoxylinoleic acid into 1-octen-3-ol
and 10-oxo-trans-8-decenoic acid [26,27]. (E)-Hept-2-en-1-ol and heptan-2-ol were observed in only obligatorily heterofermentative LAB
and not in facultatively heterofermentative or obligatorily homofermentative LAB. 3-Methylbutan-l-ol
can be formed by the reduction of 3-methylbutanal produced by the Strecker degradation of leucine,
and RTCL16 contained the most 3-methylbutan-l-ol. Vermeulen et al. reported that Lactobacillus
sanfranciscensis, which is classified as heterofermentative, reduced aldehydes to the corresponding
unsaturated alcohols, whereas Lactobacillus sakei, which is facultatively heterofermentative, did not
metabolize the unsaturated aldehydes at all [28]. In heterofermentative LAB, the reduction of aldehydes
is related to the oxidation of NADH to NAD+, which enables this heterofermentative LAB to produce
additional ATP from glucose [28]. However, LAB, which metabolize glucose via the Embden-Meyerhof
pathway, were not affected from NADH recycling during growth in sourdough [28]. (E)-Hept-2-en-1-ol and heptan-2-ol were observed in only obligatorily heterofermentative LAB
and not in facultatively heterofermentative or obligatorily homofermentative LAB. 3-Methylbutan-l-ol
can be formed by the reduction of 3-methylbutanal produced by the Strecker degradation of leucine,
and RTCL16 contained the most 3-methylbutan-l-ol. Vermeulen et al. reported that Lactobacillus
sanfranciscensis, which is classified as heterofermentative, reduced aldehydes to the corresponding
unsaturated alcohols, whereas Lactobacillus sakei, which is facultatively heterofermentative, did not
metabolize the unsaturated aldehydes at all [28]. In heterofermentative LAB, the reduction of aldehydes
is related to the oxidation of NADH to NAD+, which enables this heterofermentative LAB to produce
additional ATP from glucose [28]. However, LAB, which metabolize glucose via the Embden-Meyerhof
pathway, were not affected from NADH recycling during growth in sourdough [28]. 2-Pentylfuran, which has a beany odor note, has been reported as one of the odor-active
compounds in various rice cultivars [29] and is mainly derived from the specific oxidation of
9-hydroperoxides by lipoxygenase. It could be produced at high levels during fermentation, possibly
due to the large amount of linoleic acid and the high lipoxygenase activity in grains [30]. In addition,
furan derivatives could have been generated by thermal degradation during sample preparation in
the present study. 2.1. Volatile Compounds in Fermented Rice Samples a
0.13 ± 0.02 c
0.13 ± 0.01 c
0.20 ± 0.04 d
0.15 ± 0.03 c
0.07 ± 0.02 b
0.08 ± 0.02 b
B
44
1,2,4-
trimethylbenzene
1280
N.D. a
0.53 ± 0.01 ab
N.D. a
1.18 ± 1.02 b
N.D. a
0.56 ± 0.05 ab
N.D. a
N.D. a
N.D. a
N.D. b
N.D. a
N.D. a
A
45
1,2,3-
trimethylbenzene
1337
0.04 ± 0.00 ab
0.03 ± 0.00 ab
N.D. a
N.D. a
N.D. a
0.04 ± 0.02 ab
0.18 ± 0.03 c
0.05 ± 0.01 ab
0.07 ± 0.03 b
0.19 ± 0.05 c
0.19 ± 0.07 c
N.D. a
A
HYDROCARBONS
46
octane
780
0.98 ± 0.17 ab
0.76 ± 0.04 a
1.19 ± 0.16 bc
1.41 ± 0.08 cd
3.16 ± 0.25 e
1.06 ± 0.16 ab
1.62 ± 0.43 d
1.10 ± 0.19 ab
0.83 ± 0.16 ab
1.00 ± 0.18 ab
0.87 ± 0.08 ab
0.74 ± 0.11 a
A
TERPENES
47
1-methyl-4-prop-1-
en-2-ylcyclohexene
(limonene)
1194
N.D. a
N.D. a
0.33 ± 0.06 c
N.D. a
N.D. a
N.D. a
N.D. a
N.D. a
N.D. a
N.D. a
N.D. a
0.15 ± 0.03 b
A
1 Retention indices were determined using n-alkanes C7 to C30; 2 Mean values of relative peak area to that of internal standard ± standard deviation; 3 Identification: A, mass spectrum
agreed with the authentic compound; B, mass spectrum and retention index were consistent with those of Wiley library and literatures; 4 There were significant differences (p < 0.05)
among 12 different samples using Duncan’s multiple comparison test between the samples with the different letter in a row; 5 Not detected. 5 of 15 Molecules 2019, 24, 1183 Various alcohols and acids are produced via the pentose phosphate pathway and the TCA cycle
of LAB [24]. Ethanol was the predominant alcohol in most of the fermented rice samples in the
present study, and is mainly formed from alcoholic fermentation. In homofermentative LAB, ethanol is
derived from pyruvate via acetyl-CoA, whereas it is derived from the pentose phosphate pathway
of glucose in heterofermentative LAB [24]. The level of ethanol was much higher in samples KR10
and KR7, which are Lac. brevis strains. Annuk et al. reported that Lac. brevis produced more ethanol
than other LABs [25]. In addition, the level of ethanol differed significantly between obligatorily
heterofermentative and facultatively heterofermentative/obligatorily homofermentative LAB samples
in the present study. 2.1. Volatile Compounds in Fermented Rice Samples In particular, sample RTCL16 contained the largest amounts of furans, whereas the
amounts of furans did not differ significantly between the other samples. The amounts of aldehydes such as 3-methylbutanal, hexanal, and nonanal were larger than
those of heterofermentative LAB. Kaseleht et al. compared the formation of volatile compounds
by LAB during the fermentation of rye sourdough with an uninoculated rye sourdough control
sample [31]. They reported that aldehydes were more strongly represented in homofermentative than
heterofermentative LAB, probably due to the higher alcohol dehydrogenase activity and/or the higher
surplus of reductive power (NADH) during the growth of heterofermentative LAB. Hexanal, which contributes a fatty-green odor note, can be readily recognized by its low odor
threshold (5 ng/g) in rice [32]. Hexanal mainly derives from the oxygenation of linoleic acid via
the sequential actions of lipoxygenase and hydroperoxide lyase [33]. Hexanal is related to further
reactions such as the oxidation to hexanoic acid by aldehyde dehydrogenase and the reduction to
1-hexanol by alcohol dehydrogenase during fermentation [34]. Hexan-1-ol was much more abundant
than hexanal and hexanoic acid in all of the samples in the present study. The combined amount of
hexanal, 1-hexanol, and hexanoic acid was highest in sample RTCL16, followed by sample RTCL3. Nonanal, which can be generated by the decomposition of 9-hydroperoxy-octadecadienoic acid
derived from the oxidation of oleic acid [2], was detected only in samples RTCL79 and RTCL31. The amount of its corresponding alcohol, nonan-1-ol, was larger than that of nonanal, as was the case
for hexanal and hexan-1-ol. Czerny and Schieberle (2002) found that the concentrations of (E)-2-nonenal 6 of 15 Molecules 2019, 24, 1183 were significantly decreased during sourdough fermentation [35]. The activity of short-chain alcohol
dehydrogenase of LAB contributes to the reduction of these flavor compounds [35]. 2-Methylbutanal and 3-methylbutanal can be formed enzymatically or nonenzymatically by
the Strecker degradation of isoleucine and leucine, respectively. Only two samples in this study
(RTCL3 and RTCL16) produced both 2-methylbutanal and 3-methylbutanal. RTCL31 contained only
2-methylbutanal, whereas 3-methylbutanal was only detected in RTJL3 and LPC. Leucine produces
3-methylbutanal by Strecker degradation, and this is subsequently reduced to 3-methyl-1-butanol
by alcohol dehydrogenase. 3-Methyl-1-butanol was detected in all of the samples, whereas
3-methylbutanal was found only in three samples—RTJL3, LPC, and RTCL3. The ketones detected in fermented rice were methyl ketones including propan-2-one, butan-2-one,
6-methylhept-5-en-2-one, and nonan-2-one. 2.1. Volatile Compounds in Fermented Rice Samples Methyl ketones, which have a characteristic aroma note of
ripened cheeses, are formed from fatty acids by enzymatic oxidative decarboxylation (β-oxidation) [2]. Ketones were not detected in sample RTJL4 fermented by Lactobacillus hilgardii, while the amount of
propan-2-one was largest in sample RTCL16. A particularly interesting finding of the present study was that the amounts of 3-hydroxybutan-2-one
and butane-2,3-dione were much larger for the homofermentative/facultatively heterofermentative
LAB RTJL3, LPC, RTCL16, RTCL79, and RTCL31 than for the obligatorily heterofermentative LAB
KR10, KR7, RTJL4, JFK2-2, JKA1-6, and RTCL9. 3-Hydroxybutan-2-one and butane-2,3-dione can be
produced by the metabolism of LAB by glucose, citrate, and aspartic acid [19]. Drinan et al. found
that Lac. plantarum and Streptococcus lactis, which were classified as homofermentative LAB, could
produce larger amounts of butane-2,3-dione and 3-hydroxybutan-2-one than could Lactobacillus
viridescens, Lactobacillus fermenti, and Leuconostoc spp., which are included in heterofermentative
LAB [19]. It was also reported that heterofermentative LAB did not produce butane-2,3-dione or
3-hydroxybutan-2-one [19]. Those authors considered that citrate was utilized in some other metabolic
pathway during growth in heterofermentative LAB. The largest amounts of butane-2,3-dione and
3-hydroxybutan-2-one were found in RTJL3, followed by LPC, which are both Lactobacillus paracasei
species classified as facultatively heterofermentative LAB. RTJL3 and LPC produced much lower levels
of ethanol than did the obligatorily heterofermentative LAB KR10, KR7, RTJL4, JFK2-2, JKA1-6, and
RTCL9, and similar levels to those of the homofermentative LAB RTCL3 and RTCL31. It was considered
that low-ethanol-tolerant microorganisms could preferentially switch pyruvate utilization to an acetoin
biosynthetic pathway in order to dispose of ethanol that was present at toxic levels [36]. In addition,
relatively large amounts of lactic acid were observed in RTJL3 and LPC, while these two samples had
low levels of acetic acid. These results are consistent with Pruckler et al. reporting that Lac. plantarum
and Lactobacillus pentosus, which are facultatively heterofermentative LAB, produced a large amount of
lactic acid but little acetic acid, exhibiting a profile close to homofermentative fermentation [37]. 2.2. Nonvolatile Compounds in Fermented Rice Samples The 37 identified nonvolatile compounds with their relative peak areas are listed in Table 2. They were comprised of 17 amino acids, seven organic acids, and 13 carbohydrates. Amino acids
were most abundant in KR10 and least abundant in RTCL79. In particular, the amounts of
phenylalanine and branched-chain amino acids such as valine, leucine, and isoleucine were much
smaller in RTCL79 than in the other samples. Valine, isoleucine, leucine, and phenylalanine
can be transformed into Strecker aldehydes, leading to 2-methylpropanal, 2-methylbutanal,
3-methylbutanal, and phenylacetaldehyde, respectively [38]. These Strecker aldehydes can form
the alcohols 2-methylpropan-1-ol, 2-methylbutan-l-ol, 3-methylbutan-l-ol, and phenylethanol [38]. Phenylacetaldehyde is also the most effective precursor for the production of benzaldehyde [39]. The present study identified the presence of 2-methylbutanal, 3-methylbutanal, 3-methylbutan-l-ol,
and benzaldehyde. 7 of 15 Molecules 2019, 24, 1183 Table 2. Nonvolatile compounds identified in fermented rice according to strains of LABs. No. Compounds
Relative Peak Area 1
ID 2
RTJL3
LPC
KR10
KR7
RTCL16
RTCL79
RTJL4
RTCL3
RTCL31
JFK2-2
JKA1-6
RTCL
AMINO ACIDS
1
alanine
0.43 ± 0.05 f 3
0.36 ± 0.02 e
0.16 ± 0.02 b
0.22 ± 0.02 c
0.55 ± 0.01 cg
0.08 ± 0.02 a
0.24 ± 0.03 c
0.05 ± 0.01 a
0.44 ± 0.03 f
0.38 ± 0.02 e
0.47 ± 0.02 f
0.31 ± 0.04 d
A
2
asparagine
N.D. 4 a
N.D. a
0.03 ± 0.00 d
0.03 ± 0.01 cd
N.D. a
0.01 ± 0.00 b
0.02 ± 0.00 c
0.02 ± 0.00 c
0.02 ± 0.00 c
0.01 ± 0.00 b
0.02 ± 0.01 c
0.02 ± 0.00 b
A
3
citrulline
0.03 ± 0.00 b
0.03 ± 0.00 b
N.D. a
N.D. a
0.04 ± 0.01 c
0.04 ± 0.00 c
N.D. a
N.D. a
0.07 ± 0.01 e
0.05 ± 0.01 d
0.05 ± 0.01 d
N.D. a
A
4
γ-aminobutyric
acid
0.05 ± 0.01 a
0.09 ± 0.00 d
0.09 ± 0.00 d
0.64 ± 0.02 g
0.09 ± 0.00 de
0.07 ± 0.01 b
0.16 ± 0.01 f
0.08 ± 0.00 cd
0.08 ± 0.01 cd
0.09 ± 0.00 d
0.08 ± 0.01 cd
0.08 ± 0.00 cd
A
5
tryptophan
0.01 ± 0.00 b
N.D. a
0.03 ± 0.01 c
0.02 ± 0.00 c
0.01 ± 0.00 b
N.D. a
N.D. a
0.02 ± 0.00 c
N.D. 2.2. Nonvolatile Compounds in Fermented Rice Samples a
0.02 ± 0.01 c
0.02 ± 0.00 c
0.02 ± 0.00 c
A
6
valine
0.21 ± 0.02 e
0.11 ± 0.00 b
0.29 ± 0.01 b
0.25 ± 0.01 g
0.23 ± 0.00 f
0.01 ± 0.00 a
0.11 ± 0.00 b
0.19 ± 0.00 d
0.17 ± 0.00 c
0.16 ± 0.00 c
0.18 ± 0.01 d
0.23 ± 0.01 f
A
7
leucine
0.20 ± 0.02 c
0.14 ± 0.01 b
0.50 ± 0.01 j
0.40 ± 0.01 g
0.43 ± 0.01 i
0.01 ± 0.01 a
0.20 ± 0.00 c
0.35 ± 0.01 e
0.27 ± 0.00 d
0.37 ± 0.01 f
0.40 ± 0.00 gh
0.42 ± 0.03 hi
A
8
isoleucine
0.03 ± 0.01 c
N.D. a
0.12 ± 0.00 g
0.08 ± 0.00 ef
0.08 ± 0.00 e
N.D. a
N.D. a
0.02 ± 0.01 b
0.02 ± 0.00 b
0.03 ± 0.00 c
0.05 ± 0.00 d
0.09 ± 0.00 f
A
9
proline
0.36 ± 0.03 e
0.31 ± 0.01 d
0.46 ± 0.01 g
0.44 ± 0.01 f
0.27 ± 0.01 c
0.27 ± 0.01 c
0.23 ± 0.01 b
0.27 ± 0.01 c
0.25 ± 0.01 c
0.22 ± 0.01 ab
0.21 ± 0.01 a
0.22 ± 0.002 ab
A
10
glycine
0.13 ± 0.05 b
0.23 ± 0.02 c
0.20 ± 0.01 c
0.19 ± 0.02 c
0.10 ± 0.08 b
0.04 ± 0.01 a
0.04 ± 0.01 a
0.11 ± 0.01 b
0.09 ± 0.00 b
0.11 ± 0.00 b
0.11 ± 0.01 b
0.10 ± 0.01 b
A
11
serine
0.24 ± 0.07 efg
0.20 ± 0.02 cde
0.26 ± 0.01 g
0.26 ± 0.02 fg
0.15 ± 0.01 ab
0.15 ± 0.01 ab
0.16 ± 0.02 abc
0.12 ± 0.01 a
0.17 ± 0.02 bc
0.18 ± 0.01 bcd
0.20 ± 0.02 cde
0.22 ± 0.012 def
A
12
threonine
0.04 ± 0.01 def
0.01 ± 0.00 a
0.03 ± 0.00 cd
0.02 ± 0.00 ab
0.04 ± 0.00 gh
0.02 ± 0.01 bc
0.02 ± 0.01 ab
0.04 ± 0.01 efg
0.03 ± 0.00 de
0.05 ± 0.00 h
0.04 ± 0.00 fgh
0.05 ± 0.00 h
A
13
methionine
0.03 ± 0.00 bc
0.03 ± 0.01 bc
0.04 ± 0.00 e
0.03 ± 0.00 cd
0.03 ± 0.00 bc
N.D. 2.2. Nonvolatile Compounds in Fermented Rice Samples a
0.03 ± 0.00 bc
0.03 ± 0.00 b
0.02 ± 0.00 b
0.04 ± 0.01 e
0.04 ± 0.01 de
0.04 ± 0.00 cd
A
14
aspartic acid
0.16 ± 0.02 e
0.12 ± 0.02 d
0.09 ± 0.00 c
0.06 ± 0.01 b
0.08 ± 0.004 c
N.D. a
N.D. a
0.10 ± 0.00 c
0.06 ± 0.00 b
0.15 ± 0.01 e
0.13 ± 0.00 d
0.15 ± 0.01 e
A
15
glutamic acid
0.08 ± 0.00 c
0.08 ± 0.00 c
0.13 ± 0.00 d
0.02 ± 0.00 ab
0.07 ± 0.01 c
N.D. a
N.D. a
0.07 ± 0.01 c
0.06 ± 0.00 c
0.03 ± 0.05 b
0.05 ± 0.00 c
0.08 ± 0.01 c
A
16
phenylalanine
0.05 ± 0.00 b
0.04 ± 0.00 b
0.11 ± 0.00 e
0.10 ± 0.01 de
0.08 ± 0.01 cd
N.D. a
0.05 ± 0.01 b
0.08 ± 0.01 cd
N.D. a
0.06 ± 0.05 bc
0.08 ± 0.00 cde
0.08 ± 0.00 cde
A
17
ornithine
N.D. a
N.D. a
0.44 ± 0.03 d
0.44 ± 0.01 a
0.01 ± 0.01 a
N.D. d
0.33 ± 0.02 d
0.35 ± 0.00 a
0.27 ± 0.02 a
N.D. c
N.D. c
N.D. b
A
ORGANIC ACIDS
18
lactic acid
122.46 ± 3.81 cd
151.00 ± 10.23 d
107.17 ± 6.14 cd
991.52 ± 92.63 e
39.93 ± 0.69 a
146.83 ± 9.76 d
71.80 ± 4.81 abc
110.65 ± 8.23 cd
53.92 ± 2.68 ab
92.52 ± 8.17 bc
70.19 ± 3.22 abc
72.45 ± 5.00 abc
A
19
citric acid
0.09 ± 0.01 b
N.D. a
0.24 ± 0.00 cd
2.05 ± 0.06 f
N.D. a
N.D. a
0.21 ± 0.02 c
0.22 ± 0.01 c
N.D. 2.2. Nonvolatile Compounds in Fermented Rice Samples a
0.27 ± 0.04 de
0.30 ± 0.02 e
0.26 ± 0.01 de
A
20
propanoic acid
0.06 ± 0.01 cd
0.05 ± 0.01 cd
0.03 ± 0.00 ab
0.27 ± 0.03 f
0.04 ± 0.01 abc
0.03 ± 0.01 ab
0.04 ± 0.01 bcd
0.02 ± 0.00 a
0.02 ± 0.01 a
0.09 ± 0.02 e
0.06 ± 0.01 d
0.03 ± 0.01 ab
A
18
lactic acid
122.46 ± 3.81 cd
151.00 ± 10.23 d
107.17 ± 6.14 cd
991.52 ± 92.63 e
39.93 ± 0.69 a
146.83 ± 9.76 d
71.80 ± 4.81 abc
110.65 ± 8.23 cd
53.92 ± 2.68 ab
92.52 ± 8.17 bc
70.19 ± 3.22 abc
72.45 ± 5.00 abc
A
19
citric acid
0.09 ± 0.01 b
N.D. a
0.24 ± 0.00 cd
2.05 ± 0.06 f
N.D. a
N.D. a
0.21 ± 0.02 c
0.22 ± 0.01 c
N.D. a
0.27 ± 0.04 de
0.30 ± 0.02 e
0.26 ± 0.01 de
A
20
propanoic acid
0.06 ± 0.01 cd
0.05 ± 0.01 cd
0.03 ± 0.00 ab
0.27 ± 0.03 f
0.04 ± 0.01 abc
0.03 ± 0.01 ab
0.04 ± 0.01 bcd
0.02 ± 0.00 a
0.02 ± 0.01 a
0.09 ± 0.02 e
0.06 ± 0.01 d
0.03 ± 0.01 ab
A
21
oxalic acid
0.05 ± 0.00 bc
0.05 ± 0.00 b
0.05 ± 0.01 b
0.43 ± 0.03 d
0.05 ± 0.01 b
N.D. a
0.06 ± 0.01 bc
0.05 ± 0.00 b
0.05 ± 0.00 bc
0.06 ± 0.01 bc
0.07 ± 0.00 c
0.06 ± 0.01 bc
A
22
succinic acid
0.07 ± 0.01 ab
0.22 ± 0.12 c
0.03 ± 0.00 a
0.24 ± 0.02 c
0.06 ± 0.06 ab
0.07 ± 0.01 ab
0.10 ± 0.01 b
0.05 ± 0.01 ab
0.03 ± 0.01 ab
0.05 ± 0.01 ab
0.03 ± 0.00 ab
0.30 ± 0.01 d
A
23
fumaric acid
N.D. a
N.D. a
N.D. a
N.D. a
0.03 ± 0.00 b
N.D. a
N.D. a
N.D. a
N.D. a
0.06 ± 0.00 c
0.06 ± 0.01 d
N.D. a
A
24
malic acid
N.D. a
N.D. a
N.D. a
N.D. a
N.D. a
N.D. a
0.28 ± 0.02 b
N.D. a
N.D. a
0.27 ± 0.03 b
0.32 ± 0.02 c
N.D. a
A
CARBOHYDRATES
25
ribofuranose
N.D. a
N.D. 2.2. Nonvolatile Compounds in Fermented Rice Samples a
1.88 ± 0.33 cd
1.32 ± 0.13 b
1.70 ± 0.40 bcd
N.D. 2.2. Nonvolatile Compounds in Fermented Rice Samples a
1.84 ± 0.38 cd
1.35 ± 0.10 b
1.89 ± 0.33 cd
2.03 ± 0.29 e
1.60 ± 0.09 bc
1.63 ± 0.08 bcd
B
26
arabitol
0.02 ± 0.00 a
0.02 ± 0.00 a
0.05 ± 0.00 bc
0.05 ± 0.00 bcd
0.03 ± 0.00 a
0.04 ± 0.01 b
0.06 ± 0.01 e
0.05 ± 0.00 bcd
0.06 ± 0.00 de
0.07 ± 0.00 f
0.07 ± 0.01 f
0.05 ± 0.01 cde
A
27
xylose
0.07 ± 0.00
0.10 ± 0.04
0.14 ± 0.03
0.09 ± 0.01
0.13 ± 0.04
0.01 ± 0.00
0.13 ± 0.02
0.09 ± 0.01
0.14 ± 0.04
0.21 ± 0.07
0.12 ± 0.01
0.13 ± 0.00
A
28
myo-inositol
0.37 ± 0.09 b
0.41 ± 0.01 bcd
0.48 ± 0.01 d
0.44 ± 0.04 cd
0.45 ± 0.03 cd
0.43 ± 0.01 bcd
0.44 ± 0.06 cd
0.40 ± 0.01 bc
0.46 ± 0.017 cd
0.46 ± 0.03 cd
0.44 ± 0.00 cd
0.31 ± 0.00 a
A
29
sucrose
0.41 ± 0.03 bc
0.32 ± 0.03 a
0.55 ± 0.01 ef
0.54 ± 0.05 ef
0.45 ± 0.01 cd
0.51 ± 0.03 de
0.59 ± 0.08 f
0.59 ± 0.02 f
0.69 ± 0.04 g
0.31 ± 0.03 a
0.38 ± 0.02 ab
0.37 ± 0.07 ab
A
30
maltose
20.47 ± 1.12 b
15.34 ± 1.44 a
12.24 ± 0.82 a
21.79 ± 1.49 bc
22.45 ± 3.81 bc
24.67 ± 0.81 cd
29.39 ± 3.90 ef
24.92 ± 0.96 cd
32.78 ± 1.05 f
24.93 ± 1.60 cd
27.01 ± 3.96 de
25.28 ± 0.83 cd
A
31
trehalose
4.07 ± 0.47 b
2.67 ± 0.48 a
7.69 ± 0.25 e
7.75 ± 0.67 e
4.99 ± 0.18 c
5.70 ± 0.30 c
6.75 ± 0.89 d
6.74 ± 0.19 d
7.67 ± 0.22 e
9.86 ± 0.47 f
9.54 ± 0.09 f
10.01 ± 0.37 f
B
32
sophorose
4.99 ± 0.28 b
3.94 ± 0.658 a
7.34 ± 0.161 e
7.15 ± 0.56 de
5.60 ± 0.26 bc
5.81 ± 0.193 c
6.64 ± 0.96 de
6.40 ± 0.18 cd
7.32 ± 0.298 e
8.94 ± 0.55 f
8.56 ± 0.22 f
9.10 ± 0.39 f
B
33
mannose
82.23 ± 0.87 ab
93.91 ± 11.22 cd
85.02 ± 2.92
abcd
83.09 ± 3.32 abc
79.09 ± 5.55 a
76.00 ± 1.81 a
91.48 ± 12.40
bcd
83.10 ± 1.78 abc
93.37 ± 3.57 cd
94.44 ± 4.12 d
94.39 ± 4.28 d
93.65 ± 1.41 cd
A
34
fructose
23.30 ± 0.25 ab
26.61 ± 3.18 cd
24.09 ± 0.83
abcd
23.54 ± 0.94 abc
22.41 ± 1.57 a
21.53 ± 0.51 a
25.92 ± 3.51
bcd
23.54 ± 0.50 abc
26.46 ± 1.01 cd
26.76 ± 1.17 d
26.74 ± 1.21 d
26.53 ± 0.40 cd
A
35
galactose
17.82 ± 0.19 ab
20.35 ± 2.43 cd
18.42 ± 0.63
abcd
18.00 ± 0.72 abc
17.14 ± 1.20 a
16.47 ± 0.39 a
19.82 ± 2.69
bcd
18.00 ± 0.39 abc
20.23 ± 0.77 cd
20.46 ± 0.89 d
20.45 ± 0.93 d
20.30 ± 0.30 cd
A
36
glucose
60.56 ± 2.79 abc
70.09 ± 5.14 d
59.22 ± 2.69 ab
59.22 ± 2.05 ab
56.34 ± 3.70 a
54.54 ± 1.02 a
66.37 ± 8.78
bcd
59.43 ± 1.37 ab
67.10 ± 3.12 cd
66.68 ± 2.75
bcd
67.26 ± 4.68 cd
66.54 ± 3.10
bcd
A
37
mannitol
N.D. 2.2. Nonvolatile Compounds in Fermented Rice Samples a
N.D. a
0.39 ± 0.04 b
0.45 ± 0.26 b
N.D. a
N.D. a
0.76 ± 0.11 c
N.D. a
N.D. a
3.13 ± 0.16 e
2.09 ± 0.04 d
N.D. a
A
1 Mean values of relative peak area to that of internal standard ± standard deviation; 2 Identification: A, mass spectrum agreed with the authentic compound; B, mass spectrum agreed
with library; 3 There were significant differences (p < 0.05) among 12 different samples using Duncan’s multiple comparison test between the samples with the different letter in a row;
4 Not detected. Table 2. Nonvolatile compounds identified in fermented rice according to strains of LABs. 8 of 15 Molecules 2019, 24, 1183 Glutamic acid, which contributes to a savory and umami taste, was not detected in RTCL79 or
RTJL4. Glutamic acid can be converted into γ-aminobutyric acid (GABA) by glutamate decarboxylase
to regulate the internal pH in an acid environment [40]. GABA, which is a nonprotein amino acid,
plays an important role in the sympathetic nervous system and cardiovascular function [41]. Ornithine was found in only six samples (RTCL3, RTCL31, KR7, KR10, RTCL16, and RTJL4),
which constitute two Pediococcus sp., two Lac. brevis sp., Lac. sakei sp., and Lac. hilgardii sp. Ornithine
is a central component of the urea cycle that facilitates the disposal of excess nitrogen and can be
produced from arginine via the arginine deiminase pathway [42]. Many heterofermentative LABs
have the ability to produce energy by utilizing arginine in the formation of ornithine, NH3, CO2,
and ATP [43]. The conversion of arginine via citrulline into ornithine releases ammonia, which increases
the pH of the medium and improves the survival of bacteria under acid stress conditions [43]. Previous
studies have found that this is not only the case in obligatorily heterofermentative LAB such as
Lac. sanfranciscensis and some strains of Lactobacillus buchneri, Lac. hilgardii, Lac. reuteri, and Oenococcus
oeni, but also in the facultative heterofermentative Lac. plantarum [43]. Lactic acid derived from pyruvate by lactate dehydrogenase was the predominant organic acid in
the present study. Lactic acid not only improves the organoleptic properties of fermented foods [44]
but also inhibits the growth of spoilage bacteria in food products. In the present study, lactic acid was
much more abundant in KR7 than in rice samples fermented by other LAB. It was considered that KR7
can produce the largest amount of lactate dehydrogenase. 2.2. Nonvolatile Compounds in Fermented Rice Samples Citric acid, succinic acid, fumaric acid, and malic acid play important roles in the TCA cycle. The metabolism of citric acid is initiated by citrate permease or citrate lyase. Citrate permease leads to
the formation of succinic acid, whereas citrate lyase results in decarboxylation to pyruvate that can
be converted into α-acetolactate, which in turn is enzymatically reduced to 3-hydroxybutan-2-one or
nonenzymatically transformed into butane-2,3-dione [45]. However, the citrate conversion to succinic
acid appears to be more common in Lactobacillus strains [16,45]. The amounts of succinic acid, fumaric
acid, malic acid, and citric acid were largest in KR7, JKA1-6, RTJL4, and JFK2-2, respectively, whereas
there were only small amounts of 3-hydroxybutan-2-one and butane-2,3-dione in these four samples. LABs have a strong tendency to generate organic acids involved in the TCA cycle but rarely appear to
produce 3-hydroxybutan-2-one and butane-2,3-dione [16]. Carbohydrates are used as carbon sources to provide microbial energy for the growth of
microorganisms via carbohydrate metabolic pathways [2]. Mannitol, which is included in sugar
alcohol, can serve as an antioxidant and sweetener in foods [46]. Mannitol was detected in all of the
obligatorily heterofermentative LAB except Weissella cibaria in the present study, while it was not
found in any of the facultatively heterofermentative/homofermentative LAB samples. In particular,
JKA2-2 and JKA1-6, which are fermented by Leuconostoc mesenteroides, contained large amounts of
mannitol. Wisselink et al. demonstrated that several heterofermentative LABs produce large amounts
of mannitol using fructose as an electron acceptor, whereas homofermentative LABs only produce small
amounts of mannitol [46]. It was considered that two different key enzymes are involved in mannitol
production: (1) mannitol 1-phosphate dehydrogenase for homofermentative LAB and (2) mannitol
dehydrogenase for heterofermentative LAB. Salminen et al. reported that many heterofermentative
LAB gain additional energy by converting acetyl phosphate into acetate instead of ethanol [45]. 2.3. Principal Component Analysis of Fermented Rice Samples according to Strains of LABs The present study applied principal component analysis (PCA) to compare the differences of
volatile and nonvolatile compounds among the fermented rice samples according to strains of LABs. The PCA score plot of volatile compounds in Figure 1A shows that rice samples fermented by Lac. sakei
could be distinguished from the others along the PC1 dimension (explaining 42.5% of the variance). In addition, rice samples fermented by obligatorily homofermentative/facultatively heterofermentative
LABs, such as LPC, RTJL3, RTCL16, RTCL79, RTCL3, and RTCL31, were separated from fermented rice
samples inoculated with obligatorily heterofermentative lactic acid bacteria, such as KR10, KR7, RTJL4, 9 of 15 Molecules 2019, 24, 1183 JKA1-6, JKF2-2, and RTCL9, along the PC2 dimension (explaining 23.0% of the variance). The PCA
loading plot of volatile compounds in Figure 1B shows that 2-ethylfuran, 2-propylfuran, 2-butylfuran,
2-pentylfuran, hexanal, 2-methylbutanal, pentan-1-ol, and butan-1-ol were closely correlated with
RTCL16. On the other hand, ketones and aldehydes, such as butane-2,3-dione, 3-hydroxybutan-2-one,
6-methylhept-5-en-2-one, 2-methylbutanal, and nonanal, were related to the positive axis of the PC2
dimension. PCA analysis was performed to compare the differences among the fermented rice samples
according to strains of LABs. The PCA score plot of volatile compounds in Figure 1A shows that
rice samples fermented by LAB inoculated with Lac. sakei could be distinguished from the others
along the PC1 dimension (explaining 42.5% of the variance). In addition, rice samples fermented
by obligatorily homofermentative/facultatively heterofermentative lactic acid bacteria, such as LPC,
RTJL3, RTCL16, RTCL79, RTCL3, and RTCL31, were separated from fermented rice samples inoculated
with obligatorily heterofermentative lactic acid bacteria, such as KR10, KR7, RTJL4, JKA1-6, JKF2-2, and
RTCL9, along the PC2 dimension (explaining 23.0% of the variance). The PCA loading plot of volatile
compounds in Figure 1B shows that 2-ethylfuran, 2-propylfuran, 2-butylfuran, 2-pentylfuran, hexanal,
2-methylbutanal, pentan-1-ol, and butan-1-ol were closely correlated with RTCL16. On the other hand,
ketones and aldehydes, such as butane-2,3-dione, 3-hydroxybutan-2-one, 6-methylhept-5-en-2-one,
2-methylbutanal, and nonanal, were related to the positive axis of the PC2 dimension. Molecules 2019, 24, x
13 of 19
aldehydes,
such
as
butane-2,3-dione,
3-hydroxybutan-2-one,
6-methylhept-5-en-2-one,
2-methylbutanal, and nonanal, were related to the positive axis of the PC2 dimension. PCA analysis
was performed to compare the differences among the fermented rice samples according to strains
of LABs. The PCA score plot of volatile compounds in Figure 1A shows that rice samples fermented
by LAB inoculated with Lac. 2.3. Principal Component Analysis of Fermented Rice Samples according to Strains of LABs sakei could be distinguished from the others along the PC1 dimension
(explaining 42.5% of the variance). In addition, rice samples fermented by obligatorily
homofermentative/facultatively heterofermentative lactic acid bacteria, such as LPC, RTJL3,
RTCL16, RTCL79, RTCL3, and RTCL31, were separated from fermented rice samples inoculated
with obligatorily heterofermentative lactic acid bacteria, such as KR10, KR7, RTJL4, JKA1-6, JKF2-2,
and RTCL9, along the PC2 dimension (explaining 23.0% of the variance). The PCA loading plot of
volatile compounds in Figure 1B shows that 2-ethylfuran, 2-propylfuran, 2-butylfuran,
2-pentylfuran, hexanal, 2-methylbutanal, pentan-1-ol, and butan-1-ol were closely correlated with
RTCL16. On
the
other
hand,
ketones
and
aldehydes,
such
as
butane-2,3-dione,
3-hydroxybutan-2-one, 6-methylhept-5-en-2-one, 2-methylbutanal, and nonanal, were related to the
positive axis of the PC2 dimension Figure 1. Principal component analysis (PCA) plots of the volatile compounds identified in
fermented rice according to strains of LABs: (A) score plot, (B) loading plot. Figure 1. Principal component analysis (PCA) plots of the volatile compounds identified in fermented
rice according to strains of LABs: (A) score plot, (B) loading plot. Figure 1. Principal component analysis (PCA) plots of the volatile compounds identified in
fermented rice according to strains of LABs: (A) score plot, (B) loading plot. Figure 1. Principal component analysis (PCA) plots of the volatile compounds identified in fermented
rice according to strains of LABs: (A) score plot, (B) loading plot. The PCA score plot of nonvolatile compounds in Figure 2(A) shows that rice samples
fermented by obligatorily heterofermentative LAB such as JKA1-6, JKF2-2, and RTCL9 could be
distinguished from the other samples (except for KR10, KR7, and RTJL4) fermented with
obligatorily homofermentative/facultatively heterofermentative LABs along the PC1 axis
(explaining 25.40% of the variance). Also, in contrast to the score plot for nonvolatile compounds,
RTCL16 was not clearly separated from the other samples along the PC2 axis (explaining 22.6% of
the variance). The PCA loading plot of nonvolatile compounds in Figure 2(B) shows that most
carbohydrates (except for sucrose and maltose) were strongly associated with rice samples
fermented by obligatorily heterofermentative LABs (KR10, KR7, RTCL9, JKA1-6, and JKF2-2) on the
positive dimension of the PC1 axis of the PCA score plot. 2.3. Principal Component Analysis of Fermented Rice Samples according to Strains of LABs On the other hand, some amino acids
(serine, valine, isoleucine, phenylalanine, glycine, and ornithine) and organic acids (lactic acid,
The PCA score plot of nonvolatile compounds in Figure 2A shows that rice samples
fermented by obligatorily heterofermentative LAB such as JKA1-6, JKF2-2, and RTCL9 could be
distinguished from the other samples (except for KR10, KR7, and RTJL4) fermented with obligatorily
homofermentative/facultatively heterofermentative LABs along the PC1 axis (explaining 25.40% of
the variance). Also, in contrast to the score plot for nonvolatile compounds, RTCL16 was not clearly
separated from the other samples along the PC2 axis (explaining 22.6% of the variance). The PCA
loading plot of nonvolatile compounds in Figure 2B shows that most carbohydrates (except for sucrose
and maltose) were strongly associated with rice samples fermented by obligatorily heterofermentative
LABs (KR10, KR7, RTCL9, JKA1-6, and JKF2-2) on the positive dimension of the PC1 axis of the PCA
score plot. On the other hand, some amino acids (serine, valine, isoleucine, phenylalanine, glycine,
and ornithine) and organic acids (lactic acid, succinic acid, propanoic acid, oxalic acid, and citric acid) 10 of 15 Molecules 2019, 24, 1183 were mainly responsible for rice samples fermented by heterofermentative LABs (KR10 and KR7) on
the positive axis of the PCA score plot along the PC2 dimension. Thus, these compounds could be
major nonvolatile compounds related to rice samples fermented by the obligatorily heterofermentative
LABs (KR10, KR7, RTCL9, JKA1-6, and JKF2-2). Molecules 2019, 24, x
14 of 19
rice samples fermented by the obligatorily heterofermentative LABs (KR10, KR7, RTCL9, JKA1-6,
and JKF2-2). a
J
)
Figure 2. PCA plots of the nonvolatile volatile compounds identified in fermented rice according to
strains of LABs: (A) score plot, (B) loading plot. Figure 2. PCA plots of the nonvolatile volatile compounds identified in fermented rice according to
strains of LABs: (A) score plot, (B) loading plot. Figure 2. PCA plots of the nonvolatile volatile compounds identified in fermented rice according to
strains of LABs: (A) score plot, (B) loading plot. Figure 2. PCA plots of the nonvolatile volatile compounds identified in fermented rice according to
strains of LABs: (A) score plot, (B) loading plot. 3 1 Chemicals and Reagents
3.1. Chemicals and Reagents 3.1. Chemicals and Reagents
Three internal standard compounds (2,3,5-trimethylpyrazine, 2,3-pentanedione, and tropic
acid)
and
derivatization
reagents
[methoxyamine
hydrochloride,
pyridine,
N,O-bis(trimethylsilyl)-tirfluoroacetamide (BSTFA) containing 1% trimethylchlorosilane (TMCS)]
were purchased from Sigma-Aldrich (St. Louis, MO, USA). L-Threitol was obtained from Tokyo
Chemical Industry (Tokyo, Japan), and L-4-hydroxyproline was obtained from Fluka Chemical
(Milwaukee, WI, USA). Solvents such as methanol, chloroform, and water were of analytical grade
(J T Baker Phillipsburg NJ USA)
Three internal standard compounds (2,3,5-trimethylpyrazine, 2,3-pentanedione, and tropic
acid) and derivatization reagents [methoxyamine hydrochloride, pyridine, N,O-bis(trimethylsilyl)-
tirfluoroacetamide (BSTFA) containing 1% trimethylchlorosilane (TMCS)] were purchased from
Sigma-Aldrich (St. Louis, MO, USA). L-Threitol was obtained from Tokyo Chemical Industry
(Tokyo, Japan), and L-4-hydroxyproline was obtained from Fluka Chemical (Milwaukee, WI, USA). Solvents such as methanol, chloroform, and water were of analytical grade (J.T. Baker, Phillipsburg,
NJ, USA). (J
p
3 2 S
l P
ti
3.2. Sample Preparation 3.2. Sample Preparation
Milled rice was ground using a miller into 170 mesh. The distilled water was added at the ratio
of 2.3:7.7 (ground rice: water, w/w) before the ground samples were treated with commercial 0.05%
(w/w) α-amylase (BAN480L; Novozymes) and 0.05% (w/w) glucoamylase (AMZ1100; Novozymes,
Bagsvaerd, Denmark). The enzyme saccharification was performed by shaking at 100 rpm and 63 °C
for 20 h, following the previous method of Lee et al. [13]. Then, enzyme deactivation was performed
at 85 °C for 30 min. After enzyme deactivation, samples were inoculated with 1% (w/w) of 12
different LABs (107 CFU/mL), respectively (Table 3). For the present study, 12 LAB strains were
isolated and selected from various Korean fermented foods, considering their tolerances to acid, salt,
and glucose in a preliminary study (Table 3). Then, inoculated samples were fermented at 30 °C for
24 h. All samples were kept at about −70 °C in a deep freezer (I1 Shin Bio Base; Model No. DF8514;
D
d
h
i G
id
K
) All
i
d
d i
i li
Milled rice was ground using a miller into 170 mesh. The distilled water was added at the ratio
of 2.3:7.7 (ground rice: water, w/w) before the ground samples were treated with commercial 0.05%
(w/w) α-amylase (BAN480L; Novozymes) and 0.05% (w/w) glucoamylase (AMZ1100; Novozymes,
Bagsvaerd, Denmark). The enzyme saccharification was performed by shaking at 100 rpm and 63 ◦C
for 20 h, following the previous method of Lee et al. [13]. Then, enzyme deactivation was performed at
85 ◦C for 30 min. After enzyme deactivation, samples were inoculated with 1% (w/w) of 12 different
LABs (107 CFU/mL), respectively (Table 3). For the present study, 12 LAB strains were isolated and
selected from various Korean fermented foods, considering their tolerances to acid, salt, and glucose in
a preliminary study (Table 3). Then, inoculated samples were fermented at 30 ◦C for 24 h. All samples
were kept at about −70 ◦C in a deep freezer (I1 Shin Bio Base; Model No. DF8514; Dongdoocheon-si,
Gyeonggido, Korea). All experiments were conducted in triplicate. 11 of 15 11 of 15 Molecules 2019, 24, 1183 Table 3. The abbreviation of fermented rice samples and the strains of lactic acid bacteria used in the
present study. (J
p
3 2 S
l P
ti
3.2. Sample Preparation Samples Abbreviation
Lactic Acid Bacteria
Food Source from which Isolated
LPC
Lactobacillus paracasei (1) b
Korean traditional rice wine (makgeolli)
RTJL3
Lactobacillus paracasei (2) b
traditional rice wine (Myeoncheon Dugyeonju)
RTCL16
Lactobacillus sakei b
traditional fermented barley paste
RTCL79
Lactobacillus pentosus b
traditional fermented barley paste
KR10
Lactobacillus brevis (1) c
radish kimchi
KR7
Lactobacillus brevis (2) c
kimchi
RTJL4
Lactobacillus hilgardii c
traditional rice wine (Myeoncheon Dugyeonju)
RTCL3
Pediococcus pentoseceus a
traditional fermented barley paste
RTCL31
Pediococcus lolii a
traditional fermented barley paste
JKA1-6
Leuconostoc mesenteroides (1) c
kimchi
JKF2-2
Leuconostoc mesenteroides (2) c
radish kimchi
RTCL9
Weissella cibaria c
traditional fermented barley paste
a Obligatorily homofermentative. b Facultatively heterofermentative. c Obligatorily heterofermentative. 3.3. Extraction and Analysis of Volatile Compounds by Gas Chromatography-Mass Spectrometry Solid phase micro-extraction (SPME) was used to obtain volatile profiles of fermented rice samples. Four g of fermented rice was put into a 20 mL screw vial with a screw cap (Ultraclean 18 mm, Agilent
Technologies, Santa Clara, CA, USA). After sample preparation, the vial was maintained at 30 ◦C
for 10 min to reach an equilibrium state. Volatiles in headspace were adsorbed onto SPME fiber
coated with carboxen/polydimethylsiloxane/divinylbenzene (CAR/PDMS/DVB) (Supelco, Bellefonte,
PA, USA). The adsorption and desorption conditions were the same as those used previously [2]. 2,3,5-Trimethylpyrazine and 2,3-pentanedione (100 mg/L in methanol) were used as internal standards
of aromatic and aliphatic compounds, respectively. Gas chromatography-mass spectrometry (GC-MS) analysis was performed using an HP 7890B
GC system coupled to the 5977A mass selective detector (Agilent Technologies) and the multi-purpose
sampler MPS 2 (Gerstel, Mülheim an der Ruhr, Germany) equipped with a DB-WAX capillary
column (30 m length × 0.25 mm i.d. × 0.25 µm film thickness, J&W Scientific, Folsom, CA, USA). Other conditions for GC-MS analysis were based on a minor modification of Lee et al. [2]. 3.4. Extraction and Analysis of Nonvolatile Compounds by Gas Chromatography-Time of
Flight-Mass Spectrometry 3.4. Extraction and Analysis of Nonvolatile Compounds by Gas Chromatography-Time of
Flight-Mass Spectrometry One g of fermented rice was immersed in liquid nitrogen and then extracted with 20 mL of 80%
methanol (J. T. Baker., Phillipsburg, NJ, USA) at 70 ◦C for 25 min in an ultrasonicator (Branson, Danbury,
CT, USA). After that, it stayed in room temperature for 30 min, followed by an addition of 2 mL
chloroform (J. T. Baker., Phillipsburg, NJ, USA). It was then sonicated for 20 min prior to a centrifugation
for 10 min. Finally, 100 µL of the extracted layer was transferred to a 1.5 mL tube (Eppendorf, Hamburg,
Germany) and then injected using internal standard followed by drying overnight in a centri-vap
(Labconco Co., Kansas City, MO, USA). For methoximation, 50 µL of methoxyamine hydrochloride
(20 mg/mL in pyridine) was added to the dried extract at 30 ◦C for 90 min. After that, derivatization for
silylation was performed by 90 µL of N,O-bis (trimethylsilyl)-tirfluoroacetamide (BSTFA) containing
1% trimethylchlorosilane (TMCS), following the previous method of Son et al. [47]. Internal standard
compounds were L-threitol (100 mg/L in water) for carbohydrates, L-4-hydroxyproline (100 mg/L in
water) for amino acids, and tropic acid (100 mg/L in water) for organic acids, respectively. Gas chromatography-time-of-flight/mass spectrometry (GC-TOF/MS) analysis was performed by
the Agilent 6890N GC system (Agilent Technologies) coupled to the Leco Pegasus III mass spectrometer
(Leco, St. Joseph, MI, USA) equipped with a DB-5MS column (30 m length × 0.25 mm i.d. × 0.25 µm
film thickness, J&W Scientific). Other conditions for GC-TOF/MS analysis were based on a minor
modification of Son et al. [47]. Molecules 2019, 24, 1183 12 of 15 12 of 15 3.5. Identification and Semi-Quantification of Volatile and Nonvolatile Compounds 3.5. Identification and Semi-Quantification of Volatile and Nonvolatile Compounds Volatile compounds were identified based on a comparison of the mass spectra in the NIST08
and Wiley 9 mass spectral libraries (Agilent Technologies, Palo Alto, CA, USA) and retention index
(RI) values. The RI values of volatile compounds were calculated with an alkane mixture from C7 to
C30 as external standards. In addition, volatile compounds were positively confirmed by comparing
their mass spectrum and retention time with those of standard compounds. 3.6. Statistical Analysis The ANOVA was performed with general linear model procedure using SPSS (version 12.0,
Chicago, IL, USA). Duncan’s multiple comparison test (p < 0.05) was applied to identify statistically
significant differences. PCA was performed to discriminate fermented rice samples on the basis of their
volatile and nonvolatile compounds profiles according to microbial strains using SIMCA-P (version
11.0, Umetrics, Umea, Sweden). 3.4. Extraction and Analysis of Nonvolatile Compounds by Gas Chromatography-Time of
Flight-Mass Spectrometry Nonvolatile compounds
were identified by comparing their mass spectral data based on Fiehn library, replibrary, mainlibrary,
Wiley 9, and in-house library, and then confirmed by comparing their mass spectral data and retention
times to those of authentic standard compounds. The identification and semi-quantification procedures
were the same as those used previously [47]. Acknowledgments: This work was supported by Korea Institute of Planning and Evaluation for Technology in
Food, Agriculture, Forestry and Fisheries (IPET) through Advanced Production Technology Development Program
(grant number 316081-04) and the High Value-added Food Technology Development Program (grant number
317035-03), funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA). Author Contributions: Conceptualization, Y.-S.K.; Methodology, M.S.K. and Y.R.H.; Analysis, S.M.L. and Y.R.H.;
Investigation, Y.-S.K. and M.S.C.; Data Curation, M.S.K. and S.M.L.; Writing-Original Draft Preparation, S.M.L.
and M.S.C.; Writing-Review & Editing, Y.-S.K. and S.M.L.; Visualization, S.M.L.; Supervision, Y.-S.K. Conflicts of Interest: The authors declare no conflict of interest. References 1. Salmeron, I.; Loeza-Serrano, S.; Perez-Vega, S.; Pandiella, S.S. Headspace gas chromatography (HS-GC) analysis
of imperative flavor compounds in lactobacilli-fermented barley and malt substrates. Food Sci. Biotechnol. 2015, 24, 1363–1371. [CrossRef] 1. Salmeron, I.; Loeza-Serrano, S.; Perez-Vega, S.; Pandiella, S.S. Headspace gas chromatography (HS-GC) analysis
of imperative flavor compounds in lactobacilli-fermented barley and malt substrates. Food Sci. Biotechnol. 2015, 24, 1363–1371. [CrossRef] 2. Lee, S.M.; Oh, J.; Hurh, B.S.; Jeong, G.H.; Shin, Y.G.; Kim, Y.S. Volatile compounds produced by lactobacillus
paracasei during oat fermentation. J. Food Sci. 2016, 81, C2915–C2922. [CrossRef] [PubMed] 2. Lee, S.M.; Oh, J.; Hurh, B.S.; Jeong, G.H.; Shin, Y.G.; Kim, Y.S. Volatile compounds produced by lactobacillus
paracasei during oat fermentation. J. Food Sci. 2016, 81, C2915–C2922. [CrossRef] [PubMed] 3. Nout, M.R. Rich nutrition from the poorest–Cereal fermentations in Africa and Asia. Food Microbiol. 2009, 26,
685–692. [CrossRef] [PubMed] 3. Nout, M.R. Rich nutrition from the poorest–Cereal fermentations in Africa and Asia. Food Microbiol. 2009, 26,
685–692. [CrossRef] [PubMed] 4. Angelov, A.; Gotcheva, V.; Hristozova, T.; Gargova, S. Application of pure and mixed probiotic lactic acid
bacteria and yeast cultures for oat fermentation. J. Sci. Food Agric. 2005, 85, 2134–2141. [CrossRef] 4. Angelov, A.; Gotcheva, V.; Hristozova, T.; Gargova, S. Application of pure and mixed probiotic lactic acid
bacteria and yeast cultures for oat fermentation. J. Sci. Food Agric. 2005, 85, 2134–2141. [CrossRef] 5. Kedia, G.; Vazquez, J.A.; Pandiella, S.S. Enzymatic digestion and in vitro fermentation of oat fractions by
human lactobacillus strains. Enzyme Microb. Technol. 2008, 43, 355–361. [CrossRef] 6. Marklinder, I.; Johansson, L. Sour dough fermentation of barley flours with varied content of mixed-linked
(1→3),(1→4) β-d-glucans. Food Microbiol. 1995, 12, 363–371. [CrossRef] 7. Gupta, S.; Cox, S.; Abu-Ghannam, N. Process optimization for the development of a functional beverage
based on lactic acid fermentation of oats. Biochem. Eng. J. 2010, 52, 199–204. 8. Blandino, A.; Al-Aseeri, M.E.; Pandiella, S.S.; Cantero, D.; Webb, C. Cereal-based fermented foods and
beverages. Food Res. Int. 2003, 36, 527–543. [CrossRef] 9. Mukherjee, S.K.; Albury, M.N.; Pederson, C.S.; Van Veen, A.G.; Steinkraus, K.H. Role of Leuconostoc
mesenteroides in leavening the batter of idle, a fermented food of India. Appl. Microbiol. 1965, 13, 227–231. [PubMed] 10. Ghosh, K.; Maity, C.; Adak, A.; Halder, S.K.; Jana, A.; Das, A.; Parua, S.; Mohapatra, P.K.D.; Pati, B.R.;
Mondal, K.C. Ethnic preparation of Haria, a rice-based fermented beverage, in the province of Lateritic West
Bengal, India. Ethnobot. 4. Conclusions This study investigated differences in the volatile and nonvolatile compounds of rice samples
fermented by 12 different LABs. The level of ethanol differed significantly between obligatorily
heterofermentative and facultatively heterofermentative/obligatorily homofermentative LAB samples. In homofermentative LAB, ethanol comes from pyruvate via acetyl-CoA, whereas it is derived from
the pentose phosphate pathway of glucose in heterofermentative LAB. In addition, the amounts
of 3-hydroxybutan-2-one and butane-2,3-dione were larger for the homofermentative/facultatively
heterofermentative LABs RTJL3, LPC, RTCL16, RTCL79, and RTCL31 than for the obligatorily
heterofermentative LABs KR10, KR7, RTJL4, JFK2-2, JKA1-6, and RTCL9. This difference could at least
partially be due to low-ethanol-tolerant microorganisms preferentially switching pyruvate utilization
to the acetoin biosynthetic pathway in order to dispose of ethanol present at toxic levels. The application of PCA to data sets of the profiles of volatile and nonvolatile compounds revealed
that fermented rice samples can be distinguished according to different LAB strains. The volatile
compounds 2-ethylfuran, 2-propylfuran, 2-butylfuran, 2-pentylfuran, hexanal, 2-methylbutanal,
pentan-1-ol, and butan-1-ol were strongly correlated with the rice sample fermented by Lac. sakei
(RTCL16). On the other hand, ketones and aldehydes such as butane-2,3-dione, 3-hydroxybutan-2-one,
6-methylhept-5-en-2-one, 2-methylbutanal, and nonanal were related to fermented rice samples
inoculated with obligatorily heterofermentative LAB. These findings indicate that the profiles of
volatile and nonvolatile compounds of fermented rice inoculated with different LABs can change
significantly depending on the microbial strains present during the fermentation process. These results
can be used to improve the quality of rice-based fermented products and develop rice-based functional
foods, such as probiotic beverages. Author Contributions: Conceptualization, Y.-S.K.; Methodology, M.S.K. and Y.R.H.; Analysis, S.M.L. and Y.R.H.;
Investigation, Y.-S.K. and M.S.C.; Data Curation, M.S.K. and S.M.L.; Writing-Original Draft Preparation, S.M.L. and M.S.C.; Writing-Review & Editing, Y.-S.K. and S.M.L.; Visualization, S.M.L.; Supervision, Y.-S.K. Acknowledgments: This work was supported by Korea Institute of Planning and Evaluation for Technology in
Food, Agriculture, Forestry and Fisheries (IPET) through Advanced Production Technology Development Program
(grant number 316081-04) and the High Value-added Food Technology Development Program (grant number
317035-03), funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA). 13 of 15 13 of 15 Molecules 2019, 24, 1183 References Esterases of lactic acid bacteria and
cheese flavour: Milk fat hydrolysis, alcoholysis and esterification. Int. Dairy J. 2005, 15, 711–718. [CrossRef] 23. Holland, R.; Liu, S.; Crow, V.; Delabre, M.; Lubbers, M.; Bennett, M. Esterases of lactic acid bacteria and
cheese flavour: Milk fat hydrolysis, alcoholysis and esterification. Int. Dairy J. 2005, 15, 711–718. [CrossRef]
24. Laëtitia, G.; Pascal, D.; Yann, D. The citrate metabolism in homo-and heterofermentative LAB: A selective ,
;
,
;
,
;
,
;
,
;
,
cheese flavour: Milk fat hydrolysis, alcoholysis and esterification. Int. Dairy J. 2005, 15, 711–718. [CrossRef]
24. Laëtitia, G.; Pascal, D.; Yann, D. The citrate metabolism in homo-and heterofermentative LAB: A selective
means of becoming dominant over other microorganisms in complex ecosystems. Food Nutr. Sci. 2014, 5, 953. [CrossRef] 24. Laëtitia, G.; Pascal, D.; Yann, D. The citrate metabolism in homo-and heterofermentative LAB: A selective
means of becoming dominant over other microorganisms in complex ecosystems. Food Nutr. Sci. 2014, 5, 953. [CrossRef] 25. Annuk, H.; Shchepetova, J.; Kullisaar, T.; Songisepp, E.; Zilmer, M.; Mikelsaar, M. Characterization of
intestinal lactobacilli as putative probiotic candidates. J. Appl. Microbiol. 2003, 94, 403–412. [CrossRef] 26. Matsui, K.; Sasahara, S.; Akakabe, Y.; Kajiwara, T. Linoleic acid 10-hydroperoxide as an intermediate
during formation of 1-octen-3-ol from linoleic acid in lentinusdecadetes. Biosci. Biotechnol. Biochem. 2003, 67,
2280–2282. [CrossRef] 27. Assaf, S.; Hadar, Y.; Dosoretz, C.G. 1-octen-3-ol and 13-hydroperoxylinoleate are products of distinct
pathways in the oxidative breakdown of linoleic acid by pleurotus pulmonarius. Enzyme Microb. Technol. 1997, 21, 484–490. [CrossRef] 28. Vermeulen, N.; Czerny, M.; Gänzle, M.G.; Schieberle, P.; Vogel, R.F. Reduction of (E)-2-nonenal and
(E,E)-2,4-decadienal during sourdough fermentation. J. Cereal Sci. 2007, 45, 78–87. [CrossRef] 29. Yang, D.S.; Shewfelt, R.L.; Lee, K.; Kays, S.J. Comparison of odor-active compounds from six distinctly
different rice flavor types. J. Agric. Food Chem. 2008, 56, 2780–2787. [CrossRef] 30. Lampi, A.M.; Damerau, A.; Li, J.; Moisio, T.; Partanen, R.; Forssell, P.; Piironen, V. Changes in lipids and
volatile compounds of oat flours and extrudates during processing and storage. J. Cereal Sci. 2015, 62, 102–109. [CrossRef] 31. Kaseleht, K.; Paalme, T.; Mihhalevski, A.; Sarand, I. Analysis of volatile compounds produced by different
species of lactobacilli in rye sourdough using multiple headspace extraction. Int. J. Food Sci. Technol. 2011, 46,
1940–1946. [CrossRef] 32. Buttery, R.G.; Turnbaugh, J.G.; Ling, L.C. Contribution of volatiles to rice aroma. J. Agric. Food Chem. 1988,
36, 1006–1009. References Res. Appl. 2014, 12, 39–49. 11. Ghosh, K.; Ray, M.; Adak, A.; Dey, P.; Halder, S.K.; Das, A.; Jana, A.; Parua, S.; Mohapatra, P.K.D.;
Pati, B.R.; et al. Microbial, saccharifying and antioxidant properties of an Indian rice based fermented
beverage. Food Chem. 2015, 168, 196–202. [CrossRef] [PubMed] 12. Giraffa, G.; Chanishvili, N.; Widyastuti, Y. Importance of lactobacilli in food and feed biotechnology. Res. Microbiol. 2010, 161, 480–487. [CrossRef] 13. Lee, S.M.; Lim, H.J.; Chang, J.W.; Hurh, B.S.; Kim, Y.S. Investigation on the formations of volatile compounds,
fatty acids, and γ-lactones in white and brown rice during fermentation. Food Chem. 2018, 269, 347–354. [CrossRef] 14. Salmeron, I.; Fucinos, P.; Charalampopoulos, D.; Pandiella, S.S. Volatile compounds produced by the
probiotic strain lactobacillus plantarum NCIMB 8826 in cereal-based substrates. Food Chem. 2009, 117,
265–271. [CrossRef] 15. Audrain, B.; Farag, M.A.; Ryu, C.M.; Ghigo, J.M. Role of bacterial volatile compounds in bacterial biology. FEMS Microbiol. Rev. 2015, 39, 222–233. [CrossRef] [PubMed] 16. Xiao, Z.; Lu, J.R. Strategies for enhancing fermentative production of acetoin: A review. Biotechnol. Adv. 2014,
32, 492–503. [CrossRef] [PubMed] 17. Xiao, Z.; Xu, P. Acetoin metabolism in bacteria. C 17. Xiao, Z.; Xu, P. Acetoin metabolism in bacteria. Crit. Rev. Microbiol. 2007, 33, 127–140. [CrossRef] [PubMed] 17. Xiao, Z.; Xu, P. Acetoin metabolism in bacteria. Crit. Rev. Microbiol. 2007, 33, 127 140. [CrossRef] [PubMed]
18. Leroy, F.; De Vuyst, L. Lactic acid bacteria as functional starter cultures for the food fermentation industry. Trends Food Sci. Technol. 2004, 15, 67–78. [CrossRef] 19. Drinan, D.F.; Robin, S.; Cogan, T.M. Citric acid metabolism in hetero- and homofermentative lactic acid
bacteria. Appl. Environ. Microbiol. 1976, 31, 481–486. [PubMed] 20. Romero-Guido, C.; Belo, I.; Ta, T.M.N.; Cao-Hoang, L.; Alchihab, M.; Gomes, N.; Wache, Y. Biochemistry of
lactone formation in yeast and fungi and its utilization for the production of flavor and fragrance compounds. Appl. Microbiol. Biotechnol. 2011, 89, 535–547. [CrossRef] [PubMed] 21. Mo, X.; Fan, W.; Xu, Y. Changes in volatile compounds of Chinese rice wine wheat Qu during fermentation
and storage. J. Inst. Brew. 2009, 115, 300–307. [CrossRef] 22. Lyu, J.; Nam, P.W.; Lee, S.J.; Lee, K.G. Volatile compounds isolated from rice beers brewed with three
medicinal plants. J. Inst. Brew. 2013, 119, 271–279. [CrossRef] 14 of 15 Molecules 2019, 24, 1183 23. Holland, R.; Liu, S.; Crow, V.; Delabre, M.; Lubbers, M.; Bennett, M. References [CrossRef] 33. Boonprab, K.; Matsui, K.; Akakabe, Y.; Yotsukura, N.; Kajiwara, T. Hydroperoxy-arachidonic acid mediated
n-hexanal and (Z)-3-and (E)-2-nonenal formation in laminaria angustata. Phytochemistry 2003, 63, 669–678. [CrossRef] 34. Filipiak, W.; Sponring, A.; Baur, M.M.; Ager, C.; Filipiak, A.; Wiesenhofer, H. Characterization of volatile
metabolites taken up by or released from streptococcus pneumoniae and haemophilus influenzae by using
GC-MS. Microbiology 2012, 158, 3044–3053. [CrossRef] [PubMed] 35. Czerny, M.; Schieberle, P. Important aroma compounds in freshly ground wholemeal and white wheat
flour identification and quantitative changes during sourdough fermentation. J. Agric. Food Chem. 2002, 50,
6835–6840. [CrossRef] 36. Romano, P.; Suzzi, G. Origin and production of acetoin during wine yeast fermentation. Appl. Environ. Microbiol. 1996, 62, 309–315. 37. Prückler, M.; Lorenz, C.; Endo, A.; Kraler, M.; Dürrschmid, K.; Hendriks, K. Comparison of homo-and
heterofermentative lactic acid bacteria for implementation of fermented wheat bran in bread. Food Microbiol. 2015, 49, 211–219. [CrossRef] 38. Yvon, M.; Rijnen, L. Cheese flavour formation by amino acid catabolism. Int. Dairy J. 2001, 11, 185–201. [CrossRef] 39. Chu, F.L.; Yaylayan, V.A. Model studies on the oxygen-induced formation of benzaldehyde from
phenylacetaldehyde using pyrolysis GC-MS and FTIR. J. Agric. Food Chem. 2008, 56, 10697–10704. [CrossRef] 40. Ardo, Y.; Thage, B.; Madsen, J. Dynamics of free amino acid composition in cheese ripening. Aust. J. Dairy
Technol. 2002, 57, 109. 41. Kim, H.Y.; Hwang, I.G.; Kim, T.M.; Woo, K.S.; Park, D.S.; Kim, J.H. Chemical and functional components in
different parts of rough rice (oryza sativa L.) before and after germination. Food Chem. 2012, 134, 288–293. [CrossRef] 42. Sugino, T.; Shirai, T.; Kajimoto, Y.; Kajimoto, O. L-ornithine supplementation attenuates physical fatigue in
healthy volunteers by modulating lipid and amino acid metabolism. Nutr. Res. 2008, 28, 738–743. [CrossRef]
[PubMed] 15 of 15 Molecules 2019, 24, 1183 43. Vrancken, G.; Rimaux, T.; Weckx, S.; De Vuyst, L.; Leroy, F. Environmental pH determines citrulline and
ornithine release through the arginine deiminase pathway in Lactobacillus fermentum IMDO 130101. Int. J. Food Microbiol. 2009, 135, 216–222. [CrossRef] [PubMed] 44. Garcia-Quintans, N.; Repizo, G.; Martin, M.; Magni, C.; Lopez, P. Activation of the diacetyl/acetoin pathway
in lactococcus lactis subsp. lactis bv. diacetylactis CRL264 by acidic growth. Appl. Environ. Microbiol. 2008,
74, 1988–1996. [CrossRef] [PubMed] 45. Cselovszky, J.; Wolf, G.; Hammes, W.P. Production of formate, acetate, and succinate by anaerobic
fermentation of lactobacillus pentosus in the presence of citrate. Appl. Microbiol. Biotechnol. 1992, 37, 94–97. [CrossRef] 46. © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/). References Wisselink, H.; Weusthuis, R.; Eggink, G.; Hugenholtz, J.; Grobben, G. Mannitol production by lactic acid
bacteria: A review. Int. Dairy J. 2002, 12, 151–161. [CrossRef] 47. Son, E.Y.; Lee, S.M.; Kim, M.; Seo, J.A.; Kim, Y.S. Comparison of volatile and non-volatile metabolites in rice
wine fermented by Koji inoculated with Saccharomycopsis fibuligera and Aspergillus oryzae. Food Res. Int. 2018,
109, 596–605. [CrossRef] Sample Availability: Not Available. © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Single-cell study of the extracellular matrix effect on cell growth by <i>in situ</i> imaging of gene expression
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Chemical science
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Open Access Article. Published on 02 October 2017. Downloaded on 16/03/2018 10:47:10.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Yupeng Sun, Ruijie Deng, Kaixiang Zhang, Xiaojun Ren, Ling Zhang
d Ji
h
Li
* Yupeng Sun, Ruijie Deng, Kaixiang Zhang, Xiaojun Ren, Ling Zhang
and Jinghong Li
* Cell behaviors are known to be regulated by the cellular microenvironment. Traditional cell-population
based analysis methods need to separate cells from their extracellular matrix (ECM) and cannot resolve
the heterogeneity of cell behaviors. Herein, an in situ single-cell analysis method based on rolling circle
amplification was exploited to image gene expression in single cells for investigating the effect of ECM
stiffness on cell growth. This method enables the simultaneous quantifying of the cell phenotype and
gene expression at the single-cell level, which can help in understanding the underlying molecular
mechanism of cell growth. It is found that ECM stiffness could affect cell growth via regulating the
expression level of the cytoskeleton-assembly associated genes PFN1 and CFL1 and their co-expression
pattern. Therefore, this single-cell analysis platform may facilitate us to tap into the study of “single-cell
phenotypes” and elucidate the disease association of ECMs. Received 5th September 2017
Accepted 1st October 2017
DOI: 10.1039/c7sc03880a
rsc.li/chemical-science Received 5th September 2017
Accepted 1st October 2017 However, the mechanisms of mechanotransduction for cell
growth on different ECMs remain incompletely understood. Recently, Mooney’s group found that the effect of substrate
stress relaxation on cell spreading behavior was mediated
through similar pathways as those for substrate stiffness:
integrin adhesions, Rho activation, actomyosin-based contrac-
tility and nuclear translocation of YAP.12 However, Liu’s group
investigated the gene expression of cells in response to
mechanical stretching. They found that many genes related to
cytoskeleton formation greatly changed aer exposure to
mechanical stretching (for example, PFN1 and CFL1 increased
13.0 and 1.6 folds, respectively).13 Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry &
Chemical Biology, Tsinghua University, Beijing 100084, China. E-mail: jhli@mail.
tsinghua.edu.cn
† Electronic supplementary information (ESI) available: Sequence information, in
vitro RCA and RT-qPCR data. See DOI: 10.1039/c7sc03880a This journal is © The Royal Society of Chemistry 2017 Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry &
Chemical Biology, Tsinghua University, Beijing 100084, China. E-mail: jhli@mail.
tsinghua.edu.cn Single-cell study of the extracellular matrix effect
on cell growth by in situ imaging of gene
expression†
Yupeng Sun, Ruijie Deng, Kaixiang Zhang, Xiaojun Ren, Ling Zhang
and Jinghong Li
* Cite this: Chem. Sci., 2017, 8, 8019 † Electronic supplementary information (ESI) available: Sequence information, in
vitro RCA and RT-qPCR data. See DOI: 10.1039/c7sc03880a Introduction The extracellular matrix (ECM) not only provides a physical
support for cell adhesion but also serves an important
instructional role, providing biochemical and biomechanical
cues.1–3 Specically, the stiffness of the ECM plays an important
role in regulating cell behaviors such as cell spreading, migra-
tion, proliferation and differentiation.4–8 Cells sense their ECM
stiffness through a mechanotransduction signaling pathway
which is a cellular process that translates external mechanical
stimuli into intracellular biochemical signals.3,9 Cell growth and functions are regulated by gene expression
programs and the disturbance of gene expression can result in
many human diseases. The occurrence of cancer is not only
caused by the activation of proto-oncogene and deregulation of
cell-cycle control, but also abnormal defective mechano-
transduction signaling may lead to tumor formation and
metastatic progression.10 For example, a disturbance in ECM
mechanics stimulates the Rho-ROCK-MLC pathway, increases
cytoskeletal tension, completes a self-enforcing (positive) feed-
back loop and results in further increases in ECM stiffness,
which can promote malignant transformation, tumorigenesis
and metastasis.11 Recently, pillar arrays,14 traction force microscopy15 and
atomic force microscopy (AFM)16,17 have been successfully
applied to in situ determine cellular traction forces exerted by
the interaction of cells and their ECMs by measuring the pillar
displacement or the substrate deformations. However, detailed
descriptions of the molecular mechanisms are still missed due
to the lack of genetic information. Previous attempts to char-
acterize gene or protein expression programs and investigate
the molecular mechanisms were based on methods such as
quantitative PCR, western blotting or RNA sequencing. Never-
theless, cells must be isolated from their cultured substrates
when nucleic acids are extracted, resulting in cell–substrate
interactions missed and cell status changes. Meanwhile, the
overall average data lack the information of cell heterogeneity,
which is widespread in biological systems, and may lead to
inaccurate results.18–20 Herein, we have developed an in situ single-cell mRNA
imaging method to investigate the effect of extracellular matrix This journal is © The Royal Society of Chemistry 2017 Chem. Sci., 2017, 8, 8019–8024 | 8019 Edge Article
View Article Online Chemical Science Edge Article shown in Fig. 1A. Briey, a padlock probe was designed to
recognize the target sequence of mRNA, then specically ligate
and circularize with the mRNA as the template. In situ RCA for the analysis of cytoskeleton related gene
expression in single cells Scheme 1 illustrates the effect of extracellular matrix stiffness
on cell growth and the molecular mechanisms investigated via
an in situ single-cell platform. As illustrated in Scheme 1A, the
cells present different shapes when cultured on soand stiff
substrates, and the cells on the stiffsubstrate show a spindle
shape and obvious stress bers. A robust RNA imaging method
based on in situ rolling circle amplication (RCA) has been
developed to investigate the mRNA expression in single cells at
the
single-molecule
level.21,22
Scheme
1B
illustrates
the
proposed mechanisms of how the stiffness of the ECM affects
the cell behaviors. Briey, cells sense the ECM stiffness via an
integrin triggered mechanotransduction pathway and the
increased ECM stiffness induces specic gene expression
related to cytoskeletal rearrangement (PFN1 and CFL1), forms
actin laments and promotes cell spreading and growth.23,24 Introduction Next, the target
mRNA could be amplied by RCA, resulting in a long DNA
molecule with a large number of repeat sequences.26–28 Upon
hybridization with the detection probes, the RCA amplicon
would become visible as a diffraction-limited uorescent spot. stiffness on cell growth. In this method, the relationships of
single-cell gene expressions, morphology phenotype and the
effect of different ECMs were investigated by simultaneous in
situ imaging of the cell morphology and mRNA without
a complicated pretreatment process for the cells. The expres-
sion of cytoskeleton related mRNA (PFN1, CFL1 and ACTB) for
cells cultured on different substrates was visualized at single-
molecule levels. The multi-parameter, in situ single-cell study
of the extracellular matrix effect on cell growth indicates the
complexity and heterogeneity of cell behaviors responding to
different ECMs. Firstly, to demonstrate the feasibility of rolling circle ampli-
cation for RNA detection, the target sequence of ACTB mRNA
was amplied in vitro. According to the results of the uorescence
spectra and gel electrophoresis characterization, the padlock
probe can specically recognize and effectively amplify the target
sequence by RCA (Fig. S1–S3†). We further tested the perfor-
mance for imaging RNAs in situ in single cells. As illustrated in
Fig. 1B, the bright dots amplied from the target mRNA are easily
distinguished from the background. A control experiment was
conducted without a trigger primer which was used to initiate
DNA polymerization and no obvious uorescence signal was
observed (Fig. 1C), which conrmed that the bright dots resulted
from in situ RCA. Next, to verify the specicity of this method,
a random padlock probe was used and no distinct uorescence
signal was observed (Fig. 1D). Furthermore, a blocked probe was
designed to prevent the padlock probe from binding with the
target sequence. Few bright dots could be detected (Fig. 1E),
suggesting that the bright dot signals came from the target
mRNA. The ACTB expression ranged from approximately 84 to at
most 200 copy number per cell (the average was 115 per cell) and
presented a normal distribution (Fig. 1B, inset; Fig. S4† shows
how to quantify the copy number). The obvious variability sug-
gested that even the same batch cells would exhibit signicant
cell-to-cell variation in gene expression. Evaluation of cell behaviors on different stiffness substrates Sci., 2017, 8, 8019–8024 View Article Online Edge Article Chemical Science coated polyacrylamide (PAAm) gels with different elastic
moduli.29 The hydrogel stiffness was controlled by the ratio and
the concentration of acrylamide and bis-acrylamide (details in
ESI Table 1†) and the surface morphology of the hydrogel
substrate is similar (Fig. S5 and S6†). The cells were cultured on
gels for 12 h, then xed and stained with Alexa 488-conjugated
phalloidin and DAPI to reveal the actin lament network (green)
and the nuclei (blue), respectively. The representative uores-
cence images of the MCF-7 cells are shown in Fig. 2A. Obvious
stress bers (lamentous actin bundles) were seen in cells
grown on a stiffer substrate (30 kPa) and a glass substrate (50
GPa), but not in cells grown on sosubstrates (1 kPa, 4 kPa, and
13 kPa). Stress bers, which play an important role in cellular
cytoskeleton formation, can provide force for cells to sense and
transmit the signal of the substrate stiffness and help cells to
spread and grow.30 suggests that ECM stiffness can affect the formation of stress
bers, cell shape and spreading. Open Access Article. Published on 02 October 2017. Downloaded on 16/03/2018 10:47:10.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Next, to quantify the parameters related to cell morphology,
the cell spreading area, aspect ratio (AR) and circularity were
taken into statistical analysis. Typically, the cells on the so
substrate (1 kPa) present a round shape and smaller spreading
area (400 mm2), while the cells cultured on the stiffsubstrate
(30 kPa) show a spindle shape and larger spreading area
(1600 mm2). As presented in Fig. 2B, the cell spreading area
increased monotonically with substrate stiffness. Meanwhile,
the aspect ratio and cell circularity (as descriptors of cell shape)
were acquired from cell images using Image J. The aspect ratio
of the cells was calculated as the ratio between the cell length
and width.31 We found that the aspect ratio of the cells
increased with substrate stiffness, reaching a plateau (at about
1.5) on the 13 kPa PAAm gels. Circularity, which reects the
roundness of the cells, is dened as the spreading area multi-
plied by 4p and divided by the square of the perimeter.32
Similarly, the circularity decreased with increasing substrate
stiffness, reaching a plateau (at approximately 0.7) on the 13 kPa
PAAm gels. The distributions of these statistical parameters are
presented in Fig. S7.† Therefore, the morphological analysis Gene expression variation with ECM stiffness To investigate the effect of substrate stiffness on cell gene
expression
at
the
single-cell
level,
the
expression
of Fig. 3
Cell morphology and ACTB mRNA expression for different
culture times. (A) Imaging of cell morphology (bright field) and ACTB
mRNA by in situ RCA (dark field). Scale bars, 25 mm. (B) The average
expression level and coefficient of variation for ACTB mRNA with
different incubation times. (C) Single-cell fluctuation profiles for ACTB
mRNA with different incubation times (n > 100, C.V. ¼ s.d./mean). presented in Fig. S7.† Therefore, the morphological analysis
Fig. 2
Cell morphologies on different stiffness substrates and quan-
titative analysis. (A) Fluorescence images of MCF-7 cells on PAAm gels
with varied stiffness: 1 kPa, 4 kPa, 13 kPa, 30 kPa and glass (50 GPa). Cells were stained with phalloidin and DAPI to visualize the F-actins
(green) and nuclei (blue) after culturing on the substrate for 12 h. Scale
bar, 25 mm. Quantitative analysis of the cell morphology on the
substrate: (B) cell spreading area, (C) aspect ratio and (D) circularity (n >
100; mean s.d.; **P < 0.05, *P < 0.01, NS, not significant, compare to
glass). Fig. 3
Cell morphology and ACTB mRNA expression for different
culture times. (A) Imaging of cell morphology (bright field) and ACTB
mRNA by in situ RCA (dark field). Scale bars, 25 mm. (B) The average
expression level and coefficient of variation for ACTB mRNA with
different incubation times. (C) Single-cell fluctuation profiles for ACTB
mRNA with different incubation times (n > 100, C.V. ¼ s.d./mean). This journal is © The Royal Society of Chemistry 2017
Chem. Sci., 2017, 8, 8019–8024 | 8021 Fig. 2
Cell morphologies on different stiffness substrates and quan-
titative analysis. (A) Fluorescence images of MCF-7 cells on PAAm gels
with varied stiffness: 1 kPa, 4 kPa, 13 kPa, 30 kPa and glass (50 GPa). Cells were stained with phalloidin and DAPI to visualize the F-actins
(green) and nuclei (blue) after culturing on the substrate for 12 h. Scale
bar, 25 mm. Quantitative analysis of the cell morphology on the
substrate: (B) cell spreading area, (C) aspect ratio and (D) circularity (n >
100; mean s.d.; **P < 0.05, *P < 0.01, NS, not significant, compare to
glass). Fig. 2
Cell morphologies on different stiffness substrates and quan-
titative analysis. This journal is © The Royal Society of Chemistry 2017 Timescale of gene expression during cell growth It’s a complicated process for cells to sense the ECM stiffness
via a mechanotransduction pathway which includes sensing,
signalling and gene expression to lead cell function. The time-
scale of these events ranges from seconds to weeks.3 To inves-
tigate the timescale of cell–substrate interaction, we utilized our
single-cell mRNA imaging method to visualize the gene
expression of cells in different culture times. As shown in
Fig. 3A, the cells present varied morphologies and mRNA
expression at different time points. It was found that the
quantity of ACTB mRNA increased with the incubation time and
reached a maximum (120 copies per cell on average) aer 10 h
attachment (Fig. 3B). This may be because the cell spreading
process was nished and the cells tended to express less ACTB
mRNA.33,34 Besides, the coefficient of variation of ACTB mRNA
copy numbers per cell was more than 0.2 (Fig. 3B), indicating
that the degree of dispersion for ACTB mRNA expression in
different cells was high. These results emphasize the impor-
tance of single-cell mRNA detection due to cell heterogeneity. As
the incubation time increased, the ACTB mRNA uctuations
evolved from a narrow and peaked histogram into a widely
dispersed prole, with the average shiing to higher copy
numbers (Fig. 3C). The single-cell proling results indicate that
the distribution pattern of gene expressions in single cells can
be distinct at different stages and may help us to understand the
procedure of cell spreading and the formation of stress bers. Evaluation of cell behaviors on different stiffness substrates ACTB is a constitutive housekeeping gene which plays a critical
role in F-actin formation (generating the actin monomer as the
basic building unit of F-actin).25 A scheme of in situ RCA is To
investigate
the
inuence
of
ECM
stiffness
on
cell
morphology, MCF-7 cells were cultured on different collagen- Fig. 1
In situ imaging of the expression of ACTB mRNA in single cells. (A) The scheme of in situ RCA in single cells. Imaging of ACTB mRNA by
RCA using the target padlock probe (B), without the trigger primer (C),
using a random padlock probe (D), and after blocking the target site (E). The cell nuclei are stained by DAPI (blue), the RCA amplicons are
hybridized with the Alexa 488-fluorescence probe (green spots), and
the cell outlines are marked by a dotted line. Inset: frequency histo-
grams of RCA amplicons per cell detected. Scale bars, 20 mm. Scheme 1
An illustration of the mechanism of cell growth on the
extracellular matrix with varied stiffness via an in situ single-cell plat-
form. (A) The effect of extracellular matrix stiffness on cell growth and
the procedures of in situ RCA for detecting gene expression in single
cells. (B) The proposed mechanisms of how the stiffness of the ECM
affects the cell growth and gene expression. Fig. 1
In situ imaging of the expression of ACTB mRNA in single cells. (A) The scheme of in situ RCA in single cells. Imaging of ACTB mRNA by
RCA using the target padlock probe (B), without the trigger primer (C),
using a random padlock probe (D), and after blocking the target site (E). The cell nuclei are stained by DAPI (blue), the RCA amplicons are
hybridized with the Alexa 488-fluorescence probe (green spots), and
the cell outlines are marked by a dotted line. Inset: frequency histo-
grams of RCA amplicons per cell detected. Scale bars, 20 mm. Scheme 1
An illustration of the mechanism of cell growth on the
extracellular matrix with varied stiffness via an in situ single-cell plat-
form. (A) The effect of extracellular matrix stiffness on cell growth and
the procedures of in situ RCA for detecting gene expression in single
cells. (B) The proposed mechanisms of how the stiffness of the ECM
affects the cell growth and gene expression. This journal is © The Royal Society of Chemistry 2017 8020 | Chem. Gene expression variation with ECM stiffness (A) Fluorescence images of MCF-7 cells on PAAm gels
with varied stiffness: 1 kPa, 4 kPa, 13 kPa, 30 kPa and glass (50 GPa). Cells were stained with phalloidin and DAPI to visualize the F-actins
(green) and nuclei (blue) after culturing on the substrate for 12 h. Scale
bar, 25 mm. Quantitative analysis of the cell morphology on the
substrate: (B) cell spreading area, (C) aspect ratio and (D) circularity (n >
100; mean s.d.; **P < 0.05, *P < 0.01, NS, not significant, compare to
glass). Fig. 3
Cell morphology and ACTB mRNA expression for different
culture times. (A) Imaging of cell morphology (bright field) and ACTB
mRNA by in situ RCA (dark field). Scale bars, 25 mm. (B) The average
expression level and coefficient of variation for ACTB mRNA with
different incubation times. (C) Single-cell fluctuation profiles for ACTB
mRNA with different incubation times (n > 100, C.V. ¼ s.d./mean). This journal is © The Royal Society of Chemistry 2017 Chem. Sci., 2017, 8, 8019–8024 | 8021 Fig. 5
Gene co-expression patterns and correlation analysis. (A)
PFN1–CFL1 expression correlation analysis in a single cell on different
substrates. (B) The relationship of the single-cell spreading area and
gene co-expression pattern (PFN1/CFL1) (n > 100, mean s.d.; **P <
0.05, ‘r’ means the correlation coefficient of the single-cell gene co-
expression of PFN1 and CFL1). Edge Article
View Article Online Chemical Science cytoskeleton-related genes (ACTB, PFN1 and CFL1) in cells
cultured on substrates with varying stiffness was in situ detec-
ted. These genes play important roles in cytoskeleton remod-
eling which is a signicant process for cell growth and
metastasis.35 The spreading area on the sosubstrate was
restricted to around 600 mm2, while on the stiffsubstrate it
reached around 1600 mm2 (Fig. 4A). Specically, for the cells
cultured on the stiffsubstrate, the expression of PFN1 and CFL1
was nearly two times higher than for the cells cultured on the
soer substrate. However, the ACTB mRNA expression level was
almost consistent (1.11 fold increase compared to the so
substrate). Open Access Article. Published on 02 October 2017. Downloaded on 16/03/2018 10:47:10.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 5
Gene co-expression patterns and correlation analysis. (A)
PFN1–CFL1 expression correlation analysis in a single cell on different
substrates. (B) The relationship of the single-cell spreading area and
gene co-expression pattern (PFN1/CFL1) (n > 100, mean s.d.; **P <
0.05, ‘r’ means the correlation coefficient of the single-cell gene co-
expression of PFN1 and CFL1). expression variations of PFN1 and CFL1, rather than ACTB, play
more important roles in the cell spreading behavior when
cultured on substrates with different degrees of stiffness. As the
cell spreading process involves the rapid formation of the new
actin laments and the degradation of the remaining laments,
it demands the up-regulation of the gene expression of PFN1
and CFL1. The increased expression level of colin (encoded by
CFL1) would facilitate the depolymerization of the old actin
laments into actin monomers and prolin (encoded by PFN1)
can elongate new actin laments with the depolymerized actin
monomers to facilitate the cell spreading.37 To infer the regulatory connections between single-cell
behavior and gene expression, the single-cell gene expression
and cell spreading area were in situ investigated simultaneously
and quantitatively. A correlation coefficient is regarded as
a main parameter that quanties a type of correlation and
dependence. The correlation coefficient between the cell
spreading area and the copy number on the stiffsubstrate for
genes ACTB, PFN1, and CFL1 was 0.486, 0.580 and 0.375, and
0.482, 0.370 and 0.475 on the sosubstrate, respectively
(Fig. 4B). Compared to the sosubstrate, the correlation coef-
cient between the cell spreading area and gene PFN1 increased
and decreased for CFL1 on the stiffsubstrate, while it remained
constant for ACTB. The single-cell correlations of the cell
spreading
area
and
gene
expression
indicated
that
the g
Cell growth is a complicated process involving multiple
genes, and we hypothesize that gene co-expression patterns
have signicant inuences on cell behaviors on different ECMs. To test this hypothesis, we measured the single-cell co-
expression patterns of key genes related to cytoskeleton rear-
rangement. The single-cell correlation coefficients of PFN1 and
CFL1 on a stiffsubstrate and sosubstrate were 0.741 and
0.736, respectively (Fig. 5A). The high correlation coefficients
indicated that this gene pair could be regulated by a common
upstream gene or directly regulate each other.38 Besides, it was
found that the average ratios of PFN1 and CFL1 (PFN1/CFL1) in
single cells were 2.570 and 1.747 for cells cultured on stiffand
sosubstrates, respectively (Fig. 5B). Open Access Article. Published on 02 October 2017. Downloaded on 16/03/2018 10:47:10.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. These data indicated that
gene co-expression patterns could be regulated by substrate
stiffness. A possible mechanism for cell growth is that ACTB is
a structural gene, and PFN1 and CFL1 are regulatory genes
involved in stress ber formation, enhancing cell spreading and
promoting cell growth. It is worth emphasizing that not only the
expression level of PFN1 and CFL1 but also the gene co-
expression patterns can regulate the assembling of the cyto-
skeleton in the cell spreading process. Fig. 4
Correlation analysis of the single-cell spreading area and gene
expression. (A) Images of single-cell spreading behavior and gene
expression of ACTB, PFN1 and CFL1 on different stiffness substrates
(4 kPa and 30 kPa). Scale bars, 25 mm. Inset: frequency histograms of
RCA amplicons per cell detected. (B) The relationship of single-cell
gene expression and cell spreading area for ACTB, PFN1 and CFL1,
respectively (n > 100, mean s.d.; **P < 0.05, ‘r’ means the correlation
coefficient of the single-cell spreading area and copy number per cell). Gene expression variation with ECM stiffness Generally, actins (ACTB) can be used repeatedly as
the basic building units of F-actin in stress ber forming and
cytoskeleton remodeling, therefore the ACTB mRNA expression
level remains basically unchanged.36 As a validation, we
compared the mRNA expression of ACTB, PFN1 and CFL1
averaged over hundreds of cells by in situ RCA to those obtained
from a bulk RNA quantitative measurement (RT-qPCR) per-
formed on the same cell line. Our imaging results are in good
accordance with the RT-qPCR results in general (Fig. S8†). Conclusions Fig. 4
Correlation analysis of the single-cell spreading area and gene
expression. (A) Images of single-cell spreading behavior and gene
expression of ACTB, PFN1 and CFL1 on different stiffness substrates
(4 kPa and 30 kPa). Scale bars, 25 mm. Inset: frequency histograms of
RCA amplicons per cell detected. (B) The relationship of single-cell
gene expression and cell spreading area for ACTB, PFN1 and CFL1,
respectively (n > 100, mean s.d.; **P < 0.05, ‘r’ means the correlation
coefficient of the single-cell spreading area and copy number per cell). In summary, we report an in situ single-cell imaging method for
investigating the effect of extracellular matrix stiffness on cell
growth. In this method, there is no need to lyse large numbers
of cells to acquire enough RNA, or to separate cells from the
cultured substrate. This method can yield a simultaneous and This journal is © The Royal Society of Chemistry 2017 8022 | Chem. Sci., 2017, 8, 8019–8024 Chemical Science
View Article Online Chemical Science
View Article Online Edge Article 365 nm ultraviolet light for 10 min, washed twice with 50 mM
HEPES in PBS and then incubated in 200 mg mL1 of rat type I
collagen in HEPES overnight at 37 C. detailed description of the single-cell gene expression proles
and morphology variance under the physical microenviron-
ments. It is found that the increase of PFN1 and CFL1 mRNA
expression levels and change in the PFN1 and CFL1 co-
expression pattern, which is triggered by the stiffsubstrates,
greatly promote the assembling of the cytoskeleton in the cell
growth process. Therefore, the in situ mRNA imaging method
can help us to understand the molecular mechanism for the
inuence of ECM mechanical cues on cell growth and metas-
tasis at a deeper level. Moreover, it provides the potential to
understand the mechanisms of ECM defect related diseases. Cell staining and image analysis The MCF-7 cells on the substrates were xed with 4% PFA for
10 min and permeabilized with 0.5% Triton-X100 for 5 min at
room temperature, and then blocked with 1% BSA for 1 h for
actin lament staining. Actin staining was performed using
Alexa-Fluor 488 conjugated to phalloidin (Life Technologies,
UK). Aer post-stain washing with PBS, the cells were mounted
in 4,6-diamidino-2-5-phenylindole (DAPI, Vector Laboratories,
USA) for nuclear staining. For measurements of the cell-
spreading area in 2D, images of the phalloidin/DAPI-stained
cells were taken using a Leica TCS SP5 inverted confocal
microscope (Leica, Germany) with a 63 oil-immersion objec-
tive. Only those cells that did not exhibit any cell–cell contacts
were considered in the analysis. Images of all single cells were
then thresholded manually on the basis of the actin stain, and
the cell spreading area was determined using Image J soware. In situ visualization of mRNAs in a single-cell by RCA As in a typical in situ RCA detection experiment, the hybridiza-
tion of the target mRNA with the padlock probe was carried out
in a volume of 20 mL solution, produced by adding 2 mL padlock
probe (10 mM), 1 mL DTT (100 mM), 0.5 mL RiboLock RNase
Inhibitor (40 U mL1) and 4 mL yeast tRNA (10 mg mL1) to
12.5 mL RNase-free water, overnight at 37 C. Then, the sample
was washed using PBS-T (DEPC-PBS with 0.05% Tween-20)
three times at room temperature. The ligation process was
conducted in a volume of 10 mL containing 1 mL T4 DNA ligase
(5 U mL1), 1 mL 10 T4 DNA ligase reaction buffer, 0.25 mL
RiboLock RNase Inhibitor (40 U mL1) and RNase-free water at
37 C for 2 h. The primer hybridization reaction was then per-
formed with a 20 mL mixture containing 1 mL primer (5 mM), 1 mL
DTT (100 mM), 2 mL 20 SSC, 2 mL formamide, 13.5 mL RNase-
free water and 0.5 mL RiboLock RNase Inhibitor (40 U mL1)
for 60 min at 37 C. The RCA reaction mixture containing 1 mL
10 phi29 DNA polymerase reaction buffer, 0.5 mL phi29 DNA Materials and apparatus All synthetic oligonucleotides (Table S3†) were purchased from
Shanghai Sangon Biological Engineering Technology & Services
Co., Ltd (Shanghai, China). The RCA detection probes were
modied with Alexa488 and Cy5, and were purchased from
Thermo Fisher Scientic (Waltham, USA). The salmon sperm
DNA, 20 SSC buffer (pH 7.4) and 4% paraformaldehyde in
PBS buffer were purchased from Beijing Solarbio Science &
Technology Co., Ltd. (Beijing, China). The deoxyribonucleotides
mixture
(dNTPs)
was
purchased
from
Beijing
DingGuo
Biotechnology Co., Ltd. (Beijing, China). Tween-20, diethy
pyrocarbonate (DEPC), formamide, Triton-X100 and 3-amino-
propyl triethoxysilane were purchased from Sigma-Aldrich (St. Louis, USA). TransScript one-step gDNA removal and cDNA
synthesis were purchased from Transgen Biotech Co., Ltd. (Beijing, China). RiboLock RNase Inhibitor, T4 polynucleotide
kinase, T4 DNA ligase, phi29 DNA polymerase, RevertAid First
Strand cDNA Synthesis Kit and SYBR select master mix were
purchased from Thermo Fisher Scientic (Waltham, USA). All of
the solutions and deionized water used were treated with DEPC
and autoclaved to be protected from RNase degradation. Preparation of polyacrylamide hydrogels Briey, PAAm gel solutions containing acrylamide monomers,
cross-linker bis-acrylamide, ammonium persulphate and tetra-
methylethylenediamine (TEMED) were prepared. The ratio of
acrylamide and bis-acrylamide and the nal concentrations
were varied to control the hydrogel stiffness and porosity
(details in ESI Table 1†). Glass coverslips were activated by
Piranha solution (H2SO4 : H2O2 ¼ 3 : 1) and then functionalized
using
3-(trimethoxysilyl)propyl
methacrylate
(APTES)
and
glutaraldehyde to facilitate the covalent attachment of the
hydrogel substrates to the amino-silanated coverslips. The gel
solution was sandwiched between the functionalized coverslip
and a chloro-silanated glass slide to ensure easy detachment of
the hydrogels. Cell culture The MCF-7 cells were cultured in standard Dulbecco’s Modied
Eagle’s medium with 10% fetal bovine serum, 1% penicillin/
streptomycin, and 0.01 mg mL1 human recombinant insulin. The cells were incubated at 37
C, 5% CO2 and 95% air
humidity. Cells were seeded on 22 22 mm collagen-coated
glass coverslips (VWR, Radnor, USA) enclosed with PDMS as
a chamber (5 mm in diameter). The varying stiffness hydrogel
substrates modied with collagen were washed three times with
PBS and placed in the cell culture hood for 30 min under UV
light for sterilization before cell seeding. For cell seeding, the
cells were plated at a proper density onto the hydrogel
substrates with different degrees of stiffness, so that they had
enough space to spread and didn’t contact other cells. Cells
were allowed to spread for 12 h, and then were xed and stained
for analysis. This journal is © The Royal Society of Chemistry 2017 Acknowledgements 21 R. Deng, L. Tang, Q. Tian, Y. Wang, L. Lin and J. Li,
Angew.Chem., Int. Ed., 2014, 53, 2389–2393. This work was nancially supported by the National Natural
Science Foundation of China (No. 21621003, No. 21235004, No. 21327806), and the Tsinghua University Initiative Scientic
Research Program. 22 R. Deng, K. Zhang and J. Li, Acc. Chem. Res., 2017, 50(4),
1059–1068. 23 P. P. Provenzano and P. J. Keely, J. Cell Sci., 2011, 124, 1195–
1205. 24 K. Xu, G. Zhong and X. Zhuang, Science, 2013, 339, 452–456. Conflicts of interest 18 A. Raj and A. van Oudenaarden, Cell, 2008, 135, 216–226. 19 M. B. Elowitz, A. J. Levine, E. D. Siggia and P. S. Swain,
Science, 2002, 297, 1183–1186. There are no conicts to declare. 20 R. A. Burrell, N. McGranahan, J. Bartek and C. Swanton,
Nature, 2013, 501, 338–345. Notes and references 25 Z. B. Katz, A. L. Wells, H. Y. Park, B. Wu, S. M. Shenoy and 25 Z. B. Katz, A. L. Wells, H. Y. Park, B. Wu, S. M. Shenoy and
R. H. Singer, Genes Dev., 2012, 26, 1885–1890. 1 J. D. Humphrey, E. R. Dufresne and M. A. Schwartz, Nat. Rev. Mol. Cell Biol., 2014, 15, 802–812. 26 W. Zhao, M. M. Ali, M. A. Brook and Y. Li, Angew. Chem., Int. Ed., 2008, 47, 6330–6337. 2 Z. Zhao, H. Meng, N. Wang, M. J. Donovan, T. Fu, M. You,
Z. Chen, X. Zhang and W. Tan, Angew. Chem., Int. Ed.,
2013, 52, 7487–7491. 27 J. Ge, L.-L. Zhang, S.-J. Liu, R.-Q. Yu and X. Chu, Anal. Chem.,
2014, 86, 1808–1815. 28 Y. Cheng, X. Zhang, Z. Li, X. Jiao, Y. Wang and Y. Zhang,
Angew. Chem., Int. Ed., 2009, 121, 3318–3322. 3 T. Iskratsch, H. Wolfenson and M. P. Sheetz, Nat. Rev. Mol. Cell Biol., 2014, 15, 825–833. 29 J. H. Wen, L. G. Vincent, A. Fuhrmann, Y. S. Choi,
K. C. Hribar, H. Taylor-Weiner, S. Chen and A. J. Engler,
Nat. Mater., 2014, 13, 979–987. 4 Y. Qiu, A. C. Brown, D. R. Myers, Y. Sakurai, R. G. Mannino,
R. Tran, B. Ahn, E. T. Hardy, M. F. Kee and S. Kumar, Proc. Natl. Acad. Sci. U. S. A., 2014, 111, 14430–14435. Natl. Acad. Sci. U. S. A., 2014, 111, 14430–14435. 30 S. Walcott and S. X. Sun, Proc. Natl. Acad. Sci. U. S. A., 2010,
107, 7757–7762. 5 A. J. Engler, S. Sen, H. L. Sweeney and D. E. Discher, Cell,
2006, 126, 677–689. 31 B. Cao, R. Peng, Z. Li and J. Ding, Biomaterials, 2014, 35,
6871–6881. 6 V. Vogel and M. Sheetz, Nat. Rev. Mol. Cell Biol., 2006, 7, 265–
275. 32 S.-R. Ryoo, Y.-K. Kim, M.-H. Kim and D.-H. Min, ACS Nano,
2010, 4, 6587–6598. 7 N. D. Leipzig and M. S. Shoichet, Biomaterials, 2009, 30,
6867–6878. 33 K. Baumann, Nat. Rev. Mol. Cell Biol., 2013, 14, 688–689. 8 B. Trappmann, J. E. Gautrot, J. T. Connelly, D. G. Strange,
Y. Li, M. L. Oyen, M. A. C. Stuart, H. Boehm, B. Li and
V. Vogel, Nat. Mater., 2012, 11, 642–649. 34 C. Higashida, T. Kiuchi, Y. Akiba, H. Mizuno, M. Maruoka,
S. Narumiya, K. Mizuno and N. Watanabe, Nat. Cell Biol.,
2013, 15, 395–405. 9 S. Kumar, Nat. Access Article. Published on 02 October 2017. Downloaded on 16/03/2018 10:47:10.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 17 A. Beaussart, S. El-Kirat-Chatel, R. M. A. Sullan, D. Alsteens,
P. Herman, S. Derclaye and Y. F. Dufrˆene, Nat. Protoc., 2014,
9, 1049–1055. Preparation of PAAm gel substrates for cell seeding To allow for cell adhesion, the surface of the hydrogel was
modied with the heterobifunctional linker sulfo-SANPAH to be
conjugated to a collagen I protein. The hydrogel substrate was
incubated in 0.2 mg mL1 sulpho-SANPAH, activated with This journal is © The Royal Society of Chemistry 2017 Chem. Sci., 2017, 8, 8019–8024 | 8023 View Article Online Chemical Science polymerase, 0.25 mL RiboLock RNase Inhibitor (40 U mL1), 3 mL
dNTPs (10 mM) and 5.25 mL RNase-free water was then added
and incubated for 120 min at 37 C, followed with a wash in
PBS-T. The hybridization of the detection probes with ampli-
cons was conducted in a mixture of 100 nM uorophore-labeled
detection probes in 2 SSC, 15% formamide and 10 ng mL1
sonicated salmon sperm DNA at 37 C for 30 min. The sample
was washed three times using PBS-T. Aer being mounted with
Fluoromount-G, the slides were ready for imaging. 14 J. L. Tan, J. Tien, D. M. Pirone, D. S. Gray, K. Bhadriraju and
C. S. Chen, Proc. Natl. Acad. Sci. U. S. A., 2003, 100, 1484–
1489. 15 H. Colin-York, C. Eggeling and M. Fritzsche, Nat. Protoc.,
2017, 12, 783–796. 16 Y. F. Dufrˆene, T. Ando, R. Garcia, D. Alsteens, D. Martinez-
Martin,
A. Engel,
C. Gerber
and
D. J. M¨uller,
Nat. Nanotechnol., 2017, 12, 295–307. Notes and references Mater., 2014, 13, 918–920. 35 A. N¨urnberg, T. Kitzing and R. Grosse, Nat. Rev. Cancer, 2011,
11, 177–187. 10 D. E. Jaalouk and J. Lammerding, Nat. Rev. Mol. Cell Biol.,
2009, 10, 63–73. 36 T. D. Pollard and J. A. Cooper, Science, 2009, 326, 1208–1212. 11 S. Huang and D. E. Ingber, Cancer Cell, 2005, 8, 175–176. 37 J. J. Bravo-Cordero, M. A. Magalhaes, R. J. Eddy, L. Hodgson
and J. Condeelis, Nat. Rev. Mol. Cell Biol., 2013, 14, 405–415. 12 O. Chaudhuri,
L. Gu,
M. Darnell,
D. Klumpers,
S. A. Bencherif,
J. C. Weaver,
N. Huebsch
and
D. J. Mooney, Nat. Commun., 2015, 6, 6365. 38 S. Itzkovitz, A. Lyubimova, I. C. Blat, M. Maynard, J. Van Es,
J. Lees, T. Jacks, H. Clevers and A. Van Oudenaarden, Nat. Cell Biol., 2012, 14, 106–114. 13 H. Liu, Q. Yi, Y. Liao, J. Feng, M. Qiu and L. Tang, Gene, 2012,
501, 153–163. 8024 | Chem. Sci., 2017, 8, 8019–8024 This journal is © The Royal Society of Chemistry 2017
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Maternal and neonatal outcome of births planned in alongside midwifery units: A cohort study from a tertiary center in Germany
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Ulrich Gembruch Department of Obstetrics and Prenatal Medicine, University Hospital Bonn, Venusberg-Campus 1, D-
53127 Bonn, Germany Department of Obstetrics and Prenatal Medicine, University Hospital Bonn, Venusberg-Campus 1, D-
53127 Bonn, Germany Research article Keywords: Midwife-led care, Alongside midwifery unit, Low-risk pregnancy, Transfer rate, Maternal
healthcare provision, Obstetric service, Mode of birth, Obstetric intervention Posted Date: April 29th, 2020 DOI: https://doi.org/10.21203/rs.2.15294/v3 Waltraut Maria Merz Department of Obstetrics and Prenatal Medicine, University Hospital Bonn, Venusberg-Campus 1, D-
53127 Bonn, Germany Laura Tascon-Padron
(
laura.tascon@ukbonn.de
)
Universitatsklinikum Bonn
https://orcid.org/0000-0001-9941-9001 DOI: https://doi.org/10.21203/rs.2.15294/v3 License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. R
d F ll Li License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Page 1/20 Version of Record: A version of this preprint was published at BMC Pregnancy and Childbirth on May 6th,
2020. See the published version at https://doi.org/10.1186/s12884-020-02962-4. Version of Record: A version of this preprint was published at BMC Pregnancy and Childbirth on May 6th,
2020. See the published version at https://doi.org/10.1186/s12884-020-02962-4. Version of Record: A version of this preprint was published at BMC Pregnancy and Childbirth on May 6th,
2020. See the published version at https://doi.org/10.1186/s12884-020-02962-4. Page 2/20 Abstract Background For healthy women entering birth after uneventful pregnancy, midwife-led models of care
have the potential to reduce interventions and increase the vaginal birth rate. In Germany, 98.4% of
women are giving birth in consultant-led obstetric units. Alongside midwifery units (AMU) have been
established in 2003. We compared the outcome of women registered for planned birth in the AMU at our
hospital with a matched group of low-risk women who gave birth in standard obstetric care during the
same period of time. Methods We used a retrospective cohort study design. The study group consisted of all women admitted
to labor ward who had registered for birth in AMU from 2010 to 2017. For the control group, low-risk
women were selected; additionally, matching was performed for parity. Mode of birth was chosen as
primary outcome parameter for the mother. For the neonate, a composite primary outcome (5-minute
Apgar <7 or umbilical cord arterial pH < 7.10 or transfer to specialist neonatal care) was defined. Secondary outcomes included epidural anesthesia, duration of the second stage of labor, episiotomy,
obstetric injury, and postpartum hemorrhage. Non-inferiority was assessed, and multiple logistic
regression analysis was performed. Results 612 women were admitted for labor in AMU, the control group consisted of 612 women giving
birth in standard obstetric care. Women in the study group were on average older and had a higher BMI;
birthweight was on average 95 g higher. Non-inferiority could be established for the primary outcome
parameters. Epidural anesthesia and episiotomy rates were lower, and the mean duration of the second
stage of labor was shorter in the study group; second-degree perineal tears were less common, higher-
order obstetric lacerations occurred more frequently. Overall, 50.3% of women were transferred to
standard obstetric care. Regression analysis revealed effects of parity, age and birthweight on the chance
of transfer. Conclusion Compared to births in our consultant-led obstetric unit, the outcome of births planned in the
AMU was not inferior, and intervention rates were lower. Our results support the integration of AMU as a
complementary model of care for low-risk women. Background Advantages of Midwife-Led Care (MLC) models have been widely reported. For healthy women entering
labor after an uneventful pregnancy, various beneficial effects have been observed. These include, among
others, an increased likelihood of giving birth vaginally; a lower intervention rate, including epidural
anesthesia and instrumental vaginal birth; and a shorter duration of labor (1–6). Due to the low incidence
of severe perinatal morbidity and mortality in high-income countries, less data are available with respect
to the newborn. MLC is practiced in different settings, including home births, births in freestanding midwifery units (FMU),
and births in alongside midwifery units (AMU). Within these different settings, MLC may be restricted to Page 3/20 the time of birth or may constitute a continuity of care during pregnancy, birth and postpartum. MLC may
be organized as caseload MLC, where one midwife or a small group of midwives is attending to a woman
throughout pregnancy, birth and postpartum, or a conventional type of MLC; the latter does not imply
continuity of care by one professional. Moreover, according to the organization of the maternal healthcare
system, practices in these models of care vary between countries. These pertain for example to transfer
modalities to standard obstetric care in case of complications during or immediately after labor. Therefore, studies about MLC may yield different results. In Germany, 98.4% of women are giving birth in obstetric units. Here, the care of women in labor is shared
by the attending midwife and obstetrician. The remaining 1.6% of births take place at home or in FMUs. Outcome data of out-of-hospital births are collected on a voluntary base, and show a low rate of
complications (7). However, due to the low numbers, non-uniform eligibility criteria and non-standardized
transfer modalities, the figures do not allow an evaluation of this model of care with regard to maternal
and perinatal safety. Supported by a team of researchers, care in AMU has been introduced in Germany in 2003 (8). AMUs are
hospital-based and located within obstetric units. Births in AMUs are offered in addition to standard
obstetric care by the same team of midwives. Low-risk women who have chosen this model of care are
attended to by a midwife from the team, with transfer to standard obstetric care in case of abnormalities. Continuous, one-to-one support is being provided. Since both models of care are situated within the same
premises, transfer to standard obstetric care occurs without delay. Background Indications for transfer include, among
others, maternal request for intravenous or epidural analgesia, and necessity of oxytocin augmentation. The maternal and perinatal outcome of births in AMUs in Germany has not yet been investigated. The
German birth registry (9) does not allow for an analysis of these cases, since births in AMUs are not
specifically labelled. Additionally, obstetric departments with established AMUs have no legal obligation
to report the outcome of births in this model of care. The Department of Obstetrics at the University Bonn Medical School introduced an AMU in November
2009. Data of all eligible women (medical and obstetric history, details of the birth, transfer cause if
applicable, and maternal and newborn outcome) are entered into a computer-assisted database,
irrespective of the actual place of birth (AMU or standard obstetric care, in case of transfer during or
immediately after birth). The aims of our study were (i) to compare the maternal and perinatal outcome of women registered for
birth in the AMU at our hospital, a level three university hospital department, with births of low-risk women
in standard obstetric care, occurring in our unit during the same period of time; and (ii) to investigate
causes for and outcome of births of women who were transferred from AMU to standard obstetric care
during or immediately after birth. Methods Page 4/20 Page 4/20 Women planning to give birth in AMU were assessed by a midwife from the team during an antenatal visit
in late pregnancy. A checklist which had been developed jointly by the team of obstetricians and
midwives was applied. Likewise, a checklist with criteria for transfer to standard obstetric care during
labor was in place. A retrospective cohort study design was used. The study group consisted of all women admitted to the
labor ward at the Department of Obstetrics, University Hospital Bonn, who had registered for birth in the
AMU between January 2010 and December 2017. Only singleton term cephalic pregnancies were
included. Exclusion criteria were as follows: (1) Medical history: preexisting medical condition (e.g. type 1 diabetes, cardiac disease); BMI >35 kg/m2. (2) Obstetric history: complications during previous deliveries (e.g. shoulder dystocia; postpartum
hemorrhage); previous cesarean section (CS). (1) Medical history: preexisting medical condition (e.g. type 1 diabetes, cardiac disease); BMI >35 kg/m2. (2) Obstetric history: complications during previous deliveries (e g shoulder dystocia; postpartum (1) Medical history: preexisting medical condition (e.g. type 1 diabetes, cardiac disease); BMI >35 kg/m2. 1) Medical history: preexisting medical condition (e.g. type 1 diabetes, cardia (2) Obstetric history: complications during previous deliveries (e.g. shoulder dystocia; postpartum
hemorrhage); previous cesarean section (CS). (3) Complications during the ongoing pregnancy: maternal complications (e.g. pregnancy-induced
hypertension / preeclampsia; insulin-dependent gestational diabetes); amniotic fluid abnormalities (oligo-
or polyhydramnios). Indications for transfer to standard obstetric care during birth included delayed first or second stage;
oxytocin augmentation; fetal heart rate (FHR) abnormalities; maternal pyrexia; and request for i.v. opioid
or epidural analgesia. Indications for transfer to standard obstetric care immediately after labor included
postpartum hemorrhage and higher-degree obstetric lacerations. Indications for transfer to standard obstetric care during birth included delayed first or second stage;
oxytocin augmentation; fetal heart rate (FHR) abnormalities; maternal pyrexia; and request for i.v. opioid
or epidural analgesia. Indications for transfer to standard obstetric care immediately after labor included
postpartum hemorrhage and higher-degree obstetric lacerations. The control group consisted of women who would have been eligible for birth in the AMU due to their low-
risk profile, but who gave birth in standard obstetric care during the same period of time. Methods Additionally, the following secondary outcomes were defined: For
the mother, duration of the second stage of labor; epidural analgesia; postpartum hemorrhage; obstetric
injury; and episiotomy. For the newborn, Apgar scores at one, five and ten minutes; and Base Excess (BE)
in the umbilical cord artery. In case of transfer to standard obstetric care, causes for transfer were
recorded; likewise, indications for operative vaginal birth or emergency CS were noted. Statistical methods The analysis was performed on an intention to treat approach. Descriptive analyses were performed to
examine the basic characteristics of the study and control groups. Differences between the matched
groups were evaluated using McNemar’s tests (for categorical variables) and paired sample t tests (for
continuous variables). The risk difference between paired proportions of each primary outcome variable
(measured in percent) together with the 95% confidence interval (CI) was determined. As the intent of our
analysis was to demonstrate that the safety of AMU care with respect to the primary outcomes is not
worse than the standard obstetric care by more than a small amount, we assessed non-inferiority using
the risk differences between both models of care. Non-inferiority of AMUs to standard obstetric care was
declared if the lower bound of the confidence interval of the difference did not fall below the non-
inferiority margin of -2%. Subsequently, generalized linear mixed-effects models were used to assess the
association between the type of care and the mode of birth (operative vaginal/CS versus spontaneous
birth) or the neonatal composite outcome (yes versus no). We adjusted for parity, maternal age, maternal
BMI and birthweight. Further multiple logistic regression analysis was used to identify factors for a
transfer to standard obstetric care during or immediately after birth in the study group. Analyses were
carried out using R (version 3.5.2) and SAS® Software (version 9.4, SAS Institute Inc. Cary, NC, USA). Methods Women for the control group were selected as follows: For each woman in the study group, the
subsequent birth in the delivery book was chosen if it fulfilled the eligibility criteria for care in the AMU
(see above). Additionally, matching of cases and controls according to parity (nulliparous and parous)
was performed. Not all eligibility and exclusion criteria are mentioned in the delivery book. A detailed assessment
including medical and obstetric history as well as abnormalities during the course of the pregnancy of
each case identified in the delivery book was therefore performed. For this purpose the electronic
departmental database (Viewpoint, GE Healthcare GmbH, Solingen, Germany) was used. Here, each
woman is allocated a unique number, and each pregnancy is entered as a separate case. The database
contains all medical, prenatal and obstetric data. In case eligibility was confirmed the woman was finally
entered into the control group. Otherwise, the subsequent birth in the delivery book was identified and the
procedure repeated. The study was approved by the Ethics Committee of the University Bonn Medical School (registration
number 254/18). Women in the study group gave their written consent for inclusion into the registry at the Page 5/20 Page 5/20 Page 5/20 time of consent for birth in the AMU. For the mother, mode of birth was chosen as primary outcome. For the newborn, a composite outcome of
5-minute Apgar score < 7 and / or umbilical cord arterial pH < 7.10 and /or unplanned transfer to
specialist neonatal care was defined. For the primary outcomes, non-inferiority of care in the AMU to
standard obstetric care was calculated. Additionally, the following secondary outcomes were defined: For
the mother, duration of the second stage of labor; epidural analgesia; postpartum hemorrhage; obstetric
injury; and episiotomy. For the newborn, Apgar scores at one, five and ten minutes; and Base Excess (BE)
in the umbilical cord artery. In case of transfer to standard obstetric care, causes for transfer were
recorded; likewise, indications for operative vaginal birth or emergency CS were noted. For the mother, mode of birth was chosen as primary outcome. For the newborn, a composite outcome of
5-minute Apgar score < 7 and / or umbilical cord arterial pH < 7.10 and /or unplanned transfer to
specialist neonatal care was defined. For the primary outcomes, non-inferiority of care in the AMU to
standard obstetric care was calculated. Results During the study period, 612 women were admitted for labor in the AMU. Likewise, the control group
consisted of 612 low-risk women, matched for parity, who gave birth in standard obstetric care. (a) Outcome of births in the study and control group (a) Outcome of births in the study and control group Table 1 summarizes maternal, obstetric, and neonatal data of the study and control group. The women
planning to give birth in AMC were on average older (mean 32.9 years, SD 4.4 versus mean 32.1 years, SD
5.1). The rate of overweight or obese women (BMI 25.0 – 35.0 kg/m2) was higher in the study group
(68.8% versus 32.0%). Six women with BMI > 35 kg/m2 (maximum BMI 36.8 kg/m2) had been
erroneously recruited in the study group; they were included in the analysis. Page 6/20 There was no difference in the primary maternal outcome. The analysis of secondary maternal outcomes
yielded the following results (see Table 1): The epidural anesthesia rate was significantly lower (19.1% vs. 41.2%) and the duration of the second stage of labor was shorter in the study group (47.4 min. vs. 55.6
min.). Episiotomies were less commonly performed (4.7% vs. 8.6%). Second-degree perineal tears were
less common in the study group (34.4% vs. 46.4%), higher-order obstetric lacerations occurred more
frequently in the study group (2.3% vs. 0.9%). There was no difference in the postpartum hemorrhage rate
between the study and control group. There was no difference in the primary maternal outcome. The analysis of secondary maternal outcomes
yielded the following results (see Table 1): The epidural anesthesia rate was significantly lower (19.1% vs. 41.2%) and the duration of the second stage of labor was shorter in the study group (47.4 min. vs. 55.6
min.). Episiotomies were less commonly performed (4.7% vs. 8.6%). Second-degree perineal tears were
less common in the study group (34.4% vs. 46.4%), higher-order obstetric lacerations occurred more
frequently in the study group (2.3% vs. 0.9%). There was no difference in the postpartum hemorrhage rate
between the study and control group. The comparison of newborn data showed a significant difference in the birthweight. Newborns in the
study group were on average 95 g heavier, and fourteen (2.3%) newborns (versus four (0.7%) in the
control group) had a birthweight ≥ 4,500 g. No difference was present for the components of the
neonatal composite outcome and the secondary neonatal outcomes. Non-inferiority results are summarized in Table 2. Non-inferiority could be confirmed for the predefined
outcome parameters. Non-inferiority results are summarized in Table 2. Non-inferiority could be confirmed for the predefined
outcome parameters. (a) Outcome of births in the study and control group Second stage of labor was longer after transfer to standard obstetric care, see Figure 2 (median
46 minutes, compared to 16 minutes in the study group, and 31 minutes in the control group, Transfer to standard obstetric care was common in nulliparous women (74.7% of all nulliparous women,
representing 68.2% of all transfers), see Figure 1. 73.7% of transferred women had a BMI ≥ 25 kg/m2, see
Figure 1. Second stage of labor was longer after transfer to standard obstetric care, see Figure 2 (median
46 minutes, compared to 16 minutes in the study group, and 31 minutes in the control group,
respectively). Epidural analgesia rates were comparable for the transferred and the control group (38.0%
vs. 41.2%). Episiotomies were more frequently performed after transfer to standard obstetric care, see
Figure 3. respectively). Epidural analgesia rates were comparable for the transferred and the control group (38.0%
vs. 41.2%). Episiotomies were more frequently performed after transfer to standard obstetric care, see
Figure 3. The number of newborns with an umbilical artery pH < 7.10 was similar in the transferred group and in
the newborns of the control group (see Figure 4), as was the rate of the other neonatal outcome
parameters 5-minute Apgar score <7 and need for specialist neonatal care (data not shown). Logistic regression analysis revealed significant effects of parity, age and birthweight with higher odds
for transfer for nulliparous women, higher age and increased birthweight, see Table 6. Discussion Our retrospective analysis of births planned in the AMU at our institution, a level three university hospital,
confirms the non-inferiority of this model compared to standard obstetric care for selected maternal and
newborn outcomes. Additionally, the outcome of women transferred during or immediately after labor
serves as an indirect confirmation of the medical safety of this model of care and the appropriateness of
the transfer checklist. Our findings apply to healthy women entering labor after uneventful pregnancy. To
the best of our knowledge, this is the first study reporting on obstetric and perinatal outcomes of women
intending to give birth in an AMU in Germany. (a) Outcome of births in the study and control group The generalized linear mixed effects model did not reveal significant effects of AMU care, BMI or
birthweight on the mode of birth (CS plus instrumental birth versus spontaneous birth), nulliparity and a
higher age however showed significantly higher odds for CS/vaginal instrumental birth (Model 1, Table
3). Further, AMU care, nulliparity, age, BMI, mode of birth and birthweight did not affect the composite
neonatal outcome, see Model 2 in Table 3. Indications for CS and instrumental vaginal birth are listed in Table 4. The leading cause in the study and
control group consisted of fetal heart rate abnormalities, followed by delayed second stage of labor. (b) Causes and outcome of births transferred to standard obstetric care (b) Causes and outcome of births transferred to standard obstetric care Overall, 308 women (50.3%) were transferred from care in the AMU to standard obstetric care. Transfer
causes according to their frequency were as follows: request for regional analgesia (28.9%); fetal heart
rate abnormalities or stained amniotic fluid (24.4%); pregnancy-induced hypertension / preeclampsia,
postdate, or prelabor rupture of membranes requiring induction of labor (19.2%); incomplete placenta,
postpartum hemorrhage, or higher-degree laceration (13.6%); and delayed first or second stage of labor
(13.3%). The vaginal birth rate after transfer was 81.5%, including 12.3% instrumental vaginal deliveries. There
was no difference in the indications for instrumental vaginal birth and CS between the women after
transfer and the control group (data not shown); likewise, postpartum hemorrhage rates were
comparable. Of the 13 higher-degree tears in the study group, four occurred during care in the AMU and
resulted in transfer to standard obstetric care; the remaining nine tears occurred after transfer to standard
obstetric care, see Table 5. Five higher-degree lacerations (38.5%) were associated with instrumental
vaginal delivery. Page 7/20 Page 7/20 Transfer to standard obstetric care was common in nulliparous women (74.7% of all nulliparous women,
representing 68.2% of all transfers), see Figure 1. 73.7% of transferred women had a BMI ≥ 25 kg/m2, see
Figure 1. Second stage of labor was longer after transfer to standard obstetric care, see Figure 2 (median
46 minutes, compared to 16 minutes in the study group, and 31 minutes in the control group, Transfer to standard obstetric care was common in nulliparous women (74.7% of all nulliparous women,
representing 68.2% of all transfers), see Figure 1. 73.7% of transferred women had a BMI ≥ 25 kg/m2, see
Figure 1. (b) Causes for and outcomes of transferred births With 50.3% of women being transferred to standard obstetric care during or immediately after birth, our
transfer rate was high. Explanations include the high rate of nulliparous women, and the strict transfer
criteria. Nulliparity, higher maternal age and birthweight increased the chance of transfer to standard
obstetric care. Higher transfer rates of nulliparous women have also been described by the authors of the
Birthplace in England Study (36-45% in nulliparous vs. 9-13% in parous women) (6). The effect of parity
may also explain the low transfer rate (7.0%) of a recently published Austrian study. Here, only 27% of
women were nulliparous (10). The composition of the three groups (study group, transferred; study group, not transferred; and control
group) does not allow for a quantitative comparison of outcomes. We therefore limited our analysis to a
descriptive presentation. Overall, primary and secondary maternal and newborn outcomes were
comparable between the transferred and the control group. We take this result as indirect proof for the
safety of births in our AMU. Additionally, the comparability of maternal and newborn outcomes in the
transferred group and the control group may serve as evidence for the adequacy of the transfer checklist. Various factors limit a direct comparison of our results with other studies. These include differences in
study design and methods, e.g. with respect to randomization, analysis according to intended versus
actual place of birth, and risk assessment. Furthermore, variations exist in birth settings, since
organization of maternal healthcare provision is country-specific; this pertains to the key provider of care
during birth (midwife versus general practitioner versus specialist); the location of alongside or
freestanding midwifery units with respect to the obstetric unit; and the transfer modalities to obstetric
units in case of abnormalities occurring during or after labor. In the systematic reviews by Bohren et al. 2017 (3) and Sandall et al. 2016 (4) only randomized controlled
or cluster-randomized trials were included. One-to-one intrapartum support was compared with ‘usual’
care in any setting for its effect on various obstetric outcomes in the former, midwife-led continuity of
care models versus other models of care in the latter analysis. Among other outcome variables, higher
rates of spontaneous births, lower rates of regional analgesia, a shorter duration of labor, higher 5-minute
Apgar scores, and no difference in the perineal trauma rates were reported in these reviews (3)(4). Scarf et al. (a) Comparison between the study and control group The comparison between the study and control group revealed a non-significant trend towards higher
spontaneous and lower instrumental vaginal births. In both groups, the CS rate was below 10%,
illustrating the low risk of our cohort. We analyzed epidural anesthesia and episiotomy rates. Both interventions were less commonly
performed in the study group. This finding is in accordance with the existing literature (3)(4). Causes for the difference in the rate of higher degree perineal lacerations may include obstetric and
newborn factors: More women in the study group were overweight or obese, and more than one third of
the injuries (38.5%) that occurred after transfer to standard obstetric care were associated with
instrumental vaginal births. Additionally, newborns in the study group had a higher mean birthweight, and
newborns with birthweight ≥ 4,500 g belonged almost exclusively to the study group (91.7%). Instrumental births and macrosomia are established risk factors for higher-order obstetric lacerations. Instrumental births and macrosomia are established risk factors for higher-order obstetric lacerations. Page 8/20
Severe neonatal morbidity or even mortality are rare events in high-income countries. For example, severe
metabolic acidosis, defined as umbilical cord arterial pH < 7.00 in singleton term newborns occurred with Page 8/20 an incidence of 0.21% in Germany in 2017 (9). The size of our study group was therefore insufficient for a
valid comparison of the perinatal outcome in the two models of care. Using a composite outcome, we
showed non-inferiority of AMU to standard obstetric care in order to compensate for this shortcoming. an incidence of 0.21% in Germany in 2017 (9). The size of our study group was therefore insufficient for a
valid comparison of the perinatal outcome in the two models of care. Using a composite outcome, we
showed non-inferiority of AMU to standard obstetric care in order to compensate for this shortcoming. (b) Causes for and outcomes of transferred births (b) Causes for and outcomes of transferred births 2018 (11) in their meta-analysis compared maternal and perinatal outcomes by planned place
of birth. 28 publications with different study designs and methods were included, illustrating the above
mentioned limitations. The reported outcomes were not stratified according to core obstetric criteria
including parity, epidural analgesia, and oxytocin augmentation. The transfer rates and outcomes after
transfer were not mentioned, nor the profession of the providers looking after women who gave birth in
hospitals (11). Page 9/20 Of all non-randomized studies, the ‘Birthplace in England national prospective cohort study’ represents the
largest study of its kind with the most rigorous design (6). Maternal and perinatal outcomes of 64,538
low-risk women were prospectively analyzed according to the planned place of birth. With the exception
of planned home birth in nulliparous women, the perinatal outcome was comparable in midwifery-led
models of care compared to births in an obstetric unit, with less maternal interventions in the former
group (6). More recent European studies share our retrospective design, however, study groups or control groups are
different. Gidaszewksi et al. 2019 investigated nulliparous women only and compared caseload with
standard midwifery-led care (12). Jepsen et al. 2018 included at-risk women and compared caseload with
standard midwifery care (13). The study design applied by Bartuseviciene et al. 2018 does not allow the
calculation of the number of women who actually received standard obstetric care (14). In the above cited
study by Bodner-Adler et al. 2017, the control group was chosen for matching after (successful vaginal)
birth, thereby precluding a direct comparison with the study group. Additionally, care in AMU included
oxytocin augmentation and i.v.-opioid analgesia (10). Strengths of our study include the strict inclusion and exclusion criteria applied for both, study and
control group; the pre-specified and documented transfer criteria; and the description of the maternal and
newborn outcome after transfer from AMU to standard obstetric care. Limitations of our study pertain to its retrospective design and the size of the study and control group,
which precludes the analysis of rare maternal or newborn complications. Additionally, the high transfer
rate has to be borne in mind for comparison with other studies. Conclusion Our comparison of the maternal and perinatal outcome of births planned in AMU with standard obstetric
care revealed the non-inferiority and safety of the midwife-led model. This beneficial outcome however
requires a clear definition of low-risk pregnancy, and strict admission and transfer criteria. FMU - freestanding midwifery units
i.v. - intravenous
MLC - midwife-led care
SD - standard deviation FMU - freestanding midwifery units
i.v. - intravenous
MLC - midwife-led care
SD - standard deviation Funding The study was funded by the ‘Landeszentrum Gesundheit Nordrhein-Westfalen’. The funding body did not
have any role in the design of the study and collection, analysis and interpretation of data and in writing
the manuscript. Availability of data and materials The datasets used and analysed during the current study are available from the corresponding author on
request. Consent for publication Not applicable Ethics approval and consent to participate The study was approved by the Ethics Committee of the University Bonn Medical School (registration
number 254/18). Women in the study group gave their written consent for inclusion into the registry at the
time of consent for birth in the AMU. Competing interests The authors declare that they have no competing interests. List Of Abbreviations AMU - alongside midwifery unit Page 10/20 Page 10/20 Authors’ contributions AH: data collection
SLT: data collection AH: data collection SLT: data collection MTP: statistical analysis, manuscript writing Page 11/20 MS: study design, statistical analysis LTP: data collection, manuscript writing We confirm that the manuscript has been read and approved by all named authors and that there are no
other persons who satisfied the criteria for authorship but are not listed. We agree to be personally accountable for our contributions to this literature, for both their accuracy and
integrity. Questions regarding accuracy and integrity have been investigated, resolved and the resolution
documented in the literature. Acknowledgements Not applicable References 1. World Health Organization. WHO recommendations: Intrapartum care for a positive childbirth
experience. Geneva; 2018. 1. World Health Organization. WHO recommendations: Intrapartum care for a positive childbirth
experience. Geneva; 2018. 1. World Health Organization. WHO recommendations: Intrapartum care for a positive childbirth
experience. Geneva; 2018. 2. Oladapo OT, Tunçalp Ö, Bonet M, Lawrie TA, Portela A, Downe S et al. WHO model of intrapartum care
for a positive childbirth experience: transforming care of women and babies for improved health and
wellbeing. BJOG 2018; 125(8):918–22. 3. Bohren MA, Hofmeyr GJ, Sakala C, Fukuzawa RK, Cuthbert A. Continuous support for women during
childbirth (Review). Cochrane Database Syst Rev 2017; 7:CD003766. 4. Sandall J, Soltani H, Gates S, Shennan A, Devane D. Midwife-led continuity models versus other
models of care for childbearing women. Cochrane Database Syst Rev 2016; 4. 5. McLachlan HL, Forster DA, Davey MA, Farrell T, Gold L, Biro MA et al. Effects of continuity of care by
a primary midwife (caseload midwifery) on caesarean section rates in women of low obstetric risk:
the COSMOS randomised controlled trial. BJOG 2012; 119(12):1483–92. 5. McLachlan HL, Forster DA, Davey MA, Farrell T, Gold L, Biro MA et al. Effects of continuity of care by
a primary midwife (caseload midwifery) on caesarean section rates in women of low obstetric risk:
the COSMOS randomised controlled trial. BJOG 2012; 119(12):1483–92. 6. Brocklehurst P, Hardy P, Hollowell J, Linsell L, Macfarlane A, McCourt C et al. Perinatal and maternal
outcomes by planned place of birth for healthy women with low risk pregnancies: the Birthplace in
England national prospective cohort study: Birthplace in England Collaborative Group. BMJ 2011;
343(d7400). 6. Brocklehurst P, Hardy P, Hollowell J, Linsell L, Macfarlane A, McCourt C et al. Perinatal and maternal
outcomes by planned place of birth for healthy women with low risk pregnancies: the Birthplace in
England national prospective cohort study: Birthplace in England Collaborative Group. BMJ 2011;
343(d7400). 7. QUAG Gesellschaft für Qualität in der außerklinischen Geburtshilfe e.V. Geburtenverteilung im Jahr
2015 nach Bundesländern: Anzahl der Hausgeburten und Anzahl der Geburten in von Hebammen
geführten Einrichtungen (HgE) 2015. 8. Bauer N, Kehrbach A, Krahl, Astrid, von Rahden, Oda, zu Sayn-Wittgenstein, Friederike. Handbuch
Hebammenkreißsaal: Von der Idee zur Umsetzung. Osnabrück: Verbund Hebammenforschung; 2007. 8. Bauer N, Kehrbach A, Krahl, Astrid, von Rahden, Oda, zu Sayn-Wittgenstein, Friederike. Handbuch
Hebammenkreißsaal: Von der Idee zur Umsetzung. Osnabrück: Verbund Hebammenforschung; 2007. 9. References Institut für Qualitätssicherung und Transparenz im Gesundheitswesen. Bundesauswertung zum
Erfassungsjahr 2017 Geburtshilfe Qualitätsindikatoren 2018. 9. Institut für Qualitätssicherung und Transparenz im Gesundheitswesen. Bundesauswertung zum
Erfassungsjahr 2017 Geburtshilfe Qualitätsindikatoren 2018. Page 12/20 Page 12/20 10. Bodner-Adler B, Kimberger O, Griebaum J, Husslein P, Bodner K. A ten-year study of midwife-led care
at an Austrian tertiary care center: a retrospective analysis with special consideration of perineal
trauma. BMC Pregnancy Childbirth 2017; 17(1):357. 11. Scarf VL, Rossiter C, Vedam S, Dahlen HG, Ellwood D, Della Forster et al. Maternal and perinatal
outcomes by planned place of birth among women with low-risk pregnancies in high-income
countries: A systematic review and meta-analysis. Midwifery 2018; 62:240–55. 11. Scarf VL, Rossiter C, Vedam S, Dahlen HG, Ellwood D, Della Forster et al. Maternal and perinatal
outcomes by planned place of birth among women with low-risk pregnancies in high-income
countries: A systematic review and meta-analysis. Midwifery 2018; 62:240–55. 12. Gidaszewski B, Khajehei M, Gibbs E, Chua SC. Comparison of the effect of caseload midwifery
program and standard midwifery-led care on primiparous birth outcomes: A retrospective cohort
matching study. Midwifery 2019; 69:10–6. 12. Gidaszewski B, Khajehei M, Gibbs E, Chua SC. Comparison of the effect of caseload midwifery
program and standard midwifery-led care on primiparous birth outcomes: A retrospective cohort
matching study. Midwifery 2019; 69:10–6. 13. Jepsen I, Juul S, Foureur MJ, Sørensen EE, Nohr EA. Labour outcomes in caseload midwifery and
standard care: a register-based cohort study. BMC Pregnancy Childbirth 2018; 18(1):481. 13. Jepsen I, Juul S, Foureur MJ, Sørensen EE, Nohr EA. Labour outcomes in caseload midwifery and
standard care: a register-based cohort study. BMC Pregnancy Childbirth 2018; 18(1):481. 13. Jepsen I, Juul S, Foureur MJ, Sørensen EE, Nohr EA. Labour outcomes in caseload midwifery and
standard care: a register-based cohort study. BMC Pregnancy Childbirth 2018; 18(1):481. 14. Bartuseviciene E, Kacerauskiene J, Bartusevicius A, Paulionyte M, Nadisauskiene RJ, Kliucinskas M
et al. Comparison of midwife-led and obstetrician-led care in Lithuania: A retrospective cohort study. Midwifery 2018; (65):67–71 14. Bartuseviciene E, Kacerauskiene J, Bartusevicius A, Paulionyte M, Nadisauskiene RJ, Kliucinskas M
et al. Comparison of midwife-led and obstetrician-led care in Lithuania: A retrospective cohort study. Midwifery 2018; (65):67–71 Table 1 Basic characteristics of the study and control group (N=1224). *Missing for pairs with mode of birth = cesarean. D = standard deviation. BMI = body mass index, SD = standard deviation. Tables le 1 Basic characteristics of the study and control group (N=1224). Table 1 Basic characteristics of the study and control group (N=1224). Page 13/20 Study group
(n = 612)
Control group
(n = 612)
p value
Age (years), mean (SD)
32.9 (4.4)
32.1 (5.1)
0.003
BMI (kg/m2) ≥ 25, n (%)
421 (68.8)
196 (32.0)
<0.001
Mode of birth, n (%)
0.101
Spontaneous
517 (84.5)
502 (82.0)
Instrumental vaginal
38 (6.2)
58 (9.5)
Cesarean
57 (9.3)
52 (8.5)
Epidural anesthesia, n (%)
117 (19.1)
252 (41.2)
<0.001
Duration, second stage of labor* (min), mean (SD)
47.4 (54.1)
55.6 (59.5)
0.002ϯ
Episiotomy*, n (%)
26 (4.7)
48 (8.6)
0.066
Obstetric injury*, n (%)
0.007
First degree perineal laceration,
labia laceration
149 (26.8)
119 (21.2)
Second degree perineal laceration,
vaginal or clitoral laceration
191 (34.4)
260 (46.4)
Third or fourth degree perineal
laceration, cervical laceration
13 (2.3)
5 (0.9)
None
202 (36.4)
176 (31.4)
Post-partum hemorrhage, n (%)
43 (7.0)
55 (9.0)
0.246
Birthweight (gram), mean (SD)
3561.0 (427.3)
3466.3 (422.5)
<0.001
5-minute APGAR score < 7, n (%)
3 (0.5)
2 (0.3)
1.0
Umbilical cord arterial pH < 7.10, n (%)
8 (1.3)
16 (2.6)
0.153
Missing
12 (2.0)
3 (0.5)
Transfer to specialist neonatal care, n (%)
5 (0.8)
9 (1.5)
0.386 BMI = body mass index, SD = standard deviation. *Missing for pairs with mode of birth = cesarean. ϯWilcoxon signed-rank test. Table 2 Results of the non-inferiority analysis (with non-inferiority margin of -2%) (N=1224). Page 14/20 Page 14/20 Study
group
(n = 612)
Control
group
(n = 612)
% Difference
(95% CI)
p value
Cesarean/ instrumental vaginal birth, n
(%)
95 (15.5)
110 (18.0)
2.45
(-1.34 - 6.25)
0.010
Composite outcomeϮ, n (%)
14 (2.3)
22 (3.7)
1.34
(-0.65 - 3.40)
<0.001 Study
group
(n = 612)
Control
group
(n = 612)
% Difference
(95% CI)
p value
Cesarean/ instrumental vaginal birth, n
(%)
95 (15.5)
110 (18.0)
2.45
(-1.34 - 6.25)
0.010
Composite outcomeϮ, n (%)
14 (2.3)
22 (3.7)
1.34
(-0.65 - 3.40)
<0.001
CI = confidence interval. ϮNewborns with umbilical cord arterial pH < 7.10 and/or 5-minute APGAR < 7 and/or transfer CI = confidence interval. ϮNewborns with umbilical cord arterial pH < 7.10 and/or 5-minute APGAR < 7 and/or transfer
to specialist neonatal care; pairs with missing pH values were excluded. ϮNewborns with umbilical cord arterial pH < 7.10 and/or 5-minute APGAR < 7 and/or transfer
to specialist neonatal care; pairs with missing pH values were excluded. able 3 Results of the generalized linear mixed-effects models for the mode of birth (model 1) and
he composite outcome (model 2Ϯ). Model 1
Model 2Ϯ
aOR (95% CI)
p value
aOR (95% CI)
p value
Study group
0.86 (0.59-1.26)
0.449
0.59 (0.27-1.26)
0.171
Nulliparous
10.82 (6.55-
17.89)
<0.001
1.47 (0.66-3.24)
0.344
Age (years)
≤ 29
1.00 (-)
1.00 (-)
30-34
1.33 (0.84-2.09)
0.220
1.02 (0.42-2.51)
0.960
≥ 35
2.56 (1.56-4.20)
<0.001
1.05 (0.39-2.79)
0.927
BMI (kg/m2) < 25
1.38 (0.93-2.04)
0.115
0.97 (0.45-2.08)
0.935
Birthweight (100 gram)
1.04 (0.99-1.08)
0.090
1.04 (0.96-1.14)
0.326
Cesarean/ instrumental vaginal
birth
–
–
1.50 (0.61-3.66)
0.376 Table 3 Results of the generalized linear mixed-effects models for the mode of birth (model 1) and
the composite outcome (model 2Ϯ). aOR = Adjusted Odds Ratio; CI = Confidence interval; BMI = Body mass index. ϮPairs with missing pH values were excluded. Table 4 Medical indications for instrumental vaginal birth or emergency cesarean by study and
control group (N=205). Page 15/20 Page 15/20 Study group
(n = 95)
Control group
(n = 110)
p value
Indications, n (%)
0.930
Abnormal FHR
41 (43.2)
54 (49.1)
Delayed second stage
36 (37.9)
29 (26.4)
Delayed first stage
9 (9.5)
8 (7.3)
Other
9 (9.5)
19 (17.3) FHR = fetal heart rate. Table 5 Higher order obstetric injuries by study and control group and actual place of birth
(N=18). Study group (n=13)
Control
group
(n = 5)
During care in AMU
(n=4)
After transfer to standard obstetric
care (n=9)
Obstetric injury, n
(%)
Third degree
tear
4 (100.0)
7 (77.8)
1 (20.0)
Fourth degree
tear
0 (0.0)
2 (22.2)
0 (0.0)
Cervical tear
0 (0.0)
0 (0.0)
4 (80.0)
AMU = alongside midwifery units. Table 5 Higher order obstetric injuries by study and control group
(N=18). Table 5 Higher order obstetric injuries by study and control group and actual place of birth
(N=18). AMU = alongside midwifery units. Table 6 Results of multiple logistic regression analysis for the transfer to obstetric care during
or immediately after birth (N=612). CI = confidence interval. Table 6 Results of multiple logistic regression analysis for the transfer to obstetric care during
or immediately after birth (N=612). Page 16/20
aOR (95% CI)
p value
Nulliparous
8.70 (5.84-13.20)
<0.001
Age (years)
≤ 29
1.00 (-)
30-34
1.26 (0.77-2.05)
0.361
≥ 35
2.12 (1.25-3.65)
0.006
BMI (kg/m2) < 25
0.75 (0.51-1.12)
0.159
Birthweight (100 gram)
1.05 (1.00-1.09)
0.035 aOR = Adjusted Odds Ratio; CI = Confidence interval; BMI = Body mass index. Figures Figures Figures Figure 1
Parity and BMI by study group, transferred (n=308); study group, not transferred (n=304); study group,
total (n=612); and control group (n=612). Figure 1 Parity and BMI by study group, transferred (n=308); study group, not transferred (n=304); study group,
total (n=612); and control group (n=612). Page 17/20 Page 17/20 Page 17/20 Page 17/20 Figure 2
Duration of the second stage of labor (minutes) by study group, transferred (n=251); study group, not
transferred (n=303); study group, total (n=554); and control group (n=559). Figure 2 Figure 2 Duration of the second stage of labor (minutes) by study group, transferred (n=251); study group, not
transferred (n=303); study group, total (n=554); and control group (n=559). Page 18/20 Figure 3
Episiotomy by study group, transferred (n=251); study group, not transferred (n=304); study group, total
(n=554); and control group (n=560). Figure 3 Figure 4 Umbilical cord arterial pH value by study group, transferred (n=302); study group, not transferred (n=298);
study group, total (n=600); and control group (n=609). Umbilical cord arterial pH value by study group, transferred (n=302); study group, not transferred (n=298);
study group, total (n=600); and control group (n=609). Figure 3 Episiotomy by study group, transferred (n=251); study group, not transferred (n=304); study group, total
(n=554); and control group (n=560). Page 19/20 Page 19/20 Page 19/20 igure 4
Umbilical cord arterial pH value by study group, transferred (n=302); study group, not transferred (n=298);
tudy group, total (n=600); and control group (n=609). Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. databaseLTPWM1.xlsx STROBEchecklist210920WM.doc Abbildungen.docx TabellenWM11.9..docx Page 20/20 Page 20/20
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Rede Social de imagens e produções locais para pensar sobre um problema nacional: a pandemia da Covid-19 no Brasil
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Revista Latinoamericana de Ciencias de la Comunicación
| 2,022
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cc-by
| 6,187
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camila Leite de Araujo professora Adjunta da universidade Federal do Amazonas do curso de Jornalismo da Faculdade
de informação e comunicação. doutora em comunicação na universidade Federal do pernambuco,
mestre em comunicação pela universidade Federal do ceará, especialista em teorias da comunicação
e da imagem pela universidade Federal do ceará e graduada em comunicação social - Jornalismo pela
universidade de Fortaleza. 144 rede sociAL de imAGens e produçÕes LocAis pArA pensAr soBre umA red sociAL de máGenes Y produccionse LocALes pArA pensAr en un
proBLemA nAcionAL: LA pAndemiA deL coVid-19 en BrAsiL cristiane Barbosa professora adjunta do curso de Jornalismo da Faculdade de informação e comunicação (Fic) da
universidade Federal do Amazonas (ufam). doutora em ciências da informação com especialidade em
Jornalismo e estudos mediáticos pela universidade Fernando pessoa (uFp/portugal) com reconhecimento
no doutorado em comunicação pela universidade Federal do rio Grande do sul (uFrGs/Brasil). e-mail: crisb.jor@gmail.com RESUMO Objetiva-se analisar a função da investigação fotográfica durante pandemia da Covid-19
no Brasil em rede social de imagens. Escolhidos três ensaios, publicados no perfil @
covidphotobrazil, analisados à luz de discussões sobre as dimensões da iconologia e da
iconografia. Concluiu-se que o papel da fotografia como provocadora de conhecimento e
debates foram renovados pelo digital. As redes sociais de imagem convidam não apenas a
vê-las, mas a questioná-las e comentá-las. As imagens são instrumentos importantes para a
conscientização sobre os diferentes contextos sociais e políticos do Brasil agravados da crise
sanitária. Considera-se de extrema importância o papel da ciência social que investiga os
dados e as informações que contextualizam as imagens na história contemporânea. Objetiva-se analisar a função da investigação fotográfica durante pandemia da Covid-19
no Brasil em rede social de imagens. Escolhidos três ensaios, publicados no perfil @
covidphotobrazil, analisados à luz de discussões sobre as dimensões da iconologia e da
iconografia. Concluiu-se que o papel da fotografia como provocadora de conhecimento e
debates foram renovados pelo digital. As redes sociais de imagem convidam não apenas a
vê-las, mas a questioná-las e comentá-las. As imagens são instrumentos importantes para a
conscientização sobre os diferentes contextos sociais e políticos do Brasil agravados da crise
sanitária. Considera-se de extrema importância o papel da ciência social que investiga os
dados e as informações que contextualizam as imagens na história contemporânea. Palavras-chave: Covid-19; fotografia digital; Brasil; representação
fotográfica. Palavras-chave: Covid-19; fotografia digital; Brasil; representação
fotográfica. Abstract The objective is to analyze the role of photographic investigation during the Covid-19
pandemic in Brazil in a social network of images. Three essays were chosen, published on
the profile @covidphotobrazil, and analyzed from iconology and iconography dimensions. It
was concluded that the role of photography as a provocateur of knowledge and debates was
renewed by the digital. Social networks of images invite not only to see them, but to question
and comment on them. Images are important tools for raising awareness about the different
social and political contexts in Brazil, aggravated by the health crisis. The role of social science
that investigates the data and information that contextualizes images in contemporary history
is extremely important. 145 Keywords: Covid-19; digital photography; Brazil; photographic
representation; SOCIAL NETWORK OF IMAGES. Palabras Clave: Covid-19; fotografía digital; Brasil; representación
fotográfica; RED SOCIAL DE IMÁGENES. 1. Introdução C
o
m C
ompreender a relação entre fotografia,
memória, cidadania e respeito por vidas
frente à crise sanitária da Covid-19, exige uma
literatura visual e debates sociais sobre esses
problemas históricos e sua documentação. Assim, acreditamos ser primordial a análise
das leituras dessas imagens, que estas sejam
amplamente compartilhadas e o entendimento
dos seus simbolismos. Foram escolhidos três ensaios, de diferentes
abordagens e autores, feitas em diferentes
lugares do país. Um a partir de uma proposta
de reportagem fotográfica, outro a partir de um
viés familiar e fotodocumental e outro feito por
um ensaio reflexivo e pessoal. Também foram
considerados no estudo alguns comentários dos
espectadores. Nesse contexto, o presente estudo se propõe
a analisar algumas imagens fotográficas feitas
durante a pandemia da Covid-19, com base em
conceitos da iconografia e da iconologia propostos
por Panofsky (2012), em 1932, e posteriormente
adaptados à linguagem fotográfica por Kossoy
(1999). O trabalho nos conduz à discussão
sobre as possibilidades do digital na produção e
circulação de imagens e seu impacto na criação
da memória sobre a representação das vítimas do
novo coronavírus e sobre o poder transformador
da fotografia em alterar a história, se amplamente
vista. Para a análise das fotografias publicadas, partiu-
se das discussões das dimensões iconológicas e
iconográficas, propostas e descritas por Panofsky
(2012), em 1932, depois adaptadas por Kossoy
(1999), que acrescentou especificidades da
linguagem fotográfica. Acredita-se que esses
métodos podem contribuir para a análise
fotográfica e compreensão da representação da
memória da Covid-19 no Brasil. O objetivo geral deste estudo é discutir o
papel central da fotografia digital como mídia
provocadora de reflexões, discussões, debates,
e, possivelmente, de empatia. Para tanto,
escolhemos como objeto de análise imagens
divulgadas pelo perfil @covidphotobrazil que tem
como desafio criar uma exposição sistemática
dessas imagens, construir um imaginário sobre
a pandemia em território nacional e divulgar
o trabalho de fotógrafos atuantes durante a
pandemia. 146 A análise iconográfica refere-se à leitura plástica
da imagem, criada a partir de um ponto de vista
do autor da imagem e eternizado pelo instante
em que o obturador foi acionado. O instante
fotográfico documentado na fotografia permite
recuperar dados preciosos para a reconstituição
da memória e da história. Resumen El objetivo es analizar el papel de la investigación fotográfica durante la pandemia Covid-19
en Brasil en una red social de imágenes. Se eligieron tres ensayos, publicados en el perfil @
covidphotobrazil, analizados a la luz de discusiones sobre las dimensiones de la iconología y
la iconografía. Se concluyó que el papel de la fotografía como provocadora de conocimientos
y debates fue renovado por lo digital. Las redes sociales de imagen te invitan no solo a verlas,
sino a cuestionarlas y comentarlas. Las imágenes son herramientas importantes para crear
conciencia sobre los diferentes contextos sociales y políticos en Brasil, agravados por la
crisis de salud. El papel de las ciencias sociales que investiga los datos y la información que
contextualiza las imágenes en la historia contemporánea es sumamente importante. Trata-se de uma análise de imagens da
pandemia da Covid-19 no Brasil em conjunto
com a análise métrica das redes sociais. O estudo
articulou métodos qualitativos de observação
direta, por meio da descrição e avaliação
qualitativa das imagens fotográficas postadas no
perfil @covidphotobrazil, e alguns comentários
de espectadores atrelados a elas. 2. Instragram e pandemiai As fotografias documentais e noticiosas
são impactadas pelo digital não apenas por
possibilitar a multiplicação de imagens com a
diminuição dos gastos envolvidos, como também
pela maior circulação por meio das redes virtuais. As fotografias documentais e noticiosas
são impactadas pelo digital não apenas por
possibilitar a multiplicação de imagens com a
diminuição dos gastos envolvidos, como também
pela maior circulação por meio das redes virtuais. Permite uma maior liberdade do fotógrafo que
pode estar ausente dos laços contratuais de uma
empresa comunicacional, e ainda assim exibir seu
trabalho que, conforme o interesse das diferentes
partes, pode ser vendido para diferentes veículos. Permite uma maior liberdade do fotógrafo que
pode estar ausente dos laços contratuais de uma
empresa comunicacional, e ainda assim exibir seu
trabalho que, conforme o interesse das diferentes
partes, pode ser vendido para diferentes veículos. As redes sociais podem representar um vetor
de fortalecimento da cidadania ao permitir a
visibilidade de temas que interessam à população e
não necessariamente a empresas comunicacionais
e ao direcionamento econômico que essas
instituições estão submetidas, inviabilizando
o cumprimento de suas funções democráticas. Proporcionam um espaço de sociabilidade,
conversação e debate sobre diversos temas a partir
de múltiplos pontos de vista. As redes sociais
possibilitam maior acesso à informação e maior
diversidade de fontes informativas e de produção
de conteúdo ao incentivar a participação da
população de forma ativa no processo político
e a resistir aos empenhos externos de subverter
sua independência. Os sites de redes sociais
são capazes de inserir novos temas na esfera de
visibilidade pública1. As consequências políticas
desses processos comunicacionais a partir das
redes sociais apontam para a importância da
abertura da agenda pública a temas que não
encontravam espaços nas mídias tracionais e que
agora se inserem na esfera de visibilidade pública,
assim como funcionam como plataforma de
questionamento da mídia dominante (SANTOS,
2012). Nesse contexto, o Instagram se apresenta
como uma das mais populares redes sociais para
o compartilhamento gratuito de imagens na
contemporaneidade. Nele, cada usuário possui
um espaço para expor suas imagens, chamado de
feed. São as imagens que vão compor o “álbum”
estável do perfil. Conforme novas imagens são
postadas, elas permanecem no topo da produção,
de forma que as antigas são menos valorizadas e
ficam no final do feed. 1 A esfera de visibilidade púbica pode ser entendida como a dimen-
são social que é visível ao conhecimento e domínio público. (GO-
MES, 2008 apud SANTOS, 2012). 1. Introdução A análise iconológica
procura informações e contextos por meio de
documentos ou do relato do autor da imagem
de forma a “desvendar a trama histórica e social
da imagem, bem como avaliar sua dimensão
cultural e ideológica” (UNFRIED, 2014, p.05). Nesse contexto, as perguntas norteadoras
que conduzem este estudo são: Como ocorre
a função social da investigação fotográfica
durante a pandemia da Covid-19 no Brasil pela
plataforma do Instagram? Como a cobertura
fotográfica sobre o Covid-19 contribui como
mídia provocadora de reflexões, discussões,
debates e empatia? Para
aprofundamento
da
interpretação
iconológica das imagens, conforme Kossoy
(1999), procuramos a fala dos fotógrafos pelo
texto-legenda e dos usuários pelos comentários
nas fotografias do perfil. linha argumentativa, sobretudo ao fato de que
a internet incentiva a discussão apenas entre
pessoas que pensam de forma semelhante, o
que implica na fragmentação das audiências e
da esfera pública. Também aponta que não é
possível atribuir à internet liberdade total, já que
o capital controla a infraestrutura de conexão, não
havendo uma divisão igualitária de visibilidade
entre diferentes tipos de informação, sem
controle sobre os fluxos de informação. Defende
ser relevante a diversidade de fatores a influir
nessa visibilidade, fazendo-a parecer maior do
que no cenário comunicacional anterior formado
por monopólios e oligopólios comunicacionais. 2 O IGTV traz é uma ferramenta de armazenamento de vídeo no
Instagram. Neles os vídeos são reproduzidos em tela cheia, formato 2. Instragram e pandemiai 147 Cada imagem, ou bloco de imagens, pode ser
comentado e curtido pelos espectadores, de forma
a criar um vínculo e uma discussão coletiva sobre
as imagens. O usuário também pode acrescentar
uma legenda à imagem ao postá-la permitindo
contextualizá-la. Mas, não há dúvidas de que o
Instagram é mais focado em imagens do que em
textos. Essa rede permite que seja postada uma
sequência de imagens de forma que as narrativas
delas passam a ser entrelaçadas, aspecto muito
utilizado no compartilhamento de fotos e ensaios. Possibilita, também, o compartilhamento de
vídeos com menos de um minuto, ficando os
demais armazenados no IGTV2. A autora citada, aponta para críticas a essa 2 O IGTV traz é uma ferramenta de armazenamento de vídeo no
Instagram. Neles os vídeos são reproduzidos em tela cheia, formato Com o auxílio de tais ferramentas, o Instagram
é uma experiência colaborativa a partir de contas
de profissionais e amadores da fotografia que
expõem suas memórias, fotografias, vídeos,
histórias, coleções e visões sobre a vida cotidiana,
íntima e social. elas e registrados pelas ferramentas de interação
das mídias sociais. Ser e fomentar cidadãos
engajados aos problemas sociais evidenciados e
provocados pela pandemia exige refletir sobre
os contextos históricos que agravam a situação
brasileira. Lewis (2016) defende que a forma como nos
mantemos conectados socialmente depende
da função das imagens, uma vez que, por
meio delas, processamos mundos diferentes
dos nossos próprios. A ferramenta fotográfica
pode atravessar nossos abismos sociais, que são
irrevogavelmente alterados pela visão. Para a
autora, a afirmação da dignidade da vida humana
não pode ser empreendida sem a representação
justa. A representação das vidas perdidas e do
sofrimento social e familiar pelo coronavírus
é uma tarefa na qual a fotografia e o vídeo são
centrais e indispensáveis. A constante expansão da internet, das redes
sociais e dos dispositivos móveis gerou uma
constante evolução da indústria de informação
e de notícia. No decorrer do registro fotográfico
da pandemia, fotógrafos locais passaram a
compartilhar as imagens de como o vírus
afetava suas regiões e as vidas das pessoas pelo
Instagram, conteúdos que chamaram a atenção
de seguidores e de veículos comunicacionais de
abrangência nacional e internacional. O projeto @covidphotobrazil é uma conta do
Instagram a partir de uma perspectiva coletiva de
expor fotografias feitas por diferentes fotógrafos
durante a pandemia da Covid-19. 2. Instragram e pandemiai Seu objetivo
é reunir imagens desse momento histórico
e permitir maior visibilidade das imagens
produzidas no contexto da situação sanitária. Na
descrição do perfil, cujo editor é @daniloverpa,
define-se como o “Diário da Covid-19 no Brasil. Fotógrafos apresentam por meio de seus olhares
o cotidiano durante a Covid-19 no Brasil”. 3 vertical e sem bordas pretas. Os vídeos do IGTV não são limitados
a um minuto.
3 Tradução livre de “Photographers showing through their vision the
day to day life during the COVID-19 in Brazil. Editor: @daniloverpa” vertical e sem bordas pretas. Os vídeos do IGTV não são limitados
a um minuto. 3. Análise As fotografias carregam contextos implícitos
que fogem do olhar atento, já que carregam limites
linguísticos, cuja memória vai além do registro do
visível. Nesse sentido, a iconografia e a iconologia
se apresentam como duas metodologias que
permitem “decifrar [...] informações explícitas
e
implícitas
no
documento
fotográfico”
(UNFRIED, 2014, p.04). Essas análises visam
permitir a recuperação de diferentes camadas de
significação. 148 No
Instagram
@covidphotobrazil
foram
publicados 619 posts feitos até o final de fevereiro
de 2021. Essas imagens representam diferentes
momentos, contextos e olhares sobre a pandemia
no Brasil. Ter uma imagem publicada pelo perfil
é reconhecimento de que ela é um documento
importante e as imagens ao longo do perfil marcam
diferentes fases da pandemia, representando uma
memória visual dos acontecimentos registrados. Kossoy
(1999),
dedicado
à
pesquisa
documental
da
fotografia,
demonstrou
a
aplicabilidade dessas metodologias para uma
análise que supera os limites plásticos fotográfico
e permite a recuperação de informações que os
contextualizam. i Kossoy
(1999),
dedicado
à
pesquisa
documental
da
fotografia,
demonstrou
a
aplicabilidade dessas metodologias para uma
análise que supera os limites plásticos fotográfico
e permite a recuperação de informações que os
contextualizam. Refletir sobre o papel da fotografia nesse
momento específico exige proximidade com
essas imagens e com os comentários atrelados a A descrição iconográfica é responsável pela
reconstituição dos elementos visíveis que
compõem a imagem fotográfica. Reconhecendo
que esta dimensão é insuficiente na apreensão
das informações dos contextos sociais e
históricos, deve ser complementada com a análise
iconológica. Esta se refere ao aprofundamento 3 Tradução livre de “Photographers showing through their vision the
day to day life during the COVID-19 in Brazil. Editor: @daniloverpa” Assim, aplicamos as instruções sobre a
investigação fotográfi ca adaptada por Kossoy
(1999) para analisar as fotografi as escolhidas
sobre a pandemia da Covid-19. investigativo para a recuperação das informações
históricas e invisíveis na imagem fotográfi ca; e se
refere ao contexto histórico antes e independente
do “clique fotográfi co”. Figura 1- Ensaio produzido por Tarso Sarraf no Pará, em maio de 2020. Figura 1- Ensaio produzido por Tarso Sarraf no Pará, em maio de 2020. Figura 1- Ensaio produzido por Tarso Sarraf no Pará, em maio de 2020. 4 Ensaio fotográfi co é um conceito que se refere a uma história con-
tada a partir de uma sequência de imagens, que permite um discurso
sensível sobre o mundo e cuja edição das imagens tenha sido feita a
partir de uma refl exão e coesão. A prática do ensaio fotográfi co tem
sido apontada como marca do fotojornalismo moderno, consolidado
nos anos de 1930 nas revistas ilustradas (PERSOCHETTI, 2000). É
importante ressaltar, também, que o fotojornalismo moderno se con-
solida a partir de uma abordagem humanista e de cunho social cujo
discurso se enfraquece a partir da década de 1960, mas que sempre
retoma seu fôlego em momentos de confl ito e de crise política e social. 4 Ensaio fotográfi co é um conceito que se refere a uma história con-
tada a partir de uma sequência de imagens, que permite um discurso
sensível sobre o mundo e cuja edição das imagens tenha sido feita a
partir de uma refl exão e coesão. A prática do ensaio fotográfi co tem
sido apontada como marca do fotojornalismo moderno, consolidado
nos anos de 1930 nas revistas ilustradas (PERSOCHETTI, 2000). É
importante ressaltar, também, que o fotojornalismo moderno se con-
solida a partir de uma abordagem humanista e de cunho social cujo
discurso se enfraquece a partir da década de 1960, mas que sempre
retoma seu fôlego em momentos de confl ito e de crise política e social. 3. Análise Fonte: Tarso Sarraf, 2020/ Instagram covidphotobrazil
https://www.instagram.com/p/CBiVp-2HXOR/ Fonte: Tarso Sarraf, 2020/ Instagram covidphotobrazil
https://www.instagram.com/p/CBiVp-2HXOR/ Fonte: Tarso Sarraf, 2020/ Instagram covidphotobrazil
https://www.instagram.com/p/CBiVp-2HXOR/ https://www.instagram.com/p/CBiVp-2HXOR/ 149 149 A fi gura 1 apresenta seis das dez fotografi as
postadas como ensaio feito pelo fotojornalista
Tarso Sarraf, @sarraft arso. Trata-se de um ensaio4
que conta um pouco sobre as difi culdades que os
povos ribeirinhos enfrentam frente a Covid-19. inter-relacionamento entre estas. A exploração
das possibilidades narrativas da fotografi a por
meio da sucessão de imagens permite uma
literatura visual mais articulada e fundamentada
sobre um tema do que se contextualizada por
uma única imagem condensada de informações
(MAGALHÃES; PEREGRINO, 2004). O impacto visual de um ensaio depende da força
narrativa individual das imagens, mas também do As
imagens
podem
ser
analisadas
iconografi camente, conforme as orientações de
Kossoy (1999) a partir da leitura de seu campo
visual. A primeira imagem trata-se de uma
fotografi a feita com o ângulo aberto; produzido
por lente angular, retratando o grupo de
socorristas na ambulancha e ao mesmo tempo
contextualizando as difi culdades de acesso e
distância que os povos ribeirinhos de Marajó do caixão. O terceiro plano são as árvores da
região e céu azul. Na sexta imagem, um retrato
em close-up do rosto de uma socorrista de olhos
fechados chorando. Nos olhos, moldurados pela
touca e máscara branca, a dor e a tristeza de quem
vivencia a realidade da Covid-19 nas regiões mais
remotas e periféricas do Brasil. sofrem para conseguir atendimento. A imagem
azulada pela cor do barco e pelas condições
atmosféricas, feita a partir de um ângulo frontal,
o que remete a um retrato posado, conversado,
autorizado, no qual se estabeleceu uma relação
entre fotógrafo e fotografados. A imagem pode ser descrita a partir de seu
centro e suas bordas. Ao centro, a ambulancha
azul, a luz interna da cabine é branca e o ponto
mais iluminado da imagem. A equipe socorrista
vestida com os equipamentos de proteção evoca
a necessidade, as dificuldades e os perigos do
contato. Ao redor do barco, a escuridão das águas
do rio e da mata, por trás dela o sol nasce. Atrelada às imagens, a informação que a
fotografia compartilhada pela @covidphotobrazil
traz a marcação do Instagram do seu autor @
sarraftarso, foi curtida por mais de 744 usuários. 3. Análise Além disso, apresenta um texto que contextualiza
alguns dos aspectos iconológicos da imagem; e
apresenta uma breve reflexão sobre a pandemia
da Covid-19 no cenário dos povos ribeirinhos: A segunda imagem pode ser lida a partir de seus
planos. No primeiro, um senhor infectado pela
Covid-19, um ribeirinho, deitado, com máscara,
olhar cansado, com a blusa aberta e sendo
examinado. As mãos enluvadas da profissional de
saúde vestida com os equipamentos de proteção
individual ligam o primeiro e o segundo plano
da imagem, a sala de atendimento, e entre eles o
zíper do plástico que isola o leito. Passei quase um mês trabalhando em três
cidades do Marajo, no Pará. E registrei a rotina
dos moradores por causa da pandemia do novo
coronavírus. Nesse período, constatei como
são gigantescas as dificuldades enfrentadas
pelos moradores desse Arquipé́lago. Estive nos
municípios de Breves, Melgaço e Portel. Uma
das principais dificuldades são as distâ̂ncias
geográficas. O transporte dos ribeirinhos é́
feito por embarcações, principalmente canoas
e rabetas. E a distâ̂ncia de uma cidade para
sua zona rural e ribeirinha é́ imensa. Observei
a luta e a força do povo marajoara. Registrei,
em imagens, moradores sendo submetidos
a exames, que revelaram, na hora, que eles
estavam infectados com a covid-19. É triste ver
essa realidade, pois sabemos das dificuldades
dos moradores a um atendimento de saúde
de qualidade. Nesse período, minhas fotos
foram publicadas em veículos de comunicação
do Brasil e do exterior, o que deu visibilidade
para a realidade do povo marajoara. quero
agradecer a France Press (@afpphoto )
agência que publicou muitas fotos minhas, nas
pessoas @riostreetphotog e @mauro_pimentel
Fiquei
muito
feliz
por,
durante
essa Na terceira imagem, um plano aberto feito a
partir de uma vista aérea retrata o trânsito de
um paciente da ambulancha para a lancha do
Samu em direção ao hospital. A quarta imagem,
feita à frente de uma casa ribeirinha retrata o
momento que a equipe socorrista conversa com
uma senhora no interior da casa. Ela, no centro
da imagem, sentada em uma rede, de costas para
a porta da casa. O interior da casa é também
o ponto de maior iluminação da imagem. No
primeiro plano, do lado de fora da casa, dois
homens sentados aguardam. 150 150 Na quinta imagem, três homens carregam um
corpo no caixão. 3. Análise No primeiro plano, no canto
esquerdo da imagem, um senhor sentado, sem
blusa, sem sapatos e sem máscara, aparentemente
sem forças, observa a cena que desenrola no
segundo plano. Três homens com o mínimo de
proteção, máscaras se pano fazem o transporte cobertura jornalí stica, ter me encontrado
com os amigos @uesleimarcelinoofi cial e @
benassatto , da Reuters, que sã o profi ssionais
competentes e bons parceiros de trabalho. E també m agradeç o a ajuda que recebi do
repó rter fotográ fi co @evaristosa . Cheguei
em Breves dia 20 e retornei a Belé m em 15
de junho. Foram dias e mais dias acordando
muito cedo e, por causa do trabalho, dormindo
tarde, para que, assim, pudesse cumprir minha
missã o jornalí stica. Percebi que, nas cidades,
há um controle um pouco maior da doenç a,
por causa da adoç ã o das medidas sanitá rias. Mas, por causa das distâ ncias geográ fi cas, isso
nã o ocorre nas comunidades ribeirinhas, onde
há muitos moradores infectados. Fisicamente,
estou muito cansado. Mas, jornalisticamente,
estou satisfeito com o resultado do meu
trabalho, que, espero, possa contribuir para
melhorar um pouco a vida do batalhador povo
marajoara. Obrigado pelo Texto @dilson_
pimentel relatos em texto e imagem, @sarraft arso. isso é
um puta de um registro histórico, parabéns” e
“Trabalho memorável do @sarraft arso Parabéns! Importantíssimo o registro”. Que chamam
atenção pelo reconhecimento do trabalho do
fotojornalista e do papel dos profi ssionais do
fotojornalismo de documentar a luta dos povos
ribeirinhos pela sobrevivência no cenário da
Covid-19. O ensaio a seguir, produzido pela fotojornalista
e documentarista Isis Medeiros, retrata o dia
em que seus avós foram vacinados. Quando
compartilhados pela página @covidphotobrazil
trouxe o seguinte texto legenda: Canaã, fevereiro de 2021- Casal de idosos
centená rios no interior de Minas Gerais
recebem juntos a primeira dose da vacina
Coronavac. Dona Terezinha, 93, e Senhor
Osvaldo, 94, aguardavam ansiosos por esse
momento desde que foram privados de
receber os familiares em casa. O momento
de comemoraç ã o foi registrado pela neta que
documentou os primeiros meses que os avó s
enfrentaram a solidã o causada pela pandemia. #covidphotobrazil #vivaosus #vacina #covid_19” Entre alguns comentários dos usuários atrelados
ao ensaio chamamos atenção: “agradecido por
dedicar seu tempo e energia pra contar esses 151 151 Figura 2 – Ensaio de Isis Medeiros, @isi.medeiross, em Canaã, Minas Gerais, fevereiro de 2021. 3. Análise Fonte: Isis Medeiros, 2021/ Instagram covidphotobrazil
https://www.instagram.com/p/CLRqNn4lN86/ Figura 2 – Ensaio de Isis Medeiros, @isi.medeiross, em Canaã, Minas Gerais, fevereiro de 2021. Figura 2 – Ensaio de Isis Medeiros, @isi.medeiross, em Canaã, Minas Gerais, fevereiro de 2021. Fonte: Isis Medeiros, 2021/ Instagram covidphotobrazil
https://www.instagram.com/p/CLRqNn4lN86/ Fonte: Isis Medeiros, 2021/ Instagram covidphotobrazil
https://www.instagram.com/p/CLRqNn4lN86/ momento, alguns comentam a sensibilidade do
ensaio: “Que lindo, @isis.medeiross !!! Por mais
e mais dias assim para todos brasileiros!”; “Que
lindo e emocionante!”; “Quanta sensibilidade
visual!”; “Isis e suas fotos que transbordam
sentimentos e emoções!”; “A última foto é incrível”. Outros comentários compartilham momentos
semelhantes vividos pelos seus autores: “Minha
avó de 90 anos foi vacinada semana passada. Dá um alívio né? Que venha a segunda dose”;
“Minhas 2 avós também já vacinadas! Viva os sus
e a ciência! Viva nossas raízes!” A Figura 2, representa seis das dez fotografias
compartilhadas por Isis Medeiros. Trata-se de um
ensaio em preto e branco, cheio de afetos, de um
evento: o dia em que seus avós foram vacinados. O caminho ao centro de saúde, a ansiedade pelo
dia da vacina, a celebração da vida, a gratidão
pelo SUS. O ensaio, recém compartilhado, foi curtido por
171 pessoas e comentado por duas: “Que coisa
linda!” e “tocantes e sensíveis imagens!”. No perfil
pessoal da fotógrafa o ensaio foi comentado 84
vezes. Dentre comentários que comemoram o Figura 3 – Ensaio de Alexandre Urch, @aurch, em São Paulo, abril de 2020. Figura 3 – Ensaio de Alexandre Urch, @aurch, em São Paulo, abril de 2020. Fonte: Alexandre Urch, 2020/ Instagram covidphotobrazil
https://www.instagram.com/p/B_NY1rJDJ_y/ 152 152 Fonte: Alexandre Urch, 2020/ Instagram covidphotobrazil
https://www.instagram.com/p/B_NY1rJDJ_y/ Fonte: Alexandre Urch, 2020/ Instagram covidphotobrazil Fonte: Alexandre Urch, 2020/ Instagram covidphotobr
https://www.instagram.com/p/B_NY1rJDJ_y/ https://www.instagram.com/p/B_NY1rJDJ_y/ A
Figura
3,
pode
ser
analisada
iconograficamente, conforme as orientações de
Kossoy a partir da leitura de seu campo visual. A imagem pode ser descrita a partir de três
planos de profundidade. No primeiro plano,
meramente o chão da rua, possivelmente úmido
de detergente, em declive, trata-se de uma subida. Em segundo plano, a personagem centralizada,
fotografada de lado, em movimento subindo a
rua, detergente às mãos. Botas pretas de plástico,
roupa branca, luvas, máscara, detergente rosa, óculos de proteção. Pano de fundo uma parede
azul claro Ao lado esquerdo da imagem, a informação que
a fotografia foi curtida por mais de 300 usuários, o
texto da postagem contextualiza alguns dos seus
aspectos iconológicos. 3. Análise “Declarado que o Coronavírus Covid-19 é
oficialmente uma pandemia” Foram muitas,
são muitas, mas de alguma forma somente
uma dúzia delas ficam ecoando na minha
cabeça. Mesmo em com muitas pessoas
andando nas ruas da babilônia em meio a
essa quarentena, é possível ouvir o silêncio. É possível ver o medo em cada par de olhos
acima das máscaras, sejam elas descartáveis
ou não. O medo. Esse como o vírus, é quase
unanimidade entre as pessoas. Digo quase
porque tem pessoas que colocam o dinheiro
acima de tudo. Dinheiro, ele quem decide
quem vive ou quem morre. Dinheiro, para
que dinheiro? Em meio ao barulho do silêncio
e ao medo eu ainda consigo enxergar o caso
em todo o seu esplendor. Ali quase naquela
esquina de um hospital, onde certamente
temos pessoas com Covid-19 internadas em
busca de uma salvação um funcionário da
limpeza surge de branco, como se fosse um
anjo um salvador, higienizando todo o local. O cheiro de produto desinfetante se mistura a
outros cheiros da rua e cria um perfume único,
que só quem anda nas ruas conhece. A saudade
dos amigos da rua está difícil, hoje conversei
por uns 15 minutos com um morador de rua
enquanto eu fotografava o Viaduto do Chá. Conversamos sobre a vida, política, dinheiro,
demos risada. Falamos sobre tudo, menos
sobre o vírus, a necessidade de interagir com
outro ser era maior. Eu. com a minha máscara
e ele totalmente desprotegido. Antes de seguir
meu caminho, abri a mochila e ofereci uma
máscara descartável para ele que aceitou
prontamente. Agradeceu muito, mas eu disse
que eu não fiz nada demais, apenas ajudei
um amigo da rua. Não fiz foto, nem tudo que
marca a sua vida precisa ser registrado por A imagem é mais comentada no perfil pessoal
do fotógrafo. Dentre os comentários, alguns nos
chamaram a atenção: “Muito obrigado pelo texto,
as pessoas precisam ler isso, para quem sabe um
dia pararem de praticar esse medo, que é o vírus,
para que podemos sair de casa sem pensar no
perigo de pegar o vírus.”; “Nem tudo que marca a
sua vida precisa ser registrado por uma câmera “;
“Profundo e intenso seu texto, ajudar o próximo
sempre nos protege pense nisso”; “Porra mano! Fotão em. Você tem uma visão de respeito. 3. Análise Fiquei
olhando essa foto por um bom tempo “; “Suas
reflexões e a forma como expõe elas, são incríveis. Te acompanhava muito pelas fotos, mas parei pra
ler e me tocou! Obrigada por compartilhar!”. 3. Análise Vemos que a fotografia
compartilhada pela @covidphotobrazil traz a
marcação do Instagram do seu autor Alexandre
Urch, @aurch. Além disso, traz do autor o texto: Não me lembro mais do primeiro dia que começou a pandemia. Algumas chamadas de
notícias vem como flashes na minha mente:
“O mundo está em perigo’’. Fiquem em casa”. “Declarado que o Coronavírus Covid-19 é
oficialmente uma pandemia” Foram muitas,
são muitas, mas de alguma forma somente
uma dúzia delas ficam ecoando na minha
cabeça. Mesmo em com muitas pessoas
andando nas ruas da babilônia em meio a
essa quarentena, é possível ouvir o silêncio. É possível ver o medo em cada par de olhos
acima das máscaras, sejam elas descartáveis
ou não. O medo. Esse como o vírus, é quase
unanimidade entre as pessoas. Digo quase
porque tem pessoas que colocam o dinheiro
acima de tudo. Dinheiro, ele quem decide
quem vive ou quem morre. Dinheiro, para
que dinheiro? Em meio ao barulho do silêncio
e ao medo eu ainda consigo enxergar o caso
em todo o seu esplendor. Ali quase naquela
esquina de um hospital, onde certamente
temos pessoas com Covid-19 internadas em
busca de uma salvação um funcionário da
limpeza surge de branco, como se fosse um
anjo um salvador, higienizando todo o local. O cheiro de produto desinfetante se mistura a
outros cheiros da rua e cria um perfume único,
que só quem anda nas ruas conhece. A saudade
dos amigos da rua está difícil, hoje conversei
por uns 15 minutos com um morador de rua
enquanto eu fotografava o Viaduto do Chá. Conversamos sobre a vida, política, dinheiro,
demos risada. Falamos sobre tudo, menos
sobre o vírus, a necessidade de interagir com
outro ser era maior. Eu. com a minha máscara
e ele totalmente desprotegido. Antes de seguir
meu caminho, abri a mochila e ofereci uma
máscara descartável para ele que aceitou
prontamente. Agradeceu muito, mas eu disse
que eu não fiz nada demais, apenas ajudei
i
d
Nã fi f
d uma câmera. Essas coisas ficam registradas
no cérebro e no coração. Fui embora, seguindo
a vida, olhando aquelas ruas do Centro de
SP tão vazias e silenciosas com a pergunta
junto com uma pequena lágrima que teimou
em escorrer: até quando?#babiloniazeroonze
#saopaulo #sp#corona #coronavirus começou a pandemia. Algumas chamadas de
notícias vem como flashes na minha mente:
“O mundo está em perigo’’. Fiquem em casa”. 4. Considerações finais 153 153 Com o digital e o advento da pandemia do
novo coronavírus, constatamos a hipótese de que
a fotografia teve seus papéis sociais renovados e
confrontados pelo seu uso nas mídias sociais, em
particular, no perfil analisado @covidphotobrazil. Os
resultados
mostram
que
o
estudo
iconológico aliado à pesquisa iconográfica
permite uma leitura imagética de importância
histórica de algumas das imagens que constroem
a memória da pandemia da Covid-19 no
território brasileiro. O estudo aqui apresentado
dedicou-se a uma amostragem de três trabalhos
feitos por diferentes autores, por diferentes
abordagens e em diferentes regiões do país. Trata-se de uma amostragem pequena frente ao
arquivo de 619 imagens divulgado pelo perfil
@covidphotobrazil. A escolha das imagens foi
apenas uma dentre muitas possíveis, escolhidas pela diversidade de abordagens entre elas. da abordagem de seus autores tem sido uma
ferramenta importante para o debate e a
conscientização sobre os diferentes contextos
sociais e políticos do Brasil durante a pandemia
e sobre a gravidade da crise sanitária reforçada
pela Covid-19, apesar da presença de discursos
negacionistas. Então, respondendo às perguntas norteadoras
que conduziram esta pesquisa “Como ocorre
a função social da investigação fotográfica
durante pandemia da Covid-19 no Brasil pela
plataforma do Instagram? Como a cobertura
fotográfica sobre o Covid-19 contribui como
mídia provocadora de reflexões, discussões,
debates e empatia?”, chegamos à conclusão de
que a fotografia circula pelas redes sociais de
imagens de forma a convidar as pessoas a verem
as fotografias que estão sendo produzidas; e,
em seguida, a debater, questionar, comentar e
criticar. Essa participação, por vezes, ainda se
dá de forma tímida, apenas a partir de curtidas
e com comentários que se limitam a aplaudir o
resultado das imagens. A rede permite o espaço
e o seu uso varia de indivíduo à individuo e seus
interesses. Ainda assim, percebe-se que o perfil apresenta
poucos seguidores, talvez por falta de incentivos
comerciais para ser compartilhado em larga
escala. Além disso, constatamos que muitos
seguidores não comentam tanto nas imagens
do perfil, mas curtem as imagens e passam a
seguir seus autores. Isso explicaria o fato de os
fotógrafos agradecerem o compartilhamento
de suas imagens pelo perfil @covidphotobrazil,
evidenciando que ter uma imagem impulsionada
pelo canal é reconhecimento de seus trabalhos. As imagens representam diferentes estratégias
estéticas do fazer fotográfico durante a pandemia
da Covid-19. 4. Considerações finais As imagens são convites à reflexão
sobre as realidades da pandemia de Covid-19 no
Brasil, mas são limitadas como fontes históricas
se analisadas apenas a partir de sua iconografia. Assim, considera-se de extrema importância o
papel da ciência social que investiga os dados e as
informações que contextualizam as imagens em
um lugar na história contemporânea do Brasil. Esses dados, assim como as entrevistas feitas com
os autores das fotografias, não encontram ainda
espaço nas redes sociais, mas na própria pesquisa
científica que deve estar atenta aos fenômenos
dos repasses sociais de fotografias. As imagens representam diferentes estratégias
estéticas do fazer fotográfico durante a pandemia
da Covid-19. As imagens são convites à reflexão
sobre as realidades da pandemia de Covid-19 no
Brasil, mas são limitadas como fontes históricas
se analisadas apenas a partir de sua iconografia. Ao acessarmos a postagem original dos
fotógrafos, em seus perfis pessoais, percebemos
que havia mais interações por lá. Isso nos levou
a pensar que talvez o perfil @covidphotobrazil
seja mais seguido por fotojornalistas e por
pessoas que têm interesse em ver as imagens que
estão sendo produzidas e arquivá-las, mas não
necessariamente comentar e debater com demais
usuários o conteúdo das imagens. Evidenciando
que os debates e diálogos nas redes sociais são
limitados pela restrição do fenômeno das bolhas
sociais. 154 Entretanto, é inegável que os resultados
mostraram que a fotografia independente LEWIS, Sarah. Visão e justiça. (2016). The fifth internacional
exposition of contemporary and moder arte. Chicago. Northern
Trust
KOSSOY, Boris. Realidade e ficções na trama fotográfica. São Paulo:
Ateliê Editorial, 1999.
MAGALHÃES, Ângela; PEREGRINO, Nadja. Fotografia no Brasil: um BARCELOS, Janaina. Por um fotojornalismo que respeite a
dignidade humana: a dimensão ética como questão fundamental na
comeporaneidade. In: Discursos fotográficos, Londrina, v.10, n.16,
p.111-134, jan/jun 2014.
Covidphotobrazil. Instagram. 2020. Acessado em: https://www.
instagram.com/covidphotobrazil/ LEWIS, Sarah. Visão e justiça. (2016). The fifth internacional
exposition of contemporary and moder arte. Chicago. Northern
Trust Referências BARCELOS, Janaina. Por um fotojornalismo que respeite a
dignidade humana: a dimensão ética como questão fundamental na
comeporaneidade. In: Discursos fotográficos, Londrina, v.10, n.16,
p.111-134, jan/jun 2014. MAGALHÃES, Ângela; PEREGRINO, Nadja. Fotografia no Brasil: um original: Meaning in the visual artes. 5 reimpr. Da 3 ed. 2001. olhar das origens ao contemporâneo. Rio de Janeiro: Funarte, 2004. OLIVEIRA, Erivam. VICENTINI, Ari. Fotojornalismo: Uma viagem
entre o analógico e o digital. São Paulo: Cengage Learning, 2009. ONU. (2019, dezembro) Relatório de desenvolvimento humano do
PNUD destaca altos índices de desigualdade no Brasil. Acessado em:
https://douradosagora.com.br/noticias/brasil/pnud-destaca-altos-
indices-de-desigualdade-no-brasil original: Meaning in the visual artes. 5 reimpr. Da 3 ed. 2001. SANTOS, Nina Fernandes dos. Dinâmicas de visibilidade e sites
de redes sociais: novas possibilidades democráticas? Dissertação
(Programa de Pós-graduação em Comunicação- Universidade
Federal da Bahia- Salvador, p. 103, 2012. Link: http://repositorio. ufba.br/ri/handle/ri/12887 ONU. (2019, dezembro) Relatório de desenvolvimento humano do
PNUD destaca altos índices de desigualdade no Brasil. Acessado em:
https://douradosagora.com.br/noticias/brasil/pnud-destaca-altos-
indices-de-desigualdade-no-brasil UNFRIED, Rosana. O uso da iconografia e da iconologia para a
análise de fotografias e recuperação da história de Londrina. Trabalho
apresentado no GT 7 – Fotografia, do Encontro Nacional de Pesquisa
em Comunicação e Imagem – ENCOI, 24 e 25 de novembro de 2014,
Londrina. Opas. (2020). Entenda a infodemia e a desinformação na luta
contra a Covid-19. [Versão Eletrônica]. Acesso em 20 de
agosto de 2020. Disponível em: https://iris.paho.org/bitstream/
handle/10665.2/52054/Factsheet-Infodemic_por.pdf?sequence=14. Acesso 12 de agosto de 2020. WERNECK, Guilherme; CARVALHO, Marilia. A pandemia de
Covid-19 no Brasil: Crônica de uma crise sanitária anunciada. In:
Cadernos de Saúde Pública, 2020. PANOFSKY, Erwin. Significado nas artes visuais. Tradução Maria
Clara F. Kneese e J. Guinsburg. São Paulo: Perspectiva, 2012. Titulo 155 155
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English
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Prediction of constitutive A-to-I editing sites from human transcriptomes in the absence of genomic sequences
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BMC genomics
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cc-by
| 12,423
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* Correspondence: linglingchen@sibcb.ac.cn; liyang@picb.ac.cn
†Equal contributors
2State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell
Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of
Sciences, Shanghai 200031, China
1Key Laboratory of Computational Biology, CAS-MPG Partner Institute for
Computational Biology, Shanghai Institutes for Biological Sciences, Chinese
Academy of Sciences, Shanghai 200031, China RESEARCH ARTICLE Open Access © 2013 Zhu et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited. Zhu et al. BMC Genomics 2013, 14:206
http://www.biomedcentral.com/1471-2164/14/206 Zhu et al. BMC Genomics 2013, 14:206
http://www.biomedcentral.com/1471-2164/14/206 Prediction of constitutive A-to-I editing sites from
human transcriptomes in the absence of genomic
sequences Shanshan Zhu1†, Jian-Feng Xiang2†, Tian Chen2, Ling-Ling Chen2* and Li Yang1* Abstract Background: Adenosine-to-inosine (A-to-I) RNA editing is recognized as a cellular mechanism for generating both
RNA and protein diversity. Inosine base pairs with cytidine during reverse transcription and therefore appears as
guanosine during sequencing of cDNA. Current approaches of RNA editing identification largely depend on the
comparison between transcriptomes and genomic DNA (gDNA) sequencing datasets from the same individuals,
and it has been challenging to identify editing candidates from transcriptomes in the absence of gDNA
information. Results: We have developed a new strategy to accurately predict constitutive RNA editing sites from publicly
available human RNA-seq datasets in the absence of relevant genomic sequences. Our approach establishes new
parameters to increase the ability to map mismatches and to minimize sequencing/mapping errors and unreported
genome variations. We identified 695 novel constitutive A-to-I editing sites that appear in clusters (named “editing
boxes”) in multiple samples and which exhibit spatial and dynamic regulation across human tissues. Some of these
editing boxes are enriched in non-repetitive regions lacking inverted repeat structures and contain an extremely
high conversion frequency of As to Is. We validated a number of editing boxes in multiple human cell lines and
confirmed that ADAR1 is responsible for the observed promiscuous editing events in non-repetitive regions, further
expanding our knowledge of the catalytic substrate of A-to-I RNA editing by ADAR enzymes. Conclusions: The approach we present here provides a novel way of identifying A-to-I RNA editing events by
analyzing only RNA-seq datasets. This method has allowed us to gain new insights into RNA editing and should
also aid in the identification of more constitutive A-to-I editing sites from additional transcriptomes. Keywords: RNA-seq, RNA editing, Potential SNP score, Constitutive editing, Editing box Keywords: RNA-seq, RNA editing, Potential SNP score, Constitutive editing, Editing box of ADAR enzyme family (adenosine deaminases that act
on RNA) [1,2]. The resulting inosines preferentially base
pair with cytidines (C) and are therefore functionally gua-
nosines (G), although there has been evidence that inosine
can also pair with guanosine [3]. Thus, A-to-I editing can
have profound effects on downstream RNA processing
and function, including recoding of open reading frames,
altering the pattern of alternative splicing, interfering
with microRNA function, modulating RNAi activity, and
playing other roles in gene regulation [1,2]. A computational flow to predict clustered A-to-I editing
sites from transcriptomes only It has been challenging to discover A-to-I RNA editing
sites from RNA-seq datasets for a number of reasons. First, edited As are interpreted as Gs in sequencing
reads. This leads to problems with alignment of edited
reads to the genome. Second, random sequencing errors
and mapping errors are often problematic. Third, some
genomic polymorphisms and somatic mutations are un-
predictable from an individual genome without know-
ledge of the genomic sequence. Finally, transcriptome
and genomic DNA sequencing datasets are not always
available for single individuals. To overcome these diffi-
culties, we have developed a computational approach
consisting of four key steps (Figure 1) to identify RNA
editing from multiple RNA-seq datasets in the absence
of the relevant genomic sequence. Inosine base pairs with cytidine during reverse transcrip-
tion and therefore appears as G during sequencing of
cDNA. Thus, A-to-I editing sites can be inferred by the
presence of G at a given position in a cDNA sequence but
only A in the corresponding genomic position [1,2]. Most
recently, the application of next-generation sequencing to
cDNAs (RNA-seq) and genomic DNAs from the same hu-
man individual followed by extensive computational ana-
lyses revealed an additional large number of editing sites
in both Alu and non-Alu elements [8-10]. Thus, the emer-
gence of new technologies and approaches has enabled
the identification of a growing list of editing sites. STEP 1: a two-round unique mapping strategy with
Bowtie to improve the mapping ability and to obtain an
increased number of aligned mismatches. Multiple map-
ping pipelines have been developed to align individual
RNA-seq reads to the corresponding genomes [23-26]. However, most mappers with default setting are not suit-
able to deal effectively with mismatches that result from
RNA editing. To increase the mapping sensitivity to cap-
ture more mismatches, we applied a two-round-unique
mapping with Bowtie to analyze 18 human cell line and
tissue transcriptomes (Methods). As we found that both
ends of sequence reads contain higher sequencing errors
(Additional file 1), we trimmed 75-nt reads from both
ends to 70-nt long for the first alignment. This mapping
scheme allowed us to not only keep longer reads to map
repetitive elements in the genome, but also retrieved a
large number of mismatches. Background RNA editing is a post-transcriptional modification process
which not only expands the number of functions encoded
by our genomes but also provides additional mechanisms
of gene regulation. The most predominant form of such
editing in higher eukaryotes is adenosine-to-inosine
(A-to-I) RNA editing, which is catalyzed by members The pattern of A-to-I RNA editing, either site-specific or
promiscuous, is likely to determine the fate of an edited
RNA molecule. The majority of A-to-I editing in the hu-
man transcriptome is located within inverted-repeated Alu
elements (IRAlus) positioned within introns and UTRs as Zhu et al. BMC Genomics 2013, 14:206
http://www.biomedcentral.com/1471-2164/14/206 Page 2 of 16 revealed by the systematic comparison of cDNA or EST
libraries to genomic sequences [4-7], and by genome-wide
profiling of transcriptomes and genomic DNAs from the
same individuals [8-10]. RNAs with extensively edited
IRAlus within their 3′UTRs are retained in nuclear para-
speckles [11-13], although this retention is not always
complete [12,14]. Compared to promiscuous A-to-I RNA
editing in repetitive elements, site-specific editing in coding
regions provides a rich source of genetic recoding that can
influence protein function. The best-characterized editing
sites in mammals occur in codons of pre-mRNAs encoding
glutamate receptor B (GluR-B) and serotonin receptor 2C
(5-HT2CR) [15,16]. In addition, site-specific A-to-I RNA
editing outside coding sequences has been shown to inter-
fere with miRNA pathways by affecting microprocessor or
Dicer cleavage, RISC loading and mature miRNA function
[17-22]. Thus, it is becoming increasingly apparent that
A-to-I RNA editing plays important roles in regulating
gene expression and product function. editing sites that occur in clusters (named “editing boxes”). Some of these are enriched in non-repetitive elements and
exhibit an extremely high A-to-I conversion frequency. Importantly, editing sites located in non-repetitive editing
boxes were validated in multiple human cell lines using
conventional PCR and Sanger sequencing and were
proven to be catalyzed by ADAR1. Finally, distinct editing
ratios of RNA sites in editing boxes from different tissues/
cell lines clearly suggest a spatial and dynamic regulation
of A-to-I RNA editing across human tissues. A computational flow to predict clustered A-to-I editing
sites from transcriptomes only 30% to 70% of mismatches carrying an overall PSS
from −18 to 0 are known SNPs (black line with dots in
Figure 3C), suggesting that the remaining mismatches car-
rying an overall PSS from −18 to 0 could be unreported
genomic variations. Importantly, 100% (11 of 11) randomly
picked mismatches with a PSS from −18, -16, or −11 were
proven to be true genomic variations, but not editing
events, by Sanger sequencing (Figure 3D and Additional
file 6). On the other hand, only less than 5% of mismatches
carrying an overall PSS from 1 to 18 are known SNPs,
suggesting that we could remove over 95% of reported and
unreported genomic variations with a PSS ≥1 (Figure 3C). However, given the fact that there are a large amount of
known gSNPs carrying PSS at −2 to 2 (blue histogram in
Figure 3C), in the current analysis, we set up a even more
stringent cutoff to remove potential genomic variation sites
with PSS < 3, which filtered out over 97% expressed SNPs
(red line in Figure 3C). From the data we noted that some
well-characterized editing sites were found in a tissue-
specific manner. For example, Q/R and R/G sites in the
pre-mRNA of GluR-B were detected only in brain with the
expected editing frequencies (Additional file 7A). These
tissue-specific editing events were largely due to the brain-
specific expression of GluR-B RNAs (Additional file 7B). In this two-round-unique mapping scheme significantly im-
proved the alignment capability for mismatches, which in
turn allowed us to obtain an accurate dataset of the editing
site/ratio prediction and to identify previously unreported
A-to-I conversions in human transcriptomes. STEP 2: a series of stringent cutoffs to reduce sequencing/
mapping errors and to remove known genomic SNPs. As different samples vary in genome coverage and se-
quencing depth, we used the HPB value (Additional file 4)
to normalize the expression level for each transcribed site
across samples, and selected a relatively higher cutoff
at HPB > 5 for a given site, comparable to RPKM/FPKM >
5 for a gene, to call potential editing candidates in
highly expressed sites. In our calculation, 5 HPB represented
8 ~ 19 raw hits for each base in different transcriptomes
(Additional file 5). A computational flow to predict clustered A-to-I editing
sites from transcriptomes only For instance, the second
split-alignment
resulted
in
only
1-4%
of
increased
mapped reads compared with first alignments (Figure 2B,
top panel, Additional file 2); however, the mapped mis-
matches were increased 20%-30% in different samples
(Figure 2B, bottom panel). In addition, the application of
this two-round mapping strategy with other aligners also
dramatically increased the mismatch calling, but with a lit-
tle increase in mapped reads (Additional file 3). Clearly, Transcriptome and genomic DNA sequencing datasets
are not always available for single individuals. However,
RNA-seq data is widespread and available through pub-
lic datasets and thus represents a relevantly rich source
of yet unexplored RNA editing sites. There are two fea-
tures that currently limit the application of RNA-seq
data to identify A-to-I RNA editing without the relevant
genomic information. On one hand, the nature of nu-
cleotide mismatches reduces the ability to uniquely align
RNA-seq reads to the genome, and therefore reduces
the capability to retrieve nucleotide variants. On the
other hand, true editing events are often hidden in a
background noise caused by sequence errors, mapping
errors and genome variations, including genomic single
nucleotide polymorphisms (SNPs) and somatic muta-
tions. Thus, it has been challenging to accurately identify
editing candidates from transcriptomes in the absence of
gDNA information. To overcome the aforementioned issues, we have devel-
oped a new pipeline to accurately predict editing sites from
18 human RNA-seq datasets, even without knowledge of
relevant genomic sequences from which the RNA-seq
data were derived. We identified 2,245 constitutive A-to-I Zhu et al. BMC Genomics 2013, 14:206
http://www.biomedcentral.com/1471-2164/14/206 Page 3 of 16 Figure 1 A computational approach for the prediction of constitutive A-to-I editing sites in clusters from multiple RNA-seq datasets. STEP 1: Two-round unique mapping. STEP 2: Removal of sequencing errors and annotated gSNPs. STEP 3: Removal of unannotated gSNPs with
customized PSS. STEP 4: Identification of constitutive A-to-I editing sites clustered in editing boxes. See Materials and Methods for details. Figure 1 A computational approach for the prediction of constitutive A-to-I editing sites in clusters from multiple RNA-seq datasets. STEP 1: Two-round unique mapping. STEP 2: Removal of sequencing errors and annotated gSNPs. STEP 3: Removal of unannotated gSNPs with
customized PSS. STEP 4: Identification of constitutive A-to-I editing sites clustered in editing boxes. See Materials and Methods for details. A computational flow to predict clustered A-to-I editing
sites from transcriptomes only Reads with 4 and 5 mismatches (bold on right) were
identified with the split and 2nd-round mapping approach and would have been missed with the default mapping. (B) The two-round mapping
approach achieved a significant increase of mapped mismatches (bottom panel) and subtle changes of mapped reads (top panel). The 1st-round
mapping, light grey bars; the split and 2nd-round mapping, dark grey bars. Figure 2 A dramatic increase of nucleotide mismatch calling from a two-round unique mapping approach. (A) Multiple mapped reads
from RNA-seq data of human colon tissue were uniquely aligned to chr2: 37,328,032 -37,328,129 of the hg19 genome with the number of
mismatches shown on the right. The predicted editing sites are highlighted with arrows. Reads with 4 and 5 mismatches (bold on right) were
identified with the split and 2nd-round mapping approach and would have been missed with the default mapping. (B) The two-round mapping
approach achieved a significant increase of mapped mismatches (bottom panel) and subtle changes of mapped reads (top panel). The 1st-round
mapping, light grey bars; the split and 2nd-round mapping, dark grey bars. were most likely to be true A-to-I editing. As they were
detected from no less than three transcriptomes, we classi-
fied these A-to-G/T-to-C mismatches after STEP 4 as
constitutive A-to-I sites, and named regions containing
such sites as “editing boxes”. the current study, we focus on editing sites constitu-
tively detected from multiple human tissues (constitu-
tive editing sites), and tissue-specific expressed RNAs
and editing events (tissue/cell-specific editing sites)
were not considered. STEP 4: predicting constitutive A-to-I editing sites
that occur in clusters. Owing to the absence of relative
genomic sequence information with which to compare
RNA sequence data, we enriched high confidence
A-to-I editing sites by considering the fact that A-to-I
sites could be clustered or promiscuously edited within
specific genomic regions. In our analysis, we found that
many A-to-G/T-to-C sites, but few from other types of
nucleotide conversions or known SNPs, could be clustered
(Table 1B and 1C). A computational flow to predict clustered A-to-I editing
sites from transcriptomes only The relatively high HPB in our ana-
lysis allowed us not only to locate the position of an
editing site, but also to accurately calculate the editing
ratio of each site. STEP 3: a new parameter, PSS, to remove unreported
genomic variances by taking advantage of large numbers
of RNA-seq datasets. PSSs for known SNPs were calcu-
lated using a similar strategy and their distribution was
then plotted as a control (Figure 3C). From our analysis, Zhu et al. BMC Genomics 2013, 14:206
http://www.biomedcentral.com/1471-2164/14/206 Page 4 of 16 Figure 2 A dramatic increase of nucleotide mismatch calling from a two-round unique mapping approach. (A) Multiple mapped reads
from RNA-seq data of human colon tissue were uniquely aligned to chr2: 37,328,032 -37,328,129 of the hg19 genome with the number of
mismatches shown on the right. The predicted editing sites are highlighted with arrows. Reads with 4 and 5 mismatches (bold on right) were
identified with the split and 2nd-round mapping approach and would have been missed with the default mapping. (B) The two-round mapping
approach achieved a significant increase of mapped mismatches (bottom panel) and subtle changes of mapped reads (top panel). The 1st-round
mapping, light grey bars; the split and 2nd-round mapping, dark grey bars. Figure 2 A dramatic increase of nucleotide mismatch calling from a two-round unique mapping approach. (A) Multiple mapped reads
from RNA-seq data of human colon tissue were uniquely aligned to chr2: 37,328,032 -37,328,129 of the hg19 genome with the number of
mismatches shown on the right. The predicted editing sites are highlighted with arrows. Reads with 4 and 5 mismatches (bold on right) were
identified with the split and 2nd-round mapping approach and would have been missed with the default mapping. (B) The two-round mapping
approach achieved a significant increase of mapped mismatches (bottom panel) and subtle changes of mapped reads (top panel). The 1st-round
mapping, light grey bars; the split and 2nd-round mapping, dark grey bars. Figure 2 A dramatic increase of nucleotide mismatch calling from a two-round unique mapping approach. (A) Multiple mapped reads
from RNA-seq data of human colon tissue were uniquely aligned to chr2: 37,328,032 -37,328,129 of the hg19 genome with the number of
mismatches shown on the right. The predicted editing sites are highlighted with arrows. A computational flow to predict clustered A-to-I editing
sites from transcriptomes only (A) The
distribution of mismatch ratios of all non-gSNPs mismatches and (B) known gSNPs in H9 cells. PSS was given to test the possibility of a mismatch
for either genomic variation (PSS = −1, with mismatch ratio ≥95% or between 40% ~ 60% in grey shadow) or editing (PSS = +1, with mismatches
ratios between 5% ~ 40% or between 60% ~ 95%) in H9 cells. (C) Application of PSS to filter out previously unannotated genome variations. Over
97% of known genomic SNPs were filtered out with PSS cutoff at 3 (red dashed line). (D) Validation of previously unannotated genome variations
predicted with PSS cutoff. Three examples with Sanger sequencing results were shown with their genomic locations, types of conversion and
PSSs (full validation list is available in Additional file 6). Figure 3 Development and application of Potential SNP Scores (PSS) to filter out previously unannotated genome variations. (A) The
distribution of mismatch ratios of all non-gSNPs mismatches and (B) known gSNPs in H9 cells. PSS was given to test the possibility of a mismatch
for either genomic variation (PSS = −1, with mismatch ratio ≥95% or between 40% ~ 60% in grey shadow) or editing (PSS = +1, with mismatches
ratios between 5% ~ 40% or between 60% ~ 95%) in H9 cells. (C) Application of PSS to filter out previously unannotated genome variations. Over
97% of known genomic SNPs were filtered out with PSS cutoff at 3 (red dashed line). (D) Validation of previously unannotated genome variations
predicted with PSS cutoff. Three examples with Sanger sequencing results were shown with their genomic locations, types of conversion and
PSSs (full validation list is available in Additional file 6). In total, we identified 2245 constitutive A-to-I editing
sites clustered in 266 editing boxes (Additional file 5). Although the editing boxes were largely from Alu ele-
ments, we found 7 editing boxes from non-Alu repetitive
regions and 21 editing boxes from non-repetitive regions
(Table 1B). The average length of non-repetitive editing
boxes is 71 nt, which is shorter than that of Alu and
non-Alu repetitive editing boxes (Table 1B). However,
the average A-to-I nucleotide conversion rate in non-
repetitive editing boxes is about 51% of all As, which is
higher than Alu and non-Alu repetitive editing boxes
(Table 1B), suggesting the surprising result that promis-
cuous A-to-I editing can occur in non-repetitive regions. editing sites that existed in multiple tissues/cell lines. A computational flow to predict clustered A-to-I editing
sites from transcriptomes only These editing sites are all located in noncoding regions,
with the majority in noncoding exons and intergenic re-
gions and ~10% in introns (Figure 4A). Compared with
several other recent studies [8-10,27] and DARNED
database (Figure 4), 1550 editing sites (69%) were
reported in at least one dataset and 695 (31%) were
novel sites (Figure 4B, left panel). More interestingly,
809 reported editing sites were found in only one of the
six datasets, and only one site was present in all six
datasets (Figure 4B, right panel). The huge difference
among these datasets could be due to a variety of cells/
tissues used in individual studies as well as different
computational approaches in acquiring editing sites. These comparisons also suggested that our computa-
tional flow allowed us to efficiently predict A-to-I
editing sites across transcriptomes even without the sup-
port of relevant genomic information. A computational flow to predict clustered A-to-I editing
sites from transcriptomes only In addition, we further performed
strand specific RNA-seq with RNAs collected from H9
cells and found that 100% of the identified T-to-C sites
were transcribed from “-” strand of chromosome and
A-to-G sites were from “+” strand of chromosome in H9
cells, suggesting clustered A-to-G/T-to-C mismatches Since it is known that A-to-I editing sites are
enriched in Alu elements, we calculated the enrichment
of A-to-I conversion in Alu elements after each step of
our computational flow. As shown in Table 1A, ~ 60%
mismatches in Alu elements were A-to-G/T-to-C con-
versions after STEP 2, compared to ~ 24% before STEP
2 (data not shown). Furthermore, ~ 83% mismatches in
Alu elements were A-to-G/T-to-C conversions after
PSS cutoff, indicating PSS could greatly improve the
identification of true editing sites. Finally, 100% mis-
matches identified in Alu elements were A-to-Is after
the cluster filtering, while several regions clustered
with other types of nucleotide conversions failed to be
validated with Sanger sequencing (Table 1B and data
not shown). Zhu et al. BMC Genomics 2013, 14:206
http://www.biomedcentral.com/1471-2164/14/206 Zhu et al. BMC Genomics 2013, 14:206 Page 5 of 16 Page 5 of 16 http://www.biomedcentral.com/1471-2164/14/206 Figure 3 Development and application of Potential SNP Scores (PSS) to filter out previously unannotated genome variations. (A) The
distribution of mismatch ratios of all non-gSNPs mismatches and (B) known gSNPs in H9 cells. PSS was given to test the possibility of a mismatch
for either genomic variation (PSS = −1, with mismatch ratio ≥95% or between 40% ~ 60% in grey shadow) or editing (PSS = +1, with mismatches
ratios between 5% ~ 40% or between 60% ~ 95%) in H9 cells. (C) Application of PSS to filter out previously unannotated genome variations. Over
97% of known genomic SNPs were filtered out with PSS cutoff at 3 (red dashed line). (D) Validation of previously unannotated genome variations
predicted with PSS cutoff. Three examples with Sanger sequencing results were shown with their genomic locations, types of conversion and
PSSs (full validation list is available in Additional file 6). ent and application of Potential SNP Scores (PSS) to filter out previously unannotated genome variations. (A) The Figure 3 Development and application of Potential SNP Scores (PSS) to filter out previously unannotated ge Figure 3 Development and application of Potential SNP Scores (PSS) to filter out previously unannotated genome variations. Characterization of predicted constitutive A-to-I sites in
editing boxes Unlike tissue-specific editing, all 2,245 A-to-I sites in
editing boxes identified in this study were constitutive Zhu et al. BMC Genomics 2013, 14:206
http://www.biomedcentral.com/1471-2164/14/206 Zhu et al. BMC Genomics 2013, 14:206 Page 6 of 16 Table 1 Characterization of editing prediction pipeline Table 1 Characterization of editing prediction pipeline
A
# of all m.m. in Alu
# of A-to-I in Alu
A-to-I ration in all m.m. STEP2: w/o PSS cutoff
95,187
57,502
60.41%
STEP3: PSS cutoff
8,721
7,266
83.32%
STEP4: in editing box
1,995
1,995
100%
B
Alu
Non-Alu repetitive
Non-repetitive
A-to-I
Editing boxes (sites)
238 (1995)
7 (61)
21 (189)
Ave. length (nt)
~108 nt
~86 nt
~71 nt
Ave. conversion rate of As to Is
~31%
~40%
~51%
A-to-C
0
0
2 [14]
T-to-G
0
0
1 [6]
A-to-T
0
0
0
T-to-A
0
0
0
C-to-A
0
1 [6]
1 [5]*
G-to-T
0
0
1 [5]
C-to-G
0
0
0
G-to-C
0
0
1 [5]*
C-to-T
0
0
1 [5]
G-to-A
0
0
1 [6]*
C
in IRAIus
within 1 kb to IRAIus
> 1 kb to IRAIus
A-to-I editing boxes (sites)
208 (1763)
36 (310)
24 (172)
(A) The enrichment of A-to-I conversion in Alu elements after each step of our computational flow. (B) Editing boxes/sites distribution in Alu, non-Alu repetitive
and non-repetitive regions. Asterisk indicated non A-to-Gs (noncanonical editing) sites are validated to be false positives. (C) Editing boxes/sites distribution in
IRAlus, within 1 kb to IRAlus and over 1 kb to IRAlus. # of all m.m. in Alu
# of A-to-I in Alu
A-to-I ration in all m.m. (A) The enrichment of A-to-I conversion in Alu elements after each step of our computational flow. (B) Editing boxes/sites distribution in Alu, non-Alu repetitive
and non-repetitive regions. Asterisk indicated non A-to-Gs (noncanonical editing) sites are validated to be false positives. (C) Editing boxes/sites distribution in
IRAlus, within 1 kb to IRAlus and over 1 kb to IRAlus. We further examined genomic locations of 695 new
editing sites in editing boxes. These new sites are
located in noncoding regions, including noncoding
exons, intergenic regions and introns (Figure 4C). In
addition, many editing sites in intergenic regions were
located within 10 kb of annotated genes, suggesting
these unannotated regions could be extended 3′-UTRs
of adjacent genes. Characterization of predicted constitutive A-to-I sites in
editing boxes Although editing box sites were
largely from Alu elements, 50 and 116 editing box sites
were from non-Alu repetitive or non-repetitive re-
gions, respectively (Figure 4D). Additional analyses re-
vealed that the majority of these editing boxes were
located in or close to IRAlus (within 1 kb to IRAlus)
(Table 1C), suggesting promiscuous editing in non-Alu
editing boxes could be facilitated by the recruitment of
ADAR enzymes to nearby duplex structures. However,
111 new editing sites in non-repetitive regions (from
172 in total, Table 1C) were further than 1 kb from the
nearest IRAlus (Figure 4E), suggesting that other
mechanisms may be involved in these promiscuous
editing events. Predicted constitutive A-to-I sites from non-repetitive
editing boxes are catalyzed by ADAR1 It is known that the majority of A-to-I editing in
the human transcriptome occurs within Alu elements
[4-6,8-10,27]; however, it was unexpected to identify
promiscuous editing sites in non-repetitive sequences. Thus, we randomly selected several such editing boxes
for validation. In an intergenic region between genes CCDC75 and
EIF2AK2 in chromosome 2, two non-repetitive editing
boxes (purple bars in Figure 5A) and one Alu editing
box (one of IRAlus, pink bar in Figure 5A) are sepa-
rated by over 1 kb. We found that this intergenic region
is differentially expressed in all examined cell lines/
tissues (Additional file 8). We further checked epigen-
etic modifications of ChIP-Seq analysis from ENCODE
project, but these showed no signs of active transcrip-
tion starts adjacent to this region, suggesting this
intergenic region is more likely co-expressed with its
neighboring gene(s). More careful analysis revealed
that similar expression signals were detected in the Zhu et al. BMC Genomics 2013, 14:206 Page 7 of 16 Page 7 of 16 Zhu et al. BMC Genomics 2013, 14:206
http://www.biomedcentral.com/1471-2164/14/206 Zhu et al. BMC Genomics 2013, 14:206
http://www.biomedcentral.com/1471-2164/14/206 Figure 4 Characterization of RNA editing sites in editing boxes. (A) The genomic distribution of constitutive A-to-I editing box sites. (B)
Comparison of predicted constitutive editing box sites with other studies [8-10,27] and DARNED database. 695 (about 31%) constitutive editing
sites in clusters were previously unreported, compared with 1550 (69%) sites were overlapped with at least one dataset (left panel). Only a few
sites were reported from multiple datasets (right panel). (C) The genomic distribution of newly identified editing box sites. (E) The distribution of
newly identified editing box sites in IRAlus, within or over 1 kb to IRAlus regions. Characterization of predicted constitutive A-to-I sites in
editing boxes (D) The distribution of newly identified editing box sites in Alu,
non-Alu repetitive or non-repetitive regions. Figure 4 Characterization of RNA editing sites in editing boxes. (A) The genomic distribution of constitutive A-to-I editing box sites. (B)
Comparison of predicted constitutive editing box sites with other studies [8-10,27] and DARNED database. 695 (about 31%) constitutive editing
sites in clusters were previously unreported, compared with 1550 (69%) sites were overlapped with at least one dataset (left panel). Only a few
sites were reported from multiple datasets (right panel). (C) The genomic distribution of newly identified editing box sites. (E) The distribution of
newly identified editing box sites in IRAlus, within or over 1 kb to IRAlus regions. (D) The distribution of newly identified editing box sites in Alu,
non-Alu repetitive or non-repetitive regions. intergenic region with EIF2AK2, and stopped at a
reported (blue bars) poly(A) site in H9 cells, suggesting
this intergenic region is an extended 3′ UTR of
EIF2AK2. This was further confirmed by strand specific
RNA-seq in H9 cells. editing sites, we also investigated the performance of this
method on editing sites that are not clustered (Table 3). However, only about half of randomly selected predicted
sites could be experimentally validated in both H9 and
HeLa cells (7 out of 15, Table 3). This further indicated
that our method is more reliable for prediction of clus-
tered A-to-I editing sites than for non-clustered ones in
the absence of the relevant genomic sequences. q
The validation results from gDNAs and cDNAs of
both H9 and HeLa cells for the two editing boxes from
non-repetitive regions revealed a high correlation with
our bioinformatic predictions. Sites predicted to be
edited in H9 and/or HeLa cells (Table 2) with over 10%
editing ratios were validated by Sanger sequences
(Figure 5B and Additional file 9A-9C). In addition, the
estimated editing ratios by the two methods correlate
relatively well (r = 0.845), as indicated by Additional
file 9D. Taken together, these results suggested that our
predicted editing sites in editing boxes are highly
confident. Moreover, knockdown of ADAR1 (Figure 5C)
significantly reduced editing ratio of individual A-to-I
sites in editing boxes (Figure 5D and Additional file 10),
suggesting that editing in non-repetitive editing boxes is
catalyzed by ADAR1. Characterization of promiscuous A-to-I RNA editing from
non-repetitive editing boxes (C) Knocking down of adar1 in HeLa cells with shRNA. Both RT-qPCR (left panel) and
Western blots (right panel) showed a successful ADAR1 knockdown (sh-adar1) compared with a scramble shRNA (sh-ctrl). (D) Newly identified
promiscuous A-to-I editing sites in non-Alu elements are catalyzed by ADAR1. editing is coupled to the recruitment of ADAR enzymes
to nearby Alu-related duplex structures [29]. potentially form long dsRNA duplexes with adjacent se-
quences (Figure 6B), suggesting the promiscuous A-to-I
RNA editing in non-repetitive editing boxes may involve a
mechanism similar to that of IRAlus. However, since over
90% of these editing boxes were located in or close to
IRAlus, we could not exclude the possibility that their To further test this possibility, we cloned sequences of
editing boxes in 3′UTR of egfp or in the upstream region
of single Alu or IRAlus in 3′UTR of egfp (Figure 6C). We
have previously shown that IRAlus, but not single Alus, Table 2 Editing ratios of constitutive A-to-I sites at one editing box in 18 human samples
chr2:
37,328,008
37,328,012
37,328,013
37,328,016
37,328,034
37,328,075
37,328,082
37,328,087
37,328,100
H9
9.1%
5.6%
43.3%
26.9%
20.0%
8.3%
HeLa
11.5%
24.7%
8.5%
71.8%
45.8%
18.9%
16.7%
Adipose
30.2%
41.5%
20.7%
78.0%
74.2%
63.3%
38.1%
8.8%
Adrenal
41.9%
7.5%
32.3%
25.9%
87.8%
69.5%
53.0%
38.6%
Brain
24.3%
5.6%
42.3%
28.0%
87.2%
63.8%
46.8%
26.6%
14.6%
Breast
9.7%
14.2%
15.5%
63.8%
47.0%
50.0%
12.5%
Colon
19.3%
19.6%
18.1%
91.8%
50.8%
7.0%
26.9%
Heart
9.1%
18.4%
53.3%
13.3%
17.6%
18.4%
Kidney
22.4%
6.4%
29.1%
58.9%
45.9%
15.1%
6.5%
Liver
50.0%
54.2%
76.0%
Lung
26.4%
7.8%
19.6%
13.7%
59.5%
29.3%
Lymph Node
29.8%
14.3%
65.7%
39.1%
20.0%
48.6%
Ovary
11.2%
40.1%
15.4%
71.2%
32.5%
25.8%
23.5%
Prostate
27.8%
7.9%
43.4%
30.3%
79.7%
95.6%
46.7%
33.2%
11.4%
Skeletal Muscle
33.3%
20.9%
Testes
19.6%
16.1%
15.5%
89.0%
42.6%
38.8%
31.1%
5.5%
Thyroid
13.2%
8.2%
31.8%
20.3%
64.8%
17.6%
28.8%
32.0%
White Bllod Cell
7.6%
31.1%
24.5%
66.3%
26.3%
36.7%
PSS
13
5
8
13
10
3
5
12
7
Darned databasea
Yes
Yes
Yes
Yes
Li, et al. 200924
Yes
Yes
Yes
Yes
Bahn, et al. 2012-BC9
Yes
Yes
Yes
Yes
Yes
Bahn, et al. 2012-U87MG9
Yes
Yes
Peng, et al. 20128
Yes
Ramaswami, et al. 201210
Yes
Yes
Yes
Yes
Predicted editing ratios of nine A-to-I sites in editing box at chr2: 37,328,008 -37,328,100 are listed in all examined cell lines/tissues. Characterization of promiscuous A-to-I RNA editing from
non-repetitive editing boxes Since this work is the first demonstration of promiscuous
editing in non-repetitive regions catalyzed by ADAR1
(Table 1 and Figure 5), we further characterized these sites
in greater detail. Although there were no consensus se-
quences in all non-repetitive editing boxes, we found that
ADAR1 preferentially targets adenosines when the 5′
nearest neighbor is A ≈U > C > G (Figure 6A). This is in
the agreement with known neighbor preferences of
ADAR1 enzyme, but is slightly different from recently re-
fined predicting sites of ADAR editing for an ~800 bp
dsRNA (U > A > C > G) [28]. Moreover, structure predic-
tion revealed that some of such editing boxes could Since the filtering applied in this study achieved high
accuracy (100% validation) in predicting clustered A-to-I Zhu et al. BMC Genomics 2013, 14:206 Page 8 of 16 Zhu et al. BMC Genomics 2013, 14:206
http://www.biomedcentral.com/1471-2164/14/206 Figure 5 (See legend on next page.)
Zhu et al. BMC Genomics 2013, 14:206
Page 8 of 16
http://www.biomedcentral.com/1471-2164/14/206 http://www.biomedcentral.com/1471-2164/14/206 Figure 5 (See legend on next page.) Figure 5 (See legend on next page.) Figure 5 (See legend on next page.) Page 9 of 16 Zhu et al. BMC Genomics 2013, 14:206
http://www.biomedcentral.com/1471-2164/14/206 (See gu e o
p e ous page.)
Figure 5 Validation of constitutive A-to-I sites in non-repetitive editing boxes. (A) Three editing boxes were identified within an intergenic
region at chromosome 2. A screenshot from the UCSC genome browser for its sequencing signals in H9 cell (dark blue) and HeLa cell (light blue)
with annotated gene models (exons in thick dark blue bars, introns labeled with arrowheads as transcription direction) was shown. CCDC75 is
transcribed from the plus strand while EIF2AK2 is transcribed from the minus strand of chromosome. A new gene model of EIF2AK2 with
extended 30 UTR (red line) is drawn beneath the UCSC genome browser snapshot box. Two editing boxes in non-repetitive regions (purple bars)
are located in the extended 30 UTR region together with another editing box in Alu (pink bar). (B) Validation of constitutive A-to-I editing sites. Predicted A-to-I editing sites were indicated with underlines (shown as T-to-Cs on plus strand of chr2), and their predicted editing ratios were
shown above each site in the cDNA sequencing chromatograms. Novel editing sites were highlighted with red arrows and their genomic sites
were indicated in the bottom, reported sites were in black. Predicted editing ratios of nine A-to-I sites in editing box at chr2: 37,328,008 -37,328,100 are listed in all examined cell lines/tissues. Blank indicates either no
editing or failure of passing our stringent cutoffs (HPB > 5, etc.). Five annotated sites were reported from different datasets/analyses. aDataset of RNA editing at
http://darned ucc ie/ Characterization of promiscuous A-to-I RNA editing from
non-repetitive editing boxes Blank indicates either no
editing or failure of passing our stringent cutoffs (HPB > 5, etc.). Five annotated sites were reported from different datasets/analyses. aDataset of RNA editing at
http://darned.ucc.ie/. Table 2 Editing ratios of constitutive A-to-I sites at one editing box in 18 human samples Zhu et al. BMC Genomics 2013, 14:206
http://www.biomedcentral.com/1471-2164/14/206 Page 10 of 16 Table 3 Comparison of predicted clustered and non-clustered constitutive A-to-I sites Table 3 Comparison of predicted clustered and non-clustered constitutive A-to-I sites
A
A-to-I conversions
A-to-Is in H9 and HeLa
Numbers of validated sites
Clustered sites
2,245
296
22 of 22
Non-clustered sites
10,220
1,542
7 of 15
B
Predicted A-to-I sites
Predicted A-to-I ratios
Validation
Chr. location
Altered base
Gene location
H9
HeLa
H9
HeLa
chr1:40041484
A- > G (+)
Coding
28.1%
29.5%
−
−
chr4:184186228
A- > G (+)
Coding
30.8%
28.1%
−
−
chr6:159187882
A- > G (+)
30UTR
46.2%
50%
−
−
chr7:44841489
A- > G (+)
30UTR
78.4%
69.2%
+
+
chr8:48890109
A- > G (+)
30UTR
29.6%
32.5%
+
+
chr10:75008955
A- > G (−)
30UTR
59.5%
65.8%
+
+
chr17:4068050
A- > G (+)
30UTR
31.8%
31.7%
−
−
chr17:61898921
A- > G (−)
Coding
27.5%
16.9%
+
+
chr17:80445942
A- > G (+)
Coding
34.4%
32.1%
−
−
chr19:10755103
A- > G (−)
30UTR
95.3%
96.7%
−
−
chr19:34718735
A- > G (+)
30UTR
46.5%
27.9%
+
+
chr19:39874895
A- > G (+)
30UTR
36.1%
38.9%
−
−
chr20:30253695
A- > G (+)
30UTR
20.0%
29.9%
−
−
chrX:54589730
A- > G (+)
30UTR
30.0%
46.3%
+
+
chrX:54589774
A- > G (+)
30UTR
6.9%
8.8%
+
+
(A) Comparison of clustered and non-clustered constitutive A-to-I sites identified with or without STEP4 cutoff. Editing sites detected in HeLa and H9 cells were
further used for validation with Sanger sequencing. (B) Validation results of randomly selected non-clustered A-to-I editing sites. “+”, validated to be an editing
site; “-”, validated not to be an editing site. can be extensively edited when expressed from plasmid
vectors, even during transient transfection [12]. We rea-
soned that if the adjacent IRAlus recruit ADARs to the
nearby editing boxes, we would find more editing sites in
editing boxes in vector containing IRAlus than those
containing single Alu or no Alu. Otherwise, if editing
boxes alone are sufficient to recruit ADARs, we would ob-
serve promiscuous editing in all examined vectors. Characterization of promiscuous A-to-I RNA editing from
non-repetitive editing boxes Strik-
ingly, our analyses revealed that sequences in editing
boxes in all examined vectors were extensively edited in a
similar way as that observed in their endogenous loci
(Figure 6C and 6D). These results demonstrated that non-
repetitive editing boxes alone can be edited by ADAR1, in-
dependent of adjacent IRAlus. highly dynamic across human tissues at two levels. On
one hand, individual sites exhibit distinct patterns of
editing across human tissues and cell lines (Table 2 and
Figure 7). On the other hand, the editing efficiency of
closely located editing boxes is highly dynamic. Interest-
ingly, non-repetitive editing boxes (Figure 7, purple histo-
grams, Table 2 and Additional file 11) exhibited even more
striking differences than editing boxes of IRAlus (Figure 7,
pink histograms) among examined samples. This indicated
that different mechanisms could facilitate promiscuous
editing within the same genomic characteristics in differ-
ent tissues/cell lines and that ADAR editing is affected by
more than nearest neighbors and local RNA structures
(Figure 6). Taken together, we have developed an approach to
quantitatively profile constitutive A-to-I RNA editing
from multiple human transcriptomes in the absence of
the relevant genomic information. The application of
our approach has allowed us to identify a large number
of clustered constitutive A-to-I sites, including 695 novel
sites. Our analysis also revealed that non-repetitive
editing boxes could be promiscuously edited by ADAR1, Constitutive A-to-I sites in editing boxes are highly
dynamic across human tissues As 2,245 constitutive A-to-I sites could be found in mul-
tiple human tissues and cell lines, we were able to analyze
the spatial and dynamic regulation of A-to-I RNA editing. Surprisingly, constitutive A-to-I sites in editing boxes are Zhu et al. BMC Genomics 2013, 14:206 Page 11 of 16 Zhu et al. BMC Genomics 2013, 14:206
http://www.biomedcentral.com/1471-2164/14/206 http://www.biomedcentral.com/1471-2164/14/206 Figure 6 Characterization of promiscuous A-to-I RNA editing clustered in non-repetitive editing boxes. (A) Neighbor preferences of A-to-I
RNA editing clustered in non-repetitive or non-Alu repetitive editing boxes. Site “0” indicates the editing sites. Probabilities of two upstream and
two downstream nucleotides are indicated. (B) Structure prediction suggests a dsRNA duplex of two editing boxes in chr2. Genomic locations of
two adjacent editing boxes are highlighted by different colors. (C) Editing of editing boxes is independent of adjacent IRAlus. Sequences with
editing boxes were cloned into 30UTR of egfp mRNA with a pair of adjacent IRAlus (pEGFP-2 EB-IRAlus), a single Alu (pEGFP-2 EB-Alu) or non-Alu
(pEGFP-2 EB). (D) Editing levels in IRAlus, single Alu and editing boxes of each transfected plasmid shown in (C). “+++”, extensive editing; “-”, low
editing; “N.A.”, not detected. Figure 6 Characterization of promiscuous A-to-I RNA editing clustered in non-repetitive editing boxes. (A) Neighbor preferences of A-to-I
RNA editing clustered in non-repetitive or non-Alu repetitive editing boxes. Site “0” indicates the editing sites. Probabilities of two upstream and
two downstream nucleotides are indicated. (B) Structure prediction suggests a dsRNA duplex of two editing boxes in chr2. Genomic locations of
two adjacent editing boxes are highlighted by different colors. (C) Editing of editing boxes is independent of adjacent IRAlus. Sequences with
editing boxes were cloned into 30UTR of egfp mRNA with a pair of adjacent IRAlus (pEGFP-2 EB-IRAlus), a single Alu (pEGFP-2 EB-Alu) or non-Alu
(pEGFP-2 EB). (D) Editing levels in IRAlus, single Alu and editing boxes of each transfected plasmid shown in (C). “+++”, extensive editing; “-”, low
editing; “N.A.”, not detected. Figure 6 Characterization of promiscuous A-to-I RNA editing clustered in non-repetitive editing boxes. (A) Neighbor preferences of A-to-I
RNA editing clustered in non-repetitive or non-Alu repetitive editing boxes. Site “0” indicates the editing sites. Probabilities of two upstream and
two downstream nucleotides are indicated. (B) Structure prediction suggests a dsRNA duplex of two editing boxes in chr2. Genomic locations of
two adjacent editing boxes are highlighted by different colors. Constitutive A-to-I sites in editing boxes are highly
dynamic across human tissues (C) Editing of editing boxes is independent of adjacent IRAlus. Sequences with
editing boxes were cloned into 30UTR of egfp mRNA with a pair of adjacent IRAlus (pEGFP-2 EB-IRAlus), a single Alu (pEGFP-2 EB-Alu) or non-Alu
(pEGFP-2 EB). (D) Editing levels in IRAlus, single Alu and editing boxes of each transfected plasmid shown in (C). “+++”, extensive editing; “-”, low
editing; “N.A.”, not detected. independent of their adjacent IRAlus. Finally, although
functionally unknown, marked differences of editing ra-
tios in the same sites identified in editing boxes clearly
suggest a spatial and dynamic regulation of A-to-I RNA
editing across human tissues. independent of their adjacent IRAlus. Finally, although
functionally unknown, marked differences of editing ra-
tios in the same sites identified in editing boxes clearly
suggest a spatial and dynamic regulation of A-to-I RNA
editing across human tissues. Discussion RNA-seq datasets, widespread through currently available
public databases, are rich sources to search for A-to-I
RNA editing sites. However, RNA-DNA mismatches
between RNA-seq reads and the genome make the Zhu et al. BMC Genomics 2013, 14:206
http://www.biomedcentral.com/1471-2164/14/206 Zhu et al. BMC Genomics 2013, 14:206 Page 12 of 16 http://www.biomedcentral.com/1471-2164/14/206 Figure 7 Highly dynamic regulation of A-to-I editing in editing boxes across human tissues/cell lines. Editing ratios of two non-repetitive
(purple) and one Alu (pink) editing boxes (shown in Figure 5A) were marked with colored histograms for each site in H9 cell, HeLa cell, Adipose
and Brain. The colored dots represent no report of editing events due to the stringent cutoffs. Full dataset for these editing boxes were available
in Additional file 11. Figure 7 Highly dynamic regulation of A-to-I editing in editing boxes across human tissues/cell lines. Editing ratios of two non-repetitive
(purple) and one Alu (pink) editing boxes (shown in Figure 5A) were marked with colored histograms for each site in H9 cell, HeLa cell, Adipose
and Brain. The colored dots represent no report of editing events due to the stringent cutoffs. Full dataset for these editing boxes were available
i
Addi i
l fil 11 Ramaswami et al. [30] although much larger datasets were
used. For example, they identified 181 out of 695 from 40
human lymphoblastoid cell lines, 273 out of 695 from 50
human brain samples, and 339 out of 695 from the same
16 human tissue samples. alignment of nucleotide variations to the genome prob-
lematic. In addition, transcriptome and genomic DNA
sequencing datasets are not always available for single in-
dividuals, thus making straightforward prediction of A-to-I
editing sites from available transcriptomes even more
challenging. In this study, we developed a new compu-
tational approach to predict RNA editing from multiple
tissues in the absence of the genome information. An
additional 695 novel A-to-I editing sites have been identi-
fied compared to several other recent studies [8-10,27]
and DARNED database (Figure 4B). We expect to detect
more constitutive A-to-I RNA editing sites with additional
sets of human transcriptomes as inputs by obtaining
a higher PSS value for each A-to-G mismatch site. In
addition, discrepancies of reported editing sites could be
due to a variety of cell lines/tissues used in different studies
(Figure 4B) [8-10,27]. Discussion Since we focused on clustered A-to-Is which are
constitutive edited in at least three human tissues/
transcriptomes, limited editing sites were identified in
this study. It is also noteworthy that some limitations
exist in this approach, including the insufficiency to pre-
dict more restricted tissue-specific editing, the inad-
equacy to identify some true editing sites with 40-60%
or >95% editing ratios, and inaccuracies in identifying
non-clustered editing sites (about 47% experimental val-
idation, Table 3). For instance, true editing sites, such as
A-to-I
sites
in
pre-mRNAs
of
GluR-B,
were
not
addressed in our study. In addition, true editing sites
with low expression or low editing ratios could have
been missed due to stringent cutoffs in the computa-
tional flow. These true editing sites would be captured if
multiple RNA-seq datasets from the same tissue (to
achieve a higher PSS value) and higher depths of RNA-
seq datasets from individual samples were included in the
future analysis. While a few non A-to-Gs (noncanonical
editing) sites might be expected, none could be vali-
dated as true editing sites. These noncanonical sites Very recently, Ramaswami et al. also reported the iden-
tification of edited sites from transcriptome data only [30]. Their method was reported earlier [10] and slightly modi-
fied for identifying RNA editing sites in the absence of the
related genomic DNA sequencing datasets [30]. In our
present study, the pipeline was designed to identify clus-
tered and constitutively edited A-to-Is. In total, 2,245 such
editing sites were identified, including 695 new ones. Strikingly, these new sites were still largely missed by Zhu et al. BMC Genomics 2013, 14:206
http://www.biomedcentral.com/1471-2164/14/206 Page 13 of 16 Page 13 of 16 Other aligners (like BWA) can certainly be used for
analysis directly with high mismatch allowance, but
new parameters are needed to avoid/remove sequen-
cing and mapping errors. The split scheme allowed us
to retrieve more mismatches (up to six editing sites
within 70-nt compared with three in default), and im-
proved our capability in identifying the clustered RNA
editing sites (Figure 2). could be derived mostly from mis-mapping reads to a
highly similar genomic duplicate region, as suggested by
Piskol et al. [31]. In the future, more stringent filters are
needed for RNA editing prediction to remove this type
of mapping errors. g
Strikingly, we found that promiscuous RNA editing is
not restricted to transcribed inversely orientated repeti-
tive elements, such as IRAlus. Discussion Our analysis revealed
many predicted constitutive A-to-I editing sites that
appeared in clusters and were enriched in non-repetitive
editing boxes with an extremely high A-to-I conversion
frequency (Table 1B). A recent study suggested that
editing of non-Alu sites appeared to be dependent on
nearby edited Alu sites, likely by the recruitment of
ADAR enzymes to nearby duplex structures [10]. How-
ever, we demonstrated that editing boxes alone were suffi-
cient to be edited promiscuously by ADAR1 in expression
vectors, and adjacent IRAlus have little effect to facilitate
more editing (Figure 6). Although we could identify no
consensus sequences in non-repetitive editing boxes, they
are likely to form dsRNAs and the edited sites have similar
5′ neighbor preferences as reported recently for other
ADAR1 substrates [28]. Thus, these new substrates pre-
dicted in this study further expanded our knowledge of
the catalytic pattern of A-to-I RNA editing by ADAR1. RNA-seq datasets RNA-seq datasets from 16 human tissues sequenced by
Illumina HiSeq 2000 (Illumina Human Body Map 2.0 Pro-
ject) and two additional cell lines sequenced by Illumina
Genome Analyzer IIx (GAIIx) [32] were retrieved from
Gene Expression Omnibus (GEO:GSE30611 for tissues
and GEO:GSE24399 for cell lines). About 40 ~ 80 million
75-nt single reads from each poly(A) + RNA-seq sample
were obtained and further trimmed to 70-nt long at both
5′ and 3′ ends for 2 nt and 3 nt, respectively to reduce
high sequencing errors at read ends (Additional file 1). Removal of sequencing errors and annotated gSNPs
(STEP 2) As the strand information of these RNA-seq datasets
was not available, we referred plus strand of (“+”) chro-
mosomes as reference for mismatch calling. In addition
to trim 75-nt reads from both ends to 70-nt, we carried
out the following stringent criteria for mismatch calling:
(i): Each mismatch site must have a Hits Per Billion-
mapped-bases (HPB) > 5. Since multiple RNA-seq datasets
with different sequencing depths were used in this study,
we developed HPB to normalize the expression level
for each base across samples, and selected a HPB
> 5 for each mismatch site (comparable to RPKM/FPKM
> 5 for genes, Additional file 4) to focus on highly
expressed mismatches. (ii): To improve the predicted
editing accuracy and reduce false positives, we used mis-
match ratio > 5% as a cutoff. Mismatch ratios were calcu-
lated by using mismatched hits vs all hits on the same
sites. For example, G:(A + C + G + T + N) > 5% for A-to-G
mismatch in a corresponding genomic position as A, and
etc. (iii): To reduce random sequencing errors and to im-
prove the correct assignment of sequence reads, we used
effective signal > 95% as a cutoff. For example G:(C + G +
T + N) > 95% for A-to-G mismatch, and etc. (iv): Require at
least two individual reads with the same type of nucleotide
conversion. (v): We finally filtered out gSNPs from the com-
mon SNP database (build 135, http://hgdownload.cse.ucsc. edu/goldenPath/hg19/database/snp135Common.txt.gz) and
1000 Genome database (http://evs.gs.washington.edu/
EVS/, downloaded on July 15, 2012). Customized mapping strategy (STEP 1) The combination of
40% ~ 60% and ≥95% in current analysis is among the best
parameter for our purpose (Zhu, et al., unpublished data). A final overall PSS for each mismatch site was obtained by
adding up PSSs from multiple tissues and cell lines. PSSs
for known SNPs were calculated with a similar strategy and
their distribution was then plotted against PSS from −18 to
18. With cutoff at PES < 3, over 97.5% expressed SNPs were
filtered out. Cell culture, plasmid construction and transfection,
knockdown of ADAR1, and Western blots
HeLa cells were cultured using standard protocol pro-
vided by ATCC. Human embryonic stem cells (H9 line)
were maintained as described before [37]. Sequences of
editing box region (Additional file 13) were cloned into
the pEGFP series vectors [12] and each plasmid was
transfected into HeLa cells for 24 hours prior to harvest
total RNAs for editing analysis. Sense and antisense oli-
gonucleotides were designed based on a human ADAR1
targeting sequence (5′- GTTGACTAAGTCACATGT
AAA-3′) [38] and a control scramble sequence (5′-
GATGGCATTACGGCATGTTCA-3′) [39] and cloned
into pLVTHM vector. Lentivirus particles were pro-
duced in HEK-293FT cells with the co-transfection of
packaging vectors psPAX2 and pMD2.G. For infection,
HeLa cells were incubated with concentrated viral parti-
cles at 37°C overnight and the medium was changed to
fresh the next day. Infected HeLa cells were collected
72 hours later for Western blots with goat anti-ADAR1
(Santa Cruz Biotechnology). Characterization of constitutive A-to-I sites in editing
boxes Previously identified editing sites were retrieved from the
RNA editing database (http://darned.ucc.ie/) and different
studies [8-10,27] for comparison. RefSeq Genes and anno-
tated intron/exon boundaries were retrieved from from
UCSC (http://hgdownload.cse.ucsc.edu/goldenPath/hg19/
database/refFlat.txt.gz). Alu and non-Alu repetitive ele-
ments were retrieved from http://hgdownload.cse.ucsc. edu/goldenPath/hg19/database/rmsk.txt.gz. IRAlus were
defined as any two or more inversely oriented Alu ele-
ments located within two kilobases in their genomic loca-
tion [6,12,34]. Customized mapping strategy (STEP 1) Removal of unannotated gSNPs by customized PSS (STEP 3)
PSS was set up to further reduce unknown genomic noise
by taking advantage of multiple human tissue RNA-seq
datasets. Notably, most mismatches showed low ratios
(< 20%) from multiple human tissues, while some showed
high mismatch ratios (>60%) (Figure 3A, and Additional
file 12). In contrast, mismatch ratios of known gSNPs were
significantly enriched in two peaks: one major peak at
around 100% (homozygous) and a minor peak at around
50% (heterozygous) mismatch ratio (Figure 3B, Additional
file 12). Theoretically, genomic variations would give rise
to either ~ 50% or ~ 100% mismatch ratios depending on
whether the variation is heterozygous (Additional file 6A)
or homozygous (Additional file 6B) [33]. For a given A two-round-unique mapping strategy with Bowtie [23],
SOAP [8], or BWA [9] was applied to retrieve an in-
creased number of mismatch calling (Figure 1). First
Bowtie (v 0.12.8) mapping was performed from 70-bp
reads to the hg19 human genome/junction [32] with up
to three mismatches. After removal of multiple-aligned
reads, unmapped 70-bp reads were split into two 35-nt
fragments. 35-nt fragments from 5′ and 3′ were sequen-
tially applied for the second unique mapping with up to
three mismatches. The mapped 35-nt fragments were
then extended to the other half with no more than 6
mismatches in total. In addition, reads with a distribu-
tion bias of mismatches that indicate higher sequencing
errors at read ends are also excluded in this analysis. Zhu et al. BMC Genomics 2013, 14:206
http://www.biomedcentral.com/1471-2164/14/206 Zhu et al. BMC Genomics 2013, 14:206
http://www.biomedcentral.com/1471-2164/14/206 Page 14 of 16 structure of adjacent two editing boxes at chr2 was pre-
dicted by RNAfold from ViennaRNA Package 2.0.7 [36]. unknown mismatch site existing in multiple tissues, a PSS
was given to test its probability for either a genome vari-
ation (PSS = −1, with mismatch ratio ≥95% or between
40% ~ 60%) or an editing (PSS = 1, with mismatches ratios
between 5% ~ 40% or between 60% ~ 95%) in each sample
(Figure 3A and Additional file 12). To optimize parame-
ters for PSS cutoff by considering both efficiency of gSNPs
removal and the number of nucleotide variants remained
after the removal, we permuted all possible combinations
among 40% ~ 60% and 90% ~ 100%. Total RNA isolation, RT-PCR, and Sanger sequencing
validation Total RNA isolation, RT-PCR, and Sanger sequencing
validation Total RNAs from HeLa, ADAR1 knockdown HeLa cells,
transfected HeLa cells, and H9 cells were extracted with
Trizol Reagent (Invitrogen) according to the manufac-
turer’s protocol. After treatment with DNase I (Ambion,
DNA-free™kit), the cDNA was transcribed with Super-
Script II (Invitrogen) with oligo (dT) or random hexamer. Genomic DNAs were purified from both cell lines by
TIANamp Genomic DNA kit (Tiangen Biotech). PCR
products from cDNAs and gDNAs were amplified with
primers (Additional file 13), and predicted A-to-I editing
sites were validated in available cell lines with the conven-
tional Sanger sequencing. Editing ratios of validated A-to-I
sites by Sanger sequencing were calculated by “ImageJ”
(http://rsb.info.nih.gov/ij/index.html). Briefly, the areas of
edited and unedited signals, indicated as the signal inten-
sities at each site, were carefully selected and measured by
“ImageJ”. The editing ratio was then calculated by dividing
edited intensity with total intensity at the same site. Correlation of editing ratios calculated from Sanger sequen-
cing and RNA-seq were determined by scatter plot. Identification of constitutive A-to-I sites in editing box
regions (STEP 4) Mismatch sites were selected using the following cri-
teria: (i) predicted editing sites were constitutively tran-
scribed at least from three human tissues/cell lines; (ii)
each site is no longer than 50 bp away from the nearest
site and the minimum transcribed genomic region is
20 bp long; (iii) Each site has a greater than 20% mis-
match rate in at least one tissue; (iv) at least 5 mis-
match sites clustered in one region with at least 20%
conversion rate for each type of nucleotide. Thus, We
named these regions containing promiscuous edited A-
to-I sites as “editing boxes”. Conclusions We present an integrative approach to quantitatively
profile constitutive A-to-I RNA editing from multiple
human transcriptomes in the absence of the relevant
genomic information. The application of our approach
has allowed us to identify a large number of clustered
constitutive A-to-I sites, including 695 novel ones. We
further demonstrated that non-repetitive editing boxes
could be promiscuously edited by ADAR1, independent
of their adjacent IRAlus. Strikingly, clear differences of
editing levels in the same editing box sites but from dif-
ferent tissues/cell-lines were also observed, strongly indi-
cating a spatial and dynamic regulation of A-to-I RNA
editing across human tissues. Our work thus offers new
insights into the catalytic pattern and complex regula-
tion of A-to-I editing by ADAR1. Additional file 8: Expression of an intergenic region with two
predicted editing boxes in all 18 samples. The expression of the
intergenic region from chr2 along with its adjacent genes in all examined
RNA-seq samples. The gene models, reported and predicted poly(A) sites,
transcription level, and ENCODE epigenetic modifications of ChIP-seq
data (H3K4Me3, H3K4Me1, H3K27Ac) were retrieved from UCSC genome
browser. A new gene model of EIF2AK2 with extended 3′ UTR (red line)
was drawn beneath the UCSC genome browser snapshot. Three editing
boxes (two non-repetitive boxes in purple and one Alu box in pink) were
indicated in the extended 3′UTR region of EIF2AK2. Note that editing
boxes in this unannotated region were highly expressed in all examined
samples. Additional file 9: Validation of predicted A-to-I editing sites in
other editing boxes. Predicted A-to-I editing sites were highlighted in
red (novel sites) or black (reported ones). Predicted editing ratios were
shown above each site in the cDNA sequencing chromatograms. Validation of some A-to-I editing sites from editing boxes at (A) chr2:
37,327,644-37,327,685; (B) chr12: 69,237,506-69,237,558; (C) chr14:
23,441,376-23,441,503. Editing ratios in chr12: 69,237,529 (B) were
underestimated in our analysis compared with conventional Sanger
sequencing, probably due to more mismatches in short fragments failed
to map to reference genome. Note that predicted sites with low editing
ratio were difficult to be validated due to the limited sensitivity of the
Sanger method. (D) Scatter plot of editing ratios for 31 A-to-I editing
events (Figure 5B and Additional file 9A-9C) identified by RNA-seq and
Sanger sequencing method. Data points corresponding to false positive
or false negative predictions were shown with red dots. Conclusions R, R squares and
P value for the linear regression (black line) indicated the relatively good
correlation between these two methods. Competing interests Competing interests
The authors declare that they have no competing interests. Stranded RNA-seq analysis Neighbor preferences were calculated based on predicted
constitutive editing sites in non-repetitive or non-Alu re-
petitive regions, by extending 2 bases in both upstream
and downstream flanking regions. The neighbor prefer-
ences were drawn by software WebLogo [35]. The Strand-specific RNA-seq libraries were prepared with
prereleased Directional mRNA-seq Library Kits (Illumina)
with minor modifications. Briefly, after enriched by oligo-
dT selection,
poly(A) + RNAs
were
fragmented,
and
treated with phosphatase and polynucleotide kinase to Zhu et al. BMC Genomics 2013, 14:206
http://www.biomedcentral.com/1471-2164/14/206 Page 15 of 16 repair the ends. RNA adapters were sequentially ligated to
the 3′ and 5′ ends of RNA fragments and reverse tran-
scribed using a primer complementary to the 3′ linker. cDNA library was then amplified and sequenced on
HiSeq2000 with 1x100 bp reads. The sequence file can be
accessed from the NCBI Sequence Read Archive by GEO
Accession Number GSE44450. nucleotide conversion, and PSS. All of them were validated by Sanger
sequencing. Three heterozygous sites (A) were only in genome 1000
dataset, but not in UCSC SNP135. All other 11 homozygous site (B and C)
were not reported by either dataset. Additional file 7: Tissue-specific expression of GluR-B in brain. (A) The well characterized A-to-I editing sites at chr4:158,281,294 and
chr4:158,257,875 in the pre-mRNA of GluR-B were only detected in brain,
with editing frequencies at 69.1% and 91.7%, respectively, as predicted
with our computational flow. (B) The expression of GluR-B in all
examined samples was retrieved from UCSC genome browser and the
relative expression was listed with a normalized FPKM value for each
sample. Note that GluR-B is highly expressed in human tissue but few if
any in other samples. Additional files Additional file 1: Distribution of RNA-DNA mismatch ratios along
the reads. Reads from 18 of human tissues/cell lines were uniquely
mapped to human reference and all types of RNA-DNA mismatches were
examined at each position of 75-bp reads. Each sample was shown with
different color. Additional file 2: Numbers of total reads used for alignment,
mapped reads after the first- and second-round alignment for all
transcriptomes from 18 tissues/cell lines. Additional file 10: Validations of A-to-I sites in editing boxes with
knockdown of adar1 in HeLa cells. Editing sites in regions
chr2:37,327,656-37,327,685 (A) and chr12: 69,237,509-69,237,534 (B). Additional file 11: Editing ratios in three editing boxes in
chromosome 2 across human tissues/cell lines. Additional file 12: The distribution of mismatch ratios of known
genomic SNPs and predicted mismatches in all 18 samples. Additional file 13: Primer sets for PCR/RT-PCR, editing box cloning
and Sanger sequencing validation. Same primer sets were used for
genomic DNA and cDNA amplification unless addressed separately
(−g for genomic DNA or -c for cDNA). Forward primers were chosen for
Sanger sequencing. Primers for editing box cloning at ch2:37327479–
37328193 region were also listed. Additional file 10: Validations of A-to-I sites in editing boxes with
knockdown of adar1 in HeLa cells. Editing sites in regions
chr2:37,327,656-37,327,685 (A) and chr12: 69,237,509-69,237,534 (B). Additional file 11: Editing ratios in three editing boxes in
chromosome 2 across human tissues/cell lines. Additional file 12: The distribution of mismatch ratios of known
genomic SNPs and predicted mismatches in all 18 samples. Additional file 10: Validations of A-to-I sites in editing boxes with
knockdown of adar1 in HeLa cells. Editing sites in regions
chr2:37 327 656-37 327 685 (A) and chr12: 69 237 509-69 237 534 (B) Additional file 10: Validations of A-to-I sites in editing boxes with
knockdown of adar1 in HeLa cells. Editing sites in regions
chr2:37,327,656-37,327,685 (A) and chr12: 69,237,509-69,237,534 (B). Additional file 3: The evaluation of the two-round mapping with
other aligners, SOAP [8] (A) and BWA [9] (B). The two-round mapping
approach achieved a significant increase of mapped mismatches (bottom
panel) and subtle changes of mapped reads (top panel). The 1st-round
mapping, light grey bars; the 2nd-round mapping, dark grey bars. Additional file 3: The evaluation of the two-round mapping with
other aligners, SOAP [8] (A) and BWA [9] (B). Additional files The two-round mapping
approach achieved a significant increase of mapped mismatches (bottom
panel) and subtle changes of mapped reads (top panel). The 1st-round
mapping, light grey bars; the 2nd-round mapping, dark grey bars. Additional file 11: Editing ratios in three editing boxes in
chromosome 2 across human tissues/cell lines. Additional file 11: Editing ratios in three editing boxes in
chromosome 2 across human tissues/cell lines. Additional file 12: The distribution of mismatch ratios of known
genomic SNPs and predicted mismatches in all 18 samples. Additional file 12: The distribution of mismatch ratios of known
genomic SNPs and predicted mismatches in all 18 samples. Additional file 4: A formula to show that normalized expression
level (hits per-billion-mapped bases, HPB) of a give site is
equivalent to the value of RPKM/FPKM at one nucleotide base
resolution. Additional file 13: Primer sets for PCR/RT-PCR, editing box cloning
and Sanger sequencing validation. Same primer sets were used for
genomic DNA and cDNA amplification unless addressed separately
(−g for genomic DNA or -c for cDNA). Forward primers were chosen for
Sanger sequencing. Primers for editing box cloning at ch2:37327479–
37328193 region were also listed. Additional file 13: Primer sets for PCR/RT-PCR, editing box cloning
and Sanger sequencing validation. Same primer sets were used for
genomic DNA and cDNA amplification unless addressed separately
(−g for genomic DNA or -c for cDNA). Forward primers were chosen for
Sanger sequencing. Primers for editing box cloning at ch2:37327479–
37328193 region were also listed. Additional file 5: Constitutive A-to-I editing sites in editing boxes
(see spreadsheet). 2,245 constitutive A-to-I editing sites in editing boxes
were listed with their genomic location, expression levels in each tissue/
cell line (> 5 HPB), unique hits, editing ratios and final PSSs. Each site was
also characterized with information of the overlapped gene, strand
information, overlapping with RepeatMask sequences, genomic location
with IRAlus and comparison with other reported editing datasets
([8-10,27] and DARNED database). Sanger sequencing. Primers for editing box cloning at ch2:37327479–
37328193 region were also listed. Abbreviations
d
b EB: Editing box; ESC: Embryonic stem cell; FPKM: Fragments per kilobase per
million; gDNA: Genomic DNA; gSNP: Genomic SNP; HPB: Hits per billion-
mapped-bases; RPKM: Reads per kilobase per million; PSS: Potential SNP
score; SNP: Single nucleotide polymorphisms. Additional file 6: Validation of unannotated genome variations
filtered out with custom PSS cutoff. Sanger sequencing of gDNAs and
cDNAs from H9 cells were compared from randomly selected (A)
heterozygous or (B) homozygous sites, which were highlighted with
arrows and were proven to be real genome variations. The reference
genome sequences from hg19 human genome are listed with the
variation sites underlined. (C) Additional eight examples of unannotated
SNPs predicted with PSS were shown with genomic locations, types of Zhu et al. BMC Genomics 2013, 14:206
http://www.biomedcentral.com/1471-2164/14/206 Zhu et al. BMC Genomics 2013, 14:206
http://www.biomedcentral.com/1471-2164/14/206 analyses, XJF and CT carried our all experiments. All authors have read and
approved the manuscript for publication. 20. Kawahara Y, Megraw M, Kreider E, Iizasa H, Valente L, Hatzigeorgiou AG,
Nishikura K: Frequency and fate of microRNA editing in human brain. Nucleic Acids Res 2008, 36:5270–5280. 21. Alon S, Mor E, Vigneault F, Church G, Locatelli F, Galeano F, Gallo A,
Shomron N, Eisenberg E: Systematic identification of edited microRNAs in
the human brain. Genome Res 2012, 22:1533–1540. Acknowledgements We are grateful to Gordon Carmichael for critical reading of the manuscript
and all lab members for helpful discussion and technical support from
Huahong Fang, Zheng Wu, and Yefen Xu. H9 cells were obtained from the
WiCell Research Institute. H9 stranded RNA-seq was performed at CAS-MPG
Partner Institute for Computational Biology Omics Core. This work was
supported by CAS(XDA01010206), NSFC(31271390), the Hundred Talents
Program of CAS (2012OHTP08), the Talents Program of SIBS (2012SSTP01)
and SMSTC (11PJ1411000) to LLC and LY. 22. Vesely C, Tauber S, Sedlazeck FJ, von Haeseler A, Jantsch MF: Adenosine
deaminases that act on RNA induce reproducible changes in abundance
and sequence of embryonic miRNAs. Genome Res 2012, 22:1468–1476. 23. Langmead B, Trapnell C, Pop M, Salzberg SL: Ultrafast and memory-
efficient alignment of short DNA sequences to the human genome. Genome Biol 2009, 10:R25. 24. Trapnell C, Pachter L, Salzberg SL: TopHat: discovering splice junctions
with RNA-Seq. Bioinformatics 2009, 25:1105–1111. 25. Kent WJ: BLAT–the BLAST-like alignment tool. Genome Res 2002,
12:656–664. Received: 26 December 2012 Accepted: 21 March 2013
Published: 27 March 2013 Received: 26 December 2012 Accepted: 21 March 2013
Published: 27 March 2013 26. Li H, Durbin R: Fast and accurate short read alignment with Burrows-
Wheeler transform. Bioinformatics 2009, 25:1754–1760. 27. Li JB, Levanon EY, Yoon JK, Aach J, Xie B, Leproust E, Zhang K, Gao Y,
Church GM: Genome-wide identification of human RNA editing sites by
parallel DNA capturing and sequencing. Science 2009, 324:1210–1213. References 1. Bass BL: RNA editing by adenosine deaminases that act on RNA. Annu Rev Biochem 2002, 71:817–846. 28. Eggington JM, Greene T, Bass BL: Predicting sites of ADAR editing in
double-stranded RNA. Nat Commun 2011, 2:319. 2. Nishikura K: Functions and Regulation of RNA Editing by ADAR 2. Nishikura K: Functions and Regulation of RNA Editing by ADAR
Deaminases. Annu Rev Biochem 2010, 79:321–349. Deaminases. Annu Rev Biochem 2010, 79:321–34 29. Enstero M, Daniel C, Wahlstedt H, Major F, Ohman M: Recognition and
coupling of A-to-I edited sites are determined by the tertiary structure of
the RNA. Nucleic Acids Res 2009, 37:6916–6926. 3. Vendeix FA, Munoz AM, Agris PF: Free energy calculation of modified
base-pair formation in explicit solvent: A predictive model. RNA 2009
15:2278–2287. 30. Ramaswami G, Zhang R, Piskol R, Keegan LP, Deng P, O’Connell MA, Li JB: 30. Ramaswami G, Zhang R, Piskol R, Keegan LP, Deng P, O’Connell MA, Li JB:
Identifying RNA editing sites using RNA sequencing data alone. Nat
Methods 2013, 10:128–132. 4. Kim DD, Kim TT, Walsh T, Kobayashi Y, Matise TC, Buyske S, Gabriel A:
Widespread RNA editing of embedded alu elements in the human
transcriptome. Genome Res 2004, 14:1719–1725. Identifying RNA editing sites using RNA sequencing data alone. Nat
Methods 2013, 10:128–132. 31. Piskol R, Peng Z, Wang J, Li JB: Lack of evidence for existence of
noncanonical RNA editing. Nat Biotechnol 2013, 31:19–20. p
,
5. Levanon EY, Eisenberg E, Yelin R, Nemzer S, Hallegger M, Shemesh R,
Fligelman ZY, Shoshan A, Pollock SR, Sztybel D, et al: Systematic
identification of abundant A-to-I editing sites in the human
transcriptome. Nat Biotechnol 2004, 22:1001–1005. 6. Athanasiadis A, Rich A, Maas S: Widespread A-to-I RNA editing of Alu-
containing mRNAs in the human transcriptome. PLoS Biol 2004, 2:e391. 5. Levanon EY, Eisenberg E, Yelin R, Nemzer S, Hallegger M, Shemesh R,
Fligelman ZY, Shoshan A, Pollock SR, Sztybel D, et al: Systematic
identification of abundant A-to-I editing sites in the human
transcriptome. Nat Biotechnol 2004, 22:1001–1005. 32. Yang L, Duff MO, Graveley BR, Carmichael GG, Chen L-L: Genomewide
characterization of non-polyadenylated RNAs. Genome Biol 2011, 12:R16. characterization of non-polyadenylated RNAs. Genome Biol 201 33. Park E, Williams B, Wold BJ, Mortazavi A: RNA editing in the human
ENCODE RNA-seq data. Genome Res 2012, 22:1626–1633. 6. References doi:10.1186/1471-2164-14-206
Cite this article as: Zhu et al.: Prediction of constitutive A-to-I editing
sites from human transcriptomes in the absence of genomic sequences. BMC Genomics 2013 14:206. 13. Mao YS, Sunwoo H, Zhang B, Spector DL: Direct visualization of the co-
transcriptional assembly of a nuclear body by noncoding RNAs. Nat Cell Biol 2011, 13:95–101. 14. Hundley HA, Krauchuk AA, Bass BL: C. elegans and H. sapiens mRNAs with
edited 3′ UTRs are present on polysomes. RNA 2008, 14:2050–2060. 15. Higuchi M, Single FN, Köhler M, Sommer B, Sprengel R, Seeburg PH: RNA
editing of AMPA receptor subunit GluR-B: a base-paired intron-exon
structure determines position and efficiency. Cell 1993, 75:1361–1370. Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit Submit your next manuscript to BioMed Central
and take full advantage of: References Athanasiadis A, Rich A, Maas S: Widespread A-to-I RNA editing of Alu-
containing mRNAs in the human transcriptome. PLoS Biol 2004, 2:e391. ENCODE RNA-seq data. Genome Res 2012, 22:1626–1633. 34. Blow M, Futreal PA, Wooster R, Stratton MR: A survey of RNA editing in
human brain. Genome Res 2004, 14:2379–2387. 7. Carmi S, Borukhov I, Levanon EY: Identification of widespread ultra-edited
human RNAs. PLoS Genet 2011, 7:e1002317. human RNAs. PLoS Genet 2011, 7:e1002317. 35. Crooks GE, Hon G, Chandonia JM, Brenner SE: WebLogo: a sequence logo
generator. Genome Res 2004, 14:1188–1190. 8. Peng Z, Cheng Y, Tan BC-M, Kang L, Tian Z, Zhu Y, Zhang W, Liang Y, Hu X,
Tan X, et al: Comprehensive analysis of RNA-Seq data reveals extensive
RNA editing in a human transcriptome. Nat Biotechnol 2012, 30:253–260. 36. Lorenz R, Bernhart SH, zu Siederdissen CH, Tafer H, Flamm C, Stadler PF,
Hofacker IL: ViennaRNA Package 2.0. Algorithms Mol Biol 2011, 6:26. 9. Bahn JH, Lee JH, Li G, Greer C, Peng G, Xiao X: Accurate identification of
A-to-I RNA editing in human by transcriptome sequencing. Genome Res
2012, 22:142–150. 37. Chen LL, Carmichael GG: Altered nuclear retention of mRNAs containing
inverted repeats in human embryonic stem cells: Functional role of a
nuclear noncoding RNA. Mol Cell 2009, 35:467–478. 10. Ramaswami G, Lin W, Piskol R, Tan MH, Davis C, Li JB: Accurate
identification of human Alu and non-Alu RNA editing sites. Nat Methods
2012, 9:579–581. 38. Toth AM, Li Z, Cattaneo R, Samuel CE: RNA-specific adenosine deaminase
ADAR1 suppresses measles virus-induced apoptosis and activation of
protein kinase PKR. J Biol Chem 2009, 284:29350–29356. 11. Prasanth KV, Prasanth SG, Xuan Z, Hearn S, Freier SM, Bennett CF, Zhang
MQ, Spector DL: Regulating gene expression through RNA nuclear
retention. Cell 2005, 123:249–263. 39. Yin QF, Yang L, Zhang Y, Xiang JF, Wu YW, Carmichael GG, Chen LL: Long
Noncoding RNAs with snoRNA Ends. Mol Cell 2012, 48:219–230. 39. Yin QF, Yang L, Zhang Y, Xiang JF, Wu YW, Carmichael GG, Chen LL: Long
Noncoding RNAs with snoRNA Ends. Mol Cell 2012, 48:219–230. 12. Chen LL, DeCerbo JN, Carmichael GG: Alu element-mediated gene
silencing. EMBO J 2008, 27:1694–1705. doi:10.1186/1471-2164-14-206
Cite this article as: Zhu et al.: Prediction of constitutive A-to-I editing
sites from human transcriptomes in the absence of genomic sequences. BMC Genomics 2013 14:206. doi:10.1186/1471-2164-14-206
Cite this article as: Zhu et al.: Prediction of constitutive A-to-I editing
sites from human transcriptomes in the absence of genomic sequences.
BMC Genomics 2013 14:206. Authors’ contributions LY and LLC conceived the study, analyzed data and wrote the manuscript
from the inputs from all authors. SSZ and LY carried out the computational Page 16 of 16 Zhu et al. BMC Genomics 2013, 14:206
http://www.biomedcentral.com/1471-2164/14/206 Submit your next manuscript to BioMed Central
and take full advantage of: 16. Burns CM, Chu H, Rueter SM, Hutchinson LK, Canton H, Sanders-Bush E,
Emeson RB: Regulation of serotonin-2C receptor G-protein coupling by
RNA editing. Nature 1997, 387:303–308. • Convenient online submission • Thorough peer review 17. Yang W, Chendrimada TP, Wang Q, Higuchi M, Seeburg PH, Shiekhattar R,
Nishikura K: Modulation of microRNA processing and expression through
RNA editing by ADAR deaminases. Nat Struct Mol Biol 2006, 13:13–21. 18. Yang W, Wang Q, Howell KL, Lee JT, Cho DS, Murray JM, Nishikura K:
ADAR1 RNA deaminase limits short interfering RNA efficacy in
mammalian cells. J Biol Chem 2005, 280:3946–3953. 18. Yang W, Wang Q, Howell KL, Lee JT, Cho DS, Murray JM, Nishikura K:
ADAR1 RNA deaminase limits short interfering RNA efficacy in
mammalian cells. J Biol Chem 2005, 280:3946–3953. 19. Kawahara Y, Zinshteyn B, Sethupathy P, Iizasa H, Hatzigeorgiou AG,
Nishikura K: Redirection of silencing targets by adenosine-to-inosine
editing of miRNAs. Science 2007, 315:1137–1140. 19. Kawahara Y, Zinshteyn B, Sethupathy P, Iizasa H, Hatzigeorgiou AG,
Nishikura K: Redirection of silencing targets by adenosine-to-inosine
editing of miRNAs. Science 2007, 315:1137–1140.
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Informed recruitment in partner studies of HIV transmission: an ethical issue in couples research
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BioMed Central BioMed Central Published: 27 August 2009 Received: 16 October 2008
Accepted: 27 August 2009 MC Medical Ethics 2009, 10:14
doi:10.1186/1472-6939-10-14 This article is available from: http://www.biomedcentral.com/1472-6939/10/14 This article is available from: http://www.biomedcentral.com/1472-6939/10/14 © 2009 McNutt et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ;
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Open A
Debate
Informed recruitment in partner studies of HIV transmission: an
ethical issue in couples research
Louise-Anne McNutt*1,2, Elisa J Gordon3 and Anneli Uusküla4 Open Access p
Louise-Anne McNutt*1,2, Elisa J Gordon3 and Anneli Uusküla4 Address: 1Department of Epidemiology and Biostatistics, University at Albany, State University of New York, Rensselaer, NY, USA, 2School of
Public Health, Tbilisi State Medical University, Tbilisi, Georgia, 3Institute for Healthcare Studies, Division of Organ Transplantation, Department
of Surgery, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA and 4Department of Public Health, University of Tartu, Ravila
Tartu, Estonia Email: Louise-Anne McNutt* - lamcnutt@gmail.com; Elisa J Gordon - gordone1@northwestern.edu; Anneli Uusküla - anneli.uuskula@ut.ee
* Corresponding author * Corresponding author Abstract Background: Much attention has been devoted to ethical issues related to randomized controlled
trials for HIV treatment and prevention. However, there has been less discussion of ethical issues
surrounding families involved in observational studies of HIV transmission. This paper describes the
process of ethical deliberation about how best to obtain informed consent from sex partners of
injection drug users (IDUs) tested for HIV, within a recent HIV study in Eastern Europe. The study
aimed to assess the amount of HIV serodiscordance among IDUs and their sexual partners, identify
barriers to harm reduction, and explore ways to optimize intervention programs. Including IDUs,
either HIV-positive or at high risk for HIV, and their sexual partners would help to gain a more
complete understanding of barriers to and opportunities for intervention. Discussion: This paper focuses on the ethical dilemma regarding informed recruitment: whether
researchers should disclose to sexual partners of IDUs that they were recruited because their
partner injects drugs (i.e., their heightened risk for HIV). Disclosing risks to partners upholds the
ethical value of respect for persons through informed consent. However, disclosure compromises
the IDU's confidentiality, and potentially, the scientific validity of the research. Following a brief
literature review, we summarize the researchers' systematic evaluation of this issue from ethical,
scientific, and logistical perspectives. While the cultural context may be somewhat unique to
Eastern Europe and Central Asia, the issues raised and solutions proposed here inform
epidemiological research designs and their underlying ethical tensions. Summary: We present ethical arguments in favor of disclosure, discuss how cultural context
shapes the ethical issues, and recommend refinement of guidance for couples research of
communicable diseases to assist investigators encountering these ethical issues in the future. a host of social, cultural, and clinical issues through
important and diverse research studies to improve preven-
tion and intervention methods. While much attention has
been paid to the research ethics surrounding clinical trials Background Informing sexual partners that they are eligible for the
study because the partner is an IDU – a group with high
HIV prevalence – may immediately prompt them to
change their risk behaviors or leave the relationship
thereby biasing estimates of transmission rates. The dis-
closure of risks to sexual partners can also be justified as a
means of minimizing harms to others. Yet not informing
the partners may result in investigators watching the nat-
ural course of disease transmission in an uncontrolled
environment. Couples research thus raises important
questions: What are researchers' obligations to disclose
information about HIV risk when HIV risk is a central
component of study selection and eligibility? How much
information should be provided to potential recruits
about their risk as part of the informed consent process? How do researchers balance the rights of each individual
within a couple, and study validity, in the informed con-
sent process? The issue is further complicated by the legal issues that
surround HIV. In much of the world, including some US
states [29] and European countries, [30] disclosure by a
physician of an individual's HIV status without the
patient's consent is illegal. In other locations, physicians
have a duty to warn partners through mandated notifica-
tion programs.[31] Further, professional ethics codes may
differ from local laws, complicating these issues.[32]
Lastly, laws pertaining to medical care and observational
research conducted outside medical settings may differ. Informed consent of research study participants begins
with the recruitment process. Typically, recruiting couples
starts with recruitment of the "index-participant" – indi-
viduals either infected with HIV or at very high risk for
HIV (e.g., IDU). The sexual partner is commonly recruited
with the assistance of the index-participant. The extent of
information disclosed to sex partners as to why they are
being sought for study inclusion remains controversial. Some studies involve specific information about eligibil-
ity related to substantial HIV risk while others do not. Major initiatives, such as the HIV Prevention Trial Net-
work (HPTN), have developed formal guidelines that
address fundamental research ethics issues, but these do
not prescribe a specific approach for "informed selec-
tion."[2] Providing accurate HIV risk information during
the informed consent process to all participants is consist-
ent with the HPTN guidelines (Sugarman J, personal com-
munication, 2008). The UN AIDS guidance for HIV
prevention trials also highlights that individuals be told
the specific reason for study eligibility. Background g
An estimated 33 million people are infected with HIV
worldwide, including about 2.5 million newly infected in
the past year. [1] Efforts to curb HIV transmission address Page 1 of 8
(page number not for citation purposes) Page 1 of 8
(page number not for citation purposes) http://www.biomedcentral.com/1472-6939/10/14 http://www.biomedcentral.com/1472-6939/10/14 BMC Medical Ethics 2009, 10:14 logistical perspectives. While the cultural context may be
somewhat unique to Eastern Europe and Central Asia, the
issues raised and solutions proposed here inform epide-
miological research designs and their underlying ethical
tensions. of HIV treatment and prevention, [2-20] less discussion
has focused on observational studies of HIV transmission
involving sex partners. Couples research is crucial to inter-
vention development and raises unique ethical dilemmas
that are not specifically addressed by regulations, laws,
and ethical standards [2-4,21-23]. of HIV treatment and prevention, [2-20] less discussion
has focused on observational studies of HIV transmission
involving sex partners. Couples research is crucial to inter-
vention development and raises unique ethical dilemmas
that are not specifically addressed by regulations, laws,
and ethical standards [2-4,21-23]. The tension between confidentiality and informed recruit-
ment in HIV transmission research first received consider-
able attention with the publication of a Ugandan cohort
study conducted between 1994 and 1998. [24-28]
Researchers identified 415 HIV-discordant couples from a
large randomized trial designed to study the impact of
bacterial sexually transmitted diseases on HIV transmis-
sion. [24] The couples were then followed to identify fac-
tors associated with HIV transmission. Substantial harm
reduction education, free condoms, and voluntary, confi-
dential HIV testing and counseling were offered to all par-
ticipants. However, researchers were legally obligated not
to inform participants who were HIV-negative about their
partners' HIV-positive status. Debate ensued about the
choice of confidentiality versus disclosure of partner HIV
status. [25-28] A decade later the debate continues with
no clear resolution. In couples research, the primary ethical tension entails
balancing the risks and benefits to the participating indi-
viduals, to the couple, and to society. Complicating the
issue, the investigators' ability to conduct scientifically
valid research may be inversely related to their ability to
fully protect the well-being of individual participants. For
example, in studies of sexual transmission of HIV among
injection drug users (IDU) and their partners, we typically
recruit IDUs and ask that they recruit their sexual partners. http://www.biomedcentral.com/1472-6939/10/14 http://www.biomedcentral.com/1472-6939/10/14 http://www.biomedcentral.com/1472-6939/10/14 BMC Medical Ethics 2009, 10:14 HIV in Estonia written informed consent form after the researcher orally
disclosed the purpose of the study, procedures, risks and
benefits of participation, participants' rights, and pro-
vided an opportunity to read the form (containing the
same information on the study) and ask questions. Each
participant was interviewed separately and blood was
obtained for HIV and HCV testing. The survey tool col-
lected detailed information about drug use, sexual behav-
iors, HIV testing, knowledge about HIV transmission, and
attitudes about HIV. Anonymous codes linked records. (Anonymity was important because IDU is a crime in
Estonia [57,58] and the equivalent of a US Certificate of
Confidentiality [59,60] does not exist to protect partici-
pant data.) All participants received counseling as part of
the HIV testing and the counseling program provided in
Estonia. All participants were asked to return for test
results. Every aspect of the study was reviewed multiple
times by the investigators to ensure IDUs received ade-
quate protection. In Estonia, the primary reservoir for HIV is the IDU com-
munity. By the turn of the century, this small Baltic state
had the highest HIV incidence in Europe.[33,34] Similar
to nearby cities in Russia, the IDU community was the
epicenter of the epidemic, and HIV spread quickly during
the post-independence transition period of the late
1990s.[34,35] About half of all IDUs in Estonia are
infected with HIV and over 90% are infected with hepati-
tis C virus (HCV). [36-38] The transmission of HIV
between IDUs is well understood and some intervention
programs are available, including free voluntary coun-
seling and testing, needle exchange programs and drug
treatment. [39-50] However, improved HIV prevention
and intervention programs are critically needed to curb
the spread of HIV among IDUs in general, and between
IDUs and their sexual partners. [39,40,45,51-54] The primary mode of HIV transmission from the IDU
community to the general population is thought to be
through
sexual
contact.[45]
However,
insufficient
research has focused on improving interventions to inter-
rupt sexual transmission between IDUs and their partners. Because the IDU community is a substantial reservoir for
HIV in Eastern Europe and Central Asia, understanding
and stopping transmission from this group is essential to
slow the spread of the epidemic. These studies must be
conducted against the backdrop of a changing social struc-
ture in the region, largely related to major changes in the
political and economic environment following independ-
ence. Discussion Thus, the crux of the ethical dilemma was the informed
consent process for the sexual partners, and specifically the
explanations about partner selection and the study pur-
pose. Our team generated multiple conceptual options for
informed consent statements, with the varying levels of
detail in italics for comparison: Informed Consent Process During the informed consent process, research staff took
great care to be transparent regarding all aspects of the
research study. All participants were told we were specifi-
cally studying HIV, including it's potential to be sexually
transmitted, and provided counseling around HIV testing. We explained to IDUs the reason for recruitment and all
relevant study procedures. But it was less clear what to dis-
close to the recruited sex partners. To fully inform sexual
partners of the study's purpose and specific reason for
their study eligibility might require a disclosure of the
index-participants' drug use and heightened risk of HIV. Background [4] This paper focuses on ethical dilemmas that arise when
IDUs are recruited into behavioral research on HIV that
will involve their sexual partners. Should researchers dis-
close to these partners that they were recruited because
their partner injects drugs (i.e., their heightened risk for
HIV)? Disclosing would uphold the ethical value of
respect for persons and serves to minimize harm to them. However, in research involving couples, disclosure com-
promises the IDU's confidentiality. Investigators may
have conflicting obligations to members of the couple,
such as in this study: there is a conflict between protecting
the interests in confidentiality of the IDUs recruited for
the study and protecting the interests of their sexual part-
ners – some of whom may not know their partner is an
IDU – in being able to make informed choices. Disclosure
also potentially weakens the scientific validity of the
research if it causes some IDUs not to enroll because they
do not want this disclosure to be made. Following a brief
literature review, we summarize the researchers' system-
atic evaluation of this issue from ethical, scientific, and Page 2 of 8
(page number not for citation purposes) Page 2 of 8
(page number not for citation purposes) http://www.biomedcentral.com/1472-6939/10/14 Page 3 of 8
(page number not for citation purposes) The Estonian Study The study discussed here, a joint project of Estonian and
US researchers, took place in 2007 following Institutional
Review Board approval for the study granted by both US
and Estonian institutions. The study objectives were three-
fold: 1) assess the amount of HIV serodiscordance among
IDUs and their sexual partners, 2) identify barriers to
harm reduction, and 3) explore ways to optimize inter-
vention programs. Thus, we wanted to include IDUs,
either HIV-positive or at high risk for HIV, and their sexual
partners to gain a more complete understanding of the
barriers to and opportunities for intervention. 1. "You were selected to participate in this study on
relationships and risk behaviors because your partner vol-
unteered for the study and was asked to invite his/her
sex partner." 1. "You were selected to participate in this study on
relationships and risk behaviors because your partner vol-
unteered for the study and was asked to invite his/her
sex partner." 2. "You were selected to participate in this study of
injection drug users and their sex partners because your
partner volunteered for the study and was asked to
invite his/her sex partner." Injection drug users were recruited into research through
harm reduction programs, at known locations of drug use,
and by using respondent-driven sampling.[55,56] Once
an IDU was recruited, he/she was asked to invite both a
sexual partner(s) who does not use injection drugs and
other IDUs to participate in the study. Index-participants
were given two sets of coded cards to hand out (one set for
sexual partners and the other for IDUs as part of respond-
ent-driven sampling). Each individual was provided a 3. "You were selected to participate in this study of
injection drug users and their sex partners because your
partner volunteered for this study and was asked to
invite his/her sex partner. About half of injection drug
users are infected with HIV and we are particularly inter- Page 3 of 8
(page number not for citation purposes) http://www.biomedcentral.com/1472-6939/10/14 http://www.biomedcentral.com/1472-6939/10/14 BMC Medical Ethics 2009, 10:14 ested in understanding risk behaviors of injection drug users
who are infected with HIV and their partners. In particu-
lar, we want to learn what behaviors result in the highest
risk of HIV transmission and better ways to reduce risk of
transmission." loss of patients' ability to trust their physicians with sensi-
tive information. The Process of Ethical Deliberation Investigators originally planned to use the first statement,
that is, to not disclose the study's primary focus on IDUs
and HIV transmission risk to their sexual partners. Driven
by our prior experience with vulnerable populations, we
designed the study to fully protect IDUs while maximiz-
ing their comfort with participating in the research
project. However, further reflection on the public health
and ethical implications of this approach to disclosure led
the research team to reconsider. We sought input from
multiple sources for over a year prior to study implemen-
tation, including informal conversations with IDUs,
NGOs (e.g., needle exchange programs), researchers, eth-
icists, friends and family members of IDUs, and others Protection of Vulnerable Populations In Estonia, research ethics committees consider IDUs
among the vulnerable potential study participants. Known IDUs are marginalized for several reasons, includ-
ing: a) their (illegal) behavior, b) most are members of an
ethnic minority (about 80% are Russian-speaking), c)
many have served time in jail or prison, d) many are poor,
and e) about half are known to be HIV positive [34] The research team was unable to use the third statement,
due to concerns of the local ethics committee about such
specific, yet ambiguous communication that could further
stigmatize IDUs. While it may appear arbitrary that one
stigma (i.e., IDU status) can be shared while another (i.e.,
HIV status) can not, it is not uncommon that inconsisten-
cies in sensitivity about exposures exist in many countries. The research team debated the first two options at length. The decision would have health, scientific, and ethical
consequences. In the following we describe the process we
underwent and factors that informed our decision. Sex partners of IDUs, who are not themselves IDUs, typi-
cally share only two of these vulnerabilities: Russian-
speaking minority and low socioeconomic status. Another important factor to consider related to vulnera-
bility is that divorce in Estonia is not uncommon. [65] As
partners learn of the index-participant's HIV/IDU status,
they may consider the option of divorce. Divorce is a
socially acceptable option, and viable since sufficient
housing exists in Estonia. By contrast, leaving a sexual
partner is not a viable cultural or economic option in
many other countries. The Estonian Study [61-64] Investigators must consider
many factors when developing a research study that figure
into designing the process of informed consent. The key
factors that arose in our research deliberations included
protection of vulnerable populations, confidentiality
waivers, validity of scientific results, differential risk for
partners based on informed consent, and research logis-
tics, all of which are discussed below. loss of patients' ability to trust their physicians with sensi-
tive information. [61-64] Investigators must consider
many factors when developing a research study that figure
into designing the process of informed consent. The key
factors that arose in our research deliberations included
protection of vulnerable populations, confidentiality
waivers, validity of scientific results, differential risk for
partners based on informed consent, and research logis-
tics, all of which are discussed below. The first statement provides no information to the part-
ners recruited for the study about their risk of infection
now or in the near future. The second statement differs
from the first by specifying that the partner is an IDU. This
information is especially revealing because it is common
knowledge among residents of Estonia that many IDUs
are HIV positive. The third statement clearly states part-
ners' potential risk of HIV if they are not actively prevent-
ing transmission (e.g., using condoms). Validity of Research Findings Nevertheless, there are substantial arguments in favor of
disclosure. First, informing partners, who are study partic-
ipants, enables them to become aware of the urgency with
which to undertake precautions. It is imperative to ensure
that potential study participants make informed choices,
even if how they choose to act thereafter may not be
healthful. Thus, doing so minimizes harms to study par-
ticipants. Another justification for disclosure may be
drawn from interviews with the IDUs in Estonia. Many
partners leave the relationship upon learning about drug
use. Because HIV is substantially more prevalent in the
IDU community, leaving a relationship with an IDU does
reduce a partner's risk of HIV infection. Thus, from the
partners' perspective, this is an effective method of risk
reduction that they can take and many are willing to take. For a research study to be ethical it must be scientifically
valid. No risk is warranted if the study cannot be inform-
ative. From an epidemiologic perspective, the greatest risk
to generalizability regarding informed recruitment is
selection bias. If index-participants know that their sex
partner(s) will be told about their drug use, then only
partners who already know about this risk behavior will
likely be referred for participation. Limitations on study
participation driven by the consent process would, in
turn, limit the effectiveness of resultant interventions to
those with characteristics similar to the group studied. While our study was cross-sectional, the same issue
applies to longitudinal studies designed to estimate HIV
transmission rates more broadly. Based on interviews with IDUs, we hypothesize that drug
users who do not want their partners to know about their
drug use are unlikely to recruit them for a study on HIV
risk and transmission. It is not only the researchers who
the IDU must trust, but also the other individuals the part-
ner may encounter at the research location. Thus, the pri-
mary disadvantage of informed recruitment – selection
bias – may likely be introduced by study participants, and
that additional selection bias introduced by an in-depth
informed consent is minimal. While empirical research is
needed to formally test this hypothesis, it is supported by
other researchers' experience working with IDUs (D. Des
Jarlais, personal communication, 2008). Validity of Research Findings Moreover, nondisclosure may pose the potential for emo-
tional harm to partners and the broader community if it
became known that researchers knew some HIV-negative
participants were having unprotected sex with HIV-posi-
tive individuals. Partners may feel betrayed and the com-
munity may feel manipulated. Such feelings could easily
transform into distrust and reluctance to participate in
research in the future, as has occurred among African
Americans in the US. [67,68] http://www.biomedcentral.com/1472-6939/10/14 http://www.biomedcentral.com/1472-6939/10/14 BMC Medical Ethics 2009, 10:14 lic's health. A key argument against disclosure is that
research participation does not increase the risk of HIV
transmission/infection by participants. Even if the sex
partners were not informed of the IDU status of the index
participants during the recruitment process, all partici-
pants received state-of-the art education about behavioral
risk factors and harm reduction techniques, which should
minimize their risk of transmission. Another argument is
that providing additional risk information does not nec-
essarily enhance the study participant's protection. Empir-
ical research shows that these educational and behavioral
risk reduction interventions minimally affect sexual
behavior. [51-54] That is, many partners continue to
undertake risky sexual relations with IDUs after being
informed. Accordingly, if the study provides limited risk
reduction benefit, why introduce the potential for selec-
tion bias that would jeopardize the results? Such empiri-
cal evidence may lead some to conclude that disclosure is
not necessary. For these and other reasons, nondisclosure
of the index participant's IDU status is still commonly
practiced in the US and abroad. closure of IDU in Estonia to a partner could present
significant harms to the IDU. In Estonia, not only is IDU
illegal, but it is also illegal for a person who knows he/she
is HIV-positive to transmit HIV to a person who was not
aware of this risk, and criminal convictions have occurred. [66] closure of IDU in Estonia to a partner could present
significant harms to the IDU. In Estonia, not only is IDU
illegal, but it is also illegal for a person who knows he/she
is HIV-positive to transmit HIV to a person who was not
aware of this risk, and criminal convictions have occurred. [66] Conversely, waiving limited confidentiality is commonly
required in research. If the requirements for research par-
ticipation are unacceptable, then individuals can choose
not to participate. In Estonia, the same HIV testing is free
and available as is the counseling provided to study par-
ticipants, which is comparable to that provided in the
wider community. Thus, there is no overly compelling
benefit for individuals to participate. The study's modest
compensation was attractive to community members but
not excessive such that a typical low-income IDU would
unduly agree to unacceptable conditions. (This assess-
ment was validated by the fact that some IDUs did not
participate and others did not refer partners.) Logistics
f Paper forms were used for most data collection, causing a
delay in data entry. While the index-participant's IDU sta-
tus was known, most partners were enrolled before infor-
mation about HIV status was available. Nonetheless,
studies of HIV transmission that include diverse risk
groups and follow the transmission risk to multiple sex Confidentiality Waiver For the IDU participants, requiring disclosure about drug
use may result in feelings of vulnerability or frustration if
they want to participate but do not want disclosure. Ensuring the confidentiality of study participants is a par-
amount human subjects projection. However, other
equally pressing human subjects protections – informing
partners of why they are being recruited into the study,
must be balanced against protecting the confidentiality of
the IDU. Accordingly, research participants commonly
provide limited waivers of confidentiality for many stud-
ies. These waivers may be for protected information to be
provided to researchers (e.g., medical records) or dis-
closed among research participants (e.g., research on cou-
ples
or
family
counseling). In
these
situations,
participants typically provide waivers of confidentiality
that are limited to requirements for good scientific
research. Indeed, in our study the index-participant will-
ingly disclosed their sexual relationship with the referred
partner(s). As the discussion unfolded, substantial concern focused
on the asymmetrical knowledge between investigators
and study participants. The researchers would know more
about the risk of continued unprotected intercourse with
the index-participant (e.g., discordant HIV status, rare or
no use of condoms) than the partner. Proponents of part-
ner notification advocate a defined legal limit on confi-
dentiality of the physician-patient relationship, the value
of providing life-saving treatment for infected partners,
and overall benefit to the public's health. Challengers to
partner notification question whether mandatory partner
notification laws for HIV will have an overall negative
impact on both medical care and public health due to the Is there an ethical difference between requiring informa-
tion disclosure about a person's drug use or HIV infection
compared to disclosing information about a person's sex-
ual relationship in order to participate in the study? Dis- Page 4 of 8
(page number not for citation purposes) Page 4 of 8
(page number not for citation purposes) Page 5 of 8
(page number not for citation purposes) Resolution For our study, we could not add IDU information to the
partner's informed consent form because of the legal con-
cerns about revealing HIV status. Thus, our pragmatic
compromise was to tell all index participants to only
recruit sex partners who knew about their drug use. Authors' contributions LAM and AU discussed the issues covered in the paper for
over a year in preparation for the study and during the
study. They asked EJG to join the discussion and provide
input from a trained ethicist perspective. LAM and EJG
drafted the manuscript based on these discussions and AU
edited the manuscript in an iterative process. Risk Arguments against and in favor of disclosure of the index
participant's IDU status can be made depending on differ-
ent interpretations of risk of HIV transmission to the pub- Page 5 of 8
(page number not for citation purposes) Page 5 of 8
(page number not for citation purposes) http://www.biomedcentral.com/1472-6939/10/14 BMC Medical Ethics 2009, 10:14 http://www.biomedcentral.com/1472-6939/10/14 http://www.biomedcentral.com/1472-6939/10/14 Summary During the design phase of this study, we identified ethi-
cal tensions unique to the process of studying couples. From an epidemiological perspective, when the couple is
of central interest then it is the unit of study and analysis. However, from a research ethics perspective, the individu-
als, the couple, and the greater community all are impor-
tant to consider and warrant human subjects protections. At times what is best for one person in a couple may not
be best for the other. For couples research pertaining to
HIV sexual transmission, we posit that failing to fully
inform partners about the rationale for inclusion in the
study is ethically disconcerting. Intentionally limiting dis-
closure to sexual partners remains ethically problematic
given the potential for continued risk that exists among
HIV-discordant couples with unsafe sex behaviors. While
institutional review boards have developed sensitivity to
cultural appropriateness for research in developing coun-
tries, refinement of guidance for couples research of com-
municable diseases is needed to address ethical issues
such as those raised herein. The authors thank Dr. Don Des Jarlais from Beth Israel Medical Center,
New York, New York, and Dr. Jeremy Sugarman from Johns Hopkins Ber-
man Institute of Bioethics for the information they provided during the
drafting of the manuscript. The authors also thank Dr. Sean Philpott, the
Policy and Ethics Officer for PATH's Global Campaign for Microbicides, Dr. Andres Soosaar from Department of Health (Estonia) and Ants Nõmper
from Raidla & Partners (Estonia) for their helpful comments in revision. The
authors that Alison Krug, MPH for her thoughtful comments while proving
copy editing services for this manuscript. Acknowledgements Drs. McNutt and Uusküla are supported by Civilian Research Development
Foundation grant (ESX0-2722-TA-06). Dr. Uuskula is also supported by a
US NIH grant (R01 DA 03574) and grant No. R01 TW006990 from the
Fogarty International Center. Dr. Gordon is supported by a Career Devel-
opment Award from the National Institute of Diabetes and Digestive and
Kidney Diseases, Bethesda, MD (DK063953). Conflicting interests partners may benefit from rapid tests and real time data
processing to provide appropriate information about dis-
ease risk to recruited individuals. Without these technolo-
gies, general statements related to risk of HIV can be made
to all participants. g
The authors declare that they have no competing interests. Page 6 of 8
(page number not for citation purposes) References 1. UNAIDS/WHO: AIDS Epidemic Update: December 2007
[monograph on the Internet]. 2007 [http://data.unaids.org/pub/
EPISlides/2007/2007_epiupdate_en.pdf]. UNAIDS, Geneva p p
p ]
2. MacQueen KM, Sugarman J: HIV Prevention Trials Network
Ethics Guidance for Research [monograph on the Internet]. HPTN
Ethics
Working
Group
2003
[http://www.hptn.org/
Web%20Documents/EWG/ HPTNEthicsGuidanceFINAL15April2003.pdf]. p
p ]
3. MacQueen KM, Sugarman J: Back to the Rough Ground: Work-
ing in International HIV Prevention as Ethical Debates Con-
tinue. IRB 2003, 25:11-13. We suggest that further guidance for both cross-sectional
and longitudinal studies of HIV transmission is needed
regarding informed recruitment. While in our example
recruitment was anonymous and limited to IDUs and
their sexual partners, in longitudinal HIV transmission
studies researchers recruit diverse risk groups (e.g., IDU,
commercial sex workers, MSM) and follow transmission
for multiple waves. Thus, specific HIV risk (e.g., HIV status
of sexual partners) often will not be known at the time of
study interviews. We suggest that research participants can
be accurately told that they were recruited for the study
because they are connected to a group at higher risk for
HIV then the general population. The detailed explana-
tion of this risk may vary by wave of recruitment and by
region. Additionally, in longitudinal studies, we suggest
research participants be reminded that they were recruited
for the study due to their high risk for becoming infected
with HIV and harm reduction counseling be provided
with each follow-up blood test. 4. UNAID/WHO: Ethical considerations in biomedical HIV pre-
vention trials [monograph on the Internet]. Joint United Nations
Programme on HIV/AIDS (UNAIDS) 2007 [http://data.unaids.org/pub/
Report/2007/jc1399-ethicalconsiderations_en.pdf]. g
(
)
p
Report/2007/jc1399-ethicalconsiderations_en.pdf]. 5. Perinatal HIV Intervention Research in Developing Coun-
tries Workshop participants. Science, ethics, and the future
of research into maternal infant transmission of HIV-1. Lan-
cet 1999, 353:832-5. 6. Omene JA, Lurie P, Wolfe SM, Foex B, Isturiz RE, Greco DB: Sci-
ence, ethics, and future of research into maternal-infant
transmission of HIV-1. Lancet 1999, 353:1878-1881. 7. WHO/UNAIDS/AAVP International Expert Group, Osmanov S:
Executive summary and recommendations from WHO/
UNAIDS and AAVP consultation on: 'The inclusion of ado-
lescents in HIV vaccine trials', 16–18 March 2006 in Gabor-
one, Botswana. AIDS 2007, 21:W1-10. 8. WHO-UNAIDS Expert Group: Gender, age, and ethnicity in
HIV vaccine-related research and clinical trials: report from
a
WHO-UNAIDS
consultation,
26–28
August
2004. Lausanne, Switzerland. AIDS 2005, 19:w7-28. ,
9. References Bayer R: The debate over maternal-fetal HIV transmission
prevention trials in Africa, Asia, and the Caribbean: racist
exploitation or exploitation of racism? Am J Public Health 1998,
88:567-70. 9. Bayer R: The debate over maternal-fetal HIV transmission
prevention trials in Africa, Asia, and the Caribbean: racist
exploitation or exploitation of racism? Am J Public Health 1998,
88:567-70. Page 6 of 8
(page number not for citation purposes) Page 6 of 8
(page number not for citation purposes) BMC Medical Ethics 2009, 10:14 http://www.biomedcentral.com/1472-6939/10/14 http://www.biomedcentral.com/1472-6939/10/14 33. European Centre for Epidemiological Monitoring of AIDS
(EuroHIV). HIV/AIDS surveillance in Europe: end-year
report 2006. Volume 75. Saint-Maurice, Institut de Veille Sanitaire;
2007. 10. Abdool Karim SS: Placebo controls in HIV perinatal transmis-
sion trials: a South African's viewpoint. Am J Public Health 1998,
88:564-6. 11. Varmus H, Satcher D: Ethical complexities of conducting
research in developing countries. N Engl J Med 1997,
337:1003-5. 34. Estonian Ministry of Social Affairs. Estonia: country report on
national response to HIV/AIDS epidemic 2007 [monograph
on the Internet]. Tallinn. 2008 [http://data.unaids.org/pub/
Report/2008/estonia_2008_country_progress_report_en.pdf]. 12. Lindegger G, Richter LM: HIV vaccine trials: critical issues in
informed consent. S Afr J Sci 2000, 96:313-7. J
13. Buchanan DR, Miller FG: A public health perspective on
research ethics. J Med Ethics 2006, 32:729-733. 35. Uusküla A, Kalikova A, Zilmer K, Tammai L, DeHovitz J: The role of
injection drug use in the emergence of Human Immunodefi-
ciency Virus infection in Estonia. Int J Infect Dis 2002, 6:23-7. 14. Solbakk JH: Use and abuse of empirical knowledge in contem-
porary bioethics. Med Health Care Philos 2004, 7(1):5-16. y
J
36. Uusküla A, McNutt LA, Dehovitz J, Fischer K, Heimer R: High prev-
alence of blood-borne virus infections and high-risk behav-
iour among injecting drug users in Tallinn, Estonia. Int J STD
AIDS 2007, 18:41-6. 15. Clark PA: AIDS research in developing countries: do the ends
justify the means? Med Sci Monit 2002, 8:ED5-16. j
y
16. de Zulueta P: Randomised placebo-controlled trials and HIV-
infected pregnant women in developing countries. Ethical
imperialism or unethical exploitation? Bioethics 2001,
15:289-311. 37. Uusküla A, Kals M, Rajaleid K, Abel K, Talu A, Rüütel K, et al.: High-
prevalence and high-estimated incidence of HIV infection
among new injecting drug users in Estonia: need for large
scale prevention programs. J Public Health (Oxf) 2008 in press. 17. References Normand J, Vlahov D, Moses LE, eds, Preventing HIV Transmission:
The Role of Sterile Needles and Bleach. Washington, DC:
National Academy Press; 1995. y
44. Heimer R, Kaplan EH, Khoshnood K, Jariwala B, Cadman EC: Needle
exchange decreases the prevalence of HIV-1 proviral DNA in
returned syringes in New Haven, Connecticut. Am J Med 1993,
95:214-220. 24. Quinn TC, Wawer MJ, Sewankambo N, Serwadda D, Li C, Wabwire-
Mangen F, et al.: Viral load and heterosexual transmission of
Human Immunodeficiency Virus Type 1. Rakai Project
Study Group. N Engl J Med 2000, 342:921-929. 45. Riehman KS: Injecting Drug Use and AIDS in Developing
Countries: Determinants and Issues for Policy Consideration
[monograph on the Internet] Paper prepared for the Policy
Research Report on AIDS and Development World Bank,
Policy Research Department. 2006 [http://www.worldbank.org/
aidsecon/confront/backgrnd/riehman/index.htm]. 25. Angell M: Investigators' Responsibilities for Human Subjects
in Developing Countries. N Engl J Med 2000, 342:967-969. 26. Gray RH, Quinn TC, Serwadda D, Sewankambo NK, Wabwire-Man-
gen F, Wawer MJ, Mullings AMA, Greco DB, Hearst N, Cates W Jr,
Coates TJ, Handsfield HH, Angell M: The Ethics of Research in
Developing Countries. N Engl J Med 2000, 343:361-363. g
]
46. Friedman SR, Mateu-Gelabert P, Milagros S, Hagan H, Des Jarlais DC:
Positive deviance control-case life history: a method to
develop grounded hypotheses about successful long-term
avoidance of infection. BMC Public Health 2008, 8:94. p
g
g J
27. Loue S, Okello D: Research Bioethics in the Ugandan Context
II: Procedural and Substantive Reform. J Law Med Ethics 2000,
28:165-173. 28. Thiessen C, Ssekubugu R, Wagman J, Kiddugavu M, Wawer MJ,
Emanuel E, et al.: Personal and community benefits and harms
of research: views from Rakai, Uganda. AIDS 2007,
21:2493-501. 47. Wolfe D: Paradoxes in antiretroviral treatment for injecting
drug users: access, adherence and structural barriers in Asia
and the former Soviet Union. Int J Drug Policy 2007, 18:246-54. J
g
y
48. Spire B, Lucas GM, Carrieri MP: Adherence to HIV treatment
among IDUs and the role of opioid substitution treatment
(OST). Int J Drug Policy 2007, 18:262-70. 29. National HIV/AIDS Clinicians' Consultation Center at San
Francisco General Hospital. Compendium of State HIV
Testing Laws Quick Reference Guide for Clinicians [http://
www.nccc.ucsf.edu/StateLaws/About%20Compendium/
Quick%20Reference%20Guide.pdf] (
)
J
g
y
49. Lert F, Kazatchkine MD: Antiretroviral HIV treatment and care
for injecting drug users: an evidence-based overview. Int J
Drug Policy 2007, 18:255-61. g
y
50. References Gable L, Gamharter K, Gostin LO, Hodge JG Jr, Van Puymbroeck RV:
Legal Aspects of HIV/AIDS: A Guide for Policy and Law
Reform. Global HIV/AIDS Program and Legal Vice Presidency,
Washington DC: The International Bank for Reconstruction and
Development/The World Bank; 2001. 38. Platt L, Bobrova N, Rhodes T, Uusküla A, Parry JV, Rüütel K, et al.:
High HIV prevalence among injecting drug users in Estonia:
implications for understanding the risk environment. AIDS
2006, 20:2120-3. 39. HIV/AIDS EG ITA in coordination with the HIV/AIDS EG Chair:
Working Program/Strategy for the collaboration in the pre-
vention of HIV (within the context of Northern Dimension
Partnership programme) [monograph on the internet] HIV/
AIDS EG Strategy. [http://www.ndphs.org///documents/1259/
HIV_AIDS_9_4.2_1_Rev._1_Draft%20work%20plan%202009%20revi
sed.doc]. p
18. Abdool Karim Q, Abdool K SS, Coovadia HM, Susser M: Informed
consent for HIV testing in a South African hospital: is it truly
informed and truly voluntary? Am J Public Health 1998,
88:637-40. Erratum in: Am J Public Health 1998, 88:972 J
19. Kahn JP, Mastroianni AC, Sugarman J: Beyond consent: seeking
justice in research. New York: Oxford University Press; 1998. j
y
20. Hyder AA, Wali SA, Khan AN, Teoh NB, Kass NE, Dawson L: Ethi-
cal review of health research: a perspective from developing
country researchers. J Med Ethics 2004, 30:68-72. ]
40. Commision of the European Communities: Coordinated and inte-
grated approach to combat HIV/AIDS withing the European
Union and in its neighbourhood [monograph on the Inter-
net]. Brussels. 2004 [http://ec.europa.eu/health/ph_threats/com/
aids/docs/ev_20040916_rd01_en.pdf]. y
J
21. Margolin G, Chien D, Duman SE, Fauchier A, Gordis EB, Oliver PH,
et al.: Ethical Issues in Couple and Family Research. J Fam Psy-
chol 2005, 19(1):157-167. 22. The National Commission for the Protection of Human Subjects of
Biomedical and Behavioral Research: The Belmont Report: ethi-
cal principles and guidelines for the protection of human sub-
jects of research. 1979 [http://www.hhs.gov/ohrp/humansubjects/
guidance/belmont.htm]. 41. Watters JK: Impact of HIV risk and infection and the role of
prevention services. J Subst Abuse Treat 1996, 13:375-85. 42. Yoast R, Williams MA, Deitchman SD, Champion HC: Report of the
Council on Scientific Affairs: methadone maintenance and
needle-exchange programs to reduce the medical and public
health consequences of drug abuse. J Addict Dis 2001, 20:15-40. 23. Council for International Organizations of Medical Sciences
(CIOMS) in collaboration with the World Health Organiza-
tion (WHO) International ethical guidelines for epidemiological studies
[monograph
on
the
Internet]
[http://cioms.ch/
frame_ethical_guidelines_2009.htm]. q
g
J
43. 51.
Nguyen TA, Oosterhoff P, Hardon A, Tran HN, Coutinho RA, Wright
P: A hidden HIV epidemic among women in Vietnam. BMC
Public Health 2008, 8:37. References Amundsen EJ, Eskild A, Stigum H, Smith E, Aalen OO: Legal access
to needles and syringes/needle exchange programmes ver-
sus HIV counselling and testing to prevent transmission of
HIV among intravenous drug users: a comparative study of
Denmark, Norway and Sweden. Eur J Public Health 2003,
13:252-8. 30. EuroHIV 2006 survey on HIV and AIDS surveillance in the
WHO European Region [monograph on the Internet] Saint-
Maurice: Institut de veille sanitaire 2007 [http://www.euro
hiv.org/reports/eurohiv_2006_survey_report/
eurohiv_2006_survey_report.pdf]. 31. Bayer R, Toomey KE: HIV prevention and the two faces of part-
ner notification. Am J Public Health 1992, 82:1158-1164. 51. Nguyen TA, Oosterhoff P, Hardon A, Tran HN, Coutinho RA, Wright
P: A hidden HIV epidemic among women in Vietnam. BMC
Public Health 2008, 8:37. ner notification. Am J Public Health 1992, 82:1158-11 32. Kipnis K: A Defense of Unqualified Medical Confidentiality. Am J Bioethics 2006, 6:7-18. Page 7 of 8
(page number not for citation purposes) Page 7 of 8
(page number not for citation purposes) BMC Medical Ethics 2009, 10:14 http://www.biomedcentral.com/1472-6939/10/14 http://www.biomedcentral.com/1472-6939/10/14 http://www.biomedcentral.com/1472-6939/10/14/pre
pub 52. Abdala N, Krasnoselskikh TV, Durante AJ, Timofeeva MY, Verevo-
chkin SV, Kozlov AP: Sexually transmitted infections, sexual
risk behaviors and the risk of heterosexual spread of HIV
among and beyond IDUs in St. Petersburg, Russia. Eur Addict
Res 2008, 14:19-25. 53. Beyrer C: HIV epidemiology update and transmission factors:
risks and risk contexts – 16th International AIDS Conference
epidemiology plenary. Clin Infect Dis 2007, 44:981-7. p
gy p
y
54. Lyles CM, Kay LS, Crepaz N, Herbst JH, Passin WF, Kim AS, et al.:
HIV/AIDS Prevention Research Synthesis Team. Best-evi-
dence interventions: findings from a systematic review of
HIV behavioral interventions for US populations at high risk,
2000–2004. Am J Public Health 2007, 97:133-43. J
55. Scott G: "They got their program, and I got mine": a caution-
ary tale concerning the ethical implications of using respond-
ent-driven sampling to study injection drug users. Int J Drug
Policy 2008, 19:42-51. y
56. Semaan S, Santibanez S, Garfein RS, Heckathorn DD, Des Jarlais DC:
Ethical and regulatory considerations in HIV prevention
studies employing respondent-driven sampling. Int J Drug Pol-
icy 2009, 20(1):14-27. 57. European Monitoring Centre for Drugs and Drug Addiction: Annual
report 2004: the state of the drugs problem in the European
Union and Norway [monograph on the Internet] Luxem-
bourg: Office for Official Publications of the European Com-
munities. 2003 [http://ar2004.emcdda.europa.eu/download/ar2004-
en.pdf]. p ]
58. References Publish with BioMed Central and every
scientist can read your work free of charge
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Sir Paul Nurse, Cancer Research UK
Your research papers will be:
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BioMedcentral
Page 8 of 8
(page number not for citation purposes) Publish with BioMed Central and every
scientist can read your work free of charge
"BioMed Central will be the most significant development for
disseminating the results of biomedical research in our lifetime."
Sir Paul Nurse, Cancer Research UK
Your research papers will be:
available free of charge to the entire biomedical community
peer reviewed and published immediately upon acceptance
cited in PubMed and archived on PubMed Central
yours — you keep the copyright
Submit your manuscript here:
http://www.biomedcentral.com/info/publishing_adv.asp
BioMedcentral Publish with BioMed Central and every
scientist can read your work free of charge [
p
g pp
]
67. Hall MA, Camacho F, Lawlor JS, Depuy V, Sugarman J, Weinfurt K:
Measuring trust in medical researchers. Med Care 2006,
44:1048-53. 68. Freimuth VS, Quinn SC, Thomas SB, Cole G, Zook E, Duncan T: Afri-
can Americans' views on research and the Tuskegee Syphilis
Study. Soc Sci Med 2001, 52:797-808. References European Monitoring Centre for Drugs and Drug Addiction: Report
to the EMCDDA by the Reitox National Focal Point [mono-
graph on the Internet]. Estonia drug situation; 2003 [http://
www.just.ee/orb.aw/class=file/action=preview/id
=
20533/
DRUD_SITUATION_2003%5B1%5D.pdf]. 59
Hh
[h
h
I
] US D
f H
l h 59. Hhs.gov [homepage on the Internet]: US Department of Health
and Human Services. Office of Extramural Research. National Institutes of Health. Grants Policy. Certificate of
Confidentiality:
Background
Information. [http://
grants.nih.gov/grants/policy/coc/background.htm]. g
g
g
p
y
g
]
60. Wolf LE, Zandecki J: Sleeping better at night: investigators'
experiences with certificates of confidentiality. IRB 2006,
28:1-7. 61. Odunsi B: Should caregivers be compelled to disclose
patients' HIV infection to the patients' sex partners without
consent? Stud Fam Plann 2007, 38:297-306. 62. Dixon-Mueller R: The sexual ethics of HIV testing and the
rights and responsibilities of partners. Stud Fam Plann 2007,
38:284-96. 63. Passin WF, Kim AS, Hutchinson AB, Crepaz N, Herbst JH, Lyles CM:
HIV/AIDS Prevention Research Synthesis Project Team. A
systematic review of HIV partner counseling and referral
services: client and provider attitudes, preferences, prac-
tices, and experiences. Sex Transm Dis 2006, 33:320-8. ,
p
64. Tesoriero JM, Battles HB, Heavner K, Leung SY, Nemeth C, Pulver
W, et al.: The effect of name-based reporting and partner
notification on HIV testing in New York State. Am J Public
Health 2008, 98:728-35. 65. Sardon JP: The Demographic Situation of Europe and the
Developed Countries Overseas. An Annual Report Popul 2000,
12:293-328. 66. Global Network of People Living with HIV/AIDS Europe
(GNP+) (Europe) and Terrence Higgins Trust (THT) Crimi-
nalisation of HIV transmission in Europe [monograph on the Internet]
[http://www.gnpplus.net/criminalisation/results.shtml]. Pre-publication history p
y
The pre-publication history for this paper can be accessed
here: The pre-publication history for this paper can be accessed
here: The pre-publication history for this paper can be accessed
here:
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https://www.degruyter.com/document/doi/10.1515/opth-2020-0113/pdf
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Latin
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Ἰουδαίαν in Acts 2:9: Reverse Engineering Textual Emendations
|
Open Theology
| 2,020
|
cc-by
| 8,709
|
Vincent van Altena*, Jan Krans, Henk Bakker, Jantien Stoter
Ἰουδαίαν in Acts 2:9: Reverse Engineering
Textual Emendations https://doi.org/10.1515/opth-2020-0113
received February 29, 2020; accepted April 21, 2020 https://doi.org/10.1515/opth-2020-0113
received February 29, 2020; accepted April 21, 2020 https://doi.org/10.1515/opth-2020-0113
received February 29, 2020; accepted April 21, 2020 Abstract: Building on a plethora of conjectured emendations for IOϒΔAIAN, this article approaches the
issue once again to test the viability of a quantitative tool and to establish the possibility of
palaeographical confusion of IOϒΔAIAN with an alternative topo- or demonym. The article starts with
an experiment using Greek topo- and demonyms derived from contemporary Christian, Jewish, and
Classical sources to establish a palaeographical confusion score. Next the likeliness of the “closest”
alternatives in the geographical arrangement of Acts 2:9–11 is explored. The article ends with an
evaluation of the possible implications for the text in Acts 2:9 as well as a critical appraisal of the method
for conjectural criticism. Keywords: table of nations, palaeographic confusion, conjectural criticism, spatial analysis * Corresponding author: Vincent van Altena, Faculty of the Built Environment and Architecture, Delft University of
Technology, Delft, The Netherlands; Faculty of Religion and Theology, Vrije Universiteit Amsterdam, Amsterdam,
The Netherlands, e-mail: v.p.vanaltena-1@tudelft.nl, v.p.van.altena@vu.nl
Jan Krans: Protestantse Theologische Universiteit, Amsterdam, The Netherlands
Henk Bakker: Faculty of Religion and Theology, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands
Jantien Stoter: Faculty of the Built Environment and Architecture, Delft University of Technology, Delft, The Netherlands Open Access. © 2020 Vincent van Altena et al., published by De Gruyter.
This work is licensed under the Creative Commons
Attribution 4.0 Public License. Open Theology 2020; 6: 378–391 Regular Article Vincent van Altena*, Jan Krans, Henk Bakker, Jantien Stoter 1 Introduction In the first part of this study, “Ἰουδαίαν in Acts 2:9: a diachronic overview of its conjectured emendations”,
we demonstrated that several textual emendations have been conjectured to solve the alleged interpretive
problems with IOϒΔAIAN in Acts 2:9. In the history of interpretation, solutions have been explored in
three directions: (1) IOϒΔAIAN has been interpreted as an adjective instead of a noun, (2) IOϒΔAIAN
has been regarded as a corruption or later interpolation in the text, and (3) several toponyms have been
proposed as a solution to emend the text. None of these suggestions have been generally accepted. The first aim of this article is to contribute to the discussion by testing the hypothesis that the text
might have been corrupted during transmission due to palaeographical confusion of Greek characters, i.e. the text might originally have contained a different Greek word.¹ To test this hypothesis, we need to
identify a Greek toponym that is palaeographically, historically, and geographically suitable to replace
IOϒΔAIAN in Acts 2:9. Therefore, we use a reverse engineering approach: instead of starting from the
text itself, trying to find a fitting explanation of the interpretive problem concerning IOϒΔAIAN, we start
with a list of toponyms and research which toponyms are likely alternatives based on the criterion of 1 We are of course fully aware that palaeographical confusion is not the only mechanism of scribal change within textual
transmission. * Corresponding author: Vincent van Altena, Faculty of the Built Environment and Architecture, Delft University of
Technology, Delft, The Netherlands; Faculty of Religion and Theology, Vrije Universiteit Amsterdam, Amsterdam,
The Netherlands, e-mail: v.p.vanaltena-1@tudelft.nl, v.p.van.altena@vu.nl
Jan Krans: Protestantse Theologische Universiteit, Amsterdam, The Netherlands
Henk Bakker: Faculty of Religion and Theology, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands
Jantien Stoter: Faculty of the Built Environment and Architecture, Delft University of Technology, Delft, The Netherlands Open Access. © 2020 Vincent van Altena et al., published by De Gruyter. This work is licensed under the Creative Commons
Attribution 4.0 Public License. Ἰουδαίαν in Acts 2:9: Reverse Engineering Textual Emendations
379 palaeographical confusion.² The results of this test will be further analysed to see whether we can solve
the interpretive problem in Acts 2:9 or whether we can exclude palaeographical confusion as a possible
explanation for the alleged corruption of the text. 1 Introduction The second aim of this article is to evaluate whether our method is helpful for identifying possible
f
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reeval
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inflecon
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retrieval
geocoding
inflecon
retrieval
geocoding
inflecon
topostext
greek
inflecon
Calculate
metrics
filtering
qualitave
analyses
qualitave results
New
Testament
Septuagint
Classical
authors
Classical
authors
pleiades
TIPNR
quantave
results
Figure 1: Method and data. Figure 1: Method and data. palaeographical confusion.² The results of this test will be further analysed to see whether we can solve
the interpretive problem in Acts 2:9 or whether we can exclude palaeographical confusion as a possible
explanation for the alleged corruption of the text. palaeographical confusion.² The results of this test will be further analysed to see whether we can solve
the interpretive problem in Acts 2:9 or whether we can exclude palaeographical confusion as a possible
explanation for the alleged corruption of the text. The second aim of this article is to evaluate whether our method is helpful for identifying possible
cases of palaeographical confusion in problematic texts. The article has been organised as follows: Section 2 is concerned with the method and data used for
this study. Section 3 presents the findings after the application of the method for Acts 2. Section 4
evaluates these results by considering the implications of the experiment for Acts 2 and provides a critical
appraisal of whether our experiment recommends a wider application of the method for other cases. Section 5 finalises the article with a conclusion and outlook for further research. 2 Method, algorithm, and data To gauge the probability of confusion between a toponym and the supposedly corrupted IOϒΔAIAN, we
research how easy it might have been that during textual transmission a scribe misreads one character for
another character and that this resulted in the current reading. We propose the following method (illustrated in Figure 1): we use a computer algorithm and three data
sets comprising toponyms occurring in early Christian literature, Greek Jewish Scriptures, and Classic texts
to identify possible palaeographic alternatives. Furthermore, we test the appropriateness of the most likely
results in their wider historical and geographical contexts. 2 Reverse engineering is the terminology employed in technology-related disciplines. The Cambridge Dictionary defines it as
“the process of studying another company’s product to see how it is made, sometimes in order to be able to copy it”. In this
article, we use this concept in a more loose way: we start with the hypothesis about the textual transmission process and a data
set of semantical candidates, and subsequently prune the number of likely toponyms by adding additional criteria. 380 380
Vincent van Altena et al. 2.1 Confusion distances The confusion distance has been proposed by Van Altena et al. as a metric to calculate the probability of
palaeographical confusion between two words.³ The underlying assumption is that the text could have
been corrupted at a very early stage in the transmission process. When this corruption was unintentional,
it might have been caused by a misreading of the Vorlage, possibly due to the confusion of Greek
majuscule characters. In their study, Van Altena et al. propose that the probability of confusion of Greek majuscule
characters is not equally likely and that combinations of characters might be confused for a single
character and vice versa. They therefore propose a confusion table that contains character pairs and a digit
representing the assumed ease of confusion. Our study uses this algorithm and the accompanying
confusion table to establish confusion distances of toponyms. 2.2 Data The second ingredient for our study is a collection of place names (i.e. toponyms) with geographic
locations, also known as an onomasticon or gazetteer.⁴ For the purpose of our study, this data set should
contain Greek toponyms that correspond in grammatical function with IOϒΔAIAN, are supposed to be
familiar to the original author of Acts, and fit the geographical arrangement in Acts 2:9–11.⁵ To the best of
our knowledge, a gazetteer that fits these requirements does not exist. We therefore retrieved toponyms
from textual resources, inflected them to the proper grammatical case (i.e. accusative or nominative in
case of uninflected nouns), and assigned geographical coordinates to each toponym. 3 van Altena et al., “Spatial Analysis of New Testament Textual Emendations Utilizing Confusion Distances.”
4 In a modern sense, a gazetteer consists of “three core elements: toponyms (and their history), spatial location (in various
representations, such as points, lines, and polygons), and classification (e.g., types and categories of places)”, cf. Yuan,
“Mapping Text,” 109–23.
5 van Altena et al., “Spatial Analysis of New Testament Textual Emendations Utilizing Confusion Distances,” 48–9.
6 Louw and Nida, Greek-English Lexicon of the New Testament. We selected all entries in the range 93.389–93.615 (Places) and
exported them to a spreadsheet.
7 Van der Meer identifies five problems to temper expectations in a reconstruction of LXX topography: “[1] most of the
geography and topography of the world in which the Septuagint was made[…] is not reflected in the Greek translations of
Hebrew Scripture, [2] that the corpus for the study is a heterogeneous collection, [3] that several Greek renderings of Hebrew
toponyms rest on misinterpretations or [4] deliberate actualizations, and finally [5] that several Greek toponyms were
misunderstood in the course of textual transmission”, cf. van der Meer, “The Natural and Geographical Context of the
Septuagint: Some Preliminary Observations,” 393. However, since the aim of this article is not to reconstruct an actual
topography of the Lxx but to construct a list of toponyms that the author of Acts might have been familiar with, these caveats
will not affect our main conclusion. 2.2.3 Geographical parsing The position of each toponym on the skin of the earth has to be expressed in x,y-coordinates to enable
geographical analysis. This is achieved by geographical parsing, which could be defined as “the task of identifying
and resolving toponyms to their geographical coordinates”.¹⁵ Due to their nature, it is not equally straightforward to
acquire coordinates by geoparsing for the three data sets that we introduced in the previous section. 8 The search of the Septuagint was performed with the aid of the computer program Logos Bible Software version 8.6.0.0052. See https://www.logos.com/. We used a two-step approach. First, we created a concordance for the whole Septuagint that we
filtered on Biblical Entities and subsequently on Places. Next, we used the retrieved place names to query the Septuagint and
retrieve the Greek words and the biblical references. The Septuagint text is based on: Swete, The Old Testament in Greek. We
also used the tagged text that is provided by Tan et al., The Lexham Greek-English Interlinear Septuagint. 9 Kiesling and Aikaterini Laskaridis Foundation, “ToposText,” Gazetteer, ToposText Web Version 3.0. 10 Montanari The Brill Dictionary of Ancient Greek 11 Montanari’s dictionary is lacking an arrangement according to semantic field. Neither is there any explicit indication (e.g. the abbreviation n. pr. loc.–nomen proprium loci) that the particular lemma is dealing with a toponym. As such, the dictionary
conforms to the general practice to which Louw-Nida and Swanson appear to be an exception. To tackle this obstacle, we
retrieved toponyms from Montanari doing multiple searches for the terms cape, city, country, district, ethnic, island, mountain,
pass, people, place, port, province, region, settlement, tribe, urban, and village. 11 Montanari’s dictionary is lacking an arrangement according to semantic field. Neither is there any explicit indication (e.g. the abbreviation n. pr. loc.–nomen proprium loci) that the particular lemma is dealing with a toponym. As such, the dictionary
conforms to the general practice to which Louw-Nida and Swanson appear to be an exception. To tackle this obstacle, we
retrieved toponyms from Montanari doing multiple searches for the terms cape, city, country, district, ethnic, island, mountain,
pass, people, place, port, province, region, settlement, tribe, urban, and village. 2.2.2 Inflection of toponyms Subsequently, we had to inflect these lists of toponyms, since the data retrieved from dictionaries and the
online databases are in nominative singular form. Instead of looking up almost 10,000 individual toponyms
(Table 1), we created a computer script¹³ that inflected the toponyms to the different cases in singular and
plural forms.¹⁴ The results of these processes will be evaluated using qualitative criteria (see Section 4). Table 1: Statistics about the toponym data sets
Indeclinable
Localised
Total
Louw-Nida
35
322
322
LXX
917
1,780
1,783
Montanari
61
1,548
3,389
ToposText
123
4,454
4,454
1,136
8,104
9,948 toponyms from the Septuagint.⁸ The third group pertains to toponyms occurring in classical authors. We
used the website Topostext⁹ and the Greek-English dictionary by Montanari et al.¹⁰ as the two major
sources to compile this list of toponyms. Especially the retrieval of topo- and demonyms from a dictionary
appeared to be a tedious endeavour.¹¹ Eventually, we made the resulting lists of toponyms in singular
nominative form available online.¹² toponyms from the Septuagint.⁸ The third group pertains to toponyms occurring in classical authors. We
used the website Topostext⁹ and the Greek-English dictionary by Montanari et al.¹⁰ as the two major
sources to compile this list of toponyms. Especially the retrieval of topo- and demonyms from a dictionary
appeared to be a tedious endeavour.¹¹ Eventually, we made the resulting lists of toponyms in singular
nominative form available online.¹² 13 van Altena, Greek Noun Inflection Script. 2.2.1 Retrieval of toponyms We used three types of literary resources. For the New Testament, we created a data set from a semantic
dictionary.⁶ This data set contains 259 location-related pronouns, including toponyms, demonyms, and
derivatives such as adjectives, which occur in the 27 books of the New Testament. Furthermore, we argue
that it is reasonable to construct a list of toponyms, which appear in the Septuagint (LXX), and take them
to reflect contemporary toponyms that might have been known to an author contributing to the tradition
of early Christian literature in the time of writing the book of Acts.⁷ For this purpose, we retrieved 3 van Altena et al., “Spatial Analysis of New Testament Textual Emendations Utilizing Confusion Distances.” 3 van Altena et al., “Spatial Analysis of New Testament Textual Emendations Utilizing Confusion Distances.” 4 In a modern sense, a gazetteer consists of “three core elements: toponyms (and their history), spatial location (in various
representations, such as points, lines, and polygons), and classification (e.g., types and categories of places)”, cf. Yuan,
“Mapping Text,” 109–23. 5 van Altena et al.,
Spatial Analysis of New Testament Textual Emendations Utilizing Confusion Distances,
48–9. 6 Louw and Nida, Greek-English Lexicon of the New Testament. We selected all entries in the range 93.389–93.615 (Places) and
exported them to a spreadsheet. 7 Van der Meer identifies five problems to temper expectations in a reconstruction of LXX topography: “[1] most of the
geography and topography of the world in which the Septuagint was made[…] is not reflected in the Greek translations of
Hebrew Scripture, [2] that the corpus for the study is a heterogeneous collection, [3] that several Greek renderings of Hebrew
toponyms rest on misinterpretations or [4] deliberate actualizations, and finally [5] that several Greek toponyms were
misunderstood in the course of textual transmission”, cf. van der Meer, “The Natural and Geographical Context of the
Septuagint: Some Preliminary Observations,” 393. However, since the aim of this article is not to reconstruct an actual
topography of the Lxx but to construct a list of toponyms that the author of Acts might have been familiar with, these caveats
will not affect our main conclusion. Ἰουδαίαν in Acts 2:9: Reverse Engineering Textual Emendations
381 382
Vincent van Altena et al. New Testament toponyms are easily tied to the Tyndale Individualised Proper Names with all References
(TIPNR).¹⁶ Toponyms from the Septuagint can be linked to a toponym in TIPNR using their Hebrew equivalents
but need to be checked since some Greek toponyms seem to have been actualised during translation.¹⁷ Therefore,
the LXX toponyms have also been linked to their equivalents in the Pleiades project¹⁸ as far as possible. Toponyms from classical resources had already been given a location in Topostext.¹⁹ For the geoparsing of the
additional toponyms we retrieved from the Montanari dictionary, we used the Pleiades project. 2.2.3 Geographical parsing 12 van Altena, “New Testament Toponyms;” van Altena, “Greek Old Testament Toponyms;” van Altena, “Greek Toponyms
Collected from Classical Literature.” 12 van Altena, “New Testament Toponyms;” van Altena, “Greek Old Testament Toponyms;” van Altena, “Greek Toponyms
Collected from Classical Literature.” 14 Our inflection script worked well for regular nouns, but we needed a solution for exceptions and nouns, which theoretically
can be inflected in multiple ways if its gender is unknown (e.g. nouns ending on -ος could either be inflected to the accusative
singular ending -ος or -ον be dependent on the gender of the noun). Instead of figuring out the correct inflection for each
individual case, we implemented a solution that produces all possible variant inflections. This means that the script inflects
these nouns in multiple ways. Though we expect only one of these inflections will be correct for most of the cases, we can use
this inflected list of toponyms as an intermediate input for subsequent processing. ProbIdx = confdist × ops ProbIdx = confdist × ops The results show a small shift to the right in the frequency diagram of the operations compared to the
distribution in the histogram of the confusion distance. This can be easily explained, since the confusion
distance is the result of a weighted score of the individual operations. For the remainder of this study, we have selected the first 49 toponyms that have the closest
probability index with IOϒΔAIAN.²⁰ This is of course an arbitrary selection based on the assumption that
a maximum of four unintentional confusions might still be conceivable. 16 The TIPNR is a collection of every proper or geographical name in the Bible, which are linked to the original languages. That
is, Hebrew for names occurring in the Old Testament, and Greek for those found in the New Testament. The data set does not
contain alternative readings, neither references to Greek names found in the Septuagint nor toponyms occurring in the Aramaic
parts of the Old Testament. Geographical names have been enriched with a geolocation, which was derived from the OpenBible
data set, Smith, “Bible Geocoding – Bible Maps in Google Earth and Google Maps.” and Instone-Brewer, “Tyndale
Individualised Proper Names with All References.” 18 The Pleiades data set is a community-built gazetteer of ancient places, covering the Greek and Roman worlds extensively,
and currently broadening its scope to Ancient Near Eastern, Byzantine, Celtic, and Early Medieval geography. The data set is
maintained by the Ancient World Mapping Center and the Institute for the Study of the Ancient World and is available through
services to individual human researches as well as for consummation by computational humanities research, cf. Bagnall et al.,
“Pleiades: A Gazetteer of Past Places.” 18 The Pleiades data set is a community-built gazetteer of ancient places, covering the Greek and Roman worlds extensively,
and currently broadening its scope to Ancient Near Eastern, Byzantine, Celtic, and Early Medieval geography. The data set is
maintained by the Ancient World Mapping Center and the Institute for the Study of the Ancient World and is available through
services to individual human researches as well as for consummation by computational humanities research, cf. Bagnall et al.,
“Pleiades: A Gazetteer of Past Places.” 19 ToposText is an online database that contains location references relevant to Greek history and mythology. 3 Results The confusion distance algorithm was used to estimate the probability of palaeographical confusion with
IOϒΔAIAN for each toponym in our list. From the results shown in Figures 2 and 3 and Table 2, it is
apparent that very few toponyms are palaeographically close to IOϒΔAIAN. Only 6% of the toponyms has a confusion distance smaller than 4 and even 0.9% a confusion distance
smaller than 3. Table 3 presents the toponyms that are palaeographically closest to IOϒΔAIAN, which
means they are the most likely to be confused. Taken on its own, the confusion distance might however give a distorted picture since a low confusion
distance does not automatically imply a small number of operations to change one word into another. For
example, IEPA ΠϒΛH has a small confusion distance with IOϒΔAIAN, but this metric conceals that the
transformation needed to change IEPA ΠϒΛH into IOϒΔAIAN requires seven operations (neglecting the space). To account for this bias, we here introduce a second metric, the probability index, ProbIdx, which accounts for
bias in the confusion distance, confdist, by multiplying it with the required number of operations, ops: 4 Discussion This section discusses the semantical, geographical, and historical likeliness of the palaeographically
fitting toponyms and evaluates the applicability of the method. Figure 2: Frequency diagram of palaeographical confusion distances with IOϒΔAIAN. The horizontal axis shows the number
of toponyms in a class. Figure 2: Frequency diagram of palaeographical confusion distances with IOϒΔAIAN. The horizontal axis shows the number
of toponyms in a class. Figure 3: Histogram representing the number of toponyms with a particular number of operations to change the toponym in
IOϒΔAIAN. Figure 3: Histogram representing the number of toponyms with a particular number of operations to change the toponym in
IOϒΔAIAN. ProbIdx = confdist × ops More than 5,000
references to Greek toponyms were collected from ancient texts covering a time span starting in the Neolithic period up through
the second century CE, cf. Kiesling and Aikaterini Laskaridis Foundation, “ToposText.” 19 ToposText is an online database that contains location references relevant to Greek history and mythology. More than 5,000
references to Greek toponyms were collected from ancient texts covering a time span starting in the Neolithic period up through
the second century CE, cf. Kiesling and Aikaterini Laskaridis Foundation, “ToposText.” The 56 entries in Table 3 can be divided into 32 places (settlements, towns, or cities) 16 regions, 8 miscellaneous features
(hydrography, building structures, or landmarks), and one false positive. In the remainder of this article, these entries have
been reduced to 48 unique toponyms by eliminating the false positive ΙΕΡΕΙΑΝ and redundant toponyms. Redundancy occurs
in the cases of ΕΟΡΔΑΙΑ, ΕΟΡΔΑΙΑN, and ΕΟΡΔΙΑN; ΙΟϒΔΑΙΑ, ΙΟϒΔΑΙΟΝ, ΙΟϒΔΑΙΟΣ, and ΙΟϒΔΑΝ; ΙΣΤΙΑΙΑ and
ΙΣΤΙΑΙΑΝ; ΡΟϒΔΙΑ and ΡΟϒΔΙΑΝ. The 56 entries in Table 3 can be divided into 32 places (settlements, towns, or cities) 16 regions, 8 miscellaneous features
(hydrography, building structures, or landmarks), and one false positive. In the remainder of this article, these entries have
been reduced to 48 unique toponyms by eliminating the false positive ΙΕΡΕΙΑΝ and redundant toponyms. Redundancy occurs
in the cases of ΕΟΡΔΑΙΑ, ΕΟΡΔΑΙΑN, and ΕΟΡΔΙΑN; ΙΟϒΔΑΙΑ, ΙΟϒΔΑΙΟΝ, ΙΟϒΔΑΙΟΣ, and ΙΟϒΔΑΝ; ΙΣΤΙΑΙΑ and
ΙΣΤΙΑΙΑΝ; ΡΟϒΔΙΑ and ΡΟϒΔΙΑΝ. Ἰουδαίαν in Acts 2:9: Reverse Engineering Textual Emendations
383 4.1 Candidature for emendation 384 Table 2: The 20 closest alternatives to IOϒΔAIAN based on palaeographical confusion of characters
Toponym
Confdist
Toponym
Confdist
ΙΟϒΔΑΙΑ
1
ΠΕΤΑΛΙΑ
2.063
ΙΕΡΑ ΠϒΛΗ
1.15
ΣΤAΔΙΑ
2.063
ΙΔΟϒΜΑΙΑΝ
2
ΣΤAΔΙΟΝ
2.063
ΙΟϒΔΑΙΟΣ
2
ΙΕΒΛΑΑΜ
2.07
ΙΣΤΙΑΙΑΝ
2.043
ΙΕΡΑΚΙΑ
2.07
ΕΟΡΔΑΙΑΝ
2.05
ΓΟΡΔΙΟΝ
2.083
ΙΟΡΔΑΝΗΝ
2.05
ΓΑΛΓΑΛ
2.119
ΡΟϒΔΙΑ
2.05
ΣΙΛΛϒΟΝ
2.13
ΓϒΛΛΙΟΝ
2.053
ΓΕΡΑΡΑ
2.143
ΙΤΑΛΙΑ
2.053
ΓΕΡΑΡΩΝ
2.143
Table 3: Palaeographical close toponyms to IOϒΔAIAN
Toponym
Confdist Ops Probidx Toponym
Confdist Ops Probidx Toponym
Confdist Ops Probidx
ΙΟϒΔΑΙΑ
1.000
1
1.000
ΟϒΑΡΙΑΝ
2.010
3
6.030
ΛΕϒΚΑΤΑΝ
2.043
4
8.172
ΙΟϒΔΑΙΟΝ
1.000
1
1.000
ΟϒΕΛΙΑΝ
2.010
3
6.030
ΙΣΤΡΙΑΝ
2.043
4
8.172
ΡΟϒΔΙΑΝ
1.050
2
2.100
ΟϒΛΙΑΝ
2.010
3
6.030
ΑΣΤΡΑΙΑΝ
2.043
4
8.172
ΕΟΡΔΑΙΑΝ
1.050
2
2.100
ΝΟΜΑΔΙΑΝ
2.020
3
6.060
ΧΕΤΤΑΙΑΝ
2.043
4
8.172
ΙΣΤΙΑΙΑΝ
1.043
3
3.129
ΟϒΑΓΑΝ
2.033
3
6.099
ΙΕΡΕΙΑΝ
2.060
4
8.240
ΙΔΟϒΜΑΙΑΝ
2.000
2
4.000
ΤΟϒΙΑΝ
2.033
3
6.099
ΠΙΣΙΔΙΑΝ
2.060
4
8.240
ΙΟϒΔΑΙΟΣ
2.000
2
4.000
ΙΟΡΔΑΝΗΝ
2.050
3
6.150
ΛΙΛΑΙΑΝ
2.060
4
8.240
ΙΟϒΔΑΝ
2.000
2
4.000
ΡΟϒΔΙΑ
2.050
3
6.150
ΙΣΑΡΑΝ
2.060
4
8.240
ΒΟϒΔΕΙΑΝ
2.000
2
4.000
ΕΟΡΔΑΙΑ
2.050
3
6.150
ΣΟϒΑΤΡΑΝ
2.060
4
8.240
ΛΟϒΔΙΑΝ
2.000
2
4.000
ΟϒΓΑϒΑΝ
2.050
3
6.150
ΠΡΟΠϒΛΑΙΑΝ
2.060
4
8.240
ΜΟΝΔΑΙΑΝ
2.000
2
4.000
ΕΟΡΔΙΑΝ
2.050
3
6.150
ΓΟΡΔΙΟΝ
2.083
4
8.332
ΙΤΑΛΙΑΝ
1.053
4
4.212
ΡΟΔΙΑΝ
2.050
3
6.150
ΓΑΔΑΡΑΝ
2.083
4
8.332
ΣΤΑΔΙΑΝ
1.063
4
4.252
ΙΕΡΑ ΠϒΛΗ
1.150
7
8.050
ΓΡΑΙΑΝ
2.083
4
8.332
ΙΕΡΑΚΙΑΝ
1.070
4
4.280
ΟΙΧΑΛΙΑΝ
2.020
4
8.080
ΤΡΙΤΑΙΑΝ
2.083
4
8.332
ΠΕΤΑΛΙΑΝ
1.063
5
5.315
ΕΛΑΙΑΝ
2.020
4
8.080
ΠΟΤΕΙΔΑΙΑΝ
2.093
4
8.372
ΓΕΡΑΡΑΝ
1.143
5
5.715
ΘϒΑΜΙΑΝ
2.020
4
8.080
ΙΜΙΧΑΡΑΝ
2.100
4
8.400
ΕϒΤΑΙΑΝ
2.010
3
6.030
ΕϒΠΑΛΙΑΝ
2.030
4
8.120
ΡΙΠΑΙΑΝ
2.100
4
8.400
ΙΟϒΛΙΝ
2.010
3
6.030
ΙΣΤΙΑΙΑ
2.043
4
8.172
ϒΔΡΑΜΙΑΝ
2.110
4
8.440
ΝΟϒΑΡΙΑΝ
2.010
3
6.030
ΕΣΤΙΑΙΑΝ
2.043
4
8.172 Table 2: The 20 closest alternatives to IOϒΔAIAN based on palaeographical confusion of characters
Toponym
Confdist
Toponym
Confdist
ΙΟϒΔΑΙΑ
1
ΠΕΤΑΛΙΑ
2.063
ΙΕΡΑ ΠϒΛΗ
1.15
ΣΤAΔΙΑ
2.063
ΙΔΟϒΜΑΙΑΝ
2
ΣΤAΔΙΟΝ
2.063
ΙΟϒΔΑΙΟΣ
2
ΙΕΒΛΑΑΜ
2.07
ΙΣΤΙΑΙΑΝ
2.043
ΙΕΡΑΚΙΑ
2.07
ΕΟΡΔΑΙΑΝ
2.05
ΓΟΡΔΙΟΝ
2.083
ΙΟΡΔΑΝΗΝ
2.05
ΓΑΛΓΑΛ
2.119
ΡΟϒΔΙΑ
2.05
ΣΙΛΛϒΟΝ
2.13
ΓϒΛΛΙΟΝ
2.053
ΓΕΡΑΡΑ
2.143
ΙΤΑΛΙΑ
2.053
ΓΕΡΑΡΩΝ
2.143 Table 2: The 20 closest alternatives to IOϒΔAIAN based on palaeographical confusion of characters osest alternatives to IOϒΔAIAN based on palaeographical confusion of characters Table 3: Palaeographical close toponyms to IOϒΔAIAN
Toponym
Confdist Ops Probidx Toponym
Confdist Ops Probidx Toponym
Confdist Ops Probidx
ΙΟϒΔΑΙΑ
1.000
1
1.000
ΟϒΑΡΙΑΝ
2.010
3
6.030
ΛΕϒΚΑΤΑΝ
2.043
4
8.172
ΙΟϒΔΑΙΟΝ
1.000
1
1.000
ΟϒΕΛΙΑΝ
2.010
3
6.030
ΙΣΤΡΙΑΝ
2.043
4
8.172
ΡΟϒΔΙΑΝ
1.050
2
2.100
ΟϒΛΙΑΝ
2.010
3
6.030
ΑΣΤΡΑΙΑΝ
2.043
4
8.172
ΕΟΡΔΑΙΑΝ
1.050
2
2.100
ΝΟΜΑΔΙΑΝ
2.020
3
6.060
ΧΕΤΤΑΙΑΝ
2.043
4
8.172
ΙΣΤΙΑΙΑΝ
1.043
3
3.129
ΟϒΑΓΑΝ
2.033
3
6.099
ΙΕΡΕΙΑΝ
2.060
4
8.240
ΙΔΟϒΜΑΙΑΝ
2.000
2
4.000
ΤΟϒΙΑΝ
2.033
3
6.099
ΠΙΣΙΔΙΑΝ
2.060
4
8.240
ΙΟϒΔΑΙΟΣ
2.000
2
4.000
ΙΟΡΔΑΝΗΝ
2.050
3
6.150
ΛΙΛΑΙΑΝ
2.060
4
8.240
ΙΟϒΔΑΝ
2.000
2
4.000
ΡΟϒΔΙΑ
2.050
3
6.150
ΙΣΑΡΑΝ
2.060
4
8.240
ΒΟϒΔΕΙΑΝ
2.000
2
4.000
ΕΟΡΔΑΙΑ
2.050
3
6.150
ΣΟϒΑΤΡΑΝ
2.060
4
8.240
ΛΟϒΔΙΑΝ
2.000
2
4.000
ΟϒΓΑϒΑΝ
2.050
3
6.150
ΠΡΟΠϒΛΑΙΑΝ
2.060
4
8.240
ΜΟΝΔΑΙΑΝ
2.000
2
4.000
ΕΟΡΔΙΑΝ
2.050
3
6.150
ΓΟΡΔΙΟΝ
2.083
4
8.332
ΙΤΑΛΙΑΝ
1.053
4
4.212
ΡΟΔΙΑΝ
2.050
3
6.150
ΓΑΔΑΡΑΝ
2.083
4
8.332
ΣΤΑΔΙΑΝ
1.063
4
4.252
ΙΕΡΑ ΠϒΛΗ
1.150
7
8.050
ΓΡΑΙΑΝ
2.083
4
8.332
ΙΕΡΑΚΙΑΝ
1.070
4
4.280
ΟΙΧΑΛΙΑΝ
2.020
4
8.080
ΤΡΙΤΑΙΑΝ
2.083
4
8.332
ΠΕΤΑΛΙΑΝ
1.063
5
5.315
ΕΛΑΙΑΝ
2.020
4
8.080
ΠΟΤΕΙΔΑΙΑΝ
2.093
4
8.372
ΓΕΡΑΡΑΝ
1.143
5
5.715
ΘϒΑΜΙΑΝ
2.020
4
8.080
ΙΜΙΧΑΡΑΝ
2.100
4
8.400
ΕϒΤΑΙΑΝ
2.010
3
6.030
ΕϒΠΑΛΙΑΝ
2.030
4
8.120
ΡΙΠΑΙΑΝ
2.100
4
8.400
ΙΟϒΛΙΝ
2.010
3
6.030
ΙΣΤΙΑΙΑ
2.043
4
8.172
ϒΔΡΑΜΙΑΝ
2.110
4
8.440
ΝΟϒΑΡΙΑΝ
2.010
3
6.030
ΕΣΤΙΑΙΑΝ
2.043
4
8.172 Table 3: Palaeographical close toponyms to IOϒΔAIAN toponyms listed in Table 3 might be a plausible substitute for IOϒΔAIAN in Acts 2:9, we first discuss
considerations about the variety of attestation, the validity of temporal attestation, and the probability of
Jewish communities in a region. 4.1 Candidature for emendation The results in the previous section show that only for a few instances it is reasonable to argue for
palaeographical confusion. To advance the establishment of criteria for the evaluation whether any of the 384
Vincent van Altena et al. 4.1.1 Exclusion according to semantic subtype The majority of palaeographically probable toponyms can be discarded as likely replacements for
IOϒΔAIAN when their semantical subtype is considered in relation to the structure of the catalogue. The
function of the catalogue is to clarify the identity of the audience in Acts 2:8. The catalogue consists of four
units ordered in a chiastic structure.²³ The first and last units consist of groups of demonyms, while the
second and third groups start with an active participle, respectively, οἱκατοικοῦντες (residents) and οἱ
ἐπιδημοῦντες (sojourners). IOϒΔAIAN appears in the second group as an object of κατοικοῦντες. This
constrains the possibility of toponyms by excluding the hydrography-related toponyms Λουδιας, Iορδάνης,
and Ἰσάρας, and names pertaining to building structures, i.e. Ἱερά πύλη, Προπύλαια, Eὐπαλία, Θυαμία, or
a landmark like Λευκάτας. Besides pairing κατοικοῦντες with regions (Acts 2:9, 13), Luke combines it with city names (Jerusalem
in Acts 2:14; 13:27, and Lydda in Acts 9:35). The author of Revelation uses the nomen in a construction to
refer to “those who dwell on the earth” (Rev 11:10; 13:8; 17:2, 8). However, although a city name would
technically fit the second unit of the catalogue, this would be a surprising deviation of its pattern: in
parallel to the other regional toponyms one would also expect a region name at the spot of IOϒΔAIAN. Heuristic support for this observation is found in the historical discussion of proposed emendations, which
all describe regions.²⁴ Therefore, the palaeographical-related settlements …Ἄστραια, Γραῖα, Ἐλαία, Oἰχαλία, and Ὑδραμία;
the towns Xετταία, Σουάτρα, Tριταία, Γεραρα, and Ἰμίχαρα; and the cities Bούδεια, Γάδαρα, Γόρδιον,
Ἑστίαια, Eὔταια, Ἰουλίς, Ἱστίαια, Ἰστρία, Λῐ́λαια, Mονδαία, Nουαρία, Oὐγαυά, Oὐάγα, Oὐαρία, Oὐελία,
Oὐλία, Ποτείδαια, Ῥουδία, Στάδια, and Tουία are not found to be fitting alternatives to emend IOϒΔAIAN
in Acts 2:9. This means that 38 of the palaeographically likely unique toponyms can be safely discarded as
potential emendations of IOϒΔAIAN on the basis of their semantic subtype. presence or absence of Jewish settlements in a specific region cannot be demonstrated beyond doubt from
the partial historical evidence, nor do rhetorical statements like Philo’s “not only are the main lands full of
Jewish colonies but also the most highly esteemed of the islands” (Leg. Ad Gaium 282) contribute to settle
the issue.²¹ The statement is too generic to establish evidence for the Jewish population in a specific
location. More is to be expected from exclusion according to semantic subtype, fitting in the geographic
progression, and contextual appropriateness within Acts 2:9. The results from Table 3 will be discussed
using these three criteria.²² 21 Philo, The embassy to Gaius.
22 Toponyms, which are referred to in the discussion that follows, are taken from Kiesling and Aikaterini Laskaridis
Foundation, “ToposText;” Bagnall et al., “Pleiades;” Montanari, The Brill Dictionary of Ancient Greek, and Cancik and
Schneider, New Pauly Online. A full bibliography of the referenced toponyms is available online, cf. van Altena, “List of
Toponyms Which Are Paleographically Close to Ioudaian.”
23 Brinkman “The Literary Background of the ‘Catalogue of the Nations’ ” 4.1 Candidature for emendation Widespread attestation in Classical, Jewish, and/or Christian literary sources does not help: evidence
for the familiarity of a toponym can be used to advocate the likeliness as well as the improbability of a
specific toponym simultaneously. The ambiguity is that while a wider attestation in literary sources might
increase the probability that the author of Acts might have used the toponym, simultaneously such an
attestation might reduce the chance that a scribe would have replaced a toponym for a better known
candidate. Likewise, the time span for which a toponym is attested does not contribute much to resolve the issue. Though it is tempting to advocate that a particular toponym might have been anachronistic to the author
of Acts since we only have attestation from later sources, this argumentum ex silentio can never be
conclusive since we cannot prove that the author was unfamiliar with the toponym. We simply do
not know. On a similar vein, the observation that the catalogue in Acts 2 designates Jews from several nations
and regions (Acts 2:5, 11) does not help to further exclude toponyms as possible candidates, since the 385 Ἰουδαίαν in Acts 2:9: Reverse Engineering Textual Emendations 24 See part 1 of this study: “Ἰουδαίαν in Acts 2:9: a diachronic overview of its conjectured emendations.” 21 Philo, The embassy to Gaius. 4.1.2 Fitting in the geographic progression The remaining 11 toponyms pertain to regions that all are palaeographically close to IOϒΔAIAN. For
these regions, the question needs to be addressed whether they do fit in the geographical progression of
Acts 2:9–11, somewhere between Mesopotamia and Cappadocia. To visualise the potential search area, we used ArcGIS Pro software to create a multiple ring buffer
from the centre point of the straight line between the centre points of Mesopotamia and Cappadocia 24 See part 1 of this study: “Ἰουδαίαν in Acts 2:9: a diachronic overview of its conjectured emendations.” 386
Vincent van Altena et al. Figure 4: Search area for a region between Macedonia and Cappadocia. 386
Vincent van Altena et al. Figure 4: Search area for a region between Macedonia and Cappadocia. (Figure 4). These artificial zones can be used as a visual aid in the evaluation of the geographically fitting
of the individual regions. Unfortunately, none of the palaeographically related regions form geographically fitting alternatives
for IOϒΔAIAN. Ῥιπαῖα refers to a mythical range of mountains that were thought to form the northern
edge of the world. The region was thought to be uninhabited, and already in classical times authors
doubted its existence. The other alternatives (Figure 5) break the geographical progression and have additional issues:
Ἰστρία could be either a region or people on the north-eastern Adriatic coast as well as a city at the Black
Sea. The region Πισιδία in Asia Minor is attested both in the book of Acts and in classical works. Ἰταλία
roughly corresponds to modern Italy and is very well attested in Classical, Jewish, and Christian texts. It is
also hard to conceive in which way this toponym could fit the geographical progression and how the
overlap with Rome (Acts 2:10) should be explained. Additionally, the African region Nομαδία, the Macedonian landscape Ἐορδαία or Ἐορδία, the Aegean
islands Ἱεράκια and Ῥοδία, and the island group Πεταλία do not fit the geographical arrangement better
than IOϒΔAIAN. 25 Barthius, Adversariorum commentariorum libri LX quibus ex universa antiquitatis serie, omnis generis, ad vicies octies centum,
auctorum, plus centum quinquaginta millibus, loci, tam gentilium quam christianorum, theologorum, iureconsultorum, medicorum,
philosophorum, philologorum, oratorum, rhetorum etc. obscuri, dubii, maculati, illustrantur, constituuntur, emendantur, cum
rituum, morum, legum, sanctionum, sacrorum, ceremoniarum, pacis bellique artium, formularum, locutionum denique,
observatione et eludicatione tam locuplete et varia, ut simile ab uno homine nihil umquam in litteras missum videri possit. Eduntur
praeterea ex vetustatis monumentis praeclara hoc opere non pauca, nec visa hactenus, nec videri sperata. Cum undecim indicibus,
VII auctorum, IV rerum et verborum; Bentley, Bentleii Critica Sacra: Notes on the Greek and Latin Text of the New Testament,
Extracted from the Bentley Mss; Bloomfield, ἩKαινη Διαθηκη. The Greek Testament, with English Notes, Critical, Philological, and
Exegetical. Second Edition, Corrected, Greatly Enlarged, and Considerably Improved; Penn, Annotations to the Book of the New
Covenant: With an Expository Preface. 4.1.3 Contextual appropriateness of Ἰδουμαία The remaining toponym ΙΔΟϒΜΑΙΑ has already been proposed as an alternative to IOϒΔAIAN by
several authors.²⁵ The designation was first applied to the country of Edom and later also to the southern
part of Judah. This region had an important strategic position for controlling trade routes. 25 Barthius, Adversariorum commentariorum libri LX quibus ex universa antiquitatis serie, omnis generis, ad vicies octies centum,
auctorum, plus centum quinquaginta millibus, loci, tam gentilium quam christianorum, theologorum, iureconsultorum, medicorum,
philosophorum, philologorum, oratorum, rhetorum etc. obscuri, dubii, maculati, illustrantur, constituuntur, emendantur, cum
rituum, morum, legum, sanctionum, sacrorum, ceremoniarum, pacis bellique artium, formularum, locutionum denique, Ἰουδαίαν in Acts 2:9: Reverse Engineering Textual Emendations
387 387 Figure 5: Map of regional toponyms that are paleographically close to Ioudaian. Figure 5: Map of regional toponyms that are paleographically close to Ioudaian. In favour of ΙΔΟϒMΑΙΑΝ is that it is palaeographically close to IOϒΔAIAN.²⁶ Only two operations are
required to transform it to IOϒΔAIAN and a variant reading on Mark 3:7 transposes the two words.²⁷
Furthermore, the toponym was valid and common during the time of writing, and the region had Jews
among its population from various ethnic backgrounds (Mark 3:8; JW 2.43). If ΙΔΟϒMΑΙΑΝ is assumed as the original reading, additional support is still needed to show how the
current reading IOϒΔAIAN could have come into existence. One might speculate that a copyist wanted to
remove a reference to ΙΔΟϒMΑΙΑ especially because of the turbulent relationship between ΙΔΟϒMΑΙΑ
and IOϒΔAIA: during Nebuchadnezzar’s besiege of Jerusalem the Edomites scorned and mocked the
endangered Jews. After Jerusalem was defeated in 587 BCE, its Jewish population was deported into
Babylonian captivity and their homes and possessions were confiscated by Edomites (Am. 10–14, cf. Ps. 137:7). Decades later when the Jews returned from being exiled, they found the homes of their families
occupied by Edomites. A few centuries later, after the Arabs had invaded the territory, the Jewish high
priest John Hyrcanus forced the Idumeans to follow the Jewish law and thus had them circumcised (Jos. Ant 13.9.1). Later on, the tyrant king Herod the Great was a born Idumean.²⁸ A mutual hostility between
these two peoples is not difficult to perceive. observatione et eludicatione tam locuplete et varia, ut simile ab uno homine nihil umquam in litteras missum videri possit. Eduntur
praeterea ex vetustatis monumentis praeclara hoc opere non pauca, nec visa hactenus, nec videri sperata. 27 According to the critical apparatus of UBS5, minuscule 579 reads Ἰουδαίας in stead of Ἰδουμαίας in Mark 3:8, but what is
actually happening is the transposition of the two toponyms, cf. Aland et al., The Greek New Testament, 126.
28 Herion, Herod Philip. 26 Bloomfield, Greek Testament 1. 29 So Michelsen, in Submission to Prijsvraag G 94: een verhandeling over de toepassing van de conjecturaal-kritiek op den tekst
van de schriften des Nieuwen Testaments, II-13–II-14. 4.1.4 Summary of findings on alternatives Though it is tempting to draw firm conclusions about the likely candidates for IOϒΔAIAN in Acts 2:9,
there is no significant evidence to warrant any of them. What can be concluded is that an unintentional scribal error based on palaeographical confusion of
IOϒΔAIAN with one of the toponyms we retrieved from the New Testament, Greek Old Testament, and
Classical literature seems very unlikely. We simply did not find a fitting candidate that was
palaeographically close enough to IOϒΔAIAN and might have been altered unintentionally in only a
few operations and that–on top of this–is also meeting the historical, geographical, and contextual
requirements to fit the catalogue in Acts 2:9–11. For the moment, this leaves the problem unsolved. However, the possibility remains that a new
artefact might be discovered that contains a different spelling of an existing or even a completely unknown
toponym that fits the geographical progression, corresponds to the expected semantic subtype (i.e. being a
region), and also suits the wider historical and literary context. Such a discovery would provide an ideal
toponym to emend IOϒΔAIAN in Acts 2:9. Until such a discovery is done, it might be better to refrain from speculation about unintentional
palaeographical confusion and to accept that the catalogue of nations in Acts 2 is a peculiar list in multiple
facets. The mention of IOϒΔAIAN is just one of these peculiarities. 388
Vincent van Altena et al. 388
Vincent van Altena et al. This is however pure conjecture and it does not explain why such a well-known topographic name like
ΙΔΟϒMΑΙΑ could have been confused with ΙΟϒΔΑΙΑ, which introduces even more difficulties. Furthermore, ΙΔΟϒMΑΙΑ does not provide a better solution to the odd geographic progression²⁹ (which
however could also be argued in favour of its originality). 4.1.3 Contextual appropriateness of Ἰδουμαία Cum undecim indicibus,
VII auctorum, IV rerum et verborum; Bentley, Bentleii Critica Sacra: Notes on the Greek and Latin Text of the New Testament,
Extracted from the Bentley Mss; Bloomfield, ἩKαινη Διαθηκη. The Greek Testament, with English Notes, Critical, Philological, and
Exegetical. Second Edition, Corrected, Greatly Enlarged, and Considerably Improved; Penn, Annotations to the Book of the New
Covenant: With an Expository Preface. 26 Bl
fild G
k T
t
t 1 388 ἐπειράσθησαν. This requires an inflection of the verbs to the identical grammatical–morphological form
and a semantic discussion of the found alternatives. A third case where our method can add value is in the interpretation of 2 Pet. 3:10. Here the verb
εὑρεθήσεται (“shall be found”) is unintelligible and several other verbs with a diversity of meanings have
been conjectured.³⁰ Our algorithm might contribute to identify verbs that are palaeographically close to
εὑρεθήσεται and would make sense in the sentence. Each of these cases could greatly benefit from a grammatical–morphological data set as well as from a
semantic dictionary that should be both available in digital format. The scope of the method could also be extended to different types of confusion, for instance,
phonetical. This would require a second confusion table expressing the ease of mispronunciation,
mishearing (or mis-vocalisation) of a text. Furthermore, scenarios assuming Hebrew, Aramaic, or Syriac
sources to Greek texts can be scrutinised using our algorithm and a tailored confusion table. The same
exercise could for instance be performed, supposing a Hebrew origin for the list of nations.
30 For a discussion of the issues with the variant readings and the conjectured emendations cf. Metzger, A Textual
Commentary on the Greek New Testament, a Companion Volume to the United Bible Societies, 636–37; Krans and Peerbolte, The
Amsterdam Database of New Testament Conjectural Emendation.
31 See for instance, Keener, Acts: An Exegetical Commentary – Introduction and 1:1–2:47, 1: 844–51. 4.2 Critical appraisal of method Our assessment of the method elaborated in this research is however much more positive. Given the right
conditions (see below), this method provides new insights that otherwise are difficult to obtain or would
be cumbersome since they require an enormous amount of manual work. Using a computer algorithm to
gauge whether palaeographical confusion of words might be a solution to an interpretive problem seems
promising for several cases. A first case can be found in the gospel narratives (see Matt 3:4 and its parallel text Mark 1:6), where
the character of John the Baptist is introduced. His portrayal as someone who is wearing a camel-skin
garment and eating locusts has been interpreted as a reference to Old Testament prophets. However, some
have doubted that a man could have actually been eating locusts and therefore conjectured what John
could have been eating instead. At this point, the method presented in this research might be helpful to
identify a Greek noun belonging to the semantic field of food to test whether alternatives could be detected
that might be explained by palaeographical confusion. Similar to this study, these results should be
analysed along qualitative conditions to decide between possible and plausible alternatives. A second case where the method might be helpful is the quest for a fitting verb in Hebr. 11:37. While
most of the manuscripts here have ἐπειράσθησαν (“they were tempted”), this is strange within the
enumeration of rather specific violent deaths. To solve this puzzle 17 divergent emendations have been
proposed. It might be helpful to test which verbs are likely to be confused palaeographically with 389 Ἰουδαίαν in Acts 2:9: Reverse Engineering Textual Emendations 31 See for instance, Keener, Acts: An Exegetical Commentary – Introduction and 1:1–2:47, 1: 844–51. 390
Vincent van Altena et al. 390
Vincent van Altena et al. 390
Vincent van Altena et al. Future research should concentrate on the retrieval and subdivision of semantic dictionaries as well as
ways to implement perceived scribal habits as operations in the algorithm to refine its outcomes. Additionally, the outcomes would greatly benefit from a way to “ground-truth” the scores in the confusion
distance table; i.e. these scores are now based upon expert knowledge, but it would be beneficial to
improve the scores utilising statistical data assembled from manuscript evidence. 5 Conclusion In this study, we dealt with the alleged corruption of IOϒΔAIAN in Acts 2:9. As we summarised in part 1
of this article series, a plethora of suggestions have been proposed to solve the problem in the text and
these suggestions were argued from differing perspectives: a background in contemporary textual sources,
the intrinsic logic of the narrative, the fittingness in the geographical arrangement, and the probability of
palaeographical confusion. To offer a contribution to this discussion, this study set out to investigate the fittingness of alternative
toponyms in the text assuming palaeographical confusion. It therefore used a confusion table, which
enabled the researcher to quantify the likeliness by which a scribe would confuse particular letter
combinations, and an algorithm designed to simulate operations during confusion. The results of this
experiment showed there are only a few candidates that could be regarded as real alternatives. Due to the
ambiguity of additional qualitative historical, geographical, linguistic, and contextual arguments, we did
not produce conclusive arguments in favour of a particular conjecture. Since the possibilities for
palaeographical confusion appear to be very limited, the solution to the textual problem concerning
IOϒΔAIAN in Acts 2: 9 needs to be sought in a different direction. On the one hand, our research did not
exclude the possibility of a very early intentional scribal alteration of an unknown toponym into the
transmitted reading. But due to the lack of any manuscript evidence, an attempt to resolve the issue in this
direction easily becomes mere speculation. On the other hand, our research did neither provide conclusive
reasoning that demonstrated the originality of the transmitted reading IOϒΔAIAN. Several theories have
been proposed by respected scholars to advocate the authenticity of IOϒΔAIAN,³¹ but neither of these
has yet been convincing. Therefore, either direction needs further investigation to see whether it could
solve the issue. The second aim of this study was to scrutinise whether reverse engineering palaeographical confusion
has potential as a method. The experiment confirmed the suitability of the algorithm to test the probability
of palaeographical confusion and proposed three cases for further experimentation. To be successful in
establishing probable conjectures for these cases, there is a need for a dictionary data set arranged by
semantic fields. 390 References Aland, Barbara, Aland, Kurt, Karavidopoulos, Johannes, Martini, Carlo Maria, Metzger, Bruce Manning, & American Bible
Society, eds. The Greek New Testament. Fifth revised edition, 4th corrected printing. Stuttgart: Deutsche
Bibelgesellschaft, 2018. Bagnall, Roger, Talbert, Richard J. A., Bond, Sarah, Becker, Jeffrey, Elliott, Tom, Gillies, Sean, Horne, Ryan,
McCormick, Michael, Rabinowitz, Adam, et al. “Pleiades: A Gazetteer of Past Places.” Collection, 2016. http://pleiades
stoa.org Barthius, Caspar. Adversariorum commentariorum libri LX quibus ex universa antiquitatis serie, omnis generis, ad vicies octies
centum, auctorum, plus centum quinquaginta millibus, loci, tam gentilium quam christianorum, theologorum,
iureconsultorum, medicorum, philosophorum, philologorum, oratorum, rhetorum etc. obscuri, dubii, maculati,
illustrantur, constituuntur, emendantur, cum rituum, morum, legum, sanctionum, sacrorum, ceremoniarum, pacis bellique
artium, formularum, locutionum denique, observatione et eludicatione tam locuplete et varia, ut simile ab uno homine
nihil umquam in litteras missum videri possit. Eduntur praeterea ex vetustatis monumentis praeclara hoc opere non
pauca, nec visa hactenus, nec videri sperata. Cum undecim indicibus, VII auctorum, IV rerum et verborum. Frankfurt, Main:
Aubrius, n.d. Bentley, Richard. Bentleii Critica Sacra: Notes on the Greek and Latin Text of the New Testament, Extracted from the Bentley Mss. in Trinity College Library, edited by Arthur Ayres Ellis. Cambridge: Deighton, Bell, 1862. Bloomfield, Samuel Thomas. ἩKαινη Διαθηκη. The Greek Testament, with English Notes, Critical, Philological, and Exegetical. Second Edition, Corrected, Greatly Enlarged, and Considerably Improved. vol. 1. London: Longman, Rees & Co., 1836. B i k
J A “Th
Lit
B
k
d
f th
‘C t l
f th
N ti
’ (A t 2 9 11) ” Th C th li Bibli
l Q
t
l 25 4 Bloomfield, Samuel Thomas. ἩKαινη Διαθηκη. The Greek Testament, with English Notes, Critical, Philological, and Exegetical. Second Edition, Corrected, Greatly Enlarged, and Considerably Improved. vol. 1. London: Longman, Rees & Co., 1836. Brinkman, J. A. “The Literary Background of the ‘Catalogue of the Nations’ (Acts 2,9–11).” The Catholic Biblical Quarterly 25:4
(1963), 418–27. Second Edition, Corrected, Greatly Enlarged, and Considerably Improved. vol. 1. London: Longman, Rees & Co., 1836. Brinkman, J. A. “The Literary Background of the ‘Catalogue of the Nations’ (Acts 2,9–11).” The Catholic Biblical Quarterly 25:4
(1963), 418–27. Cancik, Hubert, Schneider, Helmuth. New Pauly Online. Leiden, The Netherlands: Brill, n.d. https://brill.com/view/
package/bnpo Milan, Pilehvar, Mohammad Taher, Limsopatham, Nut, Collier, Nigel. “What’s Missing in Geographical Parsing?”
nguage Resources and Evaluation 2017 doi: 10 1007/s10579-017-9385-8 ta, Milan, Pilehvar, Mohammad Taher, Limsopatham, Nut, Collier, Nigel. Swete, Henry Barclay. The Old Testament in Greek: According to the Septuagint. Cambridge: Cambridge University P References “What’s Missing in Geographical Parsin
Language Resources and Evaluation, 2017. doi: 10.1007/s10579-017-9385-8 g
g
,
/
Herion, Gary A. E. “Herod Philip.” In The Anchor Yale Bible Dictionary 3, edited by Freedman, David Noel, Gary A. Herion, David
F. Graf, John David Pleins, and Astrid B. Beck, 161–69. New York: Doubleday, 1992. on, Gary A. E. “Herod Philip.” In The Anchor Yale Bible Dictionary 3, edited by Freedman, David Noel, Gary A. Herio F. Graf, John David Pleins, and Astrid B. Beck, 161–69. New York: Doubleday, 1992. Instone-Brewer, David. “Tyndale Individualised Proper Names with All References.” Digital. Tyndale House Cambridge, 11
February 2019. https://github.com/tyndale/STEPBible-Data Keener, Craig S. Acts: An Exegetical Commentary–Introduction and 1:1–2:47. vol. 1. Grand Rapids: Baker Academic, 2012. Kiesling John Brady Aikaterini Laskaridis Foundation “ToposText ” Gazetteer ToposText Web Version 3 0 2019 Keener, Craig S. Acts: An Exegetical Commentary–Introduction and 1:1–2:47. vol. 1. Grand Rapids: Baker Academic, 2012. Kiesling, John Brady, Aikaterini Laskaridis Foundation. “ToposText.” Gazetteer. ToposText Web Version 3.0, 2019. htt
//t
t
t
/ Keener, Craig S. Acts: An Exegetical Commentary–Introduction and 1:1–2:47. vol. 1. Grand Rapids: Baker Academ Kiesling, John Brady, Aikaterini Laskaridis Foundation. “ToposText.” Gazetteer. ToposText Web Version 3.0, 2019
https://topostext.org/ Krans, Jan, Lietaert Peerbolte, Bert Jan. “The Amsterdam Database of New Testament Conjectural Emendation,” 2016–present. http://ntvmr.uni-muenster.de/nt-conjectures Louw, Johannes P., Nida, Eugene Albert. Greek-English Lexicon of the New Testament: Based on Semantic Domains. Electronic
ed. of the 2nd edition. New York: United Bible Societies, 1996. Metzger, Bruce M. A Textual Commentary on the Greek New Testament, a Companion Volume to the United Bible Societies’
Greek New Testament (4th Rev. Ed.). 2nd ed. London, New York: United Bible Societies, 1994. Michelsen, Jan Hendrik Adolf. In Submission to “Prijsvraag G 94: een verhandeling over de toepassing van de conjecturaal-
kritiek op den tekst van de schriften des Nieuwen Testaments (1877), II–14. ATS 1258. Haarlem: Archief Teylers
Stichting, 1879. g
Montanari, Franco. The Brill Dictionary of Ancient Greek. Boston, MA: Brill, 2015. Montanari, Franco. The Brill Dictionary of Ancient Greek. Boston, MA: Brill, 2015. Penn, Granville. Annotations to the Book of the New Covenant: With an Expository Preface. London: Duncan, Philo. The embassy to Gaius. Translated by Francis Henry Colson, G. H. Whitaker, and J. W. Earp. Vol. 10 of The Lo
Library. London, England; Cambridge, MA: William Heinemann Ltd, Harvard University Press, 1929. References S
i h S
h
“ ibl
G
di
ibl
i
G
l
h
d G
l
”
ibl
G
di
d Philo. The embassy to Gaius. Translated by Francis Henry Colson, G. H. Whitaker, and J. W. Earp. Vol. 10 of The Loeb Classical
Library. London, England; Cambridge, MA: William Heinemann Ltd, Harvard University Press, 1929. Smith, Stephen. “Bible Geocoding–Bible Maps in Google Earth and Google Maps.” Bible Geocoding n.d. http://www.openbible.info/geo/
S
H
B
l
Th Old T
i
G
k A
di
h S
i
C
b id
C
b id
U i
i
P Library. London, England; Cambridge, MA: William Heinemann Ltd, Harvard University Press, 1929. Smith, Stephen. “Bible Geocoding–Bible Maps in Google Earth and Google Maps.” Bible Geocoding n.d. http://www.openbible.info/geo/ Swete, Henry Barclay. The Old Testament in Greek: According to the Septuagint. Cambridge: Cambridge Uni 391 Ἰουδαίαν in Acts 2:9: Reverse Engineering Textual Emendations Tan, Randall K., deSilva, David A., Hoogendyk, Isaiah. The Lexham Greek-English Interlinear Septuagint: H.B. Swete Edition. Bellingham, WA: Lexham Press, 2012. van Altena, Vincent. “Greek Old Testament Toponyms.” Zenodo, 7 October 2019. doi: 10.5281/zenodo.3244386. van Altena, Vincent. “New Testament Toponyms.” Zenodo, 7 October 2019. doi: 10.5281/zenodo.3463567. van Altena, Vincent. “Greek Toponyms Collected from Classical Literature.” Zenodo, 19 February 2020. doi: 10.5281/
zenodo.3675315. van Altena, Vincent. Greek Noun Inflection Script. Python. Zenodo, 2020. doi: 10.5281/zenodo.3604255. van Altena, Vincent. “List of Toponyms Which Are Paleographically Close to Ioudaian.” Zenodo, 19 February 2020. do
zenodo.3675151. van Altena, Vincent, Krans, Jan, Bakker, Henk, Dukai, Balász, Stoter, Jantien. “Spatial Analysis of New Testament Textual
Emendations Utilizing Confusion Distances.” Open Theology 5:1 (2019), 44–65. van der Meer, Michaël N. “The Natural and Geographical Context of the Septuagint: Some Preliminary Observations.” In Die
Septuaginta. Entstehung, Sprache, Geschichte. 3. Internationale Fachtagung Veranstaltet von Septuaginta Deutsch
(LXX.D), Wuppertal 22–25 Juli 2010, edited by W. Kraus, M. Karrer, M. Sigismund, 387–421. Vol. 286 of WUNT I. Tübingen:
Mohr-Siebeck, 2012. Yuan, May. “Mapping Text.” In The Spatial Humanities: GIS and the Future of Humanities Scholarship, edited by David J. Bodenhamer, John Corrigan, Trevor M. Harris, 109–23. Spatial Humanities. Bloomington: Indiana University Press, 2010.
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English
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ABCB1 and ABCG2 Together Limit the Distribution of ABCB1/ABCG2 Substrates to the Human Retina and the ABCG2 Single Nucleotide Polymorphism Q141K (c.421C> A) May Lead to Increased Drug Exposure
|
Frontiers in pharmacology
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BRIEF RESEARCH REPORT
published: 16 June 2021
doi: 10.3389/fphar.2021.698966 Specialty section:
This article was submitted to
Pharmacogenetics and
Pharmacogenomics,
a section of the journal
Frontiers in Pharmacology Specialty section:
This article was submitted to
Pharmacogenetics and
Pharmacogenomics,
a section of the journal
Frontiers in Pharmacology
Received: 22 April 2021
Accepted: 04 June 2021
Published: 16 June 2021 Received: 22 April 2021
Accepted: 04 June 2021
Published: 16 June 2021 Keywords: ABCG2, ABCB1, blood-retinal barrier, c421C>A, single-nucleotide polymorphism, PET, human, tariquidar 1Department of Clinical Pharmacology, Medical University of Vienna, Vienna, VIE, Austria, 2Centre for Medical Statistics,
Informatics, and Intelligent Systems, Medical University of Vienna, Vienna, VIE, Austria, 3Division of Nuclear Medicine, Department
of Biomedical Imaging and Image-guided Therapy, Medical University of Vienna, Vienna, VIE, Austria, 4Department of Laboratory
Medicine, Medical University of Vienna, Vienna, VIE, Austria, 5Laboratoire d’Imagerie Biomédicale Multimodale (BioMaps), CEA,
CNRS, Inserm, Service Hospitalier Frédéric Joliot, Université Paris-Saclay, Orsay, France Edited by:
Youssef Daali,
Geneva University Hospitals (HUG),
Switzerland Edited by:
Youssef Daali,
Geneva University Hospitals (HUG),
Switzerland Edited by:
Youssef Daali,
Geneva University Hospitals (HUG),
Switzerland Reviewed by:
Vera Ribeiro,
University of Algarve, Portugal
Amit Kumar Mitra,
Auburn University, United States Reviewed by:
Vera Ribeiro,
University of Algarve, Portugal
Amit Kumar Mitra,
Auburn University, United States Reviewed by:
Vera Ribeiro,
University of Algarve, Portugal
Amit Kumar Mitra,
Auburn University, United States
*Correspondence:
Martin Bauer
martin.m.bauer@meduniwien.ac.at The widely expressed and poly-specific ABC transporters breast cancer resistance protein
(ABCG2) and P-glycoprotein (ABCB1) are co-localized at the blood-brain barrier (BBB)
and have shown to limit the brain distribution of several clinically used ABCB1/ABCG2
substrate drugs. It is currently not known to which extent these transporters, which are
also expressed at the blood-retinal barrier (BRB), may limit drug distribution to the human
eye and whether the ABCG2 reduced-function single-nucleotide polymorphism (SNP)
Q141K (c.421C > A) has an impact on retinal drug distribution. Ten healthy male volunteers
(five subjects with the c.421CC and c.421CA genotype, respectively) underwent two
consecutive positron emission tomography (PET) scans after intravenous injection of the
model ABCB1/ABCG2 substrate [11C]tariquidar. The second PET scan was performed
with concurrent intravenous infusion of unlabelled tariquidar to inhibit ABCB1 in order to
specifically reveal ABCG2 function.In response to ABCB1 inhibition with unlabelled
tariquidar, ABCG2 c.421C > A genotype carriers showed significant increases (as
compared to the baseline scan) in retinal radiotracer influx K1 (+62 ± 57%, p 0.043)
and volume of distribution VT (+86 ± 131%, p 0.043), but no significant changes were
observed in subjects with the c.421C > C genotype. Our results provide the first evidence
that ABCB1 and ABCG2 may together limit the distribution of systemically administered
ABCB1/ABCG2 substrate drugs to the human retina. Functional redundancy between
ABCB1 and ABCG2 appears to be compromised in carriers of the c.421C > A SNP who
may therefore be more susceptible to transporter-mediated drug-drug interactions at the
BRB than non-carriers. INTRODUCTION 2014). The BRB is divided in two layers: (a) the inner BRB
consisting of endothelial cells of the retinal capillaries (ECRC)
and (b) the outer BRB composed of the retinal pigmental
epithelial (RPE) cells, located between the neural retina and
choriocapillaris. The transport of specific molecules across lipid membranes is an
essential function of all living organisms (Liu, 2019). In humans,
the widespread expression and poly-specificity of the adenosine
triphosphate-binding cassette (ABC) family efflux transporter
breast
cancer
resistance
protein
(ABCG2)
makes
it
an
important determinant of the pharmacokinetics of a variety of
drugs (Mao and Unadkat, 2015). Many ABCG2 substrates are
additionally
substrates
of
another
ABC-transporter,
P-glycoprotein
(ABCB1),
so
that
the
net
effect
on
the
disposition of drugs which are dual ABCB1/ABCG2 substrates
may be attributed to the combined action of both transporters. These two transporters have been recognized by the International
Transporter Consortium to be involved in clinically relevant
transporter-mediated
drug-drug
interactions
(DDIs)
given
their
impact
on
the
disposition
of
their
substrates
(International Transporter et al., 2010). The co-localization of
ABCG2 and ABCB1 at several blood-tissue barriers suggests a
crucial role in protecting key vulnerable and/or target tissues, e.g.,
the brain or the placenta, from xenobiotics and harmful
metabolites. It is, however, difficult to predict the functional
impact of ABCB1- and ABCG2-mediated efflux on tissue
exposure from conventional plasma pharmacokinetic data
(Wijaya et al., 2017). There is evidence from preclinical studies that both, ABCB1
and ABCG2, are expressed at the BRB. ABCG2 was identified in
mouse and rat retina and in the conditionally immortalized rat
ECRC cell line TR-iBRB (Asashima et al., 2006) as well as
concomitantly with ABCB1 at the luminal side of ECRC in
rabbit and mouse eyes (Chapy et al., 2016; Pascual-Pasto et al.,
2017). The data concerning the expression of ABCG2 and ABCB1
at the outer BRB are contradictory (Liu and Liu, 2019). A
dominant protein expression of ABCG2 in pig eyes at the
inner BRB over the outer BRB (22.8 fmol/μg protein and
2.76 fmol/μg protein respectively) was demonstrated (Zhang
et al., 2017). The same study revealed that ABCG2 expression
at the inner porcine BRB is 2.6-fold higher than that of ABCB1
and that the transporter expression pattern is positively
correlated between the BBB and the inner BRB in pigs (Zhang
et al., 2017). Citation: El Biali M, Karch R, Philippe C,
Haslacher H, Tournier N, Hacker M,
Zeitlinger M, Schmidl D, Langer O and
Bauer M (2021) ABCB1 and ABCG2
Together Limit the Distribution of
ABCB1/ABCG2 Substrates to the
Human Retina and the ABCG2 Single
Nucleotide Polymorphism Q141K
(c.421C> A) May Lead to Increased
Drug Exposure. Front. Pharmacol. 12:698966. doi: 10.3389/fphar.2021.698966 June 2021 | Volume 12 | Article 698966 1 Frontiers in Pharmacology | www.frontiersin.org ABCB1/ABCG2 at the Blood-Retinal Barrier El Biali et al. Frontiers in Pharmacology | www.frontiersin.org INTRODUCTION These findings are consistent with absolute
quantification data at the human BBB, which showed a higher
expression of ABCG2 than of ABCB1 (Uchida et al., 2011;
Billington et al., 2019; Li and Zhu, 2020). For the human BRB, ABCG2 and ABCB1 protein expression
has been corroborated with a predominance of ABCG2 through
RNA expression profiling and immunohistochemistry (Dahlin
et al., 2013). All in all, it is currently not known to which extent
these two efflux transporters limit the distribution of systemically
administered drugs to the human eye. (
j y
,
)
ABCB1 and ABCG2 are co-expressed
at the luminal
membrane of brain capillary endothelial cells contributing to
the protective function of the blood-brain barrier (BBB) (Uchida
et al., 2011; Kalvass et al., 2013; Billington et al., 2019; Li and Zhu,
2020). Studies in Abcb1a/b and Abcg2 knockout mice have
provided evidence for functional redundancy between ABCB1
and ABCG2 in limiting the distribution of dual ABCB1/ABCG2
substrate drugs to the brain. In absence of either ABCB1 alone or
ABCG2 alone (Abcb1a/b(−/−) mice or Abcg2(−/−) mice) the
remaining transport capacity of the other transporter was
largely sufficient to restrict brain distribution of ABCB1/
ABCG2 substrates, for which brain distribution was only
substantially
increased
in
absence
of
both
transporters
(Abcb1a/b(−/−)Abcg2(−/−) mice) (Kodaira et al., 2010; Wijaya
et al., 2017; Robey et al., 2018). A comparable functional
redundancy between ABCB1 and ABCG2 has been confirmed
in vivo at the human BBB (Bauer et al., 2016). Transporter-
mediated DDIs at the BBB may potentially result in cerebral
uptake and toxicity of medications that normally are not targeted
to the brain without significant changes in drug plasma
concentrations (Sasongko et al., 2005; Eyal et al., 2009; Bauer
et al., 2017), although the risk for their occurrence in clinical
practice is considered relatively low (Kalvass et al., 2013). y
ABCG2 g
y
The
nonsynonymous
ABCG2
single-nucleotide
polymorphism (SNP) Q141K (c.421C > A), which affects the
stability of the ABCG2 protein in the endoplasmic reticulum and
enhances its susceptibility to proteosomal degradation (Furukawa
et al., 2009), has been shown to lead to reduced transporter
expression in different tissues (Kobayashi et al., 2005; Prasad
et al., 2013; Tanaka et al., 2015). It has been reported that c.421AA
carriers have an in vivo intestinal ABCG2 function approximately
23% of that in c.421CC subjects (Tanaka et al., 2015). The efficacy
and the toxicity of diverse ABCG2 substrates, e.g., statin drugs,
chemotherapy or allopurinol, has been found to be affected by the
c.421C > A variant (Chen et al., 2019). Our previous data
indicated that carriers of the c.421C > A SNP had diminished
activity of ABCG2 at BBB, leading to increased susceptibility to
ABCB1 inhibition (Bauer et al., 2016). It remains to be explored
whether the c.421C > A SNP has an impact on ABCG2 function
at the human BRB. Positron emission tomography (PET) with radiolabelled
transporter substrates allows to directly and non-invasively
assess the influence of transporters at the BBB on drug
distribution to the human brain (Bickel, 2005; Tournier et al.,
2018). Next to measuring ABCB1 function at the BBB (Bauer
et al., 2012; Bauer et al., 2015; Bauer et al., 2017), PET with the
radiolabelled ABCB1 substrate (R)-[11C]verapamil has also been
used to measure ABCB1 function at the human BRB (Bauer et al.,
2017). PET with the dual ABCB1/ABCG2 substrate [11C]
tariquidar (Bankstahl et al., 2013) with concurrent infusion of The eye, just like the brain, is a vulnerable organ as it requires
the strict maintenance of a stable inner environment to insure
neuro-retinal homeostasis and its sensory function (Fujii et al.,
2014). The protection of the posterior segment of the eye,
especially the retina, from systemically circulating phototoxic
endogenous and exogenous substances and the regulation of the
influx transport of vital molecules are essentially provided by the
blood-retinal barrier (BRB) (Asashima et al., 2006; Agrahari et al.,
2016), in a similar way as the BBB does for the brain (Fujii et al., Frontiers in Pharmacology | www.frontiersin.org June 2021 | Volume 12 | Article 698966 2 ABCB1/ABCG2 at the Blood-Retinal Barrier El Biali et al. y
ABCG2 outcome parameters were the radiotracer transfer rate constants
across the BRB and BBB between plasma and the first tissue
compartment (influx rate constant K1 and efflux rate constant k2)
as well as between the first and second tissue compartments
(influx rate constant k3 and efflux rate constant k4) (see
Supplementary Figure S1). The fractional arterial blood
volume in tissue (Vb) was included as a fitting parameter. Logan graphical analysis was used to estimate the total volume
of distribution (VT) in a model-independent manner (Logan
et al., 1990). VT equals the tissue-to-plasma concentration
ratio at steady state. All data are given as arithmetic mean ±
standard
deviation
(SD). Differences
in
the
outcome
parameters between scan 1 and 2 were tested using the
Wilcoxon signed rank test and between groups using the
Mann-Whitney
test
(Statistica
6.1,
StatSoft,
Tulsa,
OK,
United States). To assess correlations, the Spearman rank
correlation coefficient rs was calculated. A p value of less than
0.05 was considered statistically significant. a high dose of unlabelled tariquidar to inhibit ABCB1 was
successfully employed to reveal and measure for the first time
the transport activity of ABCG2 at the human BBB (Bauer et al.,
2016). In the present study, we aimed to extend the analysis of
data from our previously published study in healthy volunteers
(Bauer et al., 2016) to assess the impact of ABCB1 and ABCG2
and the ABCG2 c.421C > A genotype on the distribution of [11C]
tariquidar to the human retina. METHODS The study was registered with EUDRACT (number 2012-005796-
14), approved by the Ethics Committee of the Medical University
of Vienna, and conducted in accordance with the Declaration of
Helsinki. The reported data are from an extended analysis of the
previously published study of Bauer et al. (Bauer et al., 2016). Eleven out of 52 screened subjects were identified as carriers of
the ABCG2 c.421C > A SNP by means of probe-based polymerase
chain reaction as previously described (Bauer et al., 2016). The
sample management and the SNPgenotyping was performed at
the MedUni Wien Biobank according to standard operating
procedures (Haslacher et al., 2018). In total, five male subjects
who were non-carriers (c.421CC) and five male subjects who
were heterozygous carriers (c.421CA) of the ABCG2 c.421C > A
SNP and who were judged to be medication free and healthy
based on the screening examinations, were enrolled into the [11C]
tariquidar study arm. A summary table of the human subjects
included is available in the Supplementary Table S1. The
volunteers
(mean
age:
30
±
9 years)
underwent
two
consecutive
60 min
PET
scans
on
an
Advance
scanner
(General
Electric
Medical
Systems,
Milwaukee,
WI,
United States) after intravenous injection of [11C]tariquidar
(injected radioactivity amount: 388 ± 18 MBq). Serial arterial
blood samples were drawn as previously described during the
imaging sessions (Bauer et al., 2013). The second PET scan was
performed with concurrent intravenous infusion of unlabelled
tariquidar (AzaTrius Pharmaceuticals, Mumbai, India) to inhibit
ABCB1 only and reveal ABCG2 function as previously described
(Bauer et al., 2016). Tariquidar infusion was started 1 h before
and continued until the end of the image acquisition (total
infusion length: 120 ± 4 min). The total administered dose of
unlabelled tariquidar was 5.8 ± 1.0 mg/kg body weight (mean
subject weight: 80 ± 12 kg). We additionally performed an extended data analysis of a (R)-
[11C]verapamil PET data set previously published by our group
(Wagner et al., 2009) in which 5 healthy volunteers underwent
two consecutive (R)-[11C]verapamil PET scans before and after
administration of tariquidar at a dose which only partially
inhibits ABCB1 function at the BBB (2 mg/kg) in order to
investigate the effect of this tariquidar dose on ABCB1
function at the BRB. The methods used are described in the
Supplementary Material. Frontiers in Pharmacology | www.frontiersin.org RESULTS Modelling June 2021 | Volume 12 | Article 698966 Frontiers in Pharmacology | www.frontiersin.org 3 El Biali et al. ABCB1/ABCG2 at the Blood-Retinal Barrier FIGURE 1 | Axial (A), sagittal (B), and coronal (C) planes of representative MR and PET average images (0–60 min) at baseline (scan 1) and during ABCB1 inhibition
(scan 2) in one c.421CA carrier. Red rectangles on MR images indicate magnified area on PET images. A representative region of interest for retina (white contour) is
shown. Anatomical structures are labelled with arrows: R, retina; On, optical nerve; C, cornea. Radiation scale is expressed as standardized uptake value (SUV) and set
from 0 to 2.5. FIGURE 1 | Axial (A), sagittal (B), and coronal (C) planes of representative MR and PET average images (0–60 min) at baseline (scan 1) and during ABCB1 inhibition
(scan 2) in one c.421CA carrier. Red rectangles on MR images indicate magnified area on PET images. A representative region of interest for retina (white contour) is
shown. Anatomical structures are labelled with arrows: R, retina; On, optical nerve; C, cornea. Radiation scale is expressed as standardized uptake value (SUV) and set
from 0 to 2 5 BRB on controlling the distribution of a model ABCB1/ABCG2
substrate ([11C]tariquidar) to the human retina. Tariquidar is a
non-marketed, third-generation ABCB1 inhibitor, which was
originally developed to overcome multidrug resistance in
cancer patients (Fox and Bates, 2007) and which has been re-
purposed to inhibit ABCB1 at the BBB in an experimental setting
(Bauer et al., 2012; Bauer et al., 2015; Bauer et al., 2017). Interestingly, non-clinical data indicated that tariquidar highly
accumulates in the eye, which has been attributed to binding to
melanin (INVESTIGATOR BROCHURE Tariquidar, 2007),
which is abundantly expressed in RPE cells (Rimpelä et al.,
2018). At tracer doses [11C]tariquidar is transported by both
ABCB1 and ABCG2 and has been employed as a PET tracer to
measure the activity of ABCB1 and ABCG2 at the BBB (Bankstahl
et al., 2013; Bauer et al., 2016). In the present study, we extend for
the first time previous ex vivo investigations on the impact of
ABCB1 on the distribution of diverse model ABCB1 substrates to
the mouse and rat eye (Hosoya et al., 2010; Toda et al., 2011; Fujii
et al., 2014; Chapy et al., 2016) to a dual ABCB1/ABCG2 substrate
examined in vivo in humans. RESULTS We further investigated the effect of
the ABCG2 c.421C > A genotype on the retinal distribution of
[11C]tariquidar. We performed two consecutive [11C]tariquidar
PET scans in five subjects who were carriers of the fully
functioning ABCG2 allele (c.421CC) and in five subjects with c.421CA subjects, there was a trend towards a positive correlation
between the percentage change in VT Logan in the retina and in the
brain in scan 2 relative to scan 1 (rs 0.8, p 0.133, not shown). In contrast to the BRB, k3 values were not significantly increased
in the brain of c.421CA subjects following ABCB1 inhibition
(Table 1). The mean modelling outcome parameters for the retina and
the brain for the (R)-[11C]verapamil PET scans without and with
ABCB1 inhibition with a lower dose of unlabelled tariquidar
(2 mg/kg) are reported in the Supplementary Table S2. In
contrast to [11C]tariquidar, baseline K1 and VT values were for
(R)-[11C]verapamil comparable for the retina and for the brain. Following ABCB1 inhibition, K1 and VT values of (R)-[11C]
verapamil were significantly increased as compared with the
baseline scan, both for the retina and the brain (K1, retina:
+97 ± 100%, p 0.043; K1, WBGM: +49 ± 36%, p 0.043;
VT Logan, retina: +43 ± 30%, p 0.043; VT Logan, WBGM: +24 ±
15%, p 0.043). RESULTS During the experiments only mild or moderate adverse events
were recorded and are listed in reference (Bauer et al., 2016). During the experiments only mild or moderate adverse events
were recorded and are listed in reference (Bauer et al., 2016). Figure 1 shows representative [11C]tariquidar PET images for
scans without and with ABCB1 inhibition of one c.421CA carrier
with the outlined retina ROI. Mean modelling outcome
parameters for the retina and the brain for the two PET scans
in c.421CC and c.421CA carriers are given in Table 1. In
Figure 2, selected modelling outcome parameters for the
baseline scan and the scan with ABCB1 inhibition for the
retina and the brain in individual c.421CC and c.421CA
subjects are displayed. For the baseline scans, K1 and VT values of [11C]tariquidar
were 4 to 5-fold higher for the retina than for WBGM. Baseline
distribution of [11C]tariquidar to the retina as well as to WBGM
did not significantly differ between c.421CC and c.421CA
subjects. In response to ABCB1 inhibition with unlabelled
tariquidar, ABCG2 c.421C > A genotype carriers showed
significant increases as compared to the baseline scan in
retinal radiotracer influx (K1: +62 ± 57%, p 0.043), and VT
Logan (+86 ± 131%, p 0.043) (Figure 2). No significant changes
in any of the modelling outcome parameters were observed in
subjects with the c.421CC genotype (Figure 2). The BRB findings
were in good agreement with those for the BBB, for which K1
(+72 ± 35%, p 0.043) and VT Logan (+91 ± 82%, p 0.043) were
also significantly and with a similar magnitude increased
following ABCB1 inhibition in c.421CA subjects only. In Region of interest (ROI) analysis was conducted for the retina
on magnetic resonance (MR)-to-PET co-registered images based
on individual T1-weighted MR images with PMOD software
(version 3.6; PMOD Technologies Ltd., Zürich, Switzerland). Probabilistic atlas-based, whole brain grey matter (WBGM)
data were already reported in (Bauer et al., 2016). A standard
2-tissue-4-rate constant compartmental (2T4K) model (see
Supplementary Figure S1) was fitted to the time–activity
curves (TACs) of [11C]tariquidar in the retina and in WBGM
from 0 to 60 min after radiotracer injection (Bauer et al., 2013)
using an arterial plasma input function which was not corrected
for radiolabelled metabolites of [11C]tariquidar (due to the low
percentage of radiolabelled metabolites in plasma). DISCUSSION K1 (mL/(cm3.min)), rate constant for radiotracer transfer from plasma into the first tissue compartment; k2 (1/min), rate constant for radiotracer transfer from the first tissue
compartment into plasma; k3 (1/min), rate constant for radiotracer transfer from the first tissue compartment into the second tissue compartment; k4 (1/min), rate constant for radiotracer
transfer from the second tissue compartment into the first tissue compartment; VT Logan (ml/cm3), total volume of distribution estimated with Logan graphical analysis; Vb, fractional arterial
blood volume in the eyes/brain. *p < 0.05 for comparison with baseline scan using the Wilcoxon signed rank test. Values are reported as arithmetic mean ± standard deviation. The value in parentheses represents the precision of the parameter estimates (expressed as their mean standard error in
percent). K1 (mL/(cm3.min)), rate constant for radiotracer transfer from plasma into the first tissue compartment; k2 (1/min), rate constant for radiotracer transfer from the first tissue
compartment into plasma; k3 (1/min), rate constant for radiotracer transfer from the first tissue compartment into the second tissue compartment; k4 (1/min), rate constant for radiotracer
transfer from the second tissue compartment into the first tissue compartment; VT Logan (ml/cm3), total volume of distribution estimated with Logan graphical analysis; Vb, fractional arterial
blood volume in the eyes/brain. *p < 0.05 for comparison with baseline scan using the Wilcoxon signed rank test. verapamil, which may be possibly related to the presence of an
unidentified uptake transporter which mediates verapamil uptake
across the BRB (Hosoya et al., 2010; Kubo et al., 2013; Chapy
et al., 2016). the c.421CA genotype which presumably results in reduced
ABCG2 abundance and function. Following the baseline PET
scan, a second scan was performed with a concurrent high dose
infusion of unlabelled tariquidar to achieve significant ABCB1
inhibition and thereby specifically reveal ABCG2 function at the
human BRB (Bauer et al., 2015; Bauer et al., 2016). Data obtained
with [11C]tariquidar for the BRB were compared with data
previously obtained with [11C]tariquidar for the BBB (Bauer
et al., 2015; Bauer et al., 2016) and with data obtained with
the ABCB1-selective substrate radiotracer (R)-[11C]verapamil. A second important finding of our study was that the BRB
efflux transport function in c.421CC carriers remained unaltered
during ABCB1 inhibition which confirms for the first time the
presence of functional ABCG2 at the human BRB. DISCUSSION In this study, we used PET imaging to assess the functional
impact of the two efflux transporters ABCB1 and ABCG2 at the June 2021 | Volume 12 | Article 698966 Frontiers in Pharmacology | www.frontiersin.org 4 ABCB1/ABCG2 at the Blood-Retinal Barrier El Biali et al. TABLE 1 | [11C]Tariquidar modelling outcome parameters for the retina and whole brain grey matter in c.421CC and c.421CA subjects for the baseline scan and the scan
during ABCB1 inhibition with unlabelled tariquidar. Region of
interest
Group
K1 (mL/(cm3.min))
k2 (1/min)
k3 (1/min)
k4 (1/min)
VT Logan (ml/cm3)
Vb
Retina
c.421CC
baseline
0.046 ± 0.018 (28)
0.590 ± 0.382 (138)
0.593 ± 0.554 (134)
0.037 ± 0.050 (158)
1.730 ± 0.995 (53)
0.009 ± 0.002 (67)
c.421CC
ABCB1
inhibition
0.057 ± 0.022 (32)
0.503 ± 0.347 (180)
0.294 ± 0.099 (501)
0.151 ± 0.294 (119)
1.742 ± 0.490 (7)
0.010 ± 0.006 (31)
c.421CA
baseline
0.035 ± 0.007 (29)
0.239 ± 0.154 (95)
0.150 ± 0.091 (95)
0.041 ± 0.029 (75)
1.130 ± 0.939 (10)
0.010 ± 0.003 (27)
c.421CA
ABCB1
inhibition
0.056 ± 0.015 (45)*
0.503 ± 0.252 (118)
0.390 ± 0.236 (63)*
0.113 ± 0.163 (54)
1.556 ± 0.890 (19)*
0.011 ± 0.007 (81)
Whole brain
grey matter
c.421CC
baseline
0.009 ± 0.004 (26)
0.340 ± 0.209 (57)
0.152 ± 0.037 (34)
0.012 ± 0.006 (23)
0.430 ± 0.102 (8)
0.047 ± 0.005 (8)
c.421CC
ABCB1
inhibition
0.008 ± 0.002 (16)
0.176 ± 0.103 (46)
0.119 ± 0.050 (32)
0.014 ± 0.005 (25)
0.408 ± 0.090 (7)
0.046 ± 0.006 (8)
c.421CA
baseline
0.008 ± 0.002 (32)
0.193 ± 0.074 (70)
0.116 ± 0.055 (38)
0.010 ± 0.001 (41)
0.417 ± 0.112 (13)
0.055 ± 0.008 (9)
c.421CA
ABCB1
inhibition
0.013 ± 0.004 (13)*
0.195 ± 0.097 (34)
0.140 ± 0.040 (22)
0.015 ± 0.006 (18)
0.738 ± 0.196 (8)*
0.047 ± 0.008 (10)*
Values are reported as arithmetic mean ± standard deviation. The value in parentheses represents the precision of the parameter estimates (expressed as their mean standard error in Values are reported as arithmetic mean ± standard deviation. The value in parentheses represents the precision of the parameter estimates (expressed as their mean standard error in
percent). Values are reported as arithmetic mean ± standard deviation. The value in parentheses represents the precision of the parameter estimates (expressed as their mean standard error in
percent). K1 (mL/(cm3.min)), rate constant for radiotracer transfer from plasma into the first tissue compartment; k2 (1/min), rate constant for radiotracer transfer from the first tissue
compartment into plasma; k3 (1/min), rate constant for radiotracer transfer from the first tissue compartment into the second tissue compartment; k4 (1/min), rate constant for radiotracer
transfer from the second tissue compartment into the first tissue compartment; VT Logan (ml/cm3), total volume of distribution estimated with Logan graphical analysis; Vb, fractional arterial
blood volume in the eyes/brain. *p < 0.05 for comparison with baseline scan using the Wilcoxon signed rank test. DISCUSSION This suggests
that a similar functional redundancy between ABCB1 and
ABCG2 as described for the rodent and human BBB (Kodaira
et al., 2010; Bauer et al., 2016) exists at the human BRB in
controlling the retinal distribution of dual ABCB1/ABCG2
substrates. One first important finding of our study was that [11C]
tariquidar distribution across the BRB to the retina (K1, VT)
was considerably higher (4 to 5 fold) than its distribution across
the BBB to the brain, regardless of the genotype and ABCB1
inhibition condition (see Table 1). This is largely consistent with
the results from studies in rodents, which revealed a higher
distribution of prototypical ABCB1 substrates (e.g., verapamil,
quinidine and digoxin) to the retina than to the brain (Hosoya
et al., 2010; Toda et al., 2011; Fujii et al., 2014; Chapy et al., 2016). This has been interpreted in a way that ABCB1-mediated efflux is
quantitatively less important at the rodent BRB than at the rodent
BBB, while passive transcellular permeability appears to be
similar at the BRB and BBB for lipophilic compounds (Hosoya
et al., 2010). However, our results obtained with [11C]tariquidar
differed from those obtained with (R)-[11C]verapamil in humans,
for which K1 and VT values were comparable for the retina and
the brain (see Supplementary Table S2 and reference (Bauer
et al., 2017). This may point to species differences between
rodents and humans with regards to the retinal distribution of Our results showed that carriers of the c.421C > A SNP had
significant increases in retinal distribution of [11C]tariquidar
following ABCB1 inhibition, while c.421CC subjects did not. This supports that the investigated ABCG2 SNP decreased the
function of ABCG2 at the BRB, just as previously reported for the
BBB (Bauer et al., 2016). This may suggest that SNP carriers may
be more susceptible to transporter-mediated DDIs at the BRB
than non-carriers because the functional redundancy between
ABCB1 and ABCG2 is compromised. The percentage increase in retinal distribution of [11C]
tariquidar in c.421CA subjects following complete ABCB1
inhibition was of similar magnitude as the percentage increase
in its brain distribution (Table 1). This again contrasted with
results obtained with (R)-[11C]verapamil for which the increase
in brain uptake was considerably higher than the increase in
retinal uptake (3.8- vs. 1.5-fold increase in VT) following complete June 2021 | Volume 12 | Article 698966 Frontiers in Pharmacology | www.frontiersin.org 5 ABCB1/ABCG2 at the Blood-Retinal Barrier El Biali et al. DISCUSSION While previous data
suggested that administration of a pharmacological dose of
tariquidar does not affect cerebral blood flow in humans
(Kreisl et al., 2010), its effect on RCBF currently remains
unknown. The choroidal circulation provides blood supply
for the outer retina and particularly the photo-receptors
while the central retinal artery irrigates the inner retinal
layers (Vaghefi& Pontre, 2016). This dual source of retinal
blood supply complicates the estimation of the impact of
RCBF variation on [11C]tariquidar and (R)-[11C]verapamil
distribution
to
the
retina
in
particular
in
case
of
inhomogeneous expression of ABCG2/ABCB1 between the
inner and outer BRB. Hence it is not possible to exclude that
tariquidar-induced increases in RCBF could have at least
partly contributed to the observed increases in retinal
[11C]tariquidar and (R)-[11C]verapamil uptake. y
p
On one hand, transporter-mediated DDIs at the BRB due to
concomitant treatment with drugs which inhibit ABCB1 and/or
ABCB2 could contribute to enhanced ocular and in particular
retinal toxicity of drugs that normally penetrate poorly into the
eye, such as imatinib (Ho et al., 2013), other tyrosine kinase
inhibitors (Williamson and Reddy, 2021), ciprofloxacin (Ramirez
et al., 2011), tamoxifen (Griffin and Garnick, 1981; Noureddin
et al., 1999; Grzybowski et al., 2015) and methotrexate
(Balachandran et al., 2002; Iqbal et al., 2005; Sbeity et al.,
2006; Sharma and Sharma, 2011; Grzybowski et al., 2015) (see
Supplementary Table S3). On the other hand, DDIs could
generate
therapeutic
benefits
by
enhancing
the
ocular
penetration
of
systemic
treatments
for
retinal
disorders
struggling to cross the BRB, which represents so far a major
challenge for ocular drug delivery (Jordán and Ruíz-Moreno,
2013; Agrahari et al., 2016; Pascual-Pasto et al., 2017; Kim and
Woo, 2021). This may be the useful for an improved treatment of
diseases, such as neovascular age-related macular degeneration,
diabetic retinopathy, and retinal vascular disorders, which are the
leading causes of vision deterioration in most developed countries
(Bickel, 2005). There is therefore a need for methodology, such as
PET imaging, to measure the ocular disposition of drugs in
humans. Another limitation of the study is that we could only include
heterozygous (c.421CA) but no homozygous carriers (c.421AA)
of the ABCG2 c.421C > A SNP owing to the rarity of this
polymorphism in the Caucasian population. DISCUSSION The heterozygous (c.421CA) and
homozygous (c.421AA) variants occur with a frequency of
40–45%
and
8–12%,
respectively,
in
the
East
Asian
population (Chinese, Japanese, Korean), whereas c.421CA
and c.421AA carriers are rare in Caucasians (frequency of 17
and 1%, respectively) and even rarer in Africans (combined
frequency of 1.3%) (Lai et al., 2012; Li and Barton, 2018; Chen
et al., 2019). The enhanced risk for transporter-mediated DDIs
under certain pathological conditions and in SNP carriers may
play a clinical role for subjects undergoing treatment with
potentially
retinotoxic
drugs
such
as
tamoxifen
or
methotrexate (Grzybowski et al., 2015). Selected ABCG2
substrates from references (Mao and Unadkat, 2015; Fohner
et al., 2017) with potential retinotoxicity or ocular therapeutic
applications are listed in Supplementary Table S3. y
The percentage increase in (R)-[11C]verapamil K1 and VT
values at the BRB following administration of a tariquidar
dose (2 mg/kg) which only partially blocks ABCB1 at the
human BBB (Wagner et al., 2009; Bauer et al., 2015) (see
Supplementary Table S2) was comparable to the percentage
increase following administration of a tariquidar dose which
almost completely blocks ABCB1 at the human BBB (see
reference (Bauer et al., 2017). This indicated that that the low
dose of tariquidar (2 mg/kg) may have already led to complete
inhibition of ABCB1 at the BRB. This may suggest that ABCB1 at
the human BBB is less susceptible to inhibition than ABCB1 at the
human BRB. Consequently, lower doses of ABCB1 inhibitors may
lead to significant changes in retinal distribution of ABCB1
substrates than those needed for inhibition of ABCB1 at the
BBB. This could suggest that there is an overall higher risk for
ABCB1-mediated DDIs to occur at the BRB than at the BBB. These findings are in line with those of a study in mice which
revealed the need of only half of the dose of an ABCB1 inhibitor
(elacridar) to achieve full ABCB1 inhibition at the BRB than for
the BBB (Chapy et al., 2016). The authors linked this finding to
lower activity of ABCB1 at the BRB as compared to the BBB. One limitation of our study was that we did not measure
effective
retinochoroidal
blood
flow
(RCBF)
in
study
participants. Retinal uptake of [11C]tariquidar and (R)-
[11C]verapamil may partly depend on RCBF and may thus
need correction for RCBF to specifically reveal the function of
ABCB1 and ABCG2 at the BRB. DISCUSSION El Biali et al. GURE 2 | [11C]tariquidar modelling outcome parameters (K1 and k2 estimated from 2T4K model and VT estimated with Logan graphical analysis) for retina (A, C,
nd E) and whole brain grey matter (B, D, and F) in c.421CC (CC) and c.421CA (CA) subjects for baseline scan (scan 1) and scan during ABCB1 inhibition (scan 2). Brain
ata are taken from Bauer et al. (Bauer et al., 2016). *p < 0.05, Wilcoxon signed rank test. FIGURE 2 | [11C]tariquidar modelling outcome parameters (K1 and k2 estimated from 2T4K model and VT estimated with Logan graphical analysis) for retina (A, C,
and E) and whole brain grey matter (B, D, and F) in c.421CC (CC) and c.421CA (CA) subjects for baseline scan (scan 1) and scan during ABCB1 inhibition (scan 2). Brain
data are taken from Bauer et al. (Bauer et al., 2016). *p < 0.05, Wilcoxon signed rank test. ABCB1 inhibition by employing the same tariquidar infusion
protocol as in the present study (Bauer et al., 2017). This could
mean that the functional impact of ABCG2 is comparable at the
human BRB and BBB, while the functional impact of ABCB1 is lower at the human BRB than at the human BBB. However, the
presence of an uptake transport system for verapamil at the BRB,
which effect was revealed only when ABC efflux transport was
abolished, complicates the comparison of the response to ABCB1 June 2021 | Volume 12 | Article 698966 Frontiers in Pharmacology | www.frontiersin.org 6 ABCB1/ABCG2 at the Blood-Retinal Barrier El Biali et al. inhibition
between
(R)-[11C]verapamil
and
[11C]tariquidar
(Chapy et al., 2016). inhibition
between
(R)-[11C]verapamil
and
[11C]tariquidar
(Chapy et al., 2016). Consequently,
a
clinically
achievable
degree
of
ABCB1
inhibition in the brain and retina could be sufficient to lead
to significantly higher tissue exposure in ABCG2 c.421C > A
genotype carriers and thereby increase the risk of side effects for
ABCB1/ABCG2 substrate drugs with a narrow therapeutic
index. The expression of the c.421C > A SNP, which is one
of the most common reduced-function variants of ABCG2, is
highly dependent on ethnicity. Frontiers in Pharmacology | www.frontiersin.org REFERENCES Distribution of (R)-[11C]verapamil in elderlyvs.Young Subjects. Br. J. Clin. Pharmacol. 83 (9), 1991–1999. doi:10.1111/bcp.13301 Bauer, M., Zeitlinger, M., Karch, R., Matzneller, P., Stanek, J., Jäger, W., et al. (2012). Pgp-mediated Interaction between (R)-[11C]verapamil and Tariquidar
at the Human Blood-Brain Barrier: a Comparison with Rat Data. Clin. Pharmacol. Ther. 91 (2), 227–233. doi:10.1038/clpt.2011.217 Agrahari, V., Mandal, A., Agrahari, V., Trinh, H. M., Joseph, M., Ray, A., et al. (2016). A Comprehensive Insight on Ocular Pharmacokinetics. Drug Deliv. Transl. Res. 6 (6), 735–754. doi:10.1007/s13346-016-0339-2 Bickel, U. (2005). How to Measure Drug Transport across the Blood-Brain Barrier. Neurotherapeutics 2 (1), 15–26. doi:10.1602/neurorx.2.1.15 Asashima, T., Hori, S., Ohtsuki, S., Tachikawa, M., Watanabe, M., Mukai, C., et al. (2006). ATP-binding Cassette Transporter G2 Mediates the Efflux of
Phototoxins on the Luminal Membrane of Retinal Capillary Endothelial
Cells. Pharm. Res. 23 (6), 1235–1242. doi:10.1007/s11095-006-0067-2 Billington, S., Salphati, L., Hop, C. E. C. A., Chu, X., Evers, R., Burdette, D., et al. (2019). Interindividual and
Regional Variability in Drug
Transporter
Abundance at the Human Blood-Brain Barrier Measured by Quantitative
Targeted Proteomics. Clin. Pharmacol. Ther. 106 (1), 228–237. doi:10.1002/
cpt.1373 Balachandran, C., McCluskey, P. J., Champion, G. D., and Halmagyi, G. M. (2002). Methotrexate-induced Optic Neuropathy. Clin. Exp. Ophthalmol. 30 (6),
440–441. doi:10.1046/j.1442-9071.2002.00578.x Bankstahl, J. P., Bankstahl, M., Römermann, K., Wanek, T., Stanek, J., Windhorst,
A. D., et al. (2013). Tariquidar and Elacridar Are Dose-Dependently
Transported by P-Glycoprotein and Bcrp at the Blood-Brain Barrier: a
Small-Animal Positron Emission Tomography and In Vitro Study. Drug
Metab. Dispos 41 (4), 754–762. doi:10.1124/dmd.112.049148 Chapy, H., Saubaméa, B., Tournier, N., Bourasset, F., Behar-Cohen, F., Declèves, X., et al. (2016). Blood-brain and Retinal Barriers Show Dissimilar ABC Transporter Impacts
and Concealed Effect of P-Glycoprotein on a Novel Verapamil Influx Carrier. Br. J. Pharmacol. 173 (3), 497–510. doi:10.1111/bph.13376 Chen, L., Manautou, J. E., Rasmussen, T. P., and Zhong, X.-b. (2019). Development
of Precision Medicine Approaches Based on Inter-individual Variability of
BCRP/ABCG2. Acta Pharmaceutica Sinica B 9 (4), 659–674. doi:10.1016/
j.apsb.2019.01.007 Bauer, M., Bamminger, K., Pichler, V., Weber, M., Binder, S., Maier-Salamon, A.,
et al. (2021). Impaired Clearance from the Brain Increases the Brain Exposure
to Metoclopramide in Elderly Subjects. Clin. Pharmacol. Ther. 109 (3), 754–761. doi:10.1002/cpt.2052 Dahlin, A., Geier, E., Stocker, S. L., Cropp, C. D., Grigorenko, E., Bloomer, M., et al. (2013). Gene Expression Profiling of Transporters in the Solute Carrier and
ATP-Binding Cassette Superfamilies in Human Eye Substructures. Mol. ETHICS STATEMENT The authors thank study nurse Maria Weber and Johann Stanek
(Department of Clinical Pharmacology) for excellent support in
this study. The staff members of the PET center at the Division of
Nuclear Medicine are acknowledged for a smooth cooperation in
this study. The authors are grateful to the team of the local
Biobank facility for genotyping. The studies involving human participants were reviewed and
approved by the Ethics committee of the Medical University of
Vienna. The
patients/participants
provided
their
written
informed consent to participate in this study. DISCUSSION Just as for the BBB (Kalvass et al., 2013; Bauer et al., 2016),
the likelihood of clinically relevant DDIs at the human BRB for
dual ABCB1/ABCG2 substrate drugs is likely to be low if ABCB1
and ABCG2 function is preserved, since both transporters
possess mutual functional redundancy. Nevertheless, some
physiological or pathological conditions have been associated
with a reduction in the abundance of ABCB1 and ABCG2 at the
BBB as for instance healthy ageing or Alzheimer’s disease
(Kannan et al., 2017; Storelli et al., 2020), which may raise
the risk for ABCB1-mediated DDIs at the BBB and central side
effects in the elderly (Bauer et al., 2017; Bauer et al., 2021). In conclusion, our study highlights the potential utility of PET
imaging to non-invasively assess ocular disposition of drugs in
humans. We provide the first evidence that, in analogy to the
BBB, ABCB1 and ABCG2 may together limit at the BRB the
distribution
of
systemically
administered
ABCB1/ABCG2
substrate drugs to the human retina. Carriers of the c.421C >
A SNP may be more susceptible to transporter-mediated DDIs at
the BRB than non-carriers. This may play a role for subjects
undergoing treatment with potentially retinotoxic drugs such as
tamoxifen or methotrexate. June 2021 | Volume 12 | Article 698966 Frontiers in Pharmacology | www.frontiersin.org 7 ABCB1/ABCG2 at the Blood-Retinal Barrier El Biali et al. FUNDING This work was supported by the Austrian Science Fund (FWF)
(grants I 4470 and KLI 480-B30). The original contributions presented in the study are included in
the article/Supplementary Material, further inquiries can be
directed to the corresponding author. SUPPLEMENTARY MATERIAL MB, OL, NT, DS, and MEB wrote the article; MEB, OL, RK, MH,
HH, and MZ designed the research; MB, RK, CP, HH, and OL
performed the research; MB, RK, MZ, DS, NT, MEB, and OL
analyzed the data. The Supplementary Material for this article can be found online at:
https://www.frontiersin.org/articles/10.3389/fphar.2021.698966/
full#supplementary-material REFERENCES H., Baydoun, L., Schmidt, S., and Loeffler, K. U. (2006). Visual Field
Changes in Methotrexate Therapy. Case Report and Review of the Literature. J. Med. Liban 54 (3), 164–167. Kobayashi, D., Ieiri, I., Hirota, T., Takane, H., Maegawa, S., Kigawa, J., et al. (2005). Functional Assessment Ofabcg2(Bcrp) Gene Polymorphisms to Protein
Expression in Human Placenta. Drug Metab. Dispos 33 (1), 94–101. doi:10.1124/dmd.104.001628 Sharma, R., and Sharma, P. (2011). Toxic Optic Neuropathy. Indian J. Ophthalmol. 59 (2), 137–141. doi:10.4103/0301-4738.77035 Storelli, F., Billington, S., Kumar, A. R., and Unadkat, J. D. (2020). Abundance of
P-Glycoprotein and Other Drug Transporters at the Human Blood-Brain
Barrier in Alzheimer’s Disease: A Quantitative Targeted Proteomic Study. Clin. Pharmacol. Ther. Kodaira, H., Kusuhara, H., Ushiki, J., Fuse, E., and Sugiyama, Y. (2010). Kinetic
Analysis of the Cooperation of P-Glycoprotein (P-gp/Abcb1) and Breast
Cancer Resistance Protein (Bcrp/Abcg2) in Limiting the Brain and Testis
Penetration of Erlotinib, Flavopiridol, and Mitoxantrone. J. Pharmacol. Exp. Ther. 333 (3), 788–796. doi:10.1124/jpet.109.162321 Tanaka, Y., Kitamura, Y., Maeda, K., and Sugiyama, Y. (2015). Quantitative Analysis of the
ABCG2 c.421C > A Polymorphism Effect on In Vivo Transport Activity of Breast
Cancer Resistance Protein (BCRP) Using an Intestinal Absorption Model. J. Pharm. Sci. 104 (9), 3039–3048. doi:10.1002/jps.24366 Kreisl, W. C., Liow, J.-S., Kimura, N., Seneca, N., Zoghbi, S. S., Morse, C. L., et al. (2010). P-glycoprotein Function at the Blood-Brain Barrier in Humans Can Be
Quantified with the Substrate Radiotracer 11C-N-Desmethyl-Loperamide. J. Nucl. Med. 51 (4), 559–566. doi:10.2967/jnumed.109.070151 Toda, R., Kawazu, K., Oyabu, M., Miyazaki, T., and Kiuchi, Y. (2011). Comparison
of Drug Permeabilities across the Blood-Retinal Barrier, Blood-Aqueous
Humor Barrier, and Blood-Brain Barrier. J. Pharm. Sci. 100 (9), 3904–3911. doi:10.1002/jps.22610 Kubo, Y., Kusagawa, Y., Tachikawa, M., Akanuma, S.-i., and Hosoya, K.-i. (2013). Involvement of a Novel Organic Cation Transporter in Verapamil Transport
across the Inner Blood-Retinal Barrier. Pharm. Res. 30 (3), 847–856. doi:10.1007/s11095-012-0926-y Tournier, N., Stieger, B., and Langer, O. (2018). Imaging Techniques to Study Drug
Transporter Function In Vivo. Pharmacol. Ther. 189, 104–122. doi:10.1016/
j.pharmthera.2018.04.006 Lai, Y., Varma, M., Feng, B., Stephens, J. C., Kimoto, E., El-Kattan, A., et al. (2012). Impact of Drug Transporter Pharmacogenomics on Pharmacokinetic and
Pharmacodynamic Variability - Considerations for Drug Development. Expert
Opin. Drug
Metab. Toxicol. 8
(6),
723–743. doi:10.1517/
17425255.2012.678048 Uchida, Y., Ohtsuki, S., Katsukura, Y., Ikeda, C., Suzuki, T., Kamiie, J., et al. (2011). Quantitative Targeted Absolute Proteomics of Human Blood-Brain Barrier
Transporters and Receptors. J. REFERENCES Iqbal, Y., Palkar, V., Al-Sudairy, R., Al-Omari, A., Abdullah, M. F., and Al-Debasi,
T. (2005). Papilledema, Presenting as Reversible Loss of Vision, in a Child with
Acute Lymphoblastic Leukemia. Pediatr. Blood Cancer 45 (1), 72–73. doi:10.1002/pbc.20369 Prasad, B., Lai, Y., Lin, Y., and Unadkat, J. D. (2013). Interindividual Variability in
the Hepatic Expression of the Human Breast Cancer Resistance Protein (BCRP/
ABCG2): Effect of Age, Sex, and Genotype. J. Pharm. Sci. 102 (3), 787–793. doi:10.1002/jps.23436 Iqbal, Y., Palkar, V., Al-Sudairy, R., Al-Omari, A., Abdullah, M. F., and Al-Debasi,
T. (2005). Papilledema, Presenting as Reversible Loss of Vision, in a Child with
Acute Lymphoblastic Leukemia. Pediatr. Blood Cancer 45 (1), 72–73. doi:10.1002/pbc.20369 Ramirez, C. J., Minch, J. D., Gay, J. M., Lahmers, S. M., Guerra, D. J., Haldorson, G. J., et al. (2011). Molecular Genetic Basis for Fluoroquinolone-Induced Retinal
Degeneration in Cats. Pharmacogenet Genomics 21 (2), 66–75. doi:10.1097/
fpc.0b013e3283425f44 Jordán, J., and Ruíz-Moreno, J. M. (2013). Advances in the Understanding of
Retinal Drug Disposition and the Role of Blood-Ocular Barrier Transporters. Expert
Opin. Drug
Metab. Toxicol. 9
(9),
1181–1192. doi:10.1517/
17425255.2013.796928 Rimpelä, A.-K., Hagström, M., Kidron, H., and Urtti, A. (2018). Melanin Targeting
for Intracellular Drug Delivery: Quantification of Bound and Free Drug in
Retinal Pigment Epithelial
Cells. J. Controlled
Release
283, 261–268. doi:10.1016/j.jconrel.2018.05.034 Kalvass, J. C., Polli, J. W., Bourdet, D. L., Feng, B., Huang, S.-M., Liu, X., et al. (2013). Why Clinical Modulation of Efflux Transport at the Human Blood-
Brain Barrier Is Unlikely: the ITC Evidence-Based Position. Clin. Pharmacol. Ther. 94 (1), 80–94. doi:10.1038/clpt.2013.34 Robey, R. W., Pluchino, K. M., Hall, M. D., Fojo, A. T., Bates, S. E., and Gottesman,
M. M. (2018). Revisiting the Role of ABC Transporters in Multidrug-Resistant
Cancer. Nat. Rev. Cancer 18 (7), 452–464. doi:10.1038/s41568-018-0005-8 Kannan, P., Schain, M., Kretzschmar, W. W., Weidner, L., Mitsios, N., Gulyás, B.,
et al. (2017). An Automated Method Measures Variability in P-Glycoprotein
and ABCG2 Densities across Brain Regions and Brain Matter. J. Cereb. Blood
Flow Metab. 37 (6), 2062–2075. doi:10.1177/0271678x16660984 Sasongko, L., Link, J., Muzi, M., Mankoff, D., Yang, X., Collier, A., et al. (2005). Imaging P-Glycoprotein Transport Activity at the Human Blood-Brain Barrier
with Positron Emission Tomography. Clin. Pharmacol. Ther. 77 (6), 503–514. doi:10.1016/j.clpt.2005.01.022 Kim, H. M., and Woo, S. J. (2021). Ocular Drug Delivery to the Retina: Current
Innovations and Future Perspectives. Pharmaceutics 13 (1). doi:10.3390/
pharmaceutics13010108 Sbeity, Z. REFERENCES Pharmaceutics 10 (2), 650–663. doi:10.1021/mp300429e Bauer, M., Karch, R., Tournier, N., Cisternino, S., Wadsak, W., Hacker, M., et al. (2017). Assessment of P-Glycoprotein Transport Activity at the Human Blood-
Retina Barrier with (R)-11C-Verapamil PET. J. Nucl. Med. 58 (4), 678–681. doi:10.2967/jnumed.116.182147 Eyal, S., Hsiao, P., and Unadkat, J. D. (2009). Drug Interactions at the Blood-Brain
Barrier: Fact or Fantasy?q. Pharmacol. Ther. 123 (1), 80–104. doi:10.1016/
j.pharmthera.2009.03.017 Bauer, M., Karch, R., Zeitlinger, M., Philippe, C., Römermann, K., Stanek, J., et al. (2015). Approaching Complete Inhibition of P-Glycoprotein at the Human
Blood-Brain Barrier: an (R)-[11C]verapamil PET Study. J. Cereb. Blood Flow
Metab. 35 (5), 743–746. doi:10.1038/jcbfm.2015.19 Fohner, A. E., Brackman, D. J., Giacomini, K. M., Altman, R. B., and Klein, T. E. (2017). PharmGKB Summary. Pharmacogenet Genomics 27 (11), 420–427. doi:10.1097/fpc.0000000000000305 Bauer, M., Karch, R., Zeitlinger, M., Stanek, J., Philippe, C., Wadsak, W., et al. (2013). Interaction of 11C-Tariquidar and 11C-Elacridar with P-Glycoprotein
and Breast Cancer Resistance Protein at the Human Blood-Brain Barrier. J. Nucl. Med. 54 (8), 1181–1187. doi:10.2967/jnumed.112.118232 Fox, E., and Bates, S. E. (2007). Tariquidar (XR9576): a P-Glycoprotein Drug Efflux
Pump Inhibitor. Expert Rev. Anticancer Ther. 7 (4), 447–459. doi:10.1586/
14737140.7.4.447 Fujii, S., Setoguchi, C., Kawazu, K., and Hosoya, K.-i. (2014). Impact of
P-Glycoprotein on Blood-Retinal Barrier Permeability: Comparison of
Blood-Aqueous Humor and Blood-Brain Barrier UsingMdr1aKnockout
Rats. Invest. Ophthalmol. Vis. Sci. 55, 4650–4658. doi:10.1167/iovs.13-13819
Furukawa, T., Wakabayashi, K., Tamura, A., Nakagawa, H., Morishima, Y., Osawa, Bauer, M., Römermann, K., Karch, R., Wulkersdorfer, B., Stanek, J., Philippe, C.,
et al. (2016). Pilot PET Study to Assess the Functional Interplay between
ABCB1 and ABCG2 at the Human Blood-Brain Barrier. Clin. Pharmacol. Ther. 100 (2), 131–141. doi:10.1002/cpt.362 Bauer, M., Wulkersdorfer, B., Karch, R., Philippe, C., Jäger, W., Stanek, J., et al. (2017). Effect of P-Glycoprotein Inhibition at the Blood-Brain Barrier on Brain Y., et al. (2009). Major SNP (Q141K) Variant of Human ABC Transporter June 2021 | Volume 12 | Article 698966 Frontiers in Pharmacology | www.frontiersin.org 8 El Biali et al. ABCB1/ABCG2 at the Blood-Retinal Barrier ABCG2 Undergoes Lysosomal and Proteasomal Degradations. Pharm. Res. 26
(2), 469–479. doi:10.1007/s11095-008-9752-7 ABCG2 Undergoes Lysosomal and Proteasomal Degradations. Pharm. Res. 26
(2), 469–479. doi:10.1007/s11095-008-9752-7 Li, J., and Zhu, H. J. (2020). Liquid Chromatography-Tandem Mass Spectrometry
(LC-MS/MS)-Based
Proteomics
of
Drug-Metabolizing
Enzymes
and
Transporters. Molecules 25 (11). doi:10.3390/molecules25112718 Griffin, J. D., and Garnick, M. B. (1981). Eye Toxicity of Cancer Chemotherapy: A
Review of the Literature. REFERENCES Cancer 48 (7), 1539–1549. doi:10.1002/1097-
0142(19811001)48:7<1539::aid-cncr2820480713>3.0.co;2-b Li, R., and Barton, H. A. (2018). Explaining Ethnic Variability of Transporter
Substrate Pharmacokinetics in Healthy Asian and Caucasian Subjects with
Allele Frequencies of OATP1B1 and BCRP: A Mechanistic Modeling Analysis. Clin. Pharmacokinet. 57 (4), 491–503. doi:10.1007/s40262-017-0568-7 Grzybowski, A., Zülsdorff, M., Wilhelm, H., and Tonagel, F. (2015). Toxic Optic
Neuropathies: an Updated Review. Acta Ophthalmol. 93 (5), 402–410. doi:10.1111/aos.12515 Liu, L., and Liu, X. (2019). Roles of Drug Transporters in Blood-Retinal Barrier. Adv. Exp. Med. Biol. 1141, 467–504. doi:10.1007/978-981-13-7647-4_10 Haslacher, H., Gerner, M., Hofer, P., Jurkowitsch, A., Hainfellner, J., Kain, R., et al. (2018). Usage Data and Scientific Impact of the Prospectively Established Fluid
Bioresources at the Hospital-Based MedUni Wien Biobank. Biopreservation
and Biobanking 16 (6), 477–482. doi:10.1089/bio.2018.0032 Liu, X. (2019). ABC Family Transporters. Adv. Exp. Med. Biol. 1141, 13–100. doi:10.1007/978-981-13-7647-4_2 Logan, J., Fowler, J. S., Volkow, N. D., Wolf, A. P., Dewey, S. L., Schlyer, D. J., et al. (1990). Graphical
Analysis
of
Reversible
Radioligand
Binding
from
Time-Activity
Measurements Applied to [N-11c-Methyl]-(−)-Cocaine PET Studies in Human
Subjects. J. Cereb. Blood Flow Metab. 10 (5), 740–747. doi:10.1038/jcbfm.1990.127 Ho, W. L., Wong, H., and Yau, T. (2013). The Ophthalmological Complications of
Targeted Agents in Cancer Therapy: what Do We Need to Know as
Ophthalmologists? Acta Ophthalmol. 91 (7), 604–609. doi:10.1111/j.1755-
3768.2012.02518.x Mao, Q., and Unadkat, J. D. (2015). Role of the Breast Cancer Resistance Protein
(BCRP/ABCG2) in Drug Transport-An Update. AAPS J. 17 (1), 65–82. doi:10.1208/s12248-014-9668-6 Hosoya, K.-i., Yamamoto, A., Akanuma, S.-i., and Tachikawa, M. (2010). Lipophilicity
and
Transporter
Influence
on
Blood-Retinal
Barrier
Permeability: a Comparison with Blood-Brain Barrier Permeability. Pharm. Res. 27 (12), 2715–2724. doi:10.1007/s11095-010-0272-x Noureddin, B. N., Seoud, M., Bashshur, Z., Salem, Z., Shamseddin, A., and Khalil,
A. (1999). Ocular Toxicity in Low-Dose Tamoxifen: a Prospective Study. Eye
(Lond). 13 (Pt 6), 729–733. doi:10.1038/eye.1999.217 International Transporter, C., Giacomini, K. M., Huang, S. M., Tweedie, D. J., Benet, L. Z., Brouwer, K. L., et al. (2010). Membrane Transporters in
Drug Development. Nat. Rev. Drug Discov. 9 (3), 215–236. doi:10.1038/
nrd3028 Pascual-Pasto, G., Olaciregui, N. G., Opezzo, J. A. W., Castillo-Ecija, H., Cuadrado-
Vilanova, M., Paco, S., et al. (2017). Increased Delivery of Chemotherapy to the
Vitreous by Inhibition of the Blood-Retinal Barrier. J. Controlled Release 264,
34–44. doi:10.1016/j.jconrel.2017.08.018 INVESTIGATOR BROCHURE Tariquidar (2007). INVESTIGATOR BROCHURE
Tariquidar (XR9576). Mumbai, India: AVAANT Pharmaceuticals Pvt Ltd, 28
May 2007. Report No. Frontiers in Pharmacology | www.frontiersin.org June 2021 | Volume 12 | Article 698966 REFERENCES Neurochem. 117 (2), 333–345. doi:10.1111/
j.1471-4159.2011.07208.x June 2021 | Volume 12 | Article 698966 Frontiers in Pharmacology | www.frontiersin.org 9 El Biali et al. ABCB1/ABCG2 at the Blood-Retinal Barrier Protein: Comparative Quantitative Targeted Absolute Proteomics Study of
CNS Barriers in Pig. Mol. Pharmaceutics 14 (11), 3729–3738. doi:10.1021/
acs.molpharmaceut.7b00493 Vaghefi, E., and Pontre, B. (2016). Application of Arterial Spin Labelling in the
Assessment of Ocular Tissues. Biomed. Res. Int. 2016, 6240504. doi:10.1155/
2016/6240504 Wagner, C. C., Bauer, M., Karch, R., Feurstein, T., Kopp, S., Chiba, P., et al. (2009). A Pilot Study to Assess the Efficacy of Tariquidar to Inhibit P-Glycoprotein at
the Human Blood-Brain Barrier with (R)-11C-verapamil and PET. J. Nucl. Med. 50 (12), 1954–1961. doi:10.2967/jnumed.109.063289 Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest. j
Wijaya, J., Fukuda, Y., and Schuetz, J. D. (2017). Obstacles to Brain Tumor
Therapy: Key ABC Transporters. Int. J. Mol. Sci. 18 (12). doi:10.3390/
ijms18122544 Copyright © 2021 El Biali, Karch, Philippe, Haslacher, Tournier, Hacker, Zeitlinger,
Schmidl, Langer and Bauer. This is an open-access article distributed under the
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or reproduction in other forums is permitted, provided the original author(s) and the
copyright owner(s) are credited and that the original publication in this journal is
cited, in accordance with accepted academic practice. No use, distribution or
reproduction is permitted which does not comply with these terms. Williamson, B., and Pilla Reddy, V. (2021). Blood Retinal Barrier and Ocular
Pharmacokinetics: Considerations for the Development of Oncology Drugs. Biopharm. Drug Dispos 42 (4), 128–136. doi:10.1002/bdd.2276 Zhang, Z., Uchida, Y., Hirano, S., Ando, D., Kubo, Y., Auriola, S., et al. (2017). Inner Blood-Retinal Barrier Dominantly Expresses Breast Cancer Resistance June 2021 | Volume 12 | Article 698966 Frontiers in Pharmacology | www.frontiersin.org 10
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Hyperchaotic Behavior in the Novel Memristor-Based Symmetric Circuit System
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Received August 3, 2020, accepted August 13, 2020, date of publication August 17, 2020, date of current version August 28, 2020. Digital Object Identifier 10.1109/ACCESS.2020.3017159 LIN TENG
, XINGYUAN WANG
, AND XIAOLIN YE Corresponding authors: Lin Teng (tenglin@mail.dlut.edu.com) and Xingyuan Wang (wangxy@dlut.edu.cn) This work was supported in part by the National Natural Science Foundation of China under Grant 61701070 and
Grant 61672124, in part by the Doctoral Start-Up Foundation of Liaoning Province under Grant 2018540090, in part
by the Key Research and Development Projects of Liaoning Province under Grant 2019020105-JH2/103, in part by
the Liaoning Province Science and Technology Innovation Leading Talents Program Project under Grant XLYC1802013,
and in part by the Jinan City 20 Universities Funding Projects Introducing Innovation Team Program under Grant 2019GXRC031. ABSTRACT A novel five-dimensional (5D) memristor-based symmetric circuit, which consists of two
symmetric capacitors, two symmetric inductors and only one memristor is presented in this article. The
multivariable first order and multivariable second order polynomial functions are used for the internal state
function of the memristor respectively. Theoretical and simulation analyses of the novel memristive circuit
are investigated using equilibrium points, phase portraits, bifurcation diagrams and Lyapunov exponent
spectra, etc. Complex chaotic behaviors are observed and analyzed through simulation results. The first
order internal state function memristor-based symmetric circuit system can only exhibit chaotic behavior
whereas the second order internal state function memristor-based symmetric circuit system can generate not
only chaotic attractors, but also hyperchaotic attractors in proper parameters. INDEX TERMS Chaos attractor, hyperchaotic attractor, memristor, second order internal state, symmetric
circuit. is work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ I. INTRODUCTION environment changes. Hu et al. [13] proposed a dynamic
synaptic design based on memristor and experimental cali-
bration of memristor model. Liu et al. [14] given a simple
and effective method for designing autonomous memristor
chaotic system with infinite chaotic attractor. Memristor, known as the fourth basic fundamental circuital
element in electronic circuits, was first put forward the
hypothesis by Leon Chua in 1971 [1]. Not until 2008, this
postulation was proved by Hewlett-Packard (HP) laboratory. They successfully fabricated the first actual physical model
of the memristor as a TiO2 nanocomponent [2]. Since then,
various kinds of circuits with the memristor have been inves-
tigated and the nonlinear dynamical characteristic of these
circuits have been studied. Such as memristor oscillators and
memristor-based crossbar [3]–[9]. As a novel element, its
ability to combine processing and memory are expected to
bring various benefit in artificial intelligence, machine learn-
ing, neuromorphic computing and self-learning. For example,
researchers have designed some neural networks combined
with memristor. Paper [10] proposed a new circuit based on
memristor and two MOSFET transistors to realize synapse. Wen et al. [11] proposed online least mean square algorithm
for echo state network based on memristor. A simple elec-
tronic circuit composed of a LC contour and a memristor is
proposed in [12] to get series of voltage pulses that mimic Memristor-based circuits can exhibit complex dynamics
and easily generate chaotic signals due to the nonlinearity
induced by memristors. Many memristor-based circuits
have been reported to construct chaotic systems in the
last few years. Sun et al. [15] designed the autonomous
memristor chaotic systems with infinite chaotic attractors. Fan et al. [16] proposed a simplified neural network based on
fractional-order memristor with discontinuous memductance
function. Paper [17] studied a class of chaotic systems based
on piecewise linear memristor. Zhang et al. [18] designed
the Josephson Junction circuit employing memristor, and
investigated the chaos encryption as well. Paper [19] derived
a novel four-wing memristive chaotic system by bringing
a flux-controlled memristor with quadratic nonlinearity into
the Liu-Chen system as a feedback term. Hyperchaos was first published by Rössler in 1979 [20]
and has attracted more and more attention from various
scientific and engineering communities. Hyperchaotic sys-
tems with multiple positive Lyapunov exponents usually have The associate editor coordinating the review of this manuscript and
approving it for publication was Di He
. 151535 L. I. INTRODUCTION Paper [21] studied the nonlinear dynamics of the
TCMNL hyperchaotic oscillator with gyrators based on a
smooth mathematical model of the system. Sambas et al. [22]
reported a new five-dimensional four-wing hyperchaotic sys-
tem with hidden attractor. Wei et al. [23] proposed and
investigated a 5D hyperchaotic generalization of a 3D model
for a self-exciting homopolar disc dynamo without unsta-
ble equilibria but with three positive Lyapunov exponents. Paper [24] reported the finding of a new hyperchaotic tem-
perature fluctuations model and described its modelling. Li et al. [25] studied a four-dimensional (4D) memristive
system modified from the 3D chaotic system proposed by
Lü and Chen. A novel memristive hyperchaotic system is
presented in paper [26] by introducing a memristor to instead
a coupling resistor in the realization of three-dimensional
chaotic circuit system. Bao et al. [27] utilized a memris-
tor to substitute a linear resistor to expand the active band
pass filter-based memristive circuit, and presented a novel
fifth-order two memristor-based Chua’s hyperchaotic cir-
cuit. The good characteristics of chaotic systems, such as
pseudo-random behavior, sensitive on initial conditions etc.,
can be applied to cryptography and confidential communica-
tion [28]–[30]. And hyperhcaotic systems with more complex
and richer dynamic behaviors can improve the security of the
encryption schemes. more complex and richer dynamic behaviors than chaotic
systems which can enhance the randomness and higher
unpredictability of the corresponding system. Generating
hyperchaotic attractors is a very attractive work in theory, but
it is quite challenging in technology. Some researchers have
investigated the hyperchaotic attractor in multi-dimensional
circuits systems and memristor-based dynamical circuits sys-
tems. Paper [21] studied the nonlinear dynamics of the
TCMNL hyperchaotic oscillator with gyrators based on a
smooth mathematical model of the system. Sambas et al. [22]
reported a new five-dimensional four-wing hyperchaotic sys-
tem with hidden attractor. Wei et al. [23] proposed and
investigated a 5D hyperchaotic generalization of a 3D model
for a self-exciting homopolar disc dynamo without unsta-
ble equilibria but with three positive Lyapunov exponents. Paper [24] reported the finding of a new hyperchaotic tem-
perature fluctuations model and described its modelling. Li et al. [25] studied a four-dimensional (4D) memristive
system modified from the 3D chaotic system proposed by
Lü and Chen. A novel memristive hyperchaotic system is
presented in paper [26] by introducing a memristor to instead
a coupling resistor in the realization of three-dimensional
chaotic circuit system. Bao et al. I. INTRODUCTION Teng et al.: Hyperchaotic Behavior in the Novel Memristor-Based Symmetric Circuit System FIGURE 3. Bifurcation diagrams of the first order memristor-based
symmetric system. FIGURE 1. Circuit structure of the simplest chaotic circuit. FIGURE 2. Circuit structure of the memristor-based symmetric chaotic
circuit. more complex and richer dynamic behaviors than chaotic
systems which can enhance the randomness and higher
unpredictability of the corresponding system. Generating
hyperchaotic attractors is a very attractive work in theory, but
it is quite challenging in technology. Some researchers have
investigated the hyperchaotic attractor in multi-dimensional
circuits systems and memristor-based dynamical circuits sys-
tems. Paper [21] studied the nonlinear dynamics of the
TCMNL hyperchaotic oscillator with gyrators based on a
smooth mathematical model of the system. Sambas et al. [22]
reported a new five-dimensional four-wing hyperchaotic sys-
tem with hidden attractor. Wei et al. [23] proposed and
investigated a 5D hyperchaotic generalization of a 3D model
for a self-exciting homopolar disc dynamo without unsta-
ble equilibria but with three positive Lyapunov exponents. Paper [24] reported the finding of a new hyperchaotic tem-
t
fl
t
ti
d l
d d
ib d it
d lli
FIGURE 3. Bifurcation diagrams of the first order memristor-based
symmetric system. Most memristive hyperchaotic systems are generated
by introducing or substituting memristor into the classical
circuit. In this article, we extended the simplest chaotic
circuit [31] which only consists three elements and one
of them is already memristor to build a hyperchaotic sys-
tem. This simplest chaotic circuit is extended to build a
fi
di
i
l (5D)
i t
b
d
t i
i
it FIGURE 1. Circuit structure of the simplest chaotic circuit. FIGURE 2. Circuit structure of the memristor-based symmetric chaotic
circuit. FIGURE 1. Circuit structure of the simplest chaotic circuit. FIGURE 2. Circuit structure of the memristor-based symmetric chaotic
circuit. FIGURE 1. Circuit structure of the simplest chaotic circuit. FIGURE 1. Circuit structure of the simplest chaotic circuit. FIGURE 2. Circuit structure of the memristor-based symmetric chaotic
circuit. FIGURE 2. Circuit structure of the memristor-based symmetric chaotic
circuit. more complex and richer dynamic behaviors than chaotic
systems which can enhance the randomness and higher
unpredictability of the corresponding system. Generating
hyperchaotic attractors is a very attractive work in theory, but
it is quite challenging in technology. Some researchers have
investigated the hyperchaotic attractor in multi-dimensional
circuits systems and memristor-based dynamical circuits sys-
tems. I. INTRODUCTION [27] utilized a memris-
tor to substitute a linear resistor to expand the active band
pass filter-based memristive circuit, and presented a novel
fifth-order two memristor-based Chua’s hyperchaotic cir-
cuit. The good characteristics of chaotic systems, such as
pseudo-random behavior, sensitive on initial conditions etc.,
can be applied to cryptography and confidential communica-
tion [28]–[30]. And hyperhcaotic systems with more complex
and richer dynamic behaviors can improve the security of the
encryption schemes. FIGURE 3. Bifurcation diagrams of the first order memristor-based
symmetric system. FIGURE 3. Bifurcation diagrams of the first order memristor-based
symmetric system. Most memristive hyperchaotic systems are generated
by introducing or substituting memristor into the classical
circuit. In this article, we extended the simplest chaotic
circuit [31] which only consists three elements and one
of them is already memristor to build a hyperchaotic sys-
tem. This simplest chaotic circuit is extended to build a
five-dimensional (5D) memristor-based symmetric circuit
which consists of two symmetric linear passive inductors, two
symmetric linear passive capacitors and only one non-linear
active memristor. The multivariable first order and multi-
variable second order polynomial functions are used for the
internal state function of the memristor respectively. We use
the second order internal state of memristor in order to make
the chaotic attractors more complexity. The chaotic system
can be hyperchaotic when its parameters are taken appro-
priately. Chaotic behaviors are illustrated using equilibrium
points, bifurcation diagrams, Lyapunov exponent spectra
and phase portraits, etc. Simulation results show that the
first order internal state function memristor-based symmetric
circuit system can only generate chaotic attractors whereas
the second order internal state function memristor-based
symmetric circuit system can generate not only chaotic 151536 VOLUME 8, 2020 VOLUME 8, 2020 L. Teng et al.: Hyperchaotic Behavior in the Novel Memristor-Based Symmetric Circuit System FIGURE 4. The iL1(t) versus x(t) phase plots of the first order memristor-based symmetric circuit system. FIGURE 4. The iL1(t) versus x(t) phase plots of the first order memristor-based symmetric circuit system. attractors,
but
also
hyperchaotic
attractors
in
proper
parameters. In this article, we extended this circuit to build an
autonomous
memristor-based
symmetric
circuit
which
consists of two symmetric linear passive inductors, two
symmetric linear passive capacitors and one non-linear active
memristor. Memristors can be classified as flux-controlled
and charge-controlled memristors. A charge-controlled mem-
ristor is used in this article, and its circuit structure is
presented in Figure 2. This article is organized as follows. I. INTRODUCTION In section 2, a novel
memristor-based symmetric chaotic system circuit is pre-
sented. In section 3, the first order of the memristor internal
state case is analyzed. In section 4, the second order of the
memristor internal state case is analyzed, the bifurcation dia-
gram and the corresponding Lyapunov exponent spectrum are
plotted with the system parameters changed, which reveals
the dynamic behavior of hyperchaos. Section 5 concludes the
paper. This memristor-based symmetric circuit consists five
elements, so the state variables for this circuit can
defined as FIGURE 5. Lyapunov exponents spectrum versus β of the first order
memristor-based symmetric circuit system. The first two state equations to construct the dynamical
system are the current-voltage relations of two capacitors. C1dvC1(t)
dt
= iL1(t)
(2)
C2dvC2(t)
dt
= iL2(t)
(3) (2) (8) (3) where the circuit parameters are C1 = 3, L1 = 1,, C2 = 1,
L2 = 1. where the circuit parameters are C1 = 3, L1 = 1,, C2 = 1,
L2 = 1. There are two loops in this memristor-based symmetric
circuit, so the other two state equations can be obtained by
applying Kirchhoff’s voltage law around the loops. III. FIRST ORDER OF THE MEMRISTOR INTERNAL
STATE CASE The first order of the memristor internal state case is analyzed. The five-dimensional symmetric memristive chaotic system
with first order of the memristor internal state can be
constructed using (2)-(6):
dv1 (t)
dt
= i1 (t)
C1
di1 (t)
dt
= −1
L1
(v1 (t) + R (x (t)) × (i1 (t) −i2 (t)))
dx (t)
dt
= −(i1 (t) −i2 (t)) −αx (t) + (i1(t) −i2 (t)) x (t)
dv2 (t)
dt
= i2 (t)
C2
di2 (t)
dt
= 1
L2
(−v2 (t) + R (x (t)) × (i1 (t) −i2 (t)))
(8) II. MEMRISTOR-BASED SYMMETRIC CHAOTIC SYSTEM yp
p
pp
p
y
The first order internal state of the memristor defined as
dx (t)
dt
= −(i1 (t) −i2 (t)) −αx (t) + (i1(t) −i2 (t)) x (t)
(6) The first order internal state of the memristor defined as The first order internal state of the memristor defined as
dx (t)
dt
= −(i1 (t) −i2 (t)) −αx (t) + (i1(t) −i2 (t)) x (t)
(6) (6) The second order internal state of the memristor defined as
dx (t)
dt
= −(i1 (t) −i2 (t)) −αx (t) + (i1(t) −i2 (t))2 x (t)
(7) The second order internal state of the memristor defined as
dx (t)
dt
= −(i1 (t) −i2 (t)) −αx (t) + (i1(t) −i2 (t))2 x (t)
(7) The second order internal state of the memristor defined as
dx (t)
dt
= −(i1 (t) −i2 (t)) −αx (t) + (i1(t) −i2 (t))2 x (t)
(7) (7) II. MEMRISTOR-BASED SYMMETRIC CHAOTIC SYSTEM z(t) =
vC1(t)
iL1(t)
x(t)
vC2(t)
iL2(t)
(1) Paper [31] presented the simplest memristor-based circuit
which can exhibited chaotic behavior. This circuit only
consists of one linear passive inductor, one linear passive
capacitor and one non-linear active memristor in series shown
in Figure 1. (1) The circuit dynamics are described by: where vC1(t) and vC2(t) denotes the voltage across the
terminals of capacitors C1 and C2 respectively, iL1(t)
and iL2(t) represents the currents flowing through inductor
L1 and L2 respectively, and the internal state variable of the
memristor is represented by x(t).
˙x = y
C
˙y = −1
L
h
x + β(z2 −1)y
i
˙z = −y −αz + yz 151537 VOLUME 8, 2020 L. Teng et al.: Hyperchaotic Behavior in the Novel Memristor-Based Symmetric Circuit System FIGURE 5. Lyapunov exponents spectrum versus β of the first order
memristor-based symmetric circuit system
TABLE 1. Lyapunov exponents of t
symmetric circuit system. attractor and orbit more com
hyperchaotic when its param
The first order internal sta
dx (t)
dt
= −(i1 (t) −i2 (t)) −
The second order internal
dx (t)
dt
= −(i1 (t) −i2 (t))
III. FIRST ORDER OF THE M
STATE CASE
The first order of the memrist
The five-dimensional symm
with first order of the m
constructed using (2)-(6):
dv1 (t)
dt
= i1 (t)
C1
di1 (t)
dt
= −1
L1
(v1 (t) + R
dx (t)
(i (t)
i (t) TABLE 1. Lyapunov exponents of the first order memristor-based
symmetric circuit system. TABLE 1. Lyapunov exponents of the first order memristor-based
symmetric circuit system. attractor and orbit more complex, the chaotic system can be
hyperchaotic when its parameters are taken appropriately. is generated in Figure 3(b). It can be acquired that the system
can generate chaotic attractors all through the range. A. ANALYSIS OF BIFURCATION DIAGRAMS AND PHASE
PORTRAITS L1diL1(t)
dt
= −{vC1(t) + R(x(t)) × (i1 (t) −i2 (t))}
(4)
L2diL2(t)
dt
= −v2 (t) + R (x (t)) × (i1 (t) −i2 (t))
(5) Fixing system parameter as α
=
1 and the setting the
five initial condition of state variables as vC1(0) = 0.1,
iL1(0) = 0, x(0) = 0.1, vC2(0) = 0.1 and iL2(0) = 0, the
bifurcation diagram for control parameter β over the range
−1 ≤β ≤0 is generated in Figure 3(a). From Figure 3(a),
the system goes through a complex alternation of numer-
ous chaotic orbit and periodic states. Take β = −0.8 and
β
= −0.2 as examples, the system exhibits the chaotic
behavior which are shown in Figure 4(a) and Figure 4(b). The
system exhibits the periodic behavior as shown in Figure 4(c)
and Figure 4(d) when β = −0.4 and β = 0. Fixing system parameter as α
=
1 and the setting the
five initial condition of state variables as vC1(0) = 0.1,
iL1(0) = 0, x(0) = 0.1, vC2(0) = 0.1 and iL2(0) = 0, the
bifurcation diagram for control parameter β over the range (5) The memristance function of M is R(x(t)) and is defined
as R (x (t)) = 2x (t)2 −1 + β in this article. −1 ≤β ≤0 is generated in Figure 3(a). From Figure 3(a),
the system goes through a complex alternation of numer-
ous chaotic orbit and periodic states. Take β = −0.8 and
β
= −0.2 as examples, the system exhibits the chaotic
behavior which are shown in Figure 4(a) and Figure 4(b). The
system exhibits the periodic behavior as shown in Figure 4(c)
and Figure 4(d) when β = −0.4 and β = 0. The multivariable first order and multivariable second
order polynomial functions are used as the internal state
memristor function respectively. We analysis the chaotic
behaviors in these two cases and find that the exponent of
the internal state chosen second order can make the chaotic 151538 VOLUME 8, 2020 L. Teng et al.: Hyperchaotic Behavior in the Novel Memristor-Based Symmetric Circuit System FIGURE 6. Chaotic attractors of the first order internal state function symmetric system. Fixing system parameter as β
= −1, the bifurcation
diagram for control parameter α over the range 0.5 ≤α ≤1.5 is generated in Figure 3(b). It can be acquired that the system
can generate chaotic attractors all through the range. B. LYAPUNOV EXPONENT SPECTRA The
Lyapunov
exponents
are
calculated
by
wolf
method [25]. Due to the numerical error in the calculation
process
by
computer,
that
|λi|
<
0.01
can
be
considered equivalent to λi
=
0. Fixing α
=
1 and
varying β, the Lyapunov exponents spectrum is shown in
Figure 5. The evidence of chaos can be provided by Lyapunov exponent
spectra [32], [33]. For the five-dimensional system, assume
the five Lyapunov exponents are λ1, λ2, λ3, λ4, λ5, where
λ1 > λ2 > λ3 > λ4 > λ5 and λ1 + λ2 + λ3 + λ4 + λ5 < 0. The relations between the Lyapunov exponents and the orbits
of the system are described as follows: y
(1) When the system is in periodic orbits, the Lyapunov
exponents of the system are one zero and four negatives. That
is if the Lyapunov spectrum is λ1 = 0 and λ2, λ3, λ4, λ5 < 0,
system produces a stable limit cycle. (2) When the system
is in quasi-periodic orbits, the Lyapunov exponents of the
system are two zero and three negatives. That is if the Lya-
punov spectrum is λ1 = 0, λ2 = 0 and λ3, λ4, λ5 < 0,
system produces a two-dimensional closed torus. (3) When
the system is in chaotic orbits, the Lyapunov exponents of
the system are one positive, one zero and three negatives. That is if the Lyapunov spectrum is λ1 > 0, λ2 = 0 and
λ3, λ4, λ5 < 0, system is in chaotic state. (4) When the
system is in hyperchaotic orbits, the Lyapunov exponents of
the system are two positives, one zero and two negatives. That
is if the Lyapunov spectrum is λ1 > 0, λ2 > 0, λ3 = 0 and
λ4, λ5 < 0, system is in hyperchaotic state. g
Figure 5(b) shows the maximum Lyapunov exponent
spectrum and the second largest Lyapunov exponent spec-
trum. From Figure 5(b) and the bifurcation diagram
Figure 3(a), it can be seen that the spectrum of Lyapunov
exponents and the bifurcation diagram are one to one corre-
spondence. There are only one positive Lyapunov exponent
during some intervals, that means the first order memristor-
based symmetric circuit system can only generate chaotic
orbits but cannot generate hyperchaotic attractors. y
(1) When the system is in periodic orbits, the Lyapunov
exponents of the system are one zero and four negatives. A. ANALYSIS OF BIFURCATION DIAGRAMS AND PHASE
PORTRAITS 151539 VOLUME 8, 2020 L. Teng et al.: Hyperchaotic Behavior in the Novel Memristor-Based Symmetric Circuit System FIGURE 7. Memristor characteristics of the first order internal state
function symmetric system. FIGURE 8. Bifurcation diagrams of the second order memristor function
symmetric system FIGURE 8. Bifurcation diagrams of the second order memristor function
symmetric system. FIGURE 7. Memristor characteristics of the first order internal state
function symmetric system. FIGURE 8. Bifurcation diagrams of the second order memristor function
symmetric system. IV. SECOND ORDER OF THE MEMRISTOR INTERNAL
STATE CASE where the circuit parameters are C1 = 3, L1 = 1, C2 = 1,
L2 = 1. L2 = 1. L2 = 1. Hyperchaotic systems which have multiple positive Lyapunov
exponents usually exhibit more complex and richer dynamic
behaviors than chaotic systems which only have one positive
Lyapunov exponents. Because the first order memristor-
based symmetric circuit system can only generate chaotic B. LYAPUNOV EXPONENT SPECTRA That
is if the Lyapunov spectrum is λ1 = 0 and λ2, λ3, λ4, λ5 < 0,
system produces a stable limit cycle. (2) When the system
is in quasi-periodic orbits, the Lyapunov exponents of the
system are two zero and three negatives. That is if the Lya-
punov spectrum is λ1 = 0, λ2 = 0 and λ3, λ4, λ5 < 0,
system produces a two-dimensional closed torus. (3) When
the system is in chaotic orbits, the Lyapunov exponents of
the system are one positive, one zero and three negatives. That is if the Lyapunov spectrum is λ1 > 0, λ2 = 0 and
λ3, λ4, λ5 < 0, system is in chaotic state. (4) When the
system is in hyperchaotic orbits, the Lyapunov exponents of
the system are two positives, one zero and two negatives. That
is if the Lyapunov spectrum is λ1 > 0, λ2 > 0, λ3 = 0 and
λ4, λ5 < 0, system is in hyperchaotic state. The phase portraits of Figure 4 and the corresponding
Lyapunov exponents results are listed in Table 1. From data
shown in Table 1, we can see that the systems with param-
eter β selected in Figure 4(a) and Figure 4(b) display the
chaotic state, while the systems parameter corresponding to
Figure 4(c) and Figure 4(d) produce a limit cycle and in
periodic state. 151540 151540 VOLUME 8, 2020 VOLUME 8, 2020 L. Teng et al.: Hyperchaotic Behavior in the Novel Memristor-Based Symmetric Circuit System FIGURE 9. The plot of iL1(t) versus x(t) with the second order memristor function symmetric system. FIGURE 9. The plot of iL1(t) versus x(t) with the second order memristor function symmetric system. C. ANALYSIS OF CHAOTIC ATTRACTORS attractor, the multivariable second order polynomial function
is adopted as the internal state of the memristor to increase
the complexity of the chaotic attractor and attempt to get
the hyperchaos. The five-dimensional symmetric memristive
chaotic system with second order of the memristor internal
state can be constructed using (2)-(5) and (7): Set the parameters as β
= −1 and α = 1, chose the
initial conditions as vC1(0) = 0.1, iL1(0) = 0, x(0) = 0.1,
vC2(0) = 0.1 and iL2(0) = 0 to simulate the first order
memristor function symmetric system. The chaotic attractors
can be obtained as shown in Figure 6. We can see that the
system exhibits multiple-scroll attractor.
dv1 (t)
dt
= i1 (t)
C1
di1 (t)
dt
= −1
L1
(v1 (t) + R (x (t)) × (i1 (t) −i2 (t)))
dx (t)
dt
=−(i1 (t) −i2 (t))−αx (t) + (i1(t)−i2 (t))2 x (t)
dv2 (t)
dt
= i2 (t)
C2
di2 (t)
dt
= 1
L2
(−v2 (t) + R (x (t)) × (i1 (t) −i2 (t)))
(10) When the first order memristor function symmetric system
exhibits the chaotic state, the memristor DCvM- iM curve (9)
and the plot of vM- iM memristor characteristics are shown
in Figure 7.
xDC =
iM
α + iM
vM = R(xDC)iM
(9) (9) in this equation, set the iM to 10A amplitude sine-wave and
the frequency to 0.5Hz. (10) A. STABILITY ABALYSIS In order to calculate the equilibrium point of the memristive
chaotic system with second-order memristor internal state,
the left side of (10) is set to zero. Obviously, P0 = (0, 0, 0, 0, 0) 151541 VOLUME 8, 2020 L. Teng et al.: Hyperchaotic Behavior in the Novel Memristor-Based Symmetric Circuit System FIGURE 10. The dynamical analysis. is the only one equilibrium set of this system. The correspond-
ing Jacobian matrix at equilibrium P0 can be derived in (11),
as shown at the bottom of the page. The characteristic polynomial of (11) with the parameters
set above is
(λ + α)(λ4 + 2λ3(β −1) + 4
3λ2 + 4
3(β −1)λ + 1
3) = 0
(12)
where α is a positive number, denote
λ4 + 2λ3(β −1) + 4
3λ2 + 4
3(β −1)λ + 1
3 = 0
(13)
According to the Routh-Hurwitz stability conditions, not
all the real parts of the root λ of (13) are negative Therefore
FIGURE 11. Lyapunov exponents spectrum versus β of the second order
memristor function symmetric system. TABLE 2. Lyapunov exponents of the SECOND order memristor-based
symmetric circuit system. FIGURE 10. The dynamical analysis. is the only one equilibrium set of this system The correspond FIGURE 10. The dynamical analysis. is the only one equilibrium set of this system. The correspond-
ing Jacobian matrix at equilibrium P0 can be derived in (11),
as shown at the bottom of the page. The characteristic polynomial of (11) with the parameters
set above is FIGURE 11. Lyapunov exponents spectrum versus β of the second order
memristor function symmetric system. (λ + α)(λ4 + 2λ3(β −1) + 4
3λ2 + 4
3(β −1)λ + 1
3) = 0
(12) TABLE 2. Lyapunov exponents of the SECOND order memristor-based
symmetric circuit system. TABLE 2. Lyapunov exponents of the SECOND order memristor-based
symmetric circuit system. TABLE 2. Lyapunov exponents of the SECOND order memristor-based
symmetric circuit system. (12) where α is a positive number, denote λ4 + 2λ3(β −1) + 4
3λ2 + 4
3(β −1)λ + 1
3 = 0
(13) According to the Routh-Hurwitz stability conditions, not
all the real parts of the root λ of (13) are negative. Therefore,
not all roots λ of (12) have negative real parts. This means that
the equilibrium point P0 is unstable. The necessary condition
for the existence of chaotic attractor is the eigenvalue λ in
the unstable region. A. STABILITY ABALYSIS Therefore, the five-dimensional sym-
metric memristive chaotic system with second order of the memristor internal state is able to produce self-excited chaotic
attractors or self-excited hyperchaotic attractors. memristor internal state is able to produce self-excited chaotic
attractors or self-excited hyperchaotic attractors. memristor internal state is able to produce self-excited chaotic
attractors or self-excited hyperchaotic attractors. J(p0) =
0
1/C1
0
0
0
−1/L1
−1/L1 · (β −1)
0
0
1/L1 · (β −1)
0
−1
−α
0
1
0
0
0
0
1/C2
0
1/L2 · (β −1)
0
−1/L2
−1/L2 · (β −1)
(11) (11) 151542
VOLUME 8, 2020 151542 L. Teng et al.: Hyperchaotic Behavior in the Novel Memristor-Based Symmetric Circuit System FIGURE 12. Hyperchaotic attractors of the second order internal state function symmetric system. OF BIFURCATION DIAGRAMS AND
iL1(0) = 0, x(0) = 0.1, vC2(0) = 0.1 and FIGURE 12. Hyperchaotic attractors of the second order internal state function symmetric system. B. ANALYSIS OF BIFURCATION DIAGRAMS AND
PHASE PORTRAITS iL1(0) = 0, x(0) = 0.1, vC2(0) = 0.1 and iL2(0) = 0, the
bifurcation diagram for control parameter β over the range
−3 ≤β ≤0 is generated in Figure 8(a). Fixing system
parameter as β = −1.5, the bifurcation diagram for control Fixing system parameter as α = 0.5 and the setting the
five initial condition of state variables as vC1(0) = 0.1, 151543 151543 V. CONCLUSION This article proposed a novel fifth-order memristor-based
symmetric circuit system. The chaotic behavior of this arti-
cle is studied. In order to obtain the hyperchaotic state,
we use multivariable first order and multivariable second
order polynomial functions for the memristor internal state
function respectively. The chaotic system can be hyperchaotic
when its parameters are taken appropriately. The stabil-
ity analysis, bifurcation diagrams, portraits and Lyapunov
exponents spectrum are performed for theoretical analysis. Simulation results show that the first order internal state
function memristor-based symmetric circuit system can only
generate chaotic attractors whereas the second order internal
state function memristor-based symmetric circuit system can
generate not only chaotic attractors, but also hyperchaotic
attractors in proper parameters. As future work, the physically
implement and the application of this hyperchaos system in
image encryption algorithms can be investigated. parameter α over the range 0 ≤α ≤1 is generated in
Figure 8(b). As can be seen from Figure 8(a), the periodic behavior is
exhibited when −0.37 < β ≤0 while the corresponding
phase portrait is shown in Figure 9(a). When in range
−1.8 < β < −0.38 the system shows the chaotic attractor,
which is plotted in Figure 9(b). When in range −1.39 < β
< −2.4 the system exhibits the limit cycle as shown in
Figure 9(c). Then in the range −3 ≤β < −2.4, the system
reveals the chaotic attractor again and shown in Figure 9(d). As can be seen from Figure 8(b), when over the approximate
range 0.3 < α < 1, the system is in chaotic state. To investigate the complex dynamic chaotic behaviors of
the five-dimensional symmetric memristive system with sec-
ond order internal state more deeply, SE and C0 complexity
varying with two system parameters are shown in Figure 10. The darker color areas are the intervals with bigger com-
plexity. So, when utilize the symmetric memristive system,
the parameters generate lighter color areas should be avoided. REFERENCES VOLUME 8, 2020 VOLUME 8, 2020 L. Teng et al.: Hyperchaotic Behavior in the Novel Memristor-Based Symmetric Circuit System FIGURE 13. Memristor characteristics of the second order internal state
function symmetric system. maximum Lyapunov exponent spectrum and the second
largest Lyapunov exponent spectrum. From Figure 11(b) and
the bifurcation diagram Figure 8(a), we can see that when
in range −1.77 < β
< −1 the system has two posi-
tive Lyapunov exponents and that means the second order
memristor-based symmetric circuit system is in hyperchaotic
state. The phase diagrams of Figure 9 and the corresponding
Lyapunov exponents results are shown in Table 2. From data
listed in Table 2, we can see that the systems with param-
eter selected in Figure 9(b) exhibit the hyperchaotic state,
the system simulated for Figure 9(d) shows the chaotic state,
while the systems parameter corresponding to Figure 9(a) and
Figure 9(c) produce a limit cycle and in periodic state. D. ANALYSIS OF CHAOTIC ATTRACTORS Set the parameters as β = −1 and α = 0.5, chose the
initial conditions as vC1(0) = 0.1, iL1(0) = 0, x(0) = 0.1,
vC2(0) = 0.1 and iL2(0) = 0 to simulate the second order
memristor function symmetric system. The hyperchaotic
attractors can be obtained as shown in Figure 12. We can see
that the system exhibits multiple-scroll attractor. When the second order memristor function symmetric
system exhibits the hyperchaotic state, the memristor
DCvM- iM curve (14) and the plot of vM- iM memristor
characteristics are shown in Figure 13.
xDC =
iM
α −i2
M
vM = R(xDC)iM
(14) (14) in this equation, set the iM to 10A amplitude sine-wave and
the frequency to 0.5Hz. FIGURE 13. Memristor characteristics of the second order internal state
function symmetric system. REFERENCES
[1] L. Chua, ‘‘Memristor—The missing circuit element,’’ IEEE Trans. Circuit
Theory, vol. CT-18, no. 5, pp. 507–519, Sep. 1971.
[2] D. B. Strukov, G. S. Snider, D. R. Stewart, and R. S. Williams, ‘‘The
missing memristor found,’’ Nature, vol. 453, no. 7191, pp. 80–83,
May 2008. [2] D. B. Strukov, G. S. Snider, D. R. Stewart, and R. S. Williams, ‘‘The
missing memristor found,’’ Nature, vol. 453, no. 7191, pp. 80–83,
May 2008. C. LYAPUNOV EXPONENT SPECTRA Gao, ‘‘Image encryption algorithm based on the matrix
semi-tensor product with a compound secret key produced by a Boolean
network,’’ Inf. Sci., vol. 539, pp. 195–214, Oct. 2020. [9] F. Gül, ‘‘Addressing the sneak-path problem in crossbar RRAM devices
using memristor-based one Schottky diode-one resistor array,’’ Results
Phys., vol. 12, pp. 1091–1096, Mar. 2019. [31] B. Muthuswamy and L. O. Chua, ‘‘Simplest chaotic circuit,’’ Int. J. Bifur-
cation Chaos, vol. 20, no. 5, pp. 1567–1580, May 2010. [32] A. Wolf, J. B. Swift, H. L. Swinney, and J. A. Vastano, ‘‘Determining
Lyapunov exponents from a time series,’’ Phys. D, Nonlinear Phenomena,
vol. 16, no. 3, pp. 285–317, Jul. 1985. [10] S. Wen, S. Xiao, Y. Yang, Z. Yan, Z. Zeng, and T. Huang, ‘‘Adjusting
learning rate of memristor-based multilayer neural networks via fuzzy
method,’’ IEEE Trans. Comput.-Aided Design Integr. Circuits Syst., vol. 38,
no. 6, pp. 1084–1094, Jun. 2019. [33] H. Kantz, ‘‘A robust method to estimate the maximal Lyapunov expo-
nent of a time series,’’ Phys. Lett. A, vol. 185, no. 1, pp. 77–87,
Jan. 1994. [11] S. Wen, R. Hu, Y. Yang, T. Huang, Z. Zeng, and Y.-D. Song, ‘‘Memristor-
based echo state network with online least mean square,’’ IEEE Trans. Syst., Man, Cybern., Syst., vol. 49, no. 9, pp. 1787–1796, Sep. 2019. [12] Y. V. Pershin, S. La Fontaine, and M. Di Ventra, ‘‘Memristive model
of amoeba learning,’’ Phys. Rev. E, Stat. Phys. Plasmas Fluids Relat. Interdiscip. Top., vol. 80, no. 2, Aug. 2009, Art. no. 012926. LIN TENG received the B.S. degree in software
engineering, the M.S. degree in computer software
and theory, and the Ph.D. degree in computer
applied from the Dalian University of Technology,
Liaoning, China, in 2009, 2012, and 2016, respec-
tively. She is currently a Postdoctoral Researcher
with the School of Information Science and Tech-
nology, Dalian Maritime University, China. Her
research interests include chaos cryptography,
image processing, secure communications, and
memristor. [13] M. Hu, Y. Chen, J. J. Yang, Y. Wang, and H. H. Li, ‘‘A compact memristor-
based dynamic synapse for spiking neural networks,’’ IEEE Trans. Comput.-Aided Design Integr. Circuits Syst., vol. 36, no. 8, pp. 1353–1366,
Aug. 2017. [14] X. Liu, M. Mao, B. Liu, B. Li, Y. Wang, H. Jiang, M. Barnell, Q. Wu,
J. Yang, H. Li, and Y. Chen, ‘‘Harmonica: A framework of heteroge-
neous computing systems with memristor-based neuromorphic computing
accelerators,’’ IEEE Trans. Circuits Syst. C. LYAPUNOV EXPONENT SPECTRA Fixing α = 0.5 and varying β, the Lyapunov exponents
spectrum is shown in Figure 11. Figure 11(b) shows the VOLUME 8, 2020 L. Teng et al.: Hyperchaotic Behavior in the Novel Memristor-Based Symmetric Circuit System [3] M. Itoh and L. O. Chua, ‘‘Memristor oscillators,’’ Int. J. Bifurcation Chaos,
vol. 18, no. 11, pp. 3183–3206, Nov. 2008. [25] Q. Li, H. Zeng, and J. Li, ‘‘Hyperchaos in a 4D memristive circuit
with infinitely many stable equilibria,’’ Nonlinear Dyn., vol. 79, no. 4,
pp. 2295–2308, Mar. 2015. [4] F. Corinto, A. Ascoli, and M. Gilli, ‘‘Nonlinear dynamics of memristor
oscillators,’’ IEEE Trans. Circuits Syst. I, Reg. Papers, vol. 58, no. 6,
pp. 1323–1336, Jun. 2011. [26] B. C. Bao, H. Bao, N. Wang, M. Chen, and Q. Xu, ‘‘Hidden extreme mul-
tistability in memristive hyperchaotic system,’’ Chaos, Solitons Fractals,
vol. 94, pp. 102–111, Jan. 2017. [5] D. Yu, H. H.-C. Iu, A. L. Fitch, and Y. Liang, ‘‘A floating memristor
emulator based relaxation oscillator,’’ IEEE Trans. Circuits Syst. I, Reg. Papers, vol. 61, no. 10, pp. 2888–2896, Oct. 2014. [27] B. Bao, T. Jiang, G. Wang, P. Jin, H. Bao, and M. Chen, ‘‘Two-
memristor-based Chua’s hyperchaotic circuit with plane equilibrium and
its extreme multistability,’’ Nonlinear Dyn., vol. 89, no. 2, pp. 1157–1171,
Jul. 2017. [6] H. Bao, N. Wang, H. Wu, Z. Song, and B. Bao, ‘‘Bi-stability in an
improved memristor-based third-order wien-bridge oscillator,’’ IETE Tech. Rev., vol. 36, no. 2, pp. 109–116, Mar. 2019. [28] X. Wang and S. Gao, ‘‘Image encryption algorithm for synchronously
updating Boolean networks based on matrix semi-tensor product theory,’’
Inf. Sci., vol. 507, pp. 16–36, Jan. 2020. [7] S. Pi, C. Li, H. Jiang, W. Xia, H. Xin, J. J. Yang, and Q. Xia, ‘‘Memristor
crossbar arrays with 6-nm half-pitch and 2-nm critical dimension,’’ Nature
Nanotechnol., vol. 14, no. 1, pp. 35–39, Jan. 2019. [29] X. Wang, L. Feng, and H. Zhao, ‘‘Fast image encryption algorithm
based on parallel computing system,’’ Inf. Sci., vol. 486, pp. 340–358,
Jun. 2019. [8] C. Li, Z. Wang, M. Rao, D. Belkin, W. Song, H. Jiang, P. Yan, Y. Li, P. Lin,
M. Hu, N. Ge, J. P. Strachan, M. Barnell, Q. Wu, R. S. Williams, J. J. Yang,
and Q. Xia, ‘‘Long short-term memory networks in memristor crossbar
arrays,’’ Nature Mach. Intell., vol. 1, no. 1, pp. 49–57, Jan. 2019. [30] X. Wang and S. XIAOLIN YE received the M.S. degree from
Dalian Polytechnic University, China, in 2018.
He is currently pursuing the Ph.D. degree in
nonlinear system analysis and image encryption
with Dalian Maritime University, China. C. LYAPUNOV EXPONENT SPECTRA I, Reg. Papers, vol. 63, no. 5,
pp. 617–628, May 2016. [15] J. Sun, X. Zhao, J. Fang, and Y. Wang, ‘‘Autonomous memristor chaotic
systems of infinite chaotic attractors and circuitry realization,’’ Nonlinear
Dyn., vol. 94, no. 4, pp. 2879–2887, Dec. 2018. [16] Y. Fan, X. Huang, Z. Wang, and Y. Li, ‘‘Nonlinear dynamics and chaos
in a simplified memristor-based fractional-order neural network with
discontinuous memductance function,’’ Nonlinear Dyn., vol. 93, no. 2,
pp. 611–627, Jul. 2018. XINGYUAN WANG received the Ph.D. degree in
computer software and theory from Northeastern
University, China, in 1999. From 1999 to 2001,
he was a Postdoctoral Researcher with Northeast-
ern University. He is currently a Professor with the
School of Information Science and Technology,
Dalian Maritime University, China. He has pub-
lished three books and over 400 scientific articles
in refereed journals and proceedings. His research
interests include nonlinear dynamics and con-
trol, image processing, chaos cryptography, systems biology, and complex
networks. trol, image processing,
networks. [17] B. Wang, ‘‘Results on a novel piecewise-linear memristor-based chaotic
system,’’ Complexity, vol. 2019, pp. 1–6, Jan. 2019. [18] G. Zhang, J. Ma, A. Alsaedi, B. Ahmad, and F. Alzahrani, ‘‘Dynamical
behavior and application in Josephson junction coupled by memristor,’’
Appl. Math. Comput., vol. 321, pp. 290–299, Mar. 2018. [19] M. Wang, Y. Deng, X. Liao, Z. Li, M. Ma, and Y. Zeng, ‘‘Dynamics
and circuit implementation of a four-wing memristive chaotic system
with attractor rotation,’’ Int. J. Non-Linear Mech., vol. 111, pp. 149–159,
May 2019. [20] O. E. Rössler, ‘‘An equation for hyperchaos,’’ Phys. Lett. A, vol. 71,
nos. 2–3, pp. 155–157, Apr. 1979. trol, image processing,
networks. [21] J. Kengne, ‘‘Coexistence of chaos with hyperchaos, period-3 doubling
bifurcation, and transient chaos in the hyperchaotic oscillator with gyra-
tors,’’ Int. J. Bifurcation Chaos, vol. 25, no. 4, pp. 4285–4762, 2015. [22] S. Vaidyanathan, L. G. Dolvis, K. Jacques, C.-H. Lien, and A. Sambas,
‘‘A new five-dimensional four-wing hyperchaotic system with hidden
attractor, its electronic circuit realisation and synchronisation via integral
sliding mode control,’’ Int. J. Model., Identificat. Control, vol. 32, no. 1,
pp. 30–45, 2019. XIAOLIN YE received the M.S. degree from
Dalian Polytechnic University, China, in 2018. He is currently pursuing the Ph.D. degree in
nonlinear system analysis and image encryption
with Dalian Maritime University, China. XIAOLIN YE received the M.S. degree from
Dalian Polytechnic University, China, in 2018. He is currently pursuing the Ph.D. C. LYAPUNOV EXPONENT SPECTRA degree in
nonlinear system analysis and image encryption
with Dalian Maritime University, China. XIAOLIN YE received the M.S. degree from
Dalian Polytechnic University, China, in 2018. He is currently pursuing the Ph.D. degree in
nonlinear system analysis and image encryption
with Dalian Maritime University, China. [23] Z. Wei, I. Moroz, J. C. Sprott, A. Akgul, and W. Zhang, ‘‘Hidden hyper-
chaos and electronic circuit application in a 5D self-exciting homopolar
disc dynamo,’’ Chaos, vol. 27, no. 3, pp. 647–650, 2017. [24] K. Rajagopal, A. Sambas, S. Kacar, U. Cavusoglu, S. Vaidyanathan,
and A. T. Azar, ‘‘A new hyperchaotic temperature fluctuations model, its
circuit simulation, FPGA implementation and an application to image
encryption,’’ Int. J. Simul. Process Model., vol. 13, no. 3, p. 281, 2018. 151545 VOLUME 8, 2020 VOLUME 8, 2020 VOLUME 8, 2020
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Medieval demise of a Himalayan giant summit induced by mega-landslide
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Nature
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Medieval demise of a Himalayan giant summit induced
by mega-landslide
Jérôme Lavé, Cyrielle Guérin, Pierre Valla, Valery Guillou, Thomas
Rigaudier, Lucilla Benedetti, Christian France-Lanord, Ananta Prasad
Gajurel, Guillaume Morin, Jean Pascal Dumoulin, et al.
To cite this version:
Jérôme Lavé, Cyrielle Guérin, Pierre Valla, Valery Guillou, Thomas Rigaudier, et al.. Medieval
demise of a Himalayan giant summit induced by mega-landslide. Nature, 2023, 619 (7968), pp.94-101.
�10.1038/s41586-023-06040-5�. �hal-04157846�
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Title: Medieval demise of a Himalayan giant summit induced by mega-landslide.
authors
Jérôme Lavé 1,*
Cyrielle Guérin 2
Pierre G. Valla 3
Valery Guillou 4
Thomas Rigaudier 1
Lucilla Benedetti 4
Christian France Lanord 1
Ananta Prasad Gajurel 5
Guillaume Morin 1#
Jean Pascal Dumoulin 6
Christophe Moreau 6
Valier Galy 7
affiliations
1. CRPG, CNRS, Université de Lorraine, Vandoeuvre les Nancy, France
2. CEA- DAM, DIF, Arpajon, France
3. ISTerre, CNRS, Université Grenoble Alpes, Grenoble, France
4. CEREGE, CNRS, Aix Marseille Université, Aix-en-Provence, France
5. Department of Geology, Tribhuvan University, Kathmandu, Nepal
6. LMC14, LSCE, CEA, CNRS, Université Paris-Saclay, Gif-sur-Yvette, France
7. Department of Marine Chemistry and Geochemistry, Woods Hole Oceanographic
Institution, Woods Hole, USA
# now at INRAE, Aix Marseille Univ, UMR RECOVER, Aix-en-Provence, France
* corresponding author, email address : jerome.lave@univ-lorraine.fr
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Abstract
Despite numerous studies on Himalayan erosion, it is not known how the very high Himalayan
peaks erode. While valley floors are efficiently eroded by glaciers, the intensity of periglacial
processes, which erode the headwalls extending from glacial cirques to crest-lines, appears to
decrease sharply with altitude1,2. This contrast suggests that erosion is muted and much less than
regional rock uplift rates for the highest Himalayan peaks raising questions about their long-term
evolution3,4. Here we report geological evidence for a giant rockslide that occurred around 1190
AD in the Annapurna massif (central Nepal), involving a total rock volume of ~23 km3. This event
collapsed a paleosummit culminating likely above 8000 m in altitude. Our data suggest that a mode
of high-altitude erosion could be mega-rockslides, leading to the sudden reduction of ridge crest
elevation by several hundred meters and ultimately preventing the disproportionate growth of the
Himalayan peaks. This erosion mode, associated with steep slopes and high relief arises from a
greater mechanical strength of the peak substratum, probably due to the presence of permafrost at
high altitude. Giant rockslides also have implications for landscape evolution and natural hazards:
the massive supply of finely-crushed sediments can fill valleys >150 km farther downstream and
overwhelm the sediment load in Himalayan rivers for a century or more.
Main text
On the human time scales, mountain peaks appear eternal. Yet, on geological time scales,
mountain peaks are ephemeral: their shape and altitude are constantly evolving in response to the
competition between tectonic uplift and erosion. However, very few direct observations exist of
how Earth’s highest mountain peaks evolve. Whereas their growth can be considered progressive
over time, we don’t know if their erosion is also progressive or episodic, or whether summit
erosion proceeds at the same rates as valley-floor lowering5?
It has been postulated that a glacial buzzsaw mechanism places an upper limit on mountain
elevation and relief irrespective of the tectonic uplift rate3,6, confining mountain heights to within
about 1.5 km of the local Equilibrium Line Altitude7 (ELA). In this concept, efficient periglacial
weathering, frost cracking, and rock falls along steep hillslopes produce rapid retreat of glacier
cirque headwalls, limiting peak elevation at rates commensurate with rapid valley-floor
downcutting by glaciers8. In various settings, however, this concept has been challenged9-12. In the
Himalayas, one of the most active mountain range on Earth, the >8000-m peaks culminate ~3000
m above the ELA and seem to contradict the notion of a comprehensively operating glacial
buzzsaw1,12. At these extreme elevations, local measurements of cirque headwall retreat show rates
mostly lower than regional erosion rates13-16. Frozen-based ice cover, as well as a sharp decrease in
frost-cracking efficiency for increasing altitudes, due to the absence of common freeze-thaw
cycles1,2, are suspected to strongly limit the erosion of the glaciated peaks protruding far above the
ELA1. If high-elevation summit erosion is unable to match rapid valley-floor downcutting rates,
this mismatch could lead in theory to disproportionate growth of the Himalayan peaks.
In steep and active mountains, it is conjectured, however, that mechanical strength of the
mountain rock mass sets an upper limit to peak elevation and the height of oversteepened slopes4,
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such that slope adjustment might occur through landslides at a rate paced by river downcutting.
This equilibrium has been corroborated in non-glaciated and vegetated landscapes, where
landslides can be tracked from satellite-image inventory17-19. The 2021 rock-and-ice avalanche of
the Ronti peak (India)20 illustrated that landslides can also contribute dramatically to the erosion of
high-elevation glaciated Himalayan peaks. Nevertheless, the lack of systematic landslide
inventories precludes any reliable assessment of the relative contribution of this erosive process in
long-term evolution of high-elevation mountainous areas. Instead, the few coseismic landslide
inventories show an apparent sharp decrease in landslide occurrence at high elevations21. This
observation indicates either an erosion mechanism distinct from landsliding, or a size-frequency
distribution of landslides17 that would be clearly skewed in favour of very large landslides,
remaining infrequent enough not to be seen by remote sensing techniques.
In the present study, we document evidence of a recent giant rockslide in the Annapurna
region (central Himalayas, Nepal; Fig.1), which illuminates the topographic evolution of high
Himalayan peaks, their abrupt erosion by large rockslides, and the disproportionate consequences
of such collapses along downstream drainage systems.
Uncovering a giant rockslide deposit
The southern flank of the Annapurna massif, currently culminating at 8091 m a.s.l., presents
a 6 km drop in elevation from the high summits to the Lesser Himalayas hills. The area
encompasses steep slopes, active microseismicity22, and strong ground shaking by major
earthquakes23,24 (Mw≥8). Heavy monsoon precipitation prevails over its southern slopes. This
tectonic and climatic setting induces some of the highest regional erosion rates in the Himalayan
range, with values of 3-5 mm/yr25-27.
The Annapurna massif, dominated by calcareous metasediments of the Tethyan
Sedimentary Series28,29 (TSS) (Extended Data Fig. 1), is punctuated by several large glacial cirques,
separated by >7500-m glaciated peaks (Fig. 1). Between Annapurna III and IV, the Sabche cirque
is a deep and wide depression bordered by extremely steep cliffs (Fig. 1). The cirque bottom is
filled with light-colored sedimentary deposits, which have been eroded into large and tapering
penitents several hundred meters high (Fig. 1d). Inaccessibility has so far prevented precise field
description of these deposits, which have been interpreted from afar as subglacial tills or
glaciolacustrine sediments30. Up close, these massive deposits are well consolidated, especially in
the southern, most basal part, comprising finely-crushed matrix-supported breccia with centimetric
to decimetric angular clasts from various TSS carbonate lithologies (Extended Data Fig. 2 and
Supp. Info. SI-1). The coarse elements are more abundant in the upper part of the deposit and reach
sizes of several meters. The breccia color is quite uniform (white to grayish) except locally with
yellowish units reminiscent of the yellow limestones of the Annapurnas28 (Fig. 1c). The breccia
sediment infill appears as relatively continuous without any apparent unconformity but displays
incomplete mixing, internal shear zones and rare jigsaw facies (Extended Data Fig. 2). These
sedimentary features31, the intense rock fragmentation, the exceptional thickness (>400 m _ Fig.
1d), and the exclusive presence of these breccia among all cirques of the Annapurna massif,
unequivocally indicate that these breccia sediments were generated by a single rockslide granular
avalanche.
The elevation of the breccia remnants along the southern side of the cirque and of a few
preserved top surfaces in its northern part (Fig. 1) suggests that the cirque was completely filled by
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rockslide debris (Supp. Info. SI-4), with thickness locally >1 km. In addition, a debris avalanche
overflowed the cirque and spilled into the upper Seti valley where scattered outcrops of massive
breccia deposits can be observed for ~10km downstream along the valley walls or as isolated >200m-high massive spur remnants (Extended Data Fig. 3 and Supp. Info. SI-2). These remnants are
dominated by angular TSS limestone clasts in a finely-fragmented and pulverized carbonate-rich
matrix. Little mixing appears at the metric scale (as observed for Sturzströme in motion32), dyke
injections are deformed by brittle deformation, and the basal shearing contact with the gneissicbedrock valley flanks is overlain by a mixing zone incorporating fragmented vegetal debris.
Volume and Age of the rockslide deposit
Subtraction of the restored basal topography of the Sabche cirque from the initial breccia
deposit surface provides an estimated volume of the rock avalanche deposits of 23.5 +4/-3km3. An
additional volume of ~3.5 km3 is similarly inferred along the upper Seti valley (see Method,
Extended Data Fig. 4 and Supp. Info SI-4). Accounting for an average breccia porosity of 15 ±5%
(Supp. Info SI-5), this reconstructed deposit would correspond to a total volume of collapsed rock
of 23 +3.5/-3km3, constituting the largest rockslide described in the Himalayas to date33,34.
Four 2- to 5-m high blocks of Silurian marly limestone were sampled on a large blocky and
chaotic remnant of the original deposit surface (Extended Data Fig. 5) for 36Cl surface-exposure
dating (Supp. Info table SI-11-2). They display consistent 36Cl concentrations with an average
surface-exposure age of 1196 ±75(2) yr AD (fig. 2a).
In parallel, infra-red stimulated luminescence (IRSL) dating on feldspar were conducted on
three breccia samples from an internal shear zone, assuming that increased temperature and/or
shear heating35 during the rock avalanche did partially reset the IRSL signal (Extended Data Fig. 6,
and Supp. Info. SI-12). The observed partial reset is moderate for two samples with average
apparent IRSL ages of several kyr, but is highly pronounced for one sample taken in the mostdeformed levels. For the latter, the IRSL age distribution is highly-scattered with an apparent
modal age of ~1200 yr BP and an estimated minimum age of ~900 yr BP (Fig. 2a), consistent with
surface-exposure dating of the top relict surface.
Finally, eight 14C analyses on plant fragments of stripped vegetation in the contact zone
between rock-avalanche material and bedrock in the upper Seti valley (site K on Fig. 1), provided
reproducible calibrated ages (Fig. 2a, Supp. Info. Table SI-10-1). The most probable age of 1190
±26(2 AD is strikingly consistent with the 36Cl surface-exposure ages of the rockslide deposit
surface, thereby confirming the monogenic nature of the rock avalanche between the Sabche cirque
and the upper Seti valley.
Reconstruction of the collapsed paleo-peak
In order to understand the origin of the collapsed rock mass, we identified the failure
surface in the NE cirque part below the Annapurna IV (Figs. 1 and 3). There, the morphology of
the rock cliffs stands out (see Methods and Extended Data Fig. 4d) with fresh planar or prismatic
structural surfaces, devoid of any major glacier or runoff-related erosion. The proposed giant
rockslide, collapsing along this uneroded failure surface, would have beheaded the paleo-summit of
Annapurna IV.
To reconstruct the initial geometry of the paleo-Annapurna IV from the restored rock
volume of 23 +3.5/-3km3, we hypothesized that the stability of the paleo-summit hillslopes follows
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mechanical characteristics that prevail for the whole Annapurna massif, for similar altitudes and
lithologies. Stability of 82-100% of the high elevation slopes requires an apparent rock mass
cohesion of 1-2 MPa, assuming a friction angle of 35 degrees (Methods and Extended Data Fig. 7
& 8). For these geomechanical properties, we generated thousands of random possible topographies
for the Annapurna IV paleo-summit, and considered as acceptable only those fitting both the
collapsed rock volume and the regional hillslope stability (Methods and Extended Data Fig. 9).
Through this Bayesian approach, we estimated that the paleo-Annapurna IV may have peaked at an
average elevation of ~8100 m (~600 m above the modern summit and comparable to the
Annapurna I), and exceeded 8000 m with a probability of 0.65-0.76 (Fig. 3d). The eroded volume
corresponds to an average 1 km of bedrock lowering, and involves lithologies ranging from
Silurian to Cambrian (Fig. 3c) in good agreement with the diversity of colour and carbonate content
of the breccia material (Fig. 4)
Sediment export and downstream consequences
The collapse released a huge amount of potential gravitational energy of ~1.2x1018 J (Fig.
3). Significant energy release was consumed in rock-material fragmentation, which, aided by the
schistose nature of the marly-calcareous bedrock, likely explains the extreme degree of
disintegration observed in the breccia material. After the paleo-summit collapse ~830 years ago, the
erosion of the fine-grained breccia by rainwash, landsliding, pro-glacial streams, or glaciers, and
their export by the upper Seti river, were extremely efficient and rapid: of the initial 23.5 km3 of
rockslide debris, only ~10% can still be observed today in the Sabche cirque (Extended Data Fig.
4c). Assuming long-term denudation rate of ~3mm/yr in the High Himalaya26, the return time of a
giant rockslide of similar volume would be >100 kyr at the scale of the entire Sabche cirque
headwalls. The response time of the glacio-fluvial system to evacuate the Sabche breccia material
appears therefore very short in comparison.
Farther downstream of the Seti river, the Pokhara Basin (Fig. 1) is known to have been
filled very rapidly 500-1000 years ago30,36 by ~5 km3 of mostly conglomeratic sediment with
calcareous clasts, whose only possible origin is the Sabche cirque. Sedimentary fill is characterized
by the stratigraphic continuity of a succession of planar beds with varied facies (Supp Info. SI-9)
that reflect rapid aggradation by fluvial, hyperconcentrated, or turbulent, sediment-laden flows30,36.
Extensive 14C dating of organic fragments found in the fine-grained units of the Pokhara
sediments36-38, collected at different burial depths, and complemented by eight new dates (Supp.
Info. table SI-10-1), provide robust constraints on the timing of aggradation (Fig. 2b). They
indicate onset of aggradation around 1200 AD at an average rate of 1m/yr until ~1300 AD. Taken
together, the similarity in carbonate content (Fig. 4) and the age of the basal units in close
agreement with the giant rockslide age, but stratigraphically postdating the breccia (Extended Data
Fig. 3a) strongly suggest a co-genetic link between these two sedimentary units. The Pokhara
conglomerates are thus best explained by the active erosion of the Sabche rock-avalanches breccia
and by the rapid sediment transfer by Seti river down to Pokhara valley.
Fine conglomerates with calcareous pebbles irregularly crop out far downstream of
Pokhara, along the Seti, the Trisuli and even the Narayani river (Fig. 4) in the foreland (site N on
Fig. 1). There, a fine, carbonate-rich conglomerate unit, dated at 1150-1220AD (14C dating, Fig.
2a), shows how the Narayani was temporarily overwhelmed by sediments from the paleoAnnapurna IV collapse. Such sediment pulse may even have reached the mid-Ganga plain, as
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suggested by a >3-m thick unit at the top of a core drilled into an elevated Narayani megafan lobe
(site G on Fig. 1). This unit is anomalously rich in TSS carbonate (30-60%) as compared to the
average proportion of ~10% in Late-Pleistocene to modern Narayani sediments (Fig. 4, and
Extended Data Fig. 10).
The amalgamated sediment volume in the Pokhara basin, middle Seti valley, and Narayani
megafan lobe may have reached 15 km3 (Supp. Info. SI-9-C), thereby representing ≤50% of the
initial Sabche breccia volume (assuming 30% fluvial sediment porosity). The other half was
presumably partly dissolved and partly exported farther downstream as suspended load, and may
have ended up in the Bengal fan, the largest sediment sink for most Himalayan sediment39.
Sediment aggradation in Pokhara valley suggests that the most intense phase of breccia
removal in the Sabche cirque and associated sediment supply downstream lasted for a century (Fig.
2b) and impacted the whole downstream sediment routing system. During that period, the rate of
sediment yield (0.1-0.2 km3/yr exported from a 60-km2 area) was comparable to the highest
measured values of post-volcanic eruption sediment transport40 and led to an overwhelming content
of TSS material in river sediments >300 km downstream. Compared to the Narayani basin (30,000
km2), which exports annually ~0.05km3/yr of sediment41,42, post-collapse erosion of the Sabche
breccias (only 0.2% of the Narayani total basin area) would have increased this annual flux by a
factor of 3 over 100-150 years (see also carbonate-based budget in Supp. Info. SI-9-D).
The collapse has paradoxically positive and negative downstream consequences. In the long
term, the creation of a large flat surface in the Pokhara valley has undoubtedly been beneficial,
allowing the development of extensive agricultural areas, and, above all, the recent development of
the country's second largest city with all of its infrastructures. On the contrary, in the short term, if
the paleo-Annapurna IV collapse occurred today, the Pokhara metropolitan area would be
decimated, with appalling human and economic costs. Not only would the upstream parts of the
valley be swept away by the granular avalanche, but numerous sediment-laden flows would rapidly
bury the valley under tens of meters of sediment, leaving a vast gravel plain in the Seti valley. Even
the Indian side of the Ganga plain, i.e. a densely populated region, might be impacted by increased
probability of river-channel avulsions and rapid sedimentation along the Narayani megafan.
Although such events are rare, understanding the causes of the paleo-Annapurna IV collapse and its
downstream consequences appears thus fundamental to comprehend the major threat that potential
giant rockslides can pose to the local populations and infrastructures in these high-mountain
regions.
Possible causes for the Sabche rockslide
Several factors make the High Himalaya region clearly prone to large-scale slope failure.
Glacial valley deepening due to sub-glacial carving, combined with intense frost cracking close to
the ELA2, represents one of the major pre-conditioning factors that produce steep, unstable
headwalls surrounding glacier cirques. Schistosity orientation and structural planes also likely
guide slope failure of the paleo-Annapurna IV (Extended Data Fig. 4d).
By contrast, causes triggering the Sabche rockslide remain speculative. It has been
suggested previously36 that the filling of the Pokhara Basin arose from various pulses of debris
flows following numerous landslides activated by 3 large earthquakes, at 1100 ±50AD23, 1255AD
and 1344 AD24. According to 14C dating of breccia at Karuwa (site K on Fig. 1), the Sabche
rockslide occurred after 1150AD and before 1255AD at >99% confidence. Seismic triggering due
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to one of these 3 large earthquakes seems therefore excluded. Nevertheless, as the current catalog
of paleo-earthquakes is likely incomplete for medieval times, we cannot exclude the impact of an
undocumented earthquake in the region.
Independently, climate might also be involved as a preparatory or/and triggering factor for
the Sabche collapse. The Medieval Climate Anomaly43, from 900 to 1200AD, was possibly
characterized by >+1°C warmer temperatures in the High Himalaya. This could have thawed part
of the failure surface (see estimate in Supp. Info. SI-8) and favoured high-elevation rockslide(s)20.
Although the cohesion loss from thawing appears to have been likely insufficient to trigger the
paleo-Annapurna IV collapse (unless the rock mass was already close to a critical state), such
climatic factor on high-elevation bedrock destabilization calls for closer attention on the effects of
current global warming when assessing future gravitational hazards related to the high Himalayan
peaks.
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The fate of the High Himalayan peaks
The identification of a cataclysmic rockslide around 1190 AD, which beheaded what may
have been the Himalaya's 15th 8,000-m peak, provides key insights into the likely evolution of the
highest Himalayan peaks. First, this is not an isolated case. Several giant rockslides have been
documented33,34,44 in central Nepal with deposits reaching up to 10-15 km3. All are located in the
high Himalayas and originated from summits/ridges between 4500 and 6000m elevation. Although
the current catalogue is incomplete and precludes definition of a precise erosion budget, massive
summit collapses resulting in a sudden reduction of their height by several hundred meters could be
the primary denudation mode of high, glaciated Himalayan peaks.
Notably, high summits commonly display steeper slopes, with median values rising from
35° below the ELA to 50° above it2,45. Steep slopes surrounding the glacial cirques of the
Annapurnas are maintained by higher bulk material strength. The regional cohesion values (C≈12MPa) calculated for the glaciated relief cannot be considered as absolute values, because the
transient effects of pore pressure or seismic acceleration have not been taken into account46.
Nevertheless, they appear, for similar internal friction angles, ~4 times higher than the cohesion
values (C≈0.2-0.7MPa) for the fluvially-dominated High Himalayan landforms (below 4000 m,
south of the glaciated Annapurna ridges _ Supp. Info. SI-6), and an order of magnitude higher than
in other non-glaciated high mountains47 (C≤0.1MPa but using a distinct approach to derive
cohesion values). In the Himalayas, this higher rock strength is not related to a lithological effect12,
given that the steeper slopes at high altitudes in central Himalaya have values independent of the
rock type2. Instead, we surmise that the higher rock mass strength is favoured by permanently
frozen conditions at high elevations. Such climatic conditions hold rocks in place by ice-rock
interlocking48,49, while preventing regolith formation, penetrative chemical weathering, and
limiting groundwater recharge and high pore pressures at depth. If permafrost strengthens the first
tens or hundreds of meters below the ground surface (Supp. Info. SI-8), and/or if weakened
bedrock is absent near the surface, then the initiation of small and medium-sized landslides
becomes more difficult. This could explain their very rare occurrence at high altitudes, and the
need to erode the high summits through rare, but large deep-seated rockslides.
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The above considerations on the evolution of glaciated high peaks allow us to revisit the
concept of glacial buzzsaw. While topographic data suggest that the mean elevation of mountain
ranges may be directly limited by active glacial and periglacial erosion processes3,7, the glacial
buzzsaw mechanism is not sufficiently effective, where rock uplift is sustained, to prevent the
emergence of high peaks >3000 m above the snowline12, as observed in the Annapurnas massif.
On the contrary, once a mountain peak passes over the zone where rock mass is permanently frozen
and the intensity of frost cracking drops drastically, the frozen conditions provide enhanced
protection against surface erosion. This may even initiate a positive feedback. The increased
mechanical strength of the frozen, unweathered rock mass promotes headwall stability, and an
increase in both their length and steepness46. In turn, the increased steepness of the headwalls that
prevents the accumulation of ice and glacial erosion11, combined with headwall lengthening12,
allows the growth of high peaks and their further protrusion into the realm of permanently frozen
and muted periglacial erosion conditions (Fig. 5). This positive feedback has a limit, however, set
by the cohesion of the deep unweathered rock mass, and our observations suggest that deep-seated
rockslides are ultimately the controlling mechanism that limits the persistent growth of the high
Himalayan glaciated summits (Fig. 5).
Such a conceptual scenario (Fig. 5) might operate both on a regional (central Himalaya) and
global scale. Many large-scale tectonic zones undergoing local sustained uplift rate present high
peaks that protrude well above the snow line (i.e. by 2500 to 4000 m), either in the Himalayan
syntaxes (Nanga Parbat and Namche Barwa), the Karakoram, the Pamir, the Western Kunlun
Cordillera, the Gongga Shan, the central TianShan (Jengish Chokusu and Khan Tengri massifs), or,
far from the India-Asia collision, e.g. in the Denali or St Elias massifs in Alaska. Future work
should assess the erosive contribution of giant rockslides on the long-term topographic evolution of
these regions, and also quantify a likely relationship between the rock uplift rate and the amplitude
of relief above the snowline50.
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References :
1. Banerjee, A., & Wani, B. A. (2018). Exponentially decreasing erosion rates protect the
high-elevation crests of the Himalaya. Earth and Planetary Science Letters, 497, 22-28.
2. Scherler, D. (2014). Climatic limits to headwall retreat in the Khumbu Himalaya, eastern
Nepal. Geology, 42(11), 1019-1022.
3. Brozovic, N., Burbank, D. W., & Meigs, A. J. (1997). Climatic limits on landscape
development in the northwestern Himalaya. Science, 276(5312), 571-574
4. Burbank, D. W., Leland, J., Fielding, E., Anderson, R. S., Brozovic, N., Reid, M. R., &
Duncan, C. (1996). Bedrock incision, rock uplift and threshold hillslopes in the
northwestern Himalayas. Nature, 379(6565), 505-510.
5. Molnar, P., & England, P. (1990). Late Cenozoic uplift of mountain ranges and global
climate change: chicken or egg?. Nature, 346(6279), 29-34.
6. Montgomery, D. R., Balco, G., & Willett, S. D. (2001). Climate, tectonics, and the
morphology of the Andes. Geology, 29(7), 579-582.
7. Egholm, D. L., Nielsen, S. B., Pedersen, V. K., & Lesemann, J. E. (2009). Glacial effects
limiting mountain height. Nature, 460(7257), 884-887.
8. Scherler, D., Bookhagen, B., & Strecker, M. R. (2011). Hillslope‐glacier coupling: The
interplay of topography and glacial dynamics in High Asia. Journal of Geophysical
Research: Earth Surface, 116(F2)
9. Thomson, S. N., Brandon, M. T., Tomkin, J. H., Reiners, P. W., Vásquez, C., & Wilson, N.
J. (2010). Glaciation as a destructive and constructive control on mountain building. Nature,
467(7313), 313-317.
10. Valla, P. G., Shuster, D. L., & Van Der Beek, P. A. (2011). Significant increase in relief of
the European Alps during mid-Pleistocene glaciations. Nature geoscience, 4(10), 688-692;
11. Ward, D. J., Anderson, R. S., & Haeussler, P. J. (2012). Scaling the Teflon Peaks: Rock
type and the generation of extreme relief in the glaciated western Alaska Range. Journal of
Geophysical Research: Earth Surface, 117(F1).
12. Brocklehurst, S. H., & Whipple, K. X. (2007). Response of glacial landscapes to spatial
variations in rock uplift rate. Journal of Geophysical Research: Earth Surface, 112(F2).
13. Heimsath, A. M., & McGlynn, R. (2008). Quantifying periglacial erosion in the Nepal high
Himalaya. Geomorphology, 97(1-2), 5-23.
14. Seong, Y. B., Owen, L. A., Caffee, M. W., Kamp, U., Bishop, M. P., Bush, A., ... &
Shroder, J. F. (2009). Rates of basin-wide rockwall retreat in the K2 region of the Central
Karakoram defined by terrestrial cosmogenic nuclide 10Be. Geomorphology, 107(3-4),
254-262.
15. Scherler, D., & Egholm, D. L. (2020). Production and transport of supraglacial debris:
Insights from cosmogenic 10Be and numerical modeling, Chhota Shigri Glacier, Indian
Himalaya. Journal of Geophysical Research: Earth Surface, 125(10), e2020JF005586.
16. Orr, E. N., Owen, L. A., Saha, S., Hammer, S. J., & Caffee, M. W. (2021). Rockwall slope
erosion in the northwestern Himalaya. Journal of Geophysical Research: Earth Surface,
126(2), e2020JF005619.
17. Hovius, N., Stark, C. P., & Allen, P. A. (1997). Sediment flux from a mountain belt derived
by landslide mapping. Geology, 25(3), 231-234.
9
367
368
369
370
371
372
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
18. Larsen, I. J., & Montgomery, D. R. (2012). Landslide erosion coupled to tectonics and river
incision. Nature Geoscience, 5(7), 468-473.
19. Marc, O., Behling, R., Andermann, C., Turowski, J. M., Illien, L., Roessner, S., & Hovius,
N. (2019). Long-term erosion of the Nepal Himalayas by bedrock landsliding: the role of
monsoons, earthquakes and giant landslides. Earth Surface Dynamics, 7(1), 107-128.
20. Shugar, D. H., Jacquemart, M., Shean, D., Bhushan, S., Upadhyay, K., Sattar, A., ... &
Westoby, M. J. (2021). A massive rock and ice avalanche caused the 2021 disaster at
Chamoli, Indian Himalaya. Science, 373(6552), 300-306.
21. Roback, K., Clark, M. K., West, A. J., Zekkos, D., Li, G., Gallen, S. F., ... & Godt, J. W.
(2018). The size, distribution, and mobility of landslides caused by the 2015 Mw7. 8
Gorkha earthquake, Nepal. Geomorphology, 301, 121-138.
22. Pandey, M. R., Tandukar, R. P., Avouac, J. P., Lave, J., & Massot, J. P. (1995). Interseismic
strain accumulation on the Himalayan crustal ramp (Nepal). Geophysical Research Letters,
22(7), 751-754.
23. Lavé, J., Yule, D., Sapkota, S., Basenta, K., Madden, C., Attal, M., Pandey, R.,
2005.Evidencefor a Great Medieval Earthquake (~A.D. 1100) in Central Himalaya, Nepal.
Science307, 1302–1305.
24. Bollinger, L., Sapkota, S. N., Tapponnier, P., Klinger, Y., Rizza, M., Van der Woerd, J., ...
& Bes de Berc, S. (2014). Estimating the return times of great Himalayan earthquakes in
eastern Nepal: Evidence from the Patu and Bardibas strands of the Main Frontal Thrust.
Journal of Geophysical Research: Solid Earth, 119(9), 7123-7163.
25. Lavé, J., Avouac, J.P., 2001.Fluvial incision and tectonic uplift across the Himalayas of
Central Nepal. J. Geophys. Res. 106 (B11), 26561–26591.
26. Burbank, D. W., Blythe, A. E., Putkonen, J., Pratt-Sitaula, B. G. A. B. E. T., Gabet, E.,
Oskin, M., ... & Ojha, T. P. (2003). Decoupling of erosion and precipitation in the
Himalayas. Nature, 426(6967), 652-655.
27. Godard, V., Burbank, D. W., Bourlès, D. L., Bookhagen, B., Braucher, R., & Fisher, G. B.
(2012). Impact of glacial erosion on 10Be concentrations in fluvial sediments of the
Marsyandi catchment, central Nepal. Journal of Geophysical Research: Earth Surface,
117(F3).
28. Colchen, M., Le Fort, P., & Pêcher, A. (1986). Recherches geologiques dans l'Himalaya du
Nepal. CNRS.
29. Godin, L. (2003). Structural evolution of the Tethyan sedimentary sequence in the
Annapurna area, central Nepal Himalaya. Journal of Asian Earth Sciences, 22(4), 307-328.
30. Fort, M. (1987). Sporadic morphogenesis in a continental subduction setting: an example
from the Annapurna Range, Nepal Himalaya. Zeitschrift für Geomorphologie, 63(9), 36.
31. Weidinger, J. T., Korup, O., Munack, H., Altenberger, U., Dunning, S. A., Tippelt, G., &
Lottermoser, W. (2014). Giant rockslides from the inside. Earth and Planetary Science
Letters, 389, 62-73.
32. Bowman, E. T., Take, W. A., Rait, K. L., & Hann, C. (2012). Physical models of rock
avalanche spreading behaviour with dynamic fragmentation. Canadian Geotechnical
Journal, 49(4), 460-476.
10
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448
449
450
451
33. Weidinger, J.T., Schramm, J.-M., Surenian, R., 1996. On preparatory causal factors,
initiating the prehistoric Tsergo Ri landslide (Langthang Himal, Nepal). Tectonophysics
260,95–107.
34. Weidinger, J.T., 2006, Predesign, failure and displacement mechanisms of large rockslides
in the Annapurna Himalayas, Nepal. Eng. Geol. 83 (1), 201–216.
35. Bateman, M. D., Swift, D. A., Piotrowski, J. A., Rhodes, E. J., & Damsgaard, A. (2018).
Can glacial shearing of sediment reset the signal used for luminescence dating?
Geomorphology, 306, 90–101.
36. Schwanghart, W., Bernhardt, A., Stolle, A., Hoelzmann, P., Adhikari, B. R., Andermann,
C., ... & Korup, O. (2016). Repeated catastrophic valley infill following medieval
earthquakes in the Nepal Himalaya. Science, 351(6269), 147-150.
37. Stolle, A., Bernhardt, A., Schwanghart, W., Hoelzmann, P., Adhikari, B. R., Fort, M., &
Korup, O. (2017). Catastrophic valley fills record large Himalayan earthquakes, Pokhara,
Nepal. Quaternary Science Reviews, 177, 88-103.
38. Stolle, A., Schwanghart, W., Andermann, C., Bernhardt, A., Fort, M., Jansen, J. D., ... &
Korup, O. (2019). Protracted river response to medieval earthquakes. Earth Surface
Processes and Landforms, 44(1), 331-341.
39. Lupker, M., France‐Lanord, C., Lavé, J., Bouchez, J., Galy, V., Métivier, F., ... & Mugnier,
J. L. (2011). A Rouse‐based method to integrate the chemical composition of river
sediments: Application to the Ganga basin. Journal of Geophysical Research: Earth
Surface, 116(F4).
40. Hayes, S. K., Montgomery, D. R., & Newhall, C. G. (2002). Fluvial sediment transport and
deposition following the 1991 eruption of Mount Pinatubo. Geomorphology, 45(3-4), 211224.
41. Lupker, M., Blard, P.-H., Lavé, J., France-Lanord, C., Leanni, L., Puchol, N., Charreau,
J.,Bourlès, D., 2012.10Be-derived Himalayan denudation rates and sediment budgetsin the
Ganga basin. Earth Planet. Sci. Lett. 333–334, 146–156
42. Morin, G. P., Lavé, J., France‐Lanord, C., Rigaudier, T., Gajurel, A. P., & Sinha, R. (2018).
Annual sediment transport dynamics in the Narayani basin, Central Nepal: assessing the
impacts of erosion processes in the annual sediment budget. Journal of Geophysical
Research: Earth Surface, 123(10), 2341-2376.
43. Mann, M. E., Zhang, Z., Rutherford, S., Bradley, R. S., Hughes, M. K., Shindell, D., ... &
Ni, F. (2009). Global signatures and dynamical origins of the Little Ice Age and Medieval
Climate Anomaly. science, 326(5957), 1256-1260.
44. Fort, M. (2011). Two large late Quaternary rock slope failures and their geomorphic
significance, Annapurna Himalayas (Nepal). Geografia Fisica e Dinamica Quaternaria,
34(1), 5-14.
45. Gabet, E.J., Pratt-Sitaula, B.A., Burbank, D.W., 2004.Climatic controls on hillslope angle
and relief in the Himalayas. Geology 32 (7), 629–632
46. Schmidt, K. M., & Montgomery, D. R. (1995). Limits to relief. Science, 270(5236), 617620.
47. Gallen, S. F., Clark, M. K., & Godt, J. W. (2015). Coseismic landslides reveal near-surface
rock strength in a high-relief, tectonically active setting. Geology, 43(1), 11-14.
11
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485
486
487
488
489
490
491
492
493
494
48. Davies, M.C.R., Hamza, O., Lumsden, B.W., and Harris, C., 2000, Laboratory
measurement of the shear strength of ice-filled rock joints: Annals of Glaciology, v. 31, p.
463–467, doi:10.3189/172756400781819897.
49. Gruber, S., and Haeberli, W., 2007, Permafrost in steep bedrock slopes and its temperaturerelated destabilization following climate change: Journal of Geophysical Research, v. 112,
F02S18, doi:10.1029/2006JF000547.
50. Pedersen, V. K., Egholm, D. L., & Nielsen, S. B. (2010). Alpine glacial topography and the
rate of rock column uplift: a global perspective. Geomorphology, 122(1-2), 129-139.
Figure Legends :
Figure 1: Location maps of the Sabche breccia deposits. (a) Map of Nepal and Narayani basin (in
red) with the >8000m summits (triangles) (G=drill cores GR1-2 (purple symbols) in Dudahi in India;
S=Sabche; P=Pokhara; k=Kathmandu). (b) Topographic map of central Nepal focusing on the Seti
river with the rock-avalanche breccia deposit (in yellow) in the Sabche cirque (headwaters of the Seti
river) and along the upper Seti valley due to the rockslide of the Annapurna IV SW-flank. Subsequent
erosion of this brecciated material led to the downstream sediment aggradation with massive
conglomeratic terraces (in pink) in Pokhara basin and as far as south of the Main Boundary Thrust
(MBT), near Narayangad (N). (S=Sabche; K=Karuwa; P=Pokhara; MCT=Main Central Thrust;
STD=South Tibetan Detachment). (c) Zoomed map of the glaciated Sabche cirque showing contrasting
breccia facies of and sample locations. (d) Aerial view of the Sabche cirque; its breccia filling
(outlined by dashed line and ‘br’) exposed by large whitish penitents, hundreds of meters high, left by
the erosion of the rock avalanche deposits.
Figure 2: Chronological constraints on deposits related to the Sabche rockslide. (a) Dating of the
rock-avalanche deposit and Pokhara conglomeratic formation along the Seti river from the Sabche
cirque down to Narayangad (site locations according to letters on fig. 1). From top to bottom: the
probability density function (pdf) of 36Cl exposure ages on blocks located on a upper surface relic of
the rockslide deposit; zoom-in on the young tail of the feldspar IRSL age pdf showing partial signal
reset (see complete pdf and individual aliquot ages on Extended Data Fig. 6) of a shearing band toward
the base of the rockslide deposit in Sabche cirque; calibrated 14C age pdf (black or gray solid lines) of
wood debris sampled in the rock avalanche deposit in Karuwa (site K on Fig.1b) at the bedrock contact
of the Seti valley; calibrated 14C age pdf of organic fragments taken in the most basal conglomeratic
units in Pokhara basin36,38; calibrated 14C age pdf of a charcoal sampled in fine conglomerates north of
Narayangad. The red curve represents the composite pdf, combining all available dating at each site.
The numbers in parentheses indicate the number of dated samples.
(b) 14C ages of numerous organic-debris samples from the central part of Pokhara basin (published and
new dating), represented according to their stratigraphic relative elevation. Points and error bars
correspond to the median value and the 95% confidence interval of the calibrated ages pdf,
respectively. Analyses restricted to leaves, wood fragments, and tree trunks, in order to minimize the
effect of suspected reworking in particular for charcoals. A notably rapid sedimentation rate of ~1m/yr
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is documented. Note that "charcoal in buried paleosols" refers to charcoal collected from former soils
on the flanks of the Seti Valley that were buried by and predate the Pokhara conglomerate.
Figure 3: Reconstruction of the Annapurna IV paleo-summit before its collapse. (a) Thickness of
the rock-avalanche deposit estimated from breccia relics in Sabche cirque and along upper Seti valley.
The orange arrow (oriented at N245) corresponds to the optimal direction of collapse (see Extended
Data Fig. 9e). Circled black letters correspond to sites described in Fig. 1b. (b) Average shape of the
reconstructed paleo-topographies that satisfy rock volume and hillslope stability constraints (grayed
paleo-summit on top the present topography in slight transparency). (c) Cross section (location in a)
through the middle of the Sabche cirque, withaveraged paleo-topography before collapse and
approximate geometry of the post-collapse breccia deposit; rsb and bdb indicate the projection on the
section plane of the centres of mass of the rock slide and breccia deposit volumes respectively, and
whose elevation difference is used to calculate the released potential gravitational energy. (d)
Probability densities (presented as histogram and resulting from 2000 random paleo-topographies) of
the summit point elevation of the paleo-Annapurna IV satisfying increasing levels of requirement:
firstly, the collapsed volume VRS is in the range between 20 and 26.5 km3 (grey); secondly, the
summit slopes were stable regarding small to medium-size landslides (pink); thirdly, the safety factor
(SF) along the failure surface was rather minimal (red) (see Method and Extended Data Fig. 9). The
probability that the summit point could have exceeded 8000 m (thick line) is 65% and 76% if the
paleo-topographies satisfy the first two and three conditions respectively.
Figure 4: Far downstream signature of the reworked rockslide deposits evidenced by sediment
carbonate content. Comparison of the carbonate content in brecciated rock avalanche material, in
TSS units at the source of the rockslide, in Pokhara conglomeratic formation, and in modern sediment
along the Seti river (see also Supp. Info table SI-13). Unlike rockslide-derived sediments, which show
a very high and relatively constant proportion of carbonate, modern sediments display downstream
increasing dilution of the carbonate fraction, in particular after the confluence of the Seti with the Madi
or Trisuli rivers. (Rivers: Ma = Mardi; M = Madi; Tr = Trisuli; Ka = Kali Gandaki; numbers in
parenthesis = ratio of drainage areas with the Seti as denominator). Circled black letters and red
acronyms correspond to the sites and faults mentioned in Fig. 1a and 1b.
Figure 5: Evolutionary sketch of the high peak emergence well above the ELA (Equilibrium Line
Altitude) in a glaciated landscape undergoing sustained rock uplift. Starting from a fluviallydominated landscape (A) raised above the ELA (white dotted line), glacier development and subglacial
erosion leads to lengthening and steepening of headwalls (B and C). Whereas sub-glacial erosion
keeps much of the relief below the ELA (i.e. buzzsaw effect3) and frost cracking erosion above the
upper glaciated rim steepens the headwalls, further rock uplift drives the summit into the zone where
the rock mass becomes permanently frozen and undergo minimal surface erosion (C and D). The
continued growth of the peak elevation ends in a major gravitational instability (D and E), i.e. a giant
rockslide that beheads the summit and results in a thick rock-avalanche deposit (yellow shading in E)
13
84°20' E
l
l
l
l
l
l
l
Annapurna IV
l
l
l
fig.1c
l
l
l
l
l
l
l
l
l
l
l
l
l
IRSL dating
14C dating
former 14C dating
carbonate analysis
^
^ ^ rock avalanche
Sabche
^ ^
^
extent
^
Pokhara conglomeratic
^
formation ^
^
older terrace deposit
giant rockslide deposit
l l detachment fault
l l
^ ^
^ ^ ^ thrust fault
^
^ ^
^
^
^
^ ^ ^
^
^ ^
^ ^
^
^
^
^
Altitude
28°00' N
0m
Seti
0
5
head scarp of the slide
Mar
sya
10 km
nd
moraine crests
glacial and fluvioglacial formation
glaciers
i
^ ^ ^ ^
^ ^
100 km
TIBET
^
^ ^ ^ ^ ^
Nar ay
an
i
^ ^ ^ ^
^
^
T
^
2 km
N
84°00' E
br
NEPAL
S
P
k
INDIA
Na
ra
G
ya
ni
Ganga
80°E
85°E
yellow breccia
light yellow breccia
br
S
MB
aproximate extent of the
initial rockslide and
avalanche deposit
relics from the surface
of the breccia deposit
coarse breccia facies
whitish breccia
d
Trisuli
^ ^ ^ ^
bedrock
UTM 44 - WGS84
Kali G
andaki
30°N
samples for IRSL dating
9,000 m
Lesser Himalayan
metasediments
^ ^ ^ ^
samples for 36Cl dating
^ ^ ^
P
^ ^ ^ ^
fig.1d
^ ^
^
^
Annapurna IV
(7,525 m)
36Cl exposure dating
tly ed gh
gh ss rou
sli xpre ial t
e lac
g
^
^
^
l
3,160,000
^
^
l
3,157,500
K
^
^
28°20' N
^
^
l
High Himalayan
gneisses
^
^
l
l
l
^
^
l
l
l
^ ^
^
a
802,500
N
^
l
l
l
l
l
MCT
^ ^ ^
Annapurna II
S
l
l
l
l
l
l
l
800,000
l ^
l
l
797,500
l
l
STD
795,000
Manaslu
l
l
l
792,500
c
3,162,500
Annapurna III
l
l
l
Annapurna I
790,000
N
carbonate-rich Tethyan
Sedimentary Series
l
28°40' N
84°00' E
b
a
0.01
IRSL aliquot ages
distribution (x10-3)
PDF
Sabche (top) S 36Cl exposure age
0.005 (N = 4)
0
800
upstream
0.005
900
1000
1100
0.6
IR50
0.4
1200
1300
0
1400
Sabche (base) S IRSL age
(1 sample / 60 aliquots)
pIR225
0.2
0
800
0.015
900
14C
1000
1100
1200
1300
1400
0.02
age
0.01
0.005
PDF
0.015
0.01
Karuwa K breccia
(N = 8)
0
0.02
0.01
0.01
Pokhara P basal
0.005 formation (N = 8)
(Schwanghart et al., 2016)
0
0.015
0.01
0.005
Narayangad N terrace
(N = 1)
0
800
1000
1100
1200
1300
1400
Date (year AD)
698 AD
20
0.2
0
800
entati
1669 BC
0.4
tree trunk
wood
leave
humic silt
charcoal
charcoal in
buried paleosol
900
40
on ra
0.6
80
this study
Schwangart et al., 2015
Stolle et al., 2017, 2019
1000
14C
1100
1200
calibrated date (year AD)
60
1300
Depth (m) below the top surface of the
Pokhara conglomerate towards the
middle of the Pokhara Basin
0.8
0
62 AD
te ~1
m/yr
1
sedim
Relative elevation within Pokhara formation
b
900
downstream
100
1400
a
Annapurna III
(7,555 m)
b
A'
paleo-Annapurna IV
present Annapurna IV
3,160,000
A'
Annapurna IV
(7,525 m)
S
l
l
l
l
l
l
l
l
l
l
A
l
3,150,000
A
2 km
2 km
l
l
2 km
l
l
l
l
l
l
l
150
l
l
l
l
N
d
VRS = 20 - 26.5 km3
hillslopes stability
STD
rockmass proner
to collapse
100
50
Epigenic gorge
^
^
^
Reconstructed
breccia thickness (m)
1,350
^
^ ^ ^ ^ ^
^
N
^ ^ ^ ^
Rockslide deposit
eroded / remaining
^
A
^ ^
ti
Se
790,000
D
5 km
0
zo
ne
Sanctuary fm
2
4
8
7
A'
present
0°C isotherm
Sombre Fm
(Silurien)
bdb
ST
0
800,000
c
7,600 7,800 8,000 8,200 8,400 8,600
Peak elevation (m)
rsb
^ ^
^
M C T^ ^
0
6
5
4
Annapurna yellow fm
(Cambrian)
6
8
Distance (km)
Nilgiri Fm
(Ordovician)
10
12
3
Elevation (km)
^ ^
^
^ ^^
Breccia deposit
top surface
Breccia deposit
remnants
^
3,140,000
K
90
clasts (individuals)
TSS units in
rockslide
source
S
K
clasts mix
matrix
matrix (basal part)
P
rock avalanche
breccia
30
20
10
Silurian
Ordovician
Cambrian
% of carbonates
40
N
G
Pokhara
conglomeratic
formation
60
50
gravels mix
sand or matrix
river sand
suspended load
river sand (tributaries)
80
70
gravels (individuals)
GR2 [0:3m]
100
?
Seti modern sediments
M
Ma
0
STD
20
MCT
40
Tr
(3:4)
(2:5)
0
60
80
100
Ka
(5:1)
120
GR 1&2
mean
(3:5)
MBT
140
160
Distance from Sabche drainage divide (km)
MFT
270
320
370
snow avalanche
50°
permanently frozen
35°
2 km
periglacial erosion
glacial erosion
2 km
rock uplift
A
B
C
D
ELA
E
537
538
539
540
541
542
543
544
545
546
547
548
549
550
551
552
553
554
555
556
557
558
559
560
561
562
563
564
565
566
567
568
569
570
571
572
573
574
575
576
577
578
579
580
Methods
14
C dating
16 organic fragments were collected from 3 distinct areas for 14C dating: 7 wood-fragments
samples taken in the breccia deposit at Karuwa (K site on fig.1), 8 samples of charcoal and wood
fragments in the southern part of the Pokhara basin (P site), and one micro-charcoal sample found
in Pokhara type sediments outcropping at the range outlet (site N in Fig. 1b).
Organic fragment preparation includes standard cleaning and ABA treatment, and
combustion at 850°C as described in ref.51. The CO2 was converted to graphite and analysed on
the LMC14 AMS facility52 “ARTEMIS’’, except for one sample prepared and analysed at the
National Ocean Sciences Accelerator Mass Spectrometry (NOSAMS) facility at the Woods Hole
Oceanographic Institution.
Radiocarbon ages are calculated53 by correcting the fractionation with the 13C calculated
from the 13C/12C ratio measured on ARTEMIS. We used the IntCal13 curve54 for calibrating our
14
C
ages
to
calendar
years
using
Oxcal
online
calculator
(https://c14.arch.ox.ac.uk/oxcal/OxCal.html) (Supp. Info. table SI-10-1).
36
Cl exposure age dating
We sampled plurimetric blocks of Siluro-Devonian black shale and calcschists, deposited
on top of the main northern remnant of the original Sabche breccia surface. Samples were prepared
at the CEREGE (Aix-en-Provence, France), following standard procedures55,56. 36Cl and Cl
concentrations were obtained on each sample by isotope dilution accelerator mass spectrometry at
ASTER-CEREGE. Major and trace elements were measured at the French National facility for
geochemical analysis (SARM-CRPG) (Supp. Info. table SI-11-3).
36
Cl production rate was calculated according to concentration in target elements (Supp.
Info. table SI-11-2) and Lal/Stone time-dependent scaling using Chronus Online calculator
(http://hess.ess.washington.edu/math/index_dev.html). Several blocks made of finely schistosed
rocks show evidence of frost cracking and physical weathering, but the shape of the original
surface is generally still identifiable and indicates a maximum surface erosion of ~10cm (Supp.
Info. table SI-11-1). An additional correction for snow shielding was considered from snow cover
measurements on the Himalayan southern flanks 25km to the east57. Snow and erosion corrections
represent ~25 and 40 yr respectively.
Infra-red stimulated luminescence (IRSL) dating on sheared breccia deposits
TSS carbonate units may contain small amounts of quartz and feldspar grains. The luminescence
signal can be reset (called triboluminescence) by heating, mechanical crushing or shearing35
processes in intense sheared bands such as frictionnites. In the southern part of the rockslide
deposit, we collected three samples from two sites approximately ~50 m apart within an internal
basal shear zone (CA-13-273, -274, and CA-13-283); Extended Data Fig. 6). Collected samples
measured 15x12x7cm and 7x7x5cm respectively. Sample preparation followed the procedure
14
581
582
583
584
585
586
587
588
589
590
591
592
593
594
595
596
597
598
599
600
601
602
603
604
605
606
607
608
609
610
611
612
613
614
615
616
617
618
619
620
621
622
623
624
described in ref.58 to isolate K-feldspar fraction. Bulk samples were sent to Actlabs or SARMCRPG for major and trace element analysis, used to estimate (see Supp. Info. SI-12 for full details)
the total dose rate with the online Dose Rate and Age Calculator59.
Luminescence measurements have been performed at the University of Lausanne (Switzerland)
using a post-IR IRSL protocol60 at 50°C (IR50) and 225°C (pIR225), as described in the Supp.
Info. SI-12. Dose-equivalent distributions were statistically analysed61 using the Central Age
Model (CAM), and also the Minimum Age Model (MAM) for sample CA-13-283 which best
represent luminescence reset during shearing (Extended Data Fig. 6). Final ages were calculated
with individual fading corrections62.
Volume of the rockslide deposit
To calculate the total volume of the initial rock-avalanche deposit in the Sabche cirque and
the upper Seti (fig. 3), we consider the difference between the initial surface of the breccia deposit
and the original topographies of the cirque bottom and upper Seti valley before the collapse
(Extended Data Fig. 4).
The original cirque surface is based on the recognition from air views and satellite images
of the outcropping substrate areas beneath the deposits (Fig. 1 and Extended Data Fig. 4a). These
outcrops spread outwards from near the cirque centre (and after removing the ice cover in the NE
part of the cirque) and permit a rough interpolation of a smoothed initial surface. It is difficult to
assess the second order relief (mostly small ridges separating glacier streams, considering by
equivalence the cirque of the Annapurna Sanctuary located just west of that of Sabche).
Introducing an a priori moderate relief carved by glaciers suggests an uncertainty on the volume
reconstruction of the order of +3/-2 km3. Based also on observations made in the Annapurna
Sanctuary cirque, the volume of moraines and ice that could have been entrained by the rock
avalanche was estimated to be <0.1 and 0.5 km3, respectively.
The initial top surface of the rockslide deposit has been estimated based on a few remnants
in the northern and north-western parts of the cirque (Extended Data Figs. 4b, 5a) and on top of the
ridge along its southern part (Extended Data Fig. 2f). These surfaces are all located at an altitude of
~4600m, but are not sufficient to constrain the geometry of the deposit surface in its central part,
where the breccia penitents left by erosion provide only a minimum elevation (Fig. SI-4-1c).
Nevertheless, literature evidence from the geometry of large collapses characterized by frontal
confinement, as the Sabche one, suggest that this central part was likely at an elevation above 4600
m (Supp. Info. SI-4). The present course of the glacier descending from the Annapurna III, which
has been diverted westward, requires also the presence of an initially convex surface of the rockavalanche deposit (Fig. 1). Despite uncertainties related to secondary hummocky features, a central
convexity or a downward transition between the cirque and the Upper Seti valley, the mean rockavalanche surface can presumably be approximated by a nearly horizontal deposit at an elevation of
~4600 m (Extended Data Fig.4b). We estimate that these uncertainties affect the reconstruction of
the breccia volume by an error of 2-3 km3 (Supp. Info. Fig. SI-4).
Along the upper Seti, a similar approach was used to estimate the breccia volume,
assuming, for the rock-avalanche deposits’ original surface, a progressive thickness decrease along
the upper Seti valley down to the last breccia occurrence (Supp. Info. Fig. SI-1-1) and relying on
the minimum thickness indicated by the relic breccia spurs.
15
625
626
627
628
629
630
631
632
633
634
635
636
637
638
639
640
641
642
643
644
645
646
647
648
649
650
651
652
653
654
655
656
657
658
659
660
661
662
663
664
665
666
667
The combination (mean squared error) of the different sources of error on the basal and
summit surfaces of the breccia deposit results in a final uncertainty in the volume reconstruction of
+4/-3 km3.
Finally, to derive the volume of solid rock that collapsed from the Annapurna IV summit,
the breccia deposit volume was corrected for breccia porosity, measured from various rockslide
breccia sampled in the Annapurna region (Supp. Info. SI-5). An average value of 15±5% was
found, consistent with previous estimates on rock-avalanche material63.
Original failure surface of the Sabche rockslide
The entire NE and NNE area of the Sabche cirque displays numerous structural surfaces
related to stratification or schistosity, and oriented at N110° (Extended Data Fig. 1). The eastern
part of the ridge joining the Annapurnas-III and -IV (Fig. 3) shows large structural slabs with dips
of ~45° to the south, i.e., quite well-oriented to represent slip planes. Below the Annapurna IV, the
cliffs are characterized by geometric breaks (prisms, dihedrals) that connect these structural
surfaces to irregular surfaces oriented at ~N0°. In contrast with slopes that dominate the western or
eastern sides of cirque, these cliffs do not display any obvious trace of glacial or avalanche erosion,
i.e. any trace of channelizing forms. This absence suggests that their surfaces were created by
recent fracturing, and most probably correspond to the rockslide failure surface.
To corroborate these observations in a more objective way and to draw the a priori
extension of the failure surface, a morphological criterion is defined by looking at the transverse
curvature (Extended Data Fig. 4d) of the high-resolution topography (2-m Pleiades tri-stereoderived DTM, see realization details in Supp. Info. SI-3), excluding ice and snow-pack areas. The
structural surface appears with minimal curvature, whereas the slopes affected by erosion display
converging thalwegs underlined by negative curvature. Using these indicators, we identified a zone
of “fresh” planar or prismatic surfaces just south and west of the Annapurna IV summit, distinct
from the areas beyond where erosion has begun to carve out channelized or converging relief. This
first area (the upper and lateral limits of which are represented by the pink line in Fig.1c and
Extended Data Figs 1-4) is considered as delimiting the original failure surface of the rockslide.
Even if we cannot exclude the possibility that small/secondary rock slides or rock falls
would have partly rejuvenated this surface, analogous to the May 5th 2012 rockslide event64 at the
SE end of the Annapurna IV, the absence of deposits on top of the northern remnant of the original
surface (Extended Data Fig. 5) suggests that no major reshaping event has affected the initial
failure surface.
Paleo-Summit Reconstruction
To reconstruct the paleo-summit, three constrains must be verified: first, the restored rock
volume must equal the collapsed one, i.e. 23+3.5/-3km3; second, the paleo-topography has to be on
the verge of collapse; and third, the slopes and cliffs below the paleo-summit must be mechanically
stable, otherwise they would have failed before the giant mass through smaller rockslides. The
failure surface follows large portions of structural surfaces associated with the local schistosity
developed in marly limestone and possibly presenting lower cohesion and friction angles. Defining
a Safety Factor (SF) associated with this failure plane is difficult in absence of specific
16
668
669
670
671
672
673
674
675
676
677
678
679
680
681
682
683
684
685
686
687
688
689
690
691
692
measurements on these planes, and because of uncertainties on the exact geometry of the most
basal part of the failure surface (below modern glaciers and debris). Other than mass conservation,
the restoration therefore relies only on cliff average stability.
Mechanical characteristics of the Annapurna range substratum
To check the stability of headwalls below the paleo-summit, we explored the average
mechanical rock properties that ensure the stability of the regional relief in the entire high
Annapurna range. Estimating the average material properties of a region can be done by
considering the best-fitting strength parameters that reproduce the frequency-area distribution of
landslides47. Here, in the absence of such a landslide distribution in the Annapurna region, we
instead estimate the stability of the Annapurna summits by exploring the maximum value for the
cohesion that allows the stability of all regional slopes. At this scale, testing slope stability through
complex numerical approaches is computationally too demanding, and simplified limit equilibrium
analysis, despite its limitations, is usually preferred65,66.
In order to calculate the spatial distribution of the Safety Factor (SF) in the Annapurna
region, we have developed a Matlab© code largely adapted from previous approaches66. The slope
stability analysis is based on the limit equilibrium concept for a 3D model with rotational
movement on a potential ellipsoidal slip surface.
At each topographic grid point, our model evaluates the slope stability conditions for a large
number of randomly selected ellipsoidal slip surfaces (Extended data Fig. 7). These are defined by
the geographic coordinates of the centre, the length of their three half-axes a, b and c, by their
orientation in the direction of the steepest topographic slope and inclination that follows the
steepest slope plus or minus 15°, and by an offset of the ellipsoid centre above the terrain zc. For
each failure surface, the SF is computed using the 3-D sliding surface Hovland’s model67:
∑
693
𝑆𝐹
694
with 𝛽
695
696
697
698
699
700
701
702
703
704
705
706
707
708
709
.
∑
𝑡𝑎𝑛
,
,
(1)
,
tan 𝛽 . cos 𝛼
𝛼 , and 𝐴
,
where the summation sign corresponds to the pixel summation of the resisting and driving forces
(projected on each surface element) applied on each vertical rock column of thickness D, and A is
the 3-D area of the slip surface for the considered pixel, the rock density, C the apparent cohesion
of the substrate, its internal friction angle, c and c the local slope and aspect of the surface of
rupture at the base of the considered column. The driving force and sliding act in the direction of α.
βm is the apparent dip of the slip surface at the considered column in the direction of sliding. No
inter-column forces or external forces, such as seismic loading, are considered in the model.
In contrast with a previous model66, and because soil is absent above 4500m, we don't
consider potential slip at the soil-substratum interface, but assume a homogeneous substratum.
Because we are not interested in exact friction and cohesion values, but only by apparent regional
values that could be applied to the slopes of the Annapurna IV, we do not consider either seepage
forces, or pore pressure effects.
Practically (Extended Data Fig. 7), we employ a brute force procedure by exploring at
regular steps different parameters. First, we iterate on the pixels of the topography. For each pixel,
17
710
711
712
713
714
715
716
717
718
719
720
721
722
723
724
725
726
727
728
729
730
731
732
733
734
735
736
737
738
739
740
741
742
743
744
745
746
747
748
749
750
751
752
we iterate between the extreme geometric limits of the ground trace, calculating the average aspect
and steepest slope over the given scale. For each explored ground window, we iterate on the
parameters (a, b, c and ) of the ellipsoids that fit the ground trace and calculate the SF (failure
surfaces displaying an overhanging portion are rejected). For each pixel, the parameters giving the
minimum SF value are retained and the associated potential rockslide is located at the coordinates
of its center of mass. Finally, in the result grid, if a pixel contains several centres of mass of
potential rockslides, we keep only the one with the lowest SF.
Before applying our model to the glaciated Annapurna area or to the fluvially-dominated
region farther south (Supp. Info. SI-6), we tested it on a benchmark case and ensured that the
searching procedure was finding: (1) similar solution (SF value) as previous studies65,66; and (2)
optimal ellipsoidal failure geometries not too far from the semi-analytic circular68 or log-spiral69
solutions. Regarding this later point, our optimal surfaces are a bit more concave than the semianalytic solutions, but the SF values match to within <10%.
To explore the mechanic stability of the Annapurna range, we consider the steep areas
within similar elevation range and lithology as the Annapurna IV collapse, i.e., for elevations
higher than ~4000 m and for TSS units, excluding the flat cirque bottom (Extended Data Fig. 8).
For the SF calculation, a fixed value of the internal friction angle at =35° (ref. 70) is considered.
Only cohesion values were varied. For each cohesion value, we plot the distribution of SF values.
If most prominent cliffs are near the verge of failure as expected in mountains with both high uplift
and high erosion16, then the distribution mode has to be slightly above the critical value of 1, i.e. for
an apparent cohesion ≥1MPa for the high relief of the Annapurna range.
Locally, the geometry of gravity instabilities is most often related to the orientation of
fracture families and/or to schistosity: at the scale of our approach, such a consideration of local
factors is not possible, and the explored cohesion value should be viewed as an average estimate. In
addition, as neither the effects of pore pressure, seepage forces, nor seismic acceleration are taken
into account, this value can be seen as a minimum46 for the glaciated relief of the Annapurnas.
More importantly, because the internal friction angle has not been explored, the cohesion values
tested in our procedure must be viewed as an apparent value. However, the precise value is of little
importance insofar as we are looking for a relative criterion to test realistic topographies of the
paleo-Annapurna IV in a second step (Supp. Info. SI-7), or to compare apparent cohesion values
between glaciated and unglaciated areas (Supp. Info. SI-6).
Paleo-topographic reconstruction
To explore a family of possible geometries for the Annapurna IV paleo-summit, we
generated 2000 topographies from fixed ground support points along the northern ridge and from
random paths for the crest and the southern basal footprints (see also computing chart in Supp.
Info. SI-7). Whereas the elevation of the southern basal footprints is interpolated from the initial
cirque surface (Extended Data Fig. 4a), the crestline elevation is randomly varied within a fixed
range (the northern slopes between the present head scarp and the crest are allowed to vary between
35 and 50°, whereas the horizontal path is oriented in continuity with the present ridgelines at their
eastern and western terminations). Intermediate points with randomly varying elevations are also
generated before performing surface interpolation and volume calculation.
18
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760
761
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764
765
766
767
768
769
770
771
772
773
774
775
776
777
To consider a paleo-topography as acceptable, besides producing a rockslide volume
between 20 and 26.5km3, its hillslopes must be stable according to the SF criterion defined for the
Annapurna region. For each randomly generated paleo-topography, we thus apply the same
procedure described in the previous section (Extended Data Fig. 7), and derive the distribution of
minimal SF values. For the high relief of the Annapurnas and assuming = 35°, all SF values were
found to be >=1 for a cohesion value C=2MPa. Based on this absolute cut-off, any paleotopography presenting SF values lower than unity for C=2MPa were rejected. From the final set of
acceptable geometries, we calculate a mean topography, a distribution of volumes, of crest profiles
and of paleo-summit elevations (Fig. 3d and Extended Data Fig.9).
It should be noted that for the modelled paleo-topographies the optimal ellipsoidal failures
predict a steeper and shallower rupture surface than that actually observed and preserved in the
present topography beneath Annapurna IV. Applying Hovland formula67 (eq. (1)) on the actual
failure surface, and exploring various sliding directions in order to reach a minimal stability, leads
to SFfs values of 1.42±0.1 for C=2MPa (vertical red line in Extended Data Fig. 9g), well above the
collapsing threshold of 1. Although this high value results in part from the surface roughness
compared to that of a smooth ellipsoid, it also reflects the likelihood that the rupture was strongly
guided by existing schistosity planes, especially in the western part of the collapse. It is
hypothesized that these schistosity planes exhibit friction angles and cohesion significantly lower
than 35° and 1-2MPa, respectively. Nevertheless, if we attempt to reduce the set of possible paleotopographies, we could potentially add a third criterion that the SFfs along the failure surface has to
be minimal or not too high: we might, therefore, preferentially select a subset of paleo-topographies
with SFfs ≤1.42 (for C=2MPa), which would be more prone to collapse (dark red histogram in Fig.
3d).
19
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820
Methods and Extended Data references:
51. Dumoulin J.P. et al., 2017. Status report on sample preparation protocols developed at the
LMC14 Laboratory, Saclay, France: from sample collection to 14C AMS measurement.
Radiocarbon 59, 713-726.
52. Moreau, C., Caffy, I., Comby, C., Delqué-Količ, E., Dumoulin, J.-P., Hain, S., Quiles, A.,
Setti, V., Souprayen, C., Thellier, B., et al. (2013). Research and Development of the
Artemis 14C AMS Facility: Status Report. Radiocarbon; Vol 55, No 2–3 (2013).
53. Mook, W. G., & Van Der Plicht, J. (1999). Reporting 14C activities and concentrations.
Radiocarbon, 41(3), 227-239.
54. Reimer, P. J., Bard, E., Bayliss, A., Beck, J. W., Blackwell, P. G., Ramsey, C. B., ... & Van
Der Plicht, J. (2013). IntCal13 and Marine13 radiocarbon age calibration curves 0–50,000
years cal BP. radiocarbon, 55(4), 1869-1887.
55. Stone, J. O., Allan, G. L., Fifield, L. K., & Cresswell, R. G. (1996). Cosmogenic chlorine36 from calcium spallation. Geochimica et Cosmochimica Acta, 60(4), 679-692.
56. Schlagenhauf, A., Gaudemer, Y., Benedetti, L., Manighetti, I., Palumbo, L.,
Schimmelpfennig, I., ... & Pou, K. (2010). Using in situ Chlorine-36 cosmonuclide to
recover past earthquake histories on limestone normal fault scarps: a reappraisal of
methodology and interpretations. Geophysical Journal International, 182(1), 36-72.
57. Putkonen, J.K., 2004.Continuous snow and rain data at 500 to 4400 m altitude.
Arct.Antarct. Alp. Res. 36 (2), 244–248
58. Diaz, N., King, G.E., Valla, P.G., Herman, F. & Verrecchia, E., 2016: Pedogenic carbonate
nodules as soils time archives: challenges and investigations related to OSL dating. Quat.
Geochronol. 36, 120-133.
59. Durcan, J.A., King, G.E. & Duller, G.A.T. 2015: DRAC: Dose Rate and Age Calculator for
trapped charge dating. Quaternary Geochronology 28, 54–61.
60. Buylaert, J.P., Murray, A.S., Thomsen, K.J. & Jain, M. 2009: Testing the potential of an
elevated temperature IRSL signal from K-feldspar. Radiation Measurements 44, 560–565.
61. Galbraith, R.F., Roberts, R.G., Laslett, G.M., Yoshida, H. & Olley, J.M., 1999: Optical
dating of single and multiple grains of quartz from jinmium rock shelter, northern Australia:
part i, experimental design and statistical models. Archaeometry 2, 339-364.
62. Huntley, D.J. & Lamothe, M. 2011: Ubiquity of anomalous fading in K-feldspars and the
measurement and correction for it in optical dating. Canadian Journal of Earth Sciences 38,
1093–1106.
63. Govi, Gula & Nicoletti, (2002), Val Pola rock avalanche of July 28, 1987, in Valtellina
(Central Italian Alps), in Catastrophic Landslides: Effects, Occurrence, and Mechanisms
GSA rev.in Engineering Geology, 15. )
64. Oi, H., Higaki, D., Yagi, H., Usuki, N., & Yoshino, K. (2014). Report of the investigation
of the flood disaster that occurred on May 5, 2012 along the Seti River in Nepal.
International Journal of Erosion Control Engineering, 7(4), 111-117.
65. Xie, M., Esaki, T., Qiu, C., & Wang, C. Geographical information system-based
computational implementation and application of spatial three-dimensional slope stability
analysis. Computers and Geotechnics, 33(4-5), 260-274 (2006).
20
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823
824
825
826
827
828
829
830
831
832
833
834
835
836
837
838
66. Mergili, M., Marchesini, I., Rossi, M., Guzzetti, F., & Fellin, W. (2014). Spatially
distributed three-dimensional slope stability modelling in a raster GIS. Geomorphology,
206, 178-195.
67. Hovland, H. J. (1977). Three-dimensional slope stability analysis method. Journal of the
Geotechnical Engineering Division, 103(9), 971-986.
68. Hungr, O., Salgado, F.M., Byrne, P.M., 1989. Evaluation of a three-dimensional method of
slope stability analysis. Can. Geotech. J. 26, 679–686.
69. Leshchinsky, D., Baker, R., & Silver, M. L. (1985). Three dimensional analysis of slope
stability. International Journal for Numerical and Analytical Methods in Geomechanics,
9(3), 199-223.
70. Byerlee, J. (1978). Friction of rocks. In Rock friction and earthquake prediction (pp. 615626). Birkhäuser, Basel.
71. Parsons, A. J., Law, R. D., Searle, M. P., Phillips, R. J., & Lloyd, G. E. (2016). Geology of
the Dhaulagiri-Annapurna-Manaslu Himalaya, Western Region, Nepal. 1: 200,000. Journal
of Maps, 12(1), 100-110.
72. Morin, G. (2015). L’érosion et l’altération en Himalaya et leur évolution depuis le tardipléistocène: analyse des processus d’érosion à partir de sédiments de rivière actuels et
passés au Népal central (Doctoral dissertation, Université de Lorraine).
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Extended Data Figure Legends
Extended Data Fig. 1: Geologic map and cross section of the Sabche cirque area. (Upper)
Interpretative geologic map of the Sabche cirque area based on published maps28,71 West and North
of the cirque. The TSS units that outcrop along those cliffs correspond to the base of the TSS
series, namely carbonate-rich sediments, Cambrian to Silurian in age, and presenting an upward
decreasing degree of metamorphism28. The different lithologic limits were extrapolated across the
cirque based according to our observations made on photos taken from ultralight aeroplane and
helicopter, and from satellite images. Structures and bedding attitudes were also estimated from
photos or from the tri-stereo DEM. (Lower) Interpretative structural cross section AA’ (see
location on main map) built according to published sections further West28,71 and to the bedding
attitude we estimated from a large set of airplane photos or from the DEM. The northern part of the
section, which has been affected by the rockslide, is relatively well constrained since bedding are
pretty well identifiable on photos. The southern part is much less constrained because the
Sanctuary units display less identifiable beddings and because the structure is much more heckled
with numerous small-scale folding. STD = South Tibetan Detachment, DD = Deodali detachment.
Extended Data Fig. 2: Different facies of the breccia deposit in Sabche cirque (photos a to f =
taken from an ultra-light aeroplane _ See pictures location on fig. 1 in Supp. Info.): (a) compact,
homogeneous, pulverized and indurated fine grained breccia exposed over ~200 m high cliffs; (b)
and (c) package of coarse and blocky material topped by more finely pulverized fine grained
breccia material; (c) and (d) Internal contacts between grey and yellowish breccia facies (issued
from the Annapurna Yellow Limestone formation28) presenting inverted order and suggesting
limited mixing during the rockslide collapse; (d) Contact close to the southern base of the cirque
deposit in its southern part: this contact, slightly sloping towards the N or NW, presents as a thin
band of shearing and micro-crushing where IRSL samples were taken (CA-273, -274 and -283). (e)
Rare preserved phantom of limestone dismantled strata, or jigsaw facies. (f) In the southern part of
the cirque, veneers of breccia deposit are preserved on the N-dipping flank of the cirque up to
4350m altitude (z2), and at the top of the eastern ridge of Macchapuchare (fig. 1) at ~4570m
altitude (z1 = satellite image taken on 30/12/2011); (g) Saw-cut samples of compact and indurated
facies exposed in the basal part of the deposit (CA-273, site of panel d): the breccia is made of
finely crushed material with a few angular clasts.
Extended Data Fig. 3: Outcrops near Karuwa (site K on fig. 1) exposing the stratigraphy of
the granular avalanche breccia deposit.
(a) 40-m-high cliff overhanging the Seti river, exposing breccia of the granular avalanche material
and cut by a small inset terrace capped by a filling of (b) finely-layered limestone rich gravels of
Pokhara conglomerates. (c) Breccia facies with juxtaposition of parts made of limestone/marble of
different colors with pockets of whitish elements and matrix within a set of darker elements and
matrix. The size of elements, the proportion of clast vs. matrix, as well as the carbonate content
remain however similar between the different colored areas. They are interpreted as figure of
incomplete mixing at meter-scale of the avalanche material issued from the basal Ordovician units
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that display alternation of light and dark layers in the Sabche cirque along the steep SW face of the
Annapurna IV (c’). (d) Internal part of the breccia displaying dyke injection of a fluid rich phase
that coloured the breccia, and the post-injection (e) multiple brittle to ductile faulting and shearing
of this dyke and of its shoulders. (f) Schematic section of the contact between the avalanche
brecciatic deposit and the gneissic bedrock of the Seti valley flanks. This contact displays a
transition in terms of lithology and clast size: breccia with large (up to >1m) and sub-angular to
rounded boulders of local biotitic gneisses are progressively replaced by smaller angular elements
(<30cm) within a matrix-dominant material made of ~100% of TSS lithologies. The gneiss rich
unit, which corresponds to local material dragged and entrained at the base of the avalanche,
contained quantity of millimetric to sub-metric pieces of vegetal debris (root, branch and trunk (g))
ripped from the sides of the valley, and whose density decreases also rapidly away from the former
valley wall. (h) Bedrock/breccia contact in the upper part of the talweg displaying a contact zone,
0 to 40-cm thick, made of a mixture between limestone breccia, local gneissic clasts, and paleosol
pockets, including numerous wood debris, and the presence of a (i) shearing zone of a few
centimetres thickness. Green numerical codes correspond to the names of the samples (CA-xx-xx)
dated by 14C.
Extended Data Fig. 4: Elements for reconstructing the volume of collapsed rockslide material
in the Sabche cirque and the initial rockslide failure surface. Maps of the base (a) and top (b) of
the rock avalanche brecciated deposits, including the interpolation constraints, and (c) present-day
residual thickness of the breccia deposits. (d) Sabche rockslide scar identification from the
transverse curvature (m-1) computed from the high resolution (2-m pixel) topography. The recent
scar surface (3 and 4) is characterized by step like dihedrals that delineate planar faces with low
curvature along the stratification/foliation planes and irregular faces following a set of fractures at
90°. In contrast, outside the scar (1 and 2), the topography displays the first marks of erosion and
gullies organisation toward a converging network.
Extended Data Fig. 5: The main dated relic of the rockslide deposit surface in the upper
Sabche cirque
(a) Satellite image (Google Earth) of a large flat area in the northern part of the Sabche cirque. This
flat area corresponds to the top of the rockslide deposit (4600m), which was amply resurfaced by
glaciers except along its southern part where the original surface is covered with numerous multimetric blocks. (b) Picture taken from an ultra-light aeroplane of the area, south of which the multimeter blocks CA-13-260 to 265 were sampled. (A: original chaotic upper surface of the rockslide
deposit; B: gently hilly surface of the hummocky moraine deposits; C: crested moraine left by the
glacier issued from the South-Eastern face of the Annapurna III and which is now deeply
entrenched into the breccia deposit; D: recent moraine left by the glacier issued from the West face
of the Annapurna IV; E: present-day glacier issued from the West face of the Annapurna IV). (c)
Five of the six blocks sampled for cosmogenic 36Cl dating at the top of this relic surface. All the
blocks of the surface consist of carbonate rocks: limestone, marly to sandstone limestone,
sandstone marl. These lithologies are finely schistosed and their surface physically weathered.
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Extended Data Fig. 6: Internal shear zone near the basal part of the rockslide deposit in the
Sabche cirque and IRSL dating (see location on Fig. 1C). (a) The shear zone varies in thickness
from a few centimetres up to 30cm at the level of fish-like structures (to the left of the hammer). (b)
The shear zone (sample CA-13-283) appears as colored bands of relatively compact breccia made
of centimeter size angular clast within a finer yellowish matrix; (c & d) Thin sections observed
under polarizing microscope, cut into the shear zone perpendicularly to the shearing plane and
showing thin zones of homogeneous finely fragmented almost glassy material within microbreccia
facies with locally remnant of larger clasts. (e) Schematic sedimentologic description of the breccia
around shear zone at 2 sites, around 50m apart (Extended Data Fig. 2d). (f and g) Age distributions
(fading-corrected) and kernel density estimate (KDE) plots for samples CA-13-283 and -273
respectively. IR50 ages and KDE are represented by filled circles and thick line, respectively.
pIR225 ages and KDE are represented by open circles and dashed line, respectively.
Extended Data Fig. 7: Definition and validation testing of the hillslope safety factor (SF)
exploration procedure in the Annapurna Range region. (a) Definition of the variables for the
computation of the Safety Factor (SF) according to 3D-Hovland criterion67, and (b) computation
chart of the searching procedure of the ellipsoid-type failure surfaces that minimize the SF all over
the topography of a given region. (c) Validation test of the searching procedure in the case of a
topographic step, 1-m high, dipping at 60° with = 15° and C = 0.116 Pa. The optimal ellipsoids
surfaces at the four increments that present minimal SF are compared with close-formed
solutions68,69. The SF values remain within 5% of the ones associated to these solutions whatever
the nominal value (solid line) or the one calculated with 3D-Hovland criterion applied to their
geometry (dashed line) is considered.
Extended Data Fig. 8: Results of the hillslope safety factor exploration in the Annapurna
Range region. (a) Map of the minimum SF along the hillslopes of the Annapurna high relief found
using the systematic searching procedure (Extended Data fig. 7) within the area delineated by the
dashed line. Calculation is made for an internal angle of friction of 35° and a cohesion, C, of 1.15
MPa. The steepest faces surrounding the highest peaks (black triangles), like the SSE face of the
Annapurna I (A-I), one of the most challenging climbing route in High Himalaya, present a Safety
Factor lower than 1 and would be considered as unstable for this given cohesion value. Note that
for a given pixel, the search procedure may have found several optimal failures, of different sizes,
whose centers of mass related to the same pixel: in this case, the failure with the minimum SF was
chosen. A-II =Annapurna II, A-III =Annapurna III, A-IV =Annapurna IV, and Ma =
Machapuchare. (b) Histograms of the values of the SF for different values of cohesion, C, between
0.5 and 2 MPa. For C=2MPa, almost all the hillslopes are apparently stable.
Extended Data Fig. 9: Paleo-summit reconstruction
(a) Construction points used to build simplified topography of the paleosummit. This construction
is essentially based on two main lines, whose geometry is explored in a random way: the paleocrest
(in cyan) and the paleocliff SW base (in blue). These lines are built as the sum of harmonics
function (up to the 8th term) of random amplitude and phase. The two extremities of the paleocrest
have been chosen to prolong on average the direction of the present crests line further West and
East. Similarly, the extremities of the base of the SW cliff and the isolated mobile point (in cyan)
24
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were chosen to prolong the SW-facing cliffs of the Sabche cirque. Densifying points are then added
before using Matlab© interpolation function griddata. (b) Map extent of the density (in log scale)
of the random paths explored for the paleocrest and the paleobase, with one random example of
path (black solid line). (c) Same as (b) but for the elevation profile of the paleocrest. In both
graphs, purple lines indicate the 95% probability of the density of the a posteriori paths that respect
both rockslide volume and hillslope stability constraints. (d) Map and histogram (inset graph) of
the SF values obtained through the searching procedure (ED-fig. 7) applied, within dashed lines
and for a cohesion value of C=2 MPa, to one of the randomly generated paleotopography. (e)
Average shape of the topographies that satisfy rockslide volume and regional stability criterion,
including a rose diagram of the optimal sliding direction N245 (i.e. the direction that minimizes the
SF on the failure surface). (f) Histogram of the volumes of the collapsed summit explored by the
generator of random topographies. 75% of the values fall within the range of the volume estimated
for the rockslide. The topographies that satisfy the regional stability criterion are superimposed in
pink colour. (g) Histograms of the SF on the failure surface for cohesion values C=2 MPa. The
topographies that satisfy the rockslide volume are superimposed in darker colour, and the ones that
satisfy both rock volume and regional stability criterion in pink colour.
Extended Data Fig. 10: Possible Sabche rockslide signature in the Gandak fan?
(a) Stratigraphic log and carbonate content of two 50-m long cores, GR1 and GR2, drilled in the
Gandak fan72. The two boreholes GR1 and GR2 show relatively constant carbonate percentages
close to the current river values (represented by the blue and gray lines respectively). Nevertheless,
GR2 is characterized by a 25-30% anomaly between -10 and -3m, and by a very strong 35-70%
carbonate anomaly between -3m and the surface. This anomaly was corroborated by additional
measurements on samples taken in a 3-m-deep small pit dug in the close vicinity of the GR2 drill
site. Such a recent anomaly in carbonate content might correspond to deposition of the fine fraction
of the sediments issued from the Sabche cirque erosion, 300km further downstream. Whereas these
carbonate rich units are not directly dated, 14C ages in GR1 core indicate that they are probably
younger than ~1.5 kyr, which is compatible with a deposit related to the Annapurna IV collapse
and more or less synchronous of the Pokhara conglomerate deposits.
(b) Residual topography obtained by subtracting a second order polynomial fit (dashed lines =
Trend function in ArcGis) to the topography (30m SRTM) of the Narayani fan. This residual
topography enables to highlight small topographic variations on the surface of the fan. It is seen
that GR1 was drilled in the recent flood plain of the Narayani (in yellow to reddish colors), while
GR2 was drilled in a N-S lobe 3 to 5 m higher than the rest of the fan (in bluish colors). This lobe,
which was then re-incised on its eastern border by the Narayani (see zoomed image) could have
been built during the episode of strong sediment input that followed the collapse in the Sabche
cirque.
25
Extended data figure 1
Extended data figure 2
Extended data figure 3
Extended data figure 4
Extended data figure 5
Extended data figure 6
Extended data figure 7
Extended data figure 8
Extended data figure 9
Extended data figure 10
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Data availability:
All data used in this study are from the published literature as referenced36-38, or presented in Sup.
Info. tables SI-10-1,2 , SI-11-1,2,3 , SI-12-1,2 and SI-13-1.
1040
Competing interests: The authors declare no competing interests.
1041
Supplementary Information is available for this paper.
1042
Correspondence and requests for materials should be addressed to J. Lavé.
Code availability:
To calculate minimal SF maps and reconstruct Annapurna IV paleo-topography, as shown in
Extended Data Figs. 8-9, we wrote two original codes in MATLAB, described in the Methods. The
two codes are available from the corresponding author upon reasonable request.
Acknowledgements: J.L. thanks B. Sitaula for his invaluable logistic help in the field, L. Bollinger
for his help during the helicopter flight in Sabche cirque and the numerous pictures he took, Y.
Gunzburger, F. Carraro Braga, M. Manas and P. Kumar for initial discussions and modelling on
slope stability of the paleo-summit. We also thank D. Burbank, S. Gallen and N. Hovius for their
very positive and constructive reviews that greatly helped to improve our manuscript. This study
was funded by the ANR Calimero and the INSU-Syster and INSU-Artemis programs. P.G.V.
acknowledges funding from the French ANR-PIA programme (ANR-18-MPGA-0006). The
Pleiades images were acquired in the framework of the Isis collaborative program between the
CNES and INSU. We thank the LMC14 (Laboratoire de Mesure du Carbone-14), ARTEMIS
national facility (LSCE (CNRS-CEA-UVSQ)-IRD-IRSN-MC) for the 14C AMS results, and the
ASTER national facility (Cerege (CNRS, Univ Aix Marseille)) for the 36Cl AMS results.
Author contributions: J.L. designed the study, conducted the modelling and wrote the manuscript,
J.L. and A.G. collected observations and samples, C.G. realized the DTM, P.G.V. the IRSL
measurements, V.Gu. and L.B. the 36Cl measurements, C.M., JP.D. and V.Ga. conducted the 14C
measurements, and T.R., C.F-L., G.M. and J.L. handled the carbonate data and core drilling in the
Ganga plain. P.G.V., C.F.L. and V.Ga. actively participated in the manuscript polishing.
1043
26
Supplementary Information
SI-1: The breccia deposits of the Sabche cirque.
Figure SI-1-1: Additional sedimentologic description of the breccia deposit in Sabche cirque.
(a) Aerial view of the Sabche cirque towards the north west with, in the background, the Annapurna III
summit and the ridge between the Annapurna Sanctuary cirque and the Sabche cirque, made up of the
Annapurna Yellow Limestone formation28. The breccia of the rock avalanche deposis is visible on a large
area of highly dissected landforms.
(b) Outcropping contact between the underlying bedrock and the breccia deposit (green dashed line).
(c) Compact, homogeneous, pulverized and indurated fine grained breccia exposed over the cliff of a steep
penitent.
(d) Internal contact between yellowish and greyish breccia facies in the central part of the deposit, as
observed from above and suggesting limited mixing during the rockslide collapse;
(e) and (f) Coarse and blocky material close to the top, displaying reverse granular grading.
(g) and (h) Contacts close to the southern base of the cirque deposit: the yellow arrow indicates a contact
between yellowish and whitish breccia facies with a larger proportion of coarse elements embedded in the
matrix of the yellowish facies; the red arrows highlight the shearing contact where thin band of shearing and
micro-crushing were sampled for IRSL analysis (CA-13-283 is located in between the two arrows pointing
toward the right).
(i) Zoom in on a rare ten-metre block of layered marly limestone (outlined by the black dashed line), within
the breccia, in the basal part of the deposit.
(j) and (k) Characteristic matrix of the breccia deposit with millimetre (j) to centimetre (k) fragments
Photos (a) to (f) = taken from an ultra-light aeroplane or from helicopter.
SI-2: Remnants of granular flow avalanche deposit in the upper Seti valley.
Along the upper Seti, few remnants of the Sabche rock avalanche deposit are preserved along the valley
walls or as isolated >200-m-high massive spurs remnants (figure SI-2-1). The preservation of the spurs is
closely linked to the pinning of the very dynamic Seti River by epigenetic gorges carved into the gneisses of
the High Himalayan Crystalline units. These epigenic gorges have been interpreted as resulting from local
landslides deposits36. However, the fine and whitish brecciated material, rich in carbonates, as well as the
absence of gneissic elements, except at the contact of the wall of the valley, indicate without any ambiguity
an origin located in the cirque of Sabche.
Figure SI-2-1: A. Map of the main remnants of the Sabche rock avalanche deposit along the upper Seti. B.
Aerial view (looking southward) of the upper Seti showing two major breccia spurs associated to epigenetic
gorges at Nanhe (yellow arrow in the foreground) and Karuwa hill (yellow arrow in the background). C.
Picture of the south face of the breccia spur at Nanhe.
SI-3: Digital Terrain Model realization
A high-resolution DTM (Digital Terrain Model) (fig. SI-3-1) has been computed from Pleiades tristereoscopic panchromatic images acquired on December 12, 2014 and November 19, 2015 at ~0.5 m spatial
resolution.
The DTMs have been calculated through an automatic pipeline which performed the data co-registration and
the DTM generation as described in (Guerin, 2017). The data co-registration is realized as a pre-processing
step in order to ensure that the images (and hence the DTM) are finely registered between each other’s. This
operation consists in a bundle-block adjustment of all the data available that can be performed without any
Ground Control Points (GCP).
Once the registration is performed, the DTMs are generated at each date according to the methodology
described in (Guérin, 2017) and based on the so-called ground space image matching, performed with the
open source software MICMAC, developed by the IGN (French Geographic Institute) (Pierrot-Deseilligny,
2006). This methodology enables the calculation of an elevation value for each point on a grid defined on
the ground with a determined planimetric and an altimetric step. Thus, for each ground point of the grid, the
images coordinates are obtained according to the refined acquisition models of the images that links the
ground position of the point and its coordinates on the images. A correlation score is then computed between
the pixels windows selected over each image and at each altitude of the grid. The final altitude value is
chosen considering the correlation score and a regularization term. Figure SI-3-1 represents the final DTM
generated from the Pleiades tri-stereoscopic images. For this study, both DTM have been generated at a 1 m
planimetric and altimetric step and resampled to 50 m for the need of the study.
Cloudy areas partially affect the summits and crests surrounding the southern and western parts of the
cirque. In those areas, the DTM was complemented from digitized 1/50,000 topographic maps (40 m
contour elevation). However, the breccia deposit and the failure surface of the rockslide in the central and
north-eastern part of the cirque respectively are well resolved, and well adapted for reconstructing the initial
volume of the deposit, measuring bedding dip of structural cliffs or for the calculation of the transverse
curvature.
Figure SI-3-1: 2 m Tri-stereo DTM (lower), before holes filling and cloud corrections, and panchromatic
0.5 m Pleiade image draped on top of the DTM (upper).
SI-4: The geometry of the initial surface
To reconstruct the initial upper surface of the landslide deposit, we can first rely on a few remnants
in the northern part of the cirque (Fig. 1c, Extended Data Fig. 4b and 5a) and on the constraints given by the
breccias outcropping on the crest that borders the southern flank of the cirque (Extended Data Fig. 2f).
These surfaces are all located at an altitude of ~4,600 m. However, they are not sufficient to constrain the
geometry of the deposit surface in its central part, and the remnants of uneroded breccias forming the steep
penitents in the centre of the cirque provide only a minimum elevation (fig. SI-4-1c).
To try to constrain this surface, it is possible to refer to the geometry generally observed for
rockslides and rock avalanches. Depending on the fall height, basal friction, degree of internal
fragmentation, or the morphology of the valley, a rock avalanche can travel a variable distance. If an
avalanche is unimpeded, it is generally characterised by a thickness and surface elevation that decreases
downstream. On the other hand, in the case of frontal confinement by a relief that hinders the avalanche
flow, depending on its residual kinetic energy, the avalanche can go up the slope (runup), and the final
deposit can be characterised by an inverse topographic gradient (e.g. Aaron and Scott McDougall, 2019).
The most commonly used parameters characterizing the geometry of rock avalanche deposit and the
avalanche mobility are the runout distance and the ratio of the fall height over the runout distance. These
parameters have been extensively documented in the past, whether or not differentiating the type of flow
confinement (e.g. Strom et al., 2019). In contrast, the shape of rock avalanche deposits has been much less
parametrically described.
The case of the Sabche cirque is quite uncommon since the avalanche was confined both frontally
and laterally, despite the fact it could overflow the ~4,600-m-high E-W crest in the southern part and also
find a lateral exit through the Upper Seti gorges. In the absence of other well-documented examples of a
collapse with such a configuration, we consider frontally confined collapses as the closest analogous and
compare with a set of 8 giant collapses. Disregarding numerous secondary depressions and ridges that can
exceed 100m in amplitude, the average geometry of these deposits shows 3 end members: a classically
decreasing elevation gradient (schematic case A on fig. SI-4-1a, and natural cases 3, 4 and 8 on fig. SI-4-1b), a uniform elevation surface (schematic case B on fig. SI-4-1a, and natural cases 1, 5 and 6 on fig. SI-4-1b), and an inverted gradient with a strong runup (schematic case C on fig. SI-4-1a, and natural cases 3, 4 and
8 on fig. SI-4-1-b). Transversely, all deposits are characterised by a convex shape (Fig. SI-4-1-b) with a
more or less flattened central part (respectively schematic cases E and D on fig. SI-4-1a).
The comparison with frontally confined collapses suggests that the presence of a depression in the
centre of the deposit is very unlikely. Longitudinally, irrespective of the type of surface deposit geometry
(type A, B or C), the centre of the deposit would be expected to be either higher than the SW edge (type A)
or higher than the NE edge (type C). In addition, the flat surface remnants in the northern part of the cirque
(red spots on Fig. SI-4-1c) display a fairly horizontal surface without any slope in either direction.
Transversely, even if the 8 collapses presented in Table SI-4-1 and Fig. SI-4-1b are not subject to lateral
confinement, a maximum elevation in the centre of the cirque with a more or less convex shape is expected.
The centre of the deposit was therefore likely ≥4,600 m. The present position of the main glacier of the
cirque, originating from Annapurna III, to the NNW, also supports this conclusion: it is strongly deflected
along the western wall of the cirque, whereas it should have flowed towards the centre of the cirque if the
deposit had been affected by a central depression.
On the basis of the above considerations, and even if one can imagine a possible convex shape in the
centre of the deposit and a chaotic surface (secondary depressions and ridges), the simplest hypothesis is to
consider that on average the initial surface of the Sabche rock avalanche can be approximated by a subhorizontal surface at an altitude of ~4,600 m. The local geometry at the transition between the cirque and the
Upper Seti valley remains however partly unresolved: there the initial surface of the Sabche deposit plunges
toward an avalanche valley deposit, the minimum altitude of which can only be estimated from the few
remnants plastered on the walls of the Upper Seti gorge (Extended Data fig. 4b and Fig. SI-2-1). Because
this zone only concerns a few square kilometres, we estimate that the associated uncertainties remain limited
to ≤1 km3. Taking into account this uncertainty as well as the possible variations in altitude of 100 to 200 m
linked to an irregular surface (secondary depressions and ridges) or a central convexity, we assume that the
uncertainties on the volume of breccia in the cirque related to the over- and under-estimation of the deposit
upper surface reaches +2 and -3 km3 respectively.
Name
Baige
Dukur Pokhari
Daguangbao
Ringmo
Aricota
Kalopani
Kofels
Flims
Country
China
Nepal
China
Nepal
Peru
Nepal
Austria
Swiss
n° on fig.
SI-4-1b
1
2
3
4
5
6
7
8
Latitude
(°N)
31.08
28.59
31.63
29.18
-17.37
28.65
47.11
48.83
Longitude
(°E)
98.70
84.19
104.12
82.94
-70.33
83.59
10.94
9.30
Estimated volume of
the deposit (km3)
0.025
1
1.3
1.5
2
3
4
12
reference
Zhang et al. (2019)
ref. 34
Chen et al. (2014)
Weidinger and Ibetsberger (2000)
Delgado et al. (2019)
Fort (2000), ref. 34
Zangerl et al. (2021)
Polet and Schneider (2004)
Table SI-4-1: Catalogue of the frontally confined rockslides and rock avalanches used in fig. SI-4-1b.
Figure SI-4-1: (a) Schematic models of the geometry of rock avalanches deposits. (b) Geometries of
various frontally confined rockslides or rock avalanche deposits (see Table SI-4-1). (c) Longitudinal cross
section on which all outcrop areas of the Sabche rock avalanche breccias have been projected (orange and
yellow areas correspond to areas SE and NW of line AA' shown on the left map), and the hypothesized
geometry of the initial surface connecting the northern and southern relics at ~4,600 m.
SI-5: Coefficient of expansion of the breccia material
In order to derive the volume of solid rock that collapsed from the Annapurna IV ridge, the volume
of breccia deposit was corrected from breccia porosity. Porosity measurements made on various samples of
breccia taken at different places in Annapurna region were realized by measuring density through weighing
of samples plunged into water and Archimede’s principle and assuming a rock density of = 2.68±0.06
g/cm3, for the Late Cambrian to Silurian TSS rocks. The porosity or expansion rate of the breccia material
slightly depends on the burial depth (Fig. SI-5-1). Average value of 15+/-5% was found consistent with
previous estimates of 15% made on rock avalanche material66.
Figure SI-5-1: Average curve of the coefficient of expansion of the breccia material vs burial depth,
as derived from density measurements made on rock and granular avalanches deposits in Sabche cirque,
Upper Seti valley and upper Marsyandi in Bragga area on the northern part of the Annarpurna massif.
SI-6: Cohesion of the unglaciated areas along the southern flank of the High Himalaya
To demonstrate the higher apparent cohesion of the rock mass building the high-altitude peaks of the
Annapurna range (Extended Data Fig. 8a), we could compare with values of the literature47, derived in
unglaciated areas. However, these previous studies in fluvially-dominated regions, which indicate cohesion
values generally <0.1 MPa, use a distinct approach and were conducted in a different setting (Longmen Shan
characterized by lower tectonic activity than the Himalaya).
In order to provide cohesion values of rock mass in unglaciated, fluvially-dominated Himalayan
areas, we applied our method (see Methods and Extended Data Fig. 7) to the landforms south of the
Annapurna massif. We defined an area (polygon in yellow color on Fig. SI-6-1a) bounded to the north by
elevation < 4,000 m and to the south by a line that follows the major topographic break in slope and river
gradient steepening (purple dashed line on Fig. SI-6-1a). Because this southern boundary is assumed to
correspond to the top of the Main Himalayan Thrust mid-crustal ramp25, this ensures that both glaciated and
non-glaciated landforms are analysed in areas affected by similar high uplift rates (~3-4 mm/yr)25,26, and that
the comparison between both landforms is relevant. Although the influence of the tectonic variable can be
ruled out, the influence of the lithology might remain, however, possible: the glaciated zone concerns
carbonate sedimentary series, whereas the non-glaciated zone is mainly dominated by gneisses.
Nevertheless, the change in slopes with altitude observed in the Everest region2 have been identified as
independent of lithology and suggests that here too lithology might not be a major controlling variable.
Our analysis shows that the rock mass building the fluvially-dominated landforms has a much lower
strength than the glaciated high-altitude ridgelines, with a proportion of unstable slopes much higher
whatever the cohesion value explored (Fig. SI-6-1b). As a matter of example, to account for ~90% of stable
slopes in fluvially-dominated landforms, the cohesion must be reduced by a factor of 4 (from 1.2 to 0.3
MPa) (Fig. SI-6-1c).
Figure SI-6-1: (a) Map of the High Himalaya in the Annapurna region displaying the two distinct
areas of fluvially-dominated southern landforms (delineated by a yellow polygon) and highest glaciated
landforms (delineated by a blue polygon), in which the systematic searching procedure (Extended Data fig.
7) has been realized using variable cohesion values but a fixed internal angle of friction of 35°. (b)
Comparison for these two areas of the fraction of unstable slopes as a function of the imposed cohesion
value. (c) Histograms of the values of the SF for values of cohesion C = 0.29 MPa and C = 1.15 MPa that
lead to a similar proportion of unstable slopes (SF < 1) for the fluvially-dominated southern landforms and
the highest glaciated landforms respectively.
SI-7: Paleotopographic reconstruction
To reconstructed the paleotopography of the collapsed summit, as also explained in the Method section, we
have applied the following computing chart:
SI-8: Effects of a rise of the 0°C isotherm within the Annapurna massif at the end of the Medieval
Climate Anomaly
The Medieval Climate Anomaly from 900 to 1200AD is characterized in some parts of the Northern
Hemisphere by warmer temperatures43. Temperature anomalies were presumably maximal north of the
Indian sub-continent, at high elevations, and at the end of the 12th century (Zhou et al., 2011). In the High
Himalaya region, this temperature increase might have exceeded +1°C, inducing a rise of the 0°C isotherm
by ~200 m.
Assuming only heat diffusion and an average thermal diffusivity for marly limestone of 1.4x10-6
m2 s−1, after 300 yr, this temperature anomaly would have propagated (with an e-fold decrease) ~150 m into
the Annapurna massif and caused a fraction of the frozen section of the future failure surface to melt, mainly
in its basal part (Fig. 3c).
To estimate this melted fraction, we consider a highly simplified and conservative 1D diffusion
thermal model applied to the substrate of the paleo-Annapurna IV. We first estimate the thickness of
permanently frozen rock below the surface, i.e. the high altitude permafrost. For that, it is assumed that the
topography consists of ~45° slopes, that the 1D (vertical) thermal gradient in the massif substrate is of the
order of 25 °C/km, that the atmospheric lapse rate is 6 °C/km and that the 0°C isotherm is located at 5000m.
Projected onto the failure surface of the giant rockslide, the model predicts that permafrost affected a portion
of about 3 km2 along the future rupture plane (light blue area in Figure SI-8-1), or 10% of the total failure
surface. Next, we diffuse (analytical solution based on the Gauss error function) the assumed medieval
climate anomaly (1°C warming for 300 years) into the massif, and calculate the area that has thawed. The
melted area would have been ~0.1 km2: this would have changed the frozen area of the failure surface by
less than 3%, and it would have changed the cohesion of a portion that represents less than 0.3% of the total
slip surface. This is a very simplified model which does not take into account the 3D effects of topography,
nor the effects of snow or ice cover, but it suggests that the change in cohesion over the entire failure plane
is proportionately very small. Unless the massif is close to a critical state or the temporary rise in
temperature also affects fluid flow, or pore pressure via a change in the water table, it is likely that the
thermal anomaly was not sufficient to trigger the slide.
Figure SI-8-1: 3D view of the presumed topography of the giant rockslide sliding surface with the frozen
parts in light blue shading.
SI-9: The Pokhara conglomeratic deposits
A. Sedimentology of the Pokhara conglomeratic formation in the Pokhara basin
The Pokhara conglomeratic Formation is characterized by a succession of planar metric to multimetric conglomerate beds ago30,36, with different facies (Fig. SI-9-1) and extend from upper Seti (Karuwa =
site K of fig.1, down to the mountain front at Narayangad (site N)). All the units are essentially made of
angulous to sub-angulous dark limestone pebbles: the origin of the gravels and pebbles is at >95% of TSS
limestone and marly limestone, with a few rare granitic or gneissic pebbles, and whose only possible origin
is in the Sabche cirque.
Figure SI-9-1: Pictures of some of the typical facies of the Pokhara conglomeratic Formation
In the center of Pokhara valley, most facies are well stratified conglomeratic alluvium made of
alternation of relatively well sorted TSS gravels or pebbles (Figure SI-9-1-b and c) of variable median size
(0.5cm < D50 < 5cm). The clasts are subangular to subrounded and are commonly imbricated (Figure SI-9-1d). The contacts between the layers are mostly planar, without presenting any major unconformity surfaces,
except at a few places where small channel reincision by a few meters can be observed (Figure SI-9-1-e). If
typical proximal debris-flow units with poorly sorted and matrix-supported material were not observed in
the Pokhara basin, layers made of unsorted homometric material (Figure SI-9-1-f), clast- to matrixsupported, suggest clast transport by competent or hyperconcentrated flows. Even in its upstream part
(Figure SI-9-1-a), south of Karuwa, a majority of the facies correspond to dominant stratified alluvium,
interlayered with a minority of debris-flow-like units. The sorting increases downstream both in the clast and
matrix-supported facies.
We interpret these planar conglomeratic units to reflect rapid aggradation during turbulent, sedimentladen flows, with successions of debris floods, sheet and hyperconcentrated flows (Blair and McPherson,
2009), and alluvial reworking. The dominance of sediment-laden or hyperconcentrated require an initial
sediment source reach in fine fraction. Finally, it has to be noted that the clasts as well the silty matrix of the
conglomerate units display carbonate content very similar to the average material of the Sabche rockslide
breccia (fig. 4).
B. Narayangad terrace
The Pokhara conglomeratic formation can be tracked at least down to the Himalayan front, just north of
the city of Narayangad (see loc. “N” on fig. 1). There, the remnant of a peculiar fill deposit observed at a few
meters above the present river on the left bank of the Narayani river in a somehow sheltered environment.
This unit contrasts strongly with the other terrace deposits of the Narayani at that place as much on the level
of its much finer granulometry as on the level of its composition much richer in carbonates. This unit
presents two sequences of opposite grading but with constant CaCO3 content (Figure SI-9-2),: a basal unit
with normal grading unit terminated by very fine silts is overlain in continuity by an inverse grading
sequence that ranges from coarse sand to gravel clast-supported conglomerates made of small subangular,
sorted, but unstratified, dark limestone gravels of similar composition as Pokhara conglomerates. We
interpret such particular stratigraphic order as a deposition first of a suspended sediment laden front, likely
generated just in front of the rock-avalanche, followed by aggradation of the fine pebble load that travelled
more slowly as a debris or dense flow. Nevertheless the scarcity of these remnants of units indicates that
after the deposition of the sequence, the Narayani river was able to recover rapidly and to wash its banks of
these ~10-15 m deposits of fine conglomerates, except very locally. A microscopic charcoal or organic
debris (C14-68-E) was found at the middle of the silty sequence, and is synchronous of the upper Seti rock
avalanche and onset of the filling of Pokhara basin (fig. 2b).
Figure SI-9-2: Micro-conglomerate deposit north of Narayangad (site N on fig.1)
C: Volume of deposited Pokhara type sediments
The volume of conglomerates in the Pokhara and upper Seti valley was estimated to reach ~5 to ~7
km (refs. 30, 36) over a distance of 70 km. Further downstream, the volume of deposited Pokhara sediment
was estimated by considering the present geometry of the valley and a sediment filling assuming a linearly
decreasing thickness from Pokhara to the Gangetic plain, based on our field measurements: 60 m in the
southern Pokhara basin, 20 m at the confluence of the Seti with the Trisuli, 13 m at Narayangad (site “N” on
fig. 1b), and 3-5 m at the GR2 borehole (site “G” on fig. 1a). In the Gangetic plain, the long and narrow lobe
raised on top of the mega-fan (blue-colored in Extended Data fig.10) was assumed to correspond to the
deposition of sediments preferentially derived from the rockslide.
Between the southern part of the Pokhara basin and Naryangad (site “N”), the deposited volume is
~1 km3. Between Naryangad and the MFT (most frontal thrust), in the Chitwan dun (piggy-back basin), an
additional volume of ~2 km3 of fine gravels and sands could have been deposited. Downstream of the MFT,
the lobe deposited at the top in the Narayani mega-fan has represents a volume of sediment of ~6 km3 (L x
W x H ≈ 120 km x 12 km x 4 m). As a whole, 15 km3 of sediments might have been deposited between
Pokhara and the Narayani megafan, that is, considering a porosity of 30% of the sediments, ≤50% of the
initial rock volume. The other half was presumably exported further downstream as suspended load like
most Himalayan sediments that end up in the Bengal Fan39.
It has to be noted that we assume above that the downstream units were not fed by the re-erosion of
the upstream conglomerates. The synchronism of the sediment at Narayangad (downstream site “N”) with
the initial granular avalanche in the most upstream part supports such hypothesis.
3
D: Proportion of Sabche material in the Gandak river after the rockslide
The carbonate-rich lobe of the Narayani fan displays variable carbonate concentration between 30
and 60%, but on average it remains slightly lower than the Sabche breccia and Pokhara conglomerate. This
likely arises from mixing with background supply of sediments issued from the whole Narayani catchment.
Considering the average carbonate concentrations of around 55, 40, and 10% for breccia material,
sediments in the carbonate-rich lobe of the Narayani fan (upper units of GR2), and mean Narayani sediments
(in GR1, lower GR2 cores or sandy fraction of modern sediments) respectively, it is shown that the
carbonate-rich lobe of the Narayani was corresponding to the mixing of two volumes of Sabche debris and
one volume of background Narayani sediments. Following the Annapurna IV collapse, the input of Sabche
material would have tripled the Narayani sediment flux compared to average values. In order to evacuate
~15 km3 (the total rockslide volume minus the breccia remaining in the Sabche cirque and the deposited part
along the middle and lower Seti respectively) of breccia material out of the mountain range at Narayangad,
such a situation could have persisted ~150 yr, which seems to confirm accelerated erosion of the Sabche
deposit over a period of 100 yr following the summit collapse.
SI-10: 14C dating of the various organic debris (wood pieces and charcoals) samples of this study
The pdf associated to the mean age for Karuwa and the basal Pokhara conglomerates (Fig. 2) were
calculated by computing the uncertainty-weighted average of the 14C raw radiocarbon ages (8 dating for
each mean age), before applying the calibration curve of IntCal 13 (ref. 54).
Lab.No
Sample ID
Karuwa debris flow
SacACA-14-59
41772
Lat [°N]
Lon [°E]
Elevation
[m asl]
Material
stratigraphic setting
Radiocarbon
age [y BP]
Calibrated age
28°
23.24'
83° 58.51'
1460
wood
base of the granular
avalanche breccia
950 ± 30
1085 AD ± 47
SacA43078
CA-14-58
28°
23.24'
83° 58.51'
1460
wood
base of the granular
avalanche breccia
840 ± 30
1199 AD ± 29
SacA48072
CA-16-5-C
28°
23.24'
83° 58.51'
1460
bark
base of the granular
avalanche breccia
830 ± 30
1208 AD ± 30
CA-16-5-D
28°
23.24'
83° 58.51'
1460
monocots
type stem
base of the granular
avalanche breccia
870 ± 30
1139 AD ± 61
CA-16-5-D
Replicate
895 ± 30
1125 AD ± 52
SacA48714
CA-16-7
28°
23.25'
83° 58.43'
1530
wood
(root)
835 ± 30
1212 AD ± 30
SacA46541
CA-16-8-B
28°
23.25'
83° 58.43'
1530
small
branch
paleosoil buried by
the granular
avalanche breccia
base of the granular
avalanche breccia
935 ± 35
1094 AD ± 48
SacA48715
CA-16-8-E
28°
23.25'
83° 58.43'
1530
small
branch
base of the granular
avalanche breccia
825 ± 30
1229 AD ± 22
28° 6.62
84° 7.18
588
small tree
trunk
Paleosoil buried by
the Pokhara Fm
740 ± 30
1261 AD ± 15
Paleosoil buried by
the Pokhara Fm
940 ± 30
1090 AD ± 46
SacA46539
SacA48073
Pokhara basin
SacACA-13-245
41776
SacA41777
CA-13-246
28° 6.64'
84° 7.19'
605
charcoal
SacA43080
CA-13-246B
28° 6.64'
84° 7.19'
605
charcoal
SacA41773
CA-14-63-B 28° 8.05'
84° 5.70'
640
charcoal
SacA43079
CA-14-64-A 28° 7.17'
84° 6.31'
612
charcoal
SacA41774
CA-14-64-B 28° 7.17'
84° 6.31'
612
Paleosoil buried by
the Pokhara Fm
Thin sand layer at
the top of Pokhara
Fm
Paleosoil buried by
the Pokhara Fm
955 ± 30
1082 AD ± 47
1940 ± 30
57 AD ± 33
940 ± 30
1090 AD ± 46
charcoal
Paleosoil buried by
the Pokhara Fm
940 ± 30
1090 AD ± 46
SacA41775
CA-14-66
28° 5.60'
84° 4.53'
547
charcoal
Paleosoil buried by
the Pokhara Fm
3375 ± 35
1678 BC ± 45
SacA46544
CA-16-27
28° 5.57'
84° 3.89'
549
wood
slackwater deposits
at tributary damming
870 ± 30
1139 AD ± 61
27°42.83' 84°26.03'
198
Microcharcoal
Silt layer at the base
of fine
conglomerates
885 ± 25
1126 AD ± 60
Narayangad
WHOI133624
CA-14-68E
Table SI-10-1: Details of the samples of the organic debris found at Karuwa, in Pokhara basin and
Narayangad, and dated by 14C AMS dating. Several charcoals, in particular CA-14-63B and CA-14-66 in
paleosoils buried by the Pokhara conglomeratic formation, are clearly too old and suggest either long
residence time in the soil (CA-14-66) or reworking (CA-14-63B).
SampleID
River
ref.
Latitude
(°N)
Pokhara Elevation
Fm top
of the Seti Age (calibrated 14C age
elevation river
AD)
mediane
(m asl)
(m asl)
(P50%)
P2.5%
P97.5%
1246
1167
1290
Humic Silt
828
710
1193
1046
1269
Leaves
773
680
Sample
Longitude elevation Material
(°E)
(m asl)
BIJ02
Bijaypur
36
28.219
84.029
820
PH02C
Phusre
36
28.18
83.968
730
PHUSRE03
Phusre
36
28.183
83.951
740
Charcoal
773
680
1098
1016
1205
1298
SARA1-1
Saraudi
36
28.096
84.029
541
Wood
560
490
1265
1215
MADHI2
Magdi
36
28.015
84.117
495
Charcoal
500
438
1061
990
1155
1038
1224
PH6
Phusre
36
28.175
83.976
703
Wood
773
680
1160
Phusre010
Phusre
36
28.175
83.982
706
Wood
773
680
1170
1043
1241
680
1232
1169
1270
Phusre012
Phusre
36
28.175
83.982
698
Phusre013
Phusre
36
28.175
83.982
696
Wood
773
680
1199
1058
1262
Phusre14
Phusre
36
28.175
83.982
701
Charcoal
773
680
1219
1163
1264
PhusreFa
Phusre
36
28.185
83.958
728
Charcoal
773
680
1106
1033
1204
PRE14C1
Phusre
36
28.184
83.934
748
Charcoal
773
680
1347
1298
1410
1263
1388
Leaves
773
KBP03
Bijaypur
36
28.218
84.027
825
Wood
828
710
1289
TAL02
Tal
36
28.117
84.105
583
Wood
623
530
1214
1161
1264
1025
1165
TAL05
Tal
36
28.117
84.105
614
Wood
623
530
1099
Anpu02
Anpu
36
28.109
84.116
591
Humic Silt
618
530
1097
1024
1155
KBP02
Bijaypur
37
28.218
84.027
825
Humic Silt
828
710
1176
1045
1250
1049
1256
Phusre11
Phusre
37
28.175
83.982
698
Wood
773
680
1186
Tal01
Tal
37
28.117
84.105
585
Humic Silt
623
530
1224
1165
1265
1025
1165
14CAnpu11
Anpu
37
28.108
84.114
590
Charcoal
618
530
1099
14CAnpu01
Anpu
37
28.111
84.105
556
Leaves
618
530
1186
1049
1256
1161
1264
14CAnpu16
Anpu
37
28.11
BIJ03
Bijaypur
37
28.219
Ph6u_14C
Phusre
37
28.176
592
Charcoal
618
530
1214
84.029
820
Humic Silt
828
710
1299
1270
1390
83.977
704
Wood
773
680
1113
1037
1207
656
769
84.12
EBGcc01
Anpu
37
28.111
84.12
609
Charcoal
618
530
698
PhTree1
Phusre
38
28.183
83.953
725
tree trunk
773
680
1232
1169
1270
1223
1295
PhTree2
Phusre
38
28.183
83.953
725
tree trunk
773
680
1272
AnpuTrib
Anpu
38
28.118
84.105
587
tree trunk
623
530
1209
1157
1264
530
1242
1190
1279
AnpuTree1
Anpu
38
AnpuTree2
Anpu
38
28.11
84.122
608
tree trunk
618
530
1240
1189
1277
AnpuTree3
Anpu
38
28.111
84.121
591
tree trunk
618
530
1228
1168
1266
1058
1262
AnpuTree4
Anpu
AnpuTree5
Anpu
upper
Begnas
CA-14-63-B
CA-14-64-A
CA-14-64-B
CA-14-66
CA-13-245
CA-13-246
CA-13-246B
CA-16-27
Rupa
Rupa
Seti
Majuwa
Anpu
Anpu
Anpu
Rudi
28.111
84.12
601
tree trunk
618
38
28.111
84.121
591
tree trunk
618
530
1199
38
this
study
this
study
this
study
this
study
this
study
this
study
this
study
this
study
28.11
84.12
592
618
530
1224
1165
1265
28.1342
84.0950
640
640
560
62
-20
130
28.1195
84.1052
612
623
532
1098
1025
1160
28.1195
84.1052
612
623
532
1098
1025
1160
28.0933
84.0755
547
585
507
-1669
-1753
-1549
28.1103
84.1197
588
618
530
1270
1224
1291
28.1107
84.1198
605
618
530
1098
1025
1160
28.1107
84.1198
605
tree trunk
charcoal in
sand lense
charcoal in
paleosoil
charcoal in
paleosoil
charcoal in
paleosoil
small tree
trunk base
charcoal in
paleosoil
charcoal in
paleosoil
618
530
1096
1022
1155
28.0929
84.0649
549
580
500
1176
1045
1250
wood
Table SI-10-2: Published 14C dating (dated by AMS) of the various organic debris samples of the
Pokhara conglomeratic formation, including their location and their elevation as well as that of the Seti
and the top of the Pokhara formation used in fig.2b. The sample names in bold correspond to the most basal
ones, used in fig.2a.
SI-11: 36Cl dating of blocks at the rockslide deposit summit (4600m)
36
Cl exposure ages of the blocks sampled at the top of the deposit at ~4,600 m have been calculated
incorporating snow cover correction and potential erosion. The snow cover correction corresponds to about
25-30 cm of snow (in water equivalent) during 120 days (mainly from January to May) and is calculated on
the basis of 50% of the snow cover measured at several stations located between 3,200 and 4,400 m along
the ridges that dominate the Khudi Khola (25 km east of Sabche cirque in a slightly more advanced position
on the southern flank of the Himalayas) between 1999 and 2002 (ref. 57).
Using depth and duration of snow cover measured on the Himalayan southern flanks 25 km to the
east61, estimate of the shielding factor by snow would reach 0.87. However, as observed on site or on
satellite images, the top of the plurimetric blocks are cleared of their snow cover much faster than the rest of
the flat surrounding ground. Considering also that the Sabche cirque may be partially sheltered compared to
the most exposed southern flank and receives less snow precipitation, we therefore adopt a conservative
snow shielding factor of 0.95+/-0.5.
Sample
name
CA13-260
CA13-261
CA13-262
CA13-263
CA13-264
CA13-265
boulder
Bulk
height sample
density
Latitude Longitude Altitude
above thickness
ground
(° N)
(° E)
(m)
(m)
(cm)
(g/cm3)
Lithology
marly
limestone
schistosed
marly
sandstone
schistosed
marl
schistosed
marl
schistosed
marl
schistosed
marly
sandstone
Possible
erosion of
the block
surface
(cm)
Topographic
shielding
Snow
shielding
28.5352
84.0274
4,590
5
3
2.6
0
0.975
0.94±0.05
28.5359
84.0271
4,590
2.5
3
2.6
2±2
0.975
0.94±0.05
28.5365
84.0276
4,590
5
4
2.6
1±1
0.975
0.94±0.05
28.5374
84.0281
4,590
1 to 4
10
2.6
3±3
0.975
0.94±0.05
28.5382
84.0284
4,590
1.2
5
2.6
2±2
0.975
0.94±0.05
28.5394
84.0297
4,590
1.1
10
2.6
1±1
0.975
0.94±0.05
Table SI-11-1: Location and characteristics of the 6 blocks sampled on Nov. 2013 at the top of the relic
surface of the rockslide deposit.
Sample
name
%
dissolv
ed
rock
%
CA13-260
82.2
CA13-261
52.8
CA13-262
75.6
CA13-263
72.9
CA13-264
75.7
CA13-265
26.0
Blank
36
Cl/35Cl
35
Cl/37Cl
(×10- 13)
2.706 ±
0.081
1.551 ±
0.058
2.493 ±
0.075
2.157 ±
0.071
1.130 ±
0.049
0.515 ±
0.032
0.072 ±
0.012
33.387 ±
0.793
40.284 ±
1.317
24.061 ±
0.886
27.046 ±
1.115
31.172 ±
1.218
31.209 ±
1.295
427.025
± 3.187
36
Cl
(/g of
dissolved
rock)
(/m of
in
K2O
dissolved target
rock)
fraction
Cl
CaO
Exposure age Exposure age
TiO2 Fe2O3 without snow with snow
shielding
shielding
103 atoms/g [ppm]
%
%
%
168.3 ± 5.2
4.4
52.36
0
0
165.5 ± 6.5
6.2
54.28
0
170.5 ± 5.3
6.8
51.86
145.1 ± 4.9
5.9
84.7 ± 3.8
93.6 ± 6.4
%
yr
yr
0
796 ± 70
839 ± 76
0
0
758 ± 69
799 ± 74
0
0
0
816 ± 70
860 ± 80
52.69
0
0
0
749 ± 73
790 ± 76
5.7
52.65
0
0
0
423 ± 39
446 ± 43
14.0
54.41
0
0
0
446 ± 46
470 ± 49
Table SI-11-2: Sample information, AMS measurement results, and calculated 36Cl and chlorine
concentrations and concentration of Ca measured at ICP-AES (inductively coupled plasma atomic emission
spectrometry).
Exposure
ages
calculated
with
Online
Chronus
Calculator
(http://hess.ess.washington.edu/math/index_dev.html).
Reported
analytical
uncertainties
include
uncertainties in AMS counting statistics, the isotopic ratios of the standards, and on the blank chemical
measurements, an external AMS error of 0.5% (Arnold et al., 2010), and the uncertainties associated with
the cosmogenic nuclide half-lives. External uncertainties include the uncertainties associated with the
cosmogenic nuclide production rates, the sample thickness, the estimated surface and snow shielding.
Sample
Name
SiO2 TiO2 Al2O3 Fe2O3 MnO MgO CaO Na2O K2O H2O
%
%
%
%
%
%
B
Sm
Gd
U
Th
Cr
Li
%
%
%
9.65 0.09
CA13-261 31.12 0.26
CA13-262 13.70 0.13
CA13-263 15.83 0.19
1.80
0.76
0.01
2.36 45.49 0.41
0.51 0.43
10
9
0.92 0.75 0.69 2.60
4.79
0.89
0.02
2.09 31.64 1.11
1.16 0.79
10
22
2.07 1.71 1.49 8.13 15.3 12.8
2.52
0.81
0.02
2.53 42.45 0.65
0.70 0.49
10
12
1.60 1.29 0.84 3.66
3.60
1.00
0.02
2.65 40.65 0.58
1.12 0.61
10
17
2.45 1.95 0.81 5.22 14.5 11.3
CA13-264 15.62 0.19
CA13-265 45.41 0.44
3.70
1.18
0.02
1.80 41.05 0.55
1.03 0.65
10
36
2.13 1.80 0.70 5.54 16.5
9.8
8.57
2.84
0.03
3.03 19.02 1.21
2.58 1.31
25
46
4.21 3.49 1.55 11.9 33.2
23
CA13-260
%
Cl
ppm ppm ppm ppm ppm ppm ppm ppm
4.3
7.2
Table SI-11-3: Bulk rocks geochemical composition of the 6 blocks sampled at the top of the relic surface
of the rockslide deposit: Major and trace elements composition measured by ICP-MS (SARM, CRPG).
5.2
6.2
SI-12: Infra-red stimulated luminescence (IRSL) measurements and results on sheared breccia
deposits
Breccia samples were first sawed in subdued light conditions to remove the exposed outer rock surfaces.
Samples were then dissolved in HCl. and the silicate fraction was treated using H2O2 (30%) and sieved to
coarse sand fraction before density separation with LST to isolate K-felspar fraction (d<2.58). K-feldspar
separates were settled on 10-mm diameter stainless steel discs for subsequent luminescence analyses. About
50 g of bulk sample was saved (external breccia surface) and sent to Actlabs or SARM-CRPG for ICP
analysis to quantify U, Th and K contents (Table SI-8-1). Final dose rate determination, using grainsize
range and estimated water content, were obtained through the Dose Rate and Age Calculator59 (DRAC).
All conventional luminescence measurements were carried out using TL/OSLDA-20 Risø readers, equipped
with a calibrated 90Sr/90Y beta source (reader dose rate ~0.1Gy/s; Institute of Earth Surface Dynamics,
University of Lausanne, Switzerland). Luminescence signals were detected using an EMI 9235QA
photomultiplier tube, in the blue region through a Schott BG-39 filter for IRSL measurements. We used the
post-IR IRSL protocol60, applying first a preheat treatment at 250°C for 60s before a single-aliquot
regenerative-dose (SAR) protocol (Murray & Wintle 2000) with a first IRSL stimulation at 50 °C (100 s,
IR50) followed by a second IRSL stimulation at 225 °C (100 s, pIR225). For all the samples, residual doses
and dose recovery ratios were quantified. Dose–response curves were constructed using an
exponential+linear fitting (with recycling ratios within 15% of unity and recuperation within 10% of the
natural dose were used as acceptance criteria for the single-aliquot data).
We used the Luminescence R package (Kreutzer et al. 2012) to quantify the Central Age Model (CAM) for
all samples61, and based on the overdispersion in De distributions we also calculated a Minimum Age Model
(MAM) for sample CA-13-283 (ref. 61). Fading rates (g2days) were measured following Auclair et al. (2003)
(Figure SI-12-1). Final fading-corrected ages were calculated following ref. 62 fading correction procedure
(Table SI-12-2).
Sample
CA-13-273
CA-13-274
CA-13-283
Coordinates
(WGS84, °N/°E, and
elevation, m a.s.l.)
28.5058; 84.0001
3410
28.5058; 84.0001
3410
28.5055; 83.9999
3405
Radionuclide concentration1
U (ppm)
Th (ppm)
K (%)
Grain size
range2 (μm)
Total dose
rate3 (Gy ka-1)
1.69±0.06
10.8±0.38
1.82±0.06
90-180
3.83±0.21
1.64±0.06
10.3±0.36
1.90±0.07
90-180
3.85±0.21
2.15±0.08
12.6±0.44
2.04±0.07
180-250
4.39±0.17
Table SI-12-1. Details of dose-rate calculations for IRSL dating.
Samples were collected at the base of the breccia deposit (see Extended Data Figs. 2 and 6) but this was
rapidly incised after deposition. To account for this, a 0.25 Gy dose has been substracted for post-deposition
exposure to cosmic rays.
1
Radionuclide concentrations were quantified on bulk samples using ICP at Actlabs (CA-13-273/-274) and
at SARM-CRPG (CA-13-283).
2
Coarse-grain fraction of 90-180 μm was isolated for samples CA-13-273 and CA-13-274, and of 180-250
μm for CA-13-283, to ensure material abundance for IRSL measurements.
3
Dose rate calculations were performed with DRAC59, assuming water content of 2±2%, an internal Kcontent of 12.5±0.5% (Huntley and Baril, 1997) and an alpha efficiency value of 0.15±0.05 (Balescu and
Lamothe, 1994).
Sample
(aliquots)
Signal
Dose
recovery
ratio1
CA-13-273
(17)
CA-13-274
(17)
CA-13-283
(60)
IR50
pIR225
IR50
pIR225
IR50
pIR225
0.94±0.04
0.90±0.03
0.94±0.04
0.91±0.03
0.88±0.02
0.86±0.02
residuals
(Gy)
CAM2
uncorrecte
d De (Gy)
OD
(%)
MAM3
uncorrecte
d De (Gy)
0.57
2.27
0.57
2.26
0.25
0.27
84.2±9.0
156.7±25.8
90.5±8.8
155.8±26.0
7.5±2.2
8.7±2.9
9.2
15.0
8.0
15.3
28.7
32.3
/
/
/
/
5.2±0.6
6.2±1.0
1
2
Fading4
g2days
(%/decade
)
1.40±0.48
0.58±0.34
1.47±0.75
0.63±0.28
1.37±0.64
0.59±0.39
CAM or MAM*
fading-corrected
age (ka)
31.9±4.3
34.6±4.1
64.4±7.4
48.1±2.5
1.21±0.23*
1.50±0.30*
Table SI-12-2. IRSL dating of the base of the breccia deposit (see Extended Data Figs. 2d and 6) on small-aliquots,
using feldspar infra-red luminescence at 50°C (IR50) and post-infra-red infra-red luminescence at 225°C (pIR225)
signals. Analytical details and measurement protocols are given in supplementary text.
1
Dose recovery ratios have been calulated for each individual sample and using a given dose of 60Gy for 5 aliquots.
Residuals have been estimated for 4 aliquots per sample, with an additional 0.25 Gy to account for post-deposition
cosmic-ray exposure following rapid breccia incision.
2
De = Equivalent doses, CAM = Central Age Model, OD = overdispersion of De distribution61.
3
MAM = Minimum Age Model61 (MAM-3). It was applied for De distributions with OD > 25%, i.e. for sample CA13-283. Sigma-b (σb) values of 0.085 and 0.152 were used for IR50 and pIR225 signals, respectively, based on OD
values for CA-13-273/-274 samples. No MAM was run for CA-13-273/-274, due to the low OD in the De distribution
(Fig. SI-12-1).
4
Fading measurements have been performed on 12-14 aliquots per sample to evaluate the variability in fading rates for
IR50 and pIRI225 signals (Fig. SI-12-1).
Fig. SI-12-1. Fading rate (g2days values) distributions for K-feldspar IR50 (A) and pIRIR225 (B) signals.
Insets show representative examples of fading measurements. Fading rate distributions illustrate similar
behaviour between CA-13-273, -274 and -283.
SI-13: Carbonate content
Total carbonate (Table SI-13-1) content was measured by phosphoric acid dissolution at 70°C on a
gas-bench coupled to a MAT253 spectrometer using internal concentration standards. Concentrations are
expressed in equivalent CaCO3 wt% and the overall uncertainty is ± 2% relative.
Sample
Sample Type
Lithology
river / location
Latitude Longitude
distance
from
Sabche
divide
CaCO3
(km)
%
19
56.0
(°N)
(°E)
28.4200
83.9921
28.3219
83.9573
30
29.4
83.9573
83.9429
30
48.5
Rivers
Seti & downstream (Trisuli, Narayani)
HF 10
sand (bank)
sand
Seti Kh. North of
Pokhara
CA-13-232
sand (bank)
sand
Seti Kh. North of
Pokhara
CA-13-233
sand (bank)
silt
Seti Kh. North of
Pokhara
HF 11
sand (bank)
sand
Seti Khola
28.3219
28.3015
36
44.0
Seti Khola
28.1274
84.0836
69.5
44.4
84.0700
LO 313
sand (bank)
sand
MO 305
Suspended Load
silt
Seti Khola
28.0500
78.5
47.0
CA-14-72
sand (bank)
sand
Seti Khola
11.0
sand (bank)
sand
Seti Khola
84.4662
84.4635
142
LO 303
27.8273
27.8255
142
24.7
CA 11133
sand (bank)
sand
Seti Khola
27.8292
84.4667
142
17
CA13-251
sand (bank)
sand
lower Trisuli
27.7564
84.4638
157.3
8.2
CA-14-67
sand (bank)
sand
Narayani
27.7022
84.4262
166.5
9.18
LO 309
sand (bank)
sand
Narayani
27.7020
84.4260
166.5
14.7
NAG 48
sand (bank)
sand
Narayani
27.7020
84.4260
166.5
13.8
LO 741
sand (bank)
sand
Nayayani
27.7020
84.4260
166.5
15.0
CA 11-140
sand (bank)
sand
Narayani
27.7020
84.4260
166.5
9.19
Narayani
sand (mean)
sand
Narayani @
Narayangad
27.7020
84.4260
166.5
12.4
Narayani
(std)
Suspended load
(average 2010
monsoon)
2.9
silt
Narayani @
Narayangad
27.7020
84.4260
166.5
(std)
CA 11305
sand (bank)
16.4
3.9
sand
Narayani
26.8598
84.2098
337
9.3
CA 11306A sand (bank)
Bank
Narayani
27.2059
83.9300
284
10.1
CA 11306B sand (bank)
Bank
Narayani
27.2059
83.9300
284
7.8
CA 11306D sand (bank)
Bank
Narayani
27.2059
83.9300
284
7.9
CA 11307
sand (bank)
Bank
Narayani
27.1215
84.0609
301
9.4
CA 11308
sand (bank)
Bank
Narayani
26.6890
84.4333
369
11.2
Main Tributaries
CA-13-237
sand (bank)
sand
Mardi Khola
28.2996
83.9267
38
7.9
CA-13-238
sand (bank)
sand
Madi Khola
28.2341
84.0868
113
2.4
CA-13-239
silt (bank)
silt
Madi Khola
28.2243
84.0592
113
4.2
CA-13-247
sand (bank)
sand
Madi Khola
27.9783
84.2600
113
3.64
LO 305
sand (bank)
sand
Trisuli
27.8180
84.4640
144
3.2
LO 307
sand (bank)
sand
Kali Gandaki
27.7486
84.4194
161
12.3
CA11136
sand (bank)
sand
Kali Gandaki
27.7486
84.4194
161
21.2
CA-13-126
sand (bank)
sand
Kali Gandaki
27.7486
84.4194
161
15.6
CA-13-128
sand (bank)
sand
Kali Gandaki
27.7486
84.4194
161
17.9
Breccia (Sabche rockslide deposit)
CA-13-270
breccia
matrix
Sabche cirque _
base of deposit
28.5061
83.9999
5
48.6
CA-13-275
sediment in gully
sand
Sabche cirque _
base of deposit
28.5039
83.9996
5
65.1
66.7
CA13275_A
sediment in gully
clast
Sabche cirque _
base of deposit
28.5039
83.9996
5
15.9
CA13275_B
sediment in gully
clast
Sabche cirque _
base of deposit
28.5039
83.9996
5
99.0
CA13275_C
sediment in gully
clast
Sabche cirque _
base of deposit
28.5039
83.9996
5
82.6
CA13275_D
sediment in gully
clast
Sabche cirque _
base of deposit
28.5039
83.9996
5
87.7
mixed clasts
Sabche cirque _
base of deposit
28.5056
83.9999
5
79.2
CA-13-280
CA13280_A
breccia
mixed clasts
Sabche cirque _
base of deposit
28.5056
83.9999
5
58.7
CA13280_B
breccia
mixed clasts
Sabche cirque _
base of deposit
28.5056
83.9999
5
75.1
matrix
Sabche cirque _
base of deposit
28.5056
83.9999
5
41.2
CA-13-281
CA13281_A
breccia
clast
Sabche cirque _
base of deposit
28.5056
83.9999
5
55.6
CA13281_B
breccia
clast
Sabche cirque _
base of deposit
28.5056
83.9999
5
20.8
CA13281_C
breccia
clast
Sabche cirque _
base of deposit
28.5056
83.9999
5
88.5
CA13281_D
breccia
clast
Sabche cirque _
base of deposit
28.5056
83.9999
5
71.0
55.9
Breccia (upper Seti rock avalanche deposit)
CA-14-57
avalanche deposit
(top)
matrix
upper Seti Kh. _
Nanhe spur
28.4112
83.9871
21
CA-14-56
fluid injection vein
greenish part
upper Seti Kh. _
Karuwa
28.3870
83.9751
24
CA-14-60
42.9
pinkish part
43.8
whitish part
19.8
breccia of light tone
clasts
upper Seti Kh. _
Karuwa
28.3870
83.9751
24
46.5
41.3
CA-16-9
avalanche deposit
(base)
matrix
upper Seti Kh. _
Karuwa
28.3875
83.9738
24
21.8
CA-16-10
avalanche deposit
(base)
matrix
upper Seti Kh. _
Karuwa
28.3875
83.9738
24
27.4
CA-16-11
avalanche deposit
(base)
matrix
upper Seti Kh. _
Karuwa
28.3875
83.9738
24
35.4
CA-16-12
avalanche deposit
(base)
matrix
upper Seti Kh. _
Karuwa
28.3875
83.9738
24
gravels
33.2
39.9
CA-16-13
avalanche deposit
(base)
matrix
upper Seti Kh. _
Karuwa
28.3875
83.9738
24
29.7
CA-16-14
avalanche deposit
(base)
matrix
upper Seti Kh. _
Karuwa
28.3875
83.9738
24
55.9
CA-16-15
avalanche deposit
(base)
upper Seti Kh. _
light tone gravels Karuwa
28.3875
83.9738
24
39.6
CA-16-17
avalanche deposit
(base)
CA-16-18
avalanche deposit
(base)
dark gravels
33.3
matrix
41.5
matrix
upper Seti Kh. _
Karuwa
upper Seti Kh. _
matrix
Karuwa
light tone gravels
28.3873
83.9750
24
32.3
28.3873
83.9750
24
45.5
26.9
dark gravels
13.6
CA-16-19
avalanche deposit
(base)
matrix
upper Seti Kh. _
Karuwa
28.3873
83.9750
24
30.3
CA-16-20
avalanche deposit
(base)
matrix
upper Seti Kh. _
Karuwa
28.3869
83.9750
24
30.9
CA-16-4
avalanche deposit
(base)
matrix
upper Seti Kh. _
Karuwa
28.3846
83.9757
24.5
38.2
CA-16-21
avalanche deposit
(base)
matrix
upper Seti Kh. _
Karuwa
28.3828
83.9754
24.8
28.6
CA-16-2
avalanche deposit
(middle height)
gravels
upper Seti Kh. _
Karuwa
28.3806
83.9733
matrix
25
59.8
25
68.3
CA-16-3
avalanche deposit
(middle height)
matrix
upper Seti Kh. _
Karuwa
28.3825
83.9732
25
70.5
CA-16-22
avalanche deposit
(middle height)
matrix
upper Seti Kh.
28.3594
83.9571
29
59.6
CA-13-234
avalanche deposit
(middle height)
matrix
Seti Kh. _ North
of Pokhara
28.3203
83.9567
34
28.2
CA-14-62
isolated breccia
remnant
matrix
Seti Kh. _ South
of Pokhara
28.1229
84.0849
68.5
40.3
gravels
upper Seti Kh. _
Karuwa
28.3860
83.9750
24
32.7
CA19-18_A conglomerate
upper Seti Kh. _
individual gravel Karuwa
28.3860
83.9750
25
14.3
CA19-18_B conglomerate
upper Seti Kh. _
individual gravel Karuwa
28.3860
83.9750
25
75.0
CA19-18_C conglomerate
upper Seti Kh. _
individual gravel Karuwa
28.3860
83.9750
25
58.0
CA19-18_D conglomerate
upper Seti Kh. _
individual gravel Karuwa
28.3860
83.9750
25
74.4
CA-16-24
gravels
28.3591
83.9566
29
59.1
29
52.7
Pokhara formation
CA-19-18
conglomerate
conglomerate
upper Seti Kh.
matrix
CA-13-236
conglomerate
CA-14-61
fine conglomerate
matrix
Seti Kh. _ north
of Pokhara
Seti Kh. _ north
of Pokhara
28.3096
83.9406
37
58.5
28.1340
84.0731
66.3
55.7
CA-13-240
CA-13-241
slackwater deposit
unstratified angular
clasts unit
silt
gravels
Seti Kh. _ south
of Pokhara
Seti Kh._ south
of Pokhara
28.1088
28.1083
84.1151
84.1156
matrix
70
58.1
70
57.1
70
58.9
70
63.1
70
62.2
70
59.9
70
58.6
70
62.6
70
62.5
CA-14-65
unstratified angular
clasts unit
matrix
Seti Kh.
28.0933
84.0755
73
63.7
CA-16-30
upper part of
Pokhara Fm
silt
Seti Kh. _ south
of Pokhara basin
28.0926
84.0688
73.5
55.1
CA-16-31
unstratified angular
clasts unit
matrix
Seti Kh. _ south
of Pokhara
28.0182
84.0967
83
53.8
CA-13-249
fine conglomerate
lower Trisuli
27.7564
84.4638
157.3
51.5
157.3
52.8
CA-13-252
fine conglomerate
gravels
Narayani at
Narayangad
27.7137
84.4339
matrix
CA-14-69
conglomerate
matrix
Narayani at
Narayangad
27.7140
84.4339
mixed gravels
164
54.5
164
58.4
164
50.4
164
50.4
164
45.6
164
52.5
CA14-69_A conglomerate
gravel
Narayani at
Narayangad
27.7140
84.4339
164
79.1
CA14-69_B conglomerate
gravel
Narayani at
Narayangad
27.7140
84.4339
164
38.7
CA14-69_C conglomerate
gravel
Narayani at
Narayangad
27.7140
84.4339
164
65.2
CA14-69_D conglomerate
gravel
Narayani at
Narayangad
27.7140
84.4339
164
68.7
CA14-69_E conglomerate
gravel
Narayani at
Narayangad
27.7140
84.4339
164
10.5
CA-14-70
sand
Narayani at
Narayangad
27.7140
84.4339
164
48.6
164
47.5
164
45.3
164
45.2
337
73.1
CA-14-71
coarse sand unit
silty unit
silt
Narayani at
Narayangad
27.7140
84.4339
pit near GR2 core (Gandak fan)
GR2 16-5
sediment @ -2.85m
below surface
sand
GR2 16-6
sediment @ -2.45m
below surface
fine sand
-
-
-
337
32.5
GR2 16-7
sediment @ -2m
below surface
micaceous sand
-
-
-
337
18.5
GR2 16-8
sediment @ -1.7m
below surface
silt / fine sand
-
-
-
337
33.7
Dudahi (India)
26.8161
84.1458
GR2 16-9
sediment @ -1.2m
below surface
silt / fine sand
-
-
-
337
41.9
GR2 16-2
sediment @ -0.9m
below surface
silt / fine sand
-
-
-
337
36.6
GR2 16-10
sediment @ -0.65m
below surface
silt
-
-
-
337
44.4
block on surface of
rockslide deposit
Sombre Fm
(Silurian)
TSS Rocks
CA-13-260
Seti (upper)
28.5352
84.0274
90.7
86.6
85.6
CA-13-261
block on surface of
rockslide deposit
Sombre Fm
(Silurian)
Seti (upper)
28.5359
84.0271
48.7
44.9
48.7
CA-13-265
block on surface of
rockslide deposit
Sombre Fm
(Silurian)
Seti (upper)
28.5394
84.0297
41.3
40.7
CA-14-29
morainic block
CA-14-32
morainic block
Sombre Fm
(Silurian)
Sombre Fm
(Silurian)
CA-14-36
glacial striated
surface
lower Nilgiri Fm
(ordovicien)
Upper Marsyandi
28.6069
84.0525
62.4
Upper Marsyandi
28.6042
84.0516
41.4
40.5
Upper Marsyandi
28.6012
84.0508
49.0
51.3
CA-14-38
morainic block
CA-14-40
morainic block
Sombre Fm
(Silurian)
Sombre Fm
(Silurian)
CA-14-44
morainic block
UM-18-3
Upper Marsyandi
28.5977
84.0510
59.3
Upper Marsyandi
28.5957
84.0513
88.1
Sombre Fm
(Silurian)
Upper Marsyandi
28.5985
84.0563
81.9
bedrock
annapurna lm
(cambrien)
Upper Marsyandi
28.5590
84.2220
58.0
UM-18-4
bedrock
annapurna lm
(cambrien)
Upper Marsyandi
28.5758
84.1870
55.2
UM-18-8
bedrock
top of annapurna
lm (cambrien)
Upper Marsyandi
28.5905
84.1949
59.8
UM-18-10
bedrock
top of annapurna
lm (cambrien)
Upper Marsyandi
28.5923
84.1949
60.9
UM-18-11
bedrock
lower Nilgiri Fm
(ordovicien)
Upper Marsyandi
28.6029
84.1742
78.6
UM-18-12
bedrock
Nilgiri Fm
(ordovicien)
Upper Marsyandi
28.6105
84.1514
32.0
UM-18-13
bedrock
top of Nilgiri Fm
(Silurian)
Upper Marsyandi
28.6252
84.1331
56.2
Table SI-13-1: Summary of the CaCO3 content of Sabche rocklside deposit, related sediments further
downstream, modern river sediment of the area, and of the Silurian to Cambrian TSS units typical of
rockslide source.
References:
Aaron, J., & McDougall, S. (2019). Rock avalanche mobility: the role of path material. Engineering Geology, 257,
105126.
Arnold, M., Merchel, S., Bourlès, D. L., Braucher, R., Benedetti, L., Finkel, R. C., ... & Klein, M. (2010). The French
accelerator mass spectrometry facility ASTER: improved performance and developments. Nuclear
Instruments and Methods in Physics Research Section B: Beam Interactions with Materials and Atoms,
268(11-12), 1954-1959.
Auclair, M., Lamothe, M. & Huot, S. 2003: Measurement of anomalous fading for feldspar IRSL using SAR.
Radiation Measurements 37, 487–492.
Balescu, S. & Lamothe, M., 1994: Comparison of TL and IRSL age estimates of feldspar coarse grains from waterlain
sediments. Quat. Sci. Rev. 13, 437-444.
Blair, T. C., & McPherson, J. G. (2009). Processes and forms of alluvial fans. In Geomorphology of desert
environments (pp. 413-467). Springer, Dordrecht.
Buylaert, J.P., Murray, A.S., Thomsen, K.J. & Jain, M. 2009: Testing the potential of an elevated temperature IRSL
signal from K-feldspar. Radiation Measurements 44, 560–565.
Chen, Q., Cheng, H., Yang, Y., Liu, G., & Liu, L. (2014). Quantification of mass wasting volume associated with the
giant landslide Daguangbao induced by the 2008 Wenchuan earthquake from persistent scatterer InSAR.
Remote sensing of environment, 152, 125-135.
Delgado, F., Zerathe, S., Audin, L., Schwartz, S., Benavente, C., Carcaillet, J., ... & Team, A. (2020). Giant landslide
triggerings and paleoprecipitations in the Central Western Andes: The aricota rockslide dam (South Peru).
Geomorphology, 350, 106932.
Durcan, J.A., King, G.E. & Duller, G.A.T. 2015: DRAC: Dose Rate and Age Calculator for trapped charge dating.
Quaternary Geochronology 28, 54–61.
Fort, M. (2000). Glaciers and mass wasting processes: their influence on the shaping of the Kali Gandaki valley
(higher Himalaya of Nepal). Quaternary International, 65, 101-119.
Galbraith, R.F., Roberts, R.G., Laslett, G.M., Yoshida, H. & Olley, J.M., 1999: Optical dating of single and multiple
grains of quartz from jinmium rock shelter, northern Australia: part i, experimental design and statistical
models. Archaeometry 2, 339-364.
Guérin, C. (2017). Effect of the DTM quality on the bundle block adjustment and orthorectification process without
GCP: Exemple on a steep area. In 2017 IEEE International Geoscience and Remote Sensing Symposium
(IGARSS) (pp. 1067-1070). IEEE.Huntley, D.J. & Baril, M.R., 1997: The K content of the K-feldspars being
measured in optical dating or in thermoluminescence dating. Anc. TL 15, 11-13.
Kreutzer, S., Schmidt, C., Fuchs, M.& Dietze, M. 2012: Introducing an R package for luminescence dating analysis.
Ancient TL 30, 1–8.
Murray, A.S. & Wintle, A.G. 2000: Dating quartz using an improved single-aliquot regenerative-dose (SAR) protocol,
Radiation Measurements 32, 57–73.
Pierrot-Deseilligny, M., & Paparoditis, N. (2006). A multiresolution and optimization-based image matching
approach: An application to surface reconstruction from SPOT5-HRS stereo imagery. Archives of
Photogrammetry, Remote Sensing and Spatial Information Sciences, 36(1/W41), 1-5.
Pollet, N., & Schneider, J. L. (2004). Dynamic disintegration processes accompanying transport of the Holocene Flims
sturzstrom (Swiss Alps). Earth and Planetary Science Letters, 221(1-4), 433-448.
Strom, A., Li, L., & Lan, H. (2019). Rock avalanche mobility: optimal characterization and the effects of confinement.
Landslides, 16(8), 1437-1452.
Weidinger, J. T., & Ibetsberger, H. J. (2000). Landslide dams of Tal, Latamrang, Ghatta Khola, Ringmo, and Darbang
in the Nepal Himalayas and related hazards. Journal of Nepal Geological Society, 22, 371-380.
Zangerl, C., Schneeberger, A., Steiner, G., & Mergili, M. (2021). Geographic-information-system-based topographic
reconstruction and geomechanical modelling of the Köfels rockslide. Natural Hazards and Earth System
Sciences, 21(8), 2461-2483.
Zhang, L., Xiao, T., He, J., & Chen, C. (2019). Erosion-based analysis of breaching of Baige landslide dams on the
Jinsha River, China, in 2018. Landslides, 16(10), 1965-1979.
Zhou, T. J., Li, B., Man, W. M., Zhang, L. X., Zhang, J., 2011. A comparison of the Medieval Warm Period, Little Ice
Age and 20th century warming simulated by the FGOALS climate system model. Chinese Science Bulletin 56
(28-29), 3028–3041.
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